Tenascin C in early breast cancer - Helda - University of Helsinki

��

��

��

��

��

To be presented, with the assent of the Medical Faculty of the University of Helsinki, forpublic examination in the Auditorium of the Surgical Hospital, Helsinki University Central

Hospital, Kasarmikatu 11-13, on August 27th, 1999, at 12 noon

�� !!!

Supervised by:

Docent Karl von Smitten, M.D. Department of Surgery, University of HelsinkiDocent Ilkka Saario, M.D. Department of Surgery, University of Helsinki

Reviewed by:

Docent Kaija Holli, M.D. Department of Oncology, University of TampereDocent Ylermi Soini, M.D. Department of Pathology, University of Oulu

Printed version ISBN 952-91-1115-0Tummavuoren Kirjapaino Oy, Vantaa 1999

ISBN 951-45-8679-4 (PDF version) Helsingin yliopiston verkkojulkaisut, Helsinki 1999

To my Family and ”all Rabbit’s friends and relations”

Contents 5

��"#$��%�&�$&��

#'��(')'&�#�*�"#'%��'�&�+++++++++++++++++++++++++++++++++++++++++++++++++++++,

�""($-'��'�&� +++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++.

'&�(��%�'�& +++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++!

($-'$/��$�#'�$(��($+++++++++++++++++++++++++++++++++++++++++++++++++++++++++++ 0

��Intraductal carcinoma (DCIS)..................................................................................................10Lobular carcinoma in situ (LCIS) ............................................................................................10Invasive ductal carcinoma (not otherwise specified) ...............................................................11Invasive lobular carcinoma ......................................................................................................11Other special types of breast carcinoma...................................................................................11

�� Breast-conserving surgery........................................................................................................11Radiotherapy after breast conserving surgery ..........................................................................12Adjuvant chemotherapy ...........................................................................................................12Adjuvant hormonal therapy......................................................................................................13

��

�� Tumour size .............................................................................................................................15DNA flow cytometry................................................................................................................15Ki-67 proliferation antigen ......................................................................................................16erbB-2 oncogene and protein ...................................................................................................16p53 protein...............................................................................................................................17Cathepsin-D .............................................................................................................................17uPA and PAI-1.........................................................................................................................17Therapeutic implications of prognostic factors........................................................................18

� ��!� ��!��"�� #

��#Structure, functions and regulation of Tn-C.............................................................................20Tn-C in embryogenesis ............................................................................................................22Tn-C in reparative processes....................................................................................................22Tn-C in normal, dysplastic and neoplasic tissues.....................................................................23Tn-C and cancer prognosis ......................................................................................................25

�'1��$��2+++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++3,

6 Contents

*��'$&��&��1$��++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++3.

��!�"��"��$�� %&Study I ..................................................................................................................................... 28Studies II and III...................................................................................................................... 28Study IV .................................................................................................................................. 29

��'�!�� %#Tn-C immunohistochemistry (studies I-IV)............................................................................. 29Immunohistochemical analysis of p53 protein, Ki-67 antigen and erbB-2 protein (study II) .. 30DNA flow cytometry (study II) ............................................................................................... 30Immunohistochemical analysis of cath-D , uPA and PAI-1 (study III) ................................... 31ER and PR immunohistochemistry (study IV)......................................................................... 32Ki-67 immunohistochemistry (study IV) ................................................................................. 32Statistical analysis.................................................................................................................... 32

($��#�� ++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++45

�'�!��"��!��(��'��)��"! �* ��

+��(��'��!��!��'��)��"!��*��

,��!��!��!��"��)��"!��!��* ��

��)��"!��!��* �� -

��"��)��"!��!��* �� -

��!��'��)��"! �.* �� -

�'�%��'�&+++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++46

%�&%#��'�&� ++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++4!

��11�(2+++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++50

�%7&�/#$�)$1$&��++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++5

($�$($&%$� ++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++55

Original publications 7

#'��(')'&�#�*�"#'%��'�&�

The present thesis is based on the following original articles that are referred to in the text bytheir Roman numerals.

I Tiina Jahkola, Terttu Toivonen, Karl von Smitten, Carl Blomqvist and Ismo Virtanen: Ex-pression of tenascin in invasion border of early breast cancer correlates with higher risk ofdistant metastasis. Int J Cancer (Pred Oncol) 69: 445-447, 1996.

II Tiina Jahkola, Terttu Toivonen, Ismo Virtanen, Karl von Smitten, Stig Nordling, Kristina vonBoguslawski, Caj Haglund, Heli Nevanlinna and Carl Blomqvist: Tenascin-C expression ininvasion border of early breast cancer - a predictor of local and distant recurrence. Br J Can-cer 78: 1507-1513, 1998.

III Tiina Jahkola, Terttu Toivonen, Karl von Smitten, Veli-Matti Wasenius and Carl Blomqvist:Cathepsin D, urokinase plasminogen activator and type-1 plasminogen activator inhibitor inearly breast cancer - an immunohistochemical study of prognostic value and relations totenascin-C and other factors. Br J Cancer 80: 167-174, 1999.

IV Tiina Jahkola, Terttu Toivonen, Stig Nordling, Karl von Smitten and Ismo Virtanen: Expres-sion of tenascin-C in intraductal carcinoma of human breast: relationship to invasion. Eur JCancer 34: 1687-1692, 1998.

8 Abbreviations

�""($-'��'�&�

Term Explanation

BM basement membrane

cath-D cathepsin-D

CI confidence interval

CMF cyclophosphamide methotrexate fluorouracil

DCIS carcinoma ductale in situ, intraductal carcinoma

DI DNA index

DNA deoxyribonucleic acid

ECM extracellular matrix

EGF epidermal growth factor

EIC extensive intraductal component

ER oestrogen receptor

Gy gray

HR hazard ratio

LRFS local recurrence-free survival

MAb monoclonal antibody

MFS metastasis-free survival

mRNA messenger ribonucleic acid

PAI-1 type-1 plasminogen activator inhibitor

PR progesterone receptor

s-phase synthesis phase

SPF synthesis-phase fraction

TGF-β transforming growth factor-β

Tn-C tenascin-C

TNM tumour node metastasis (staging)

uPA urokinase plasminogen activator

Introduction 9

'&�(��%�'�&

Improved diagnostics and population-based mammography screening programmes have changedthe profile of breast cancer. Tumours are treated at an early stage, when no lymph node metasta-ses are found. The majority of these women will be cured with surgery alone but 10-30% willeventually die of the disease (EBCTCG, 1992). Adjuvant treatment improves the prognosis but isuseful only for those who would have a recurrence, if treated with surgery without chemotherapyor hormonal therapy (Johnson� �� , 1998). Testing of the biological features of tumours asprognostic factors has been one of the main focuses in clinical breast cancer research, but noconsensus has been established about their use in treatment decisions. At present, it is not possi-ble to identify those patients who are already cured without adjuvant chemotherapy (Powles,1997; Dowsett, 1998).

As a result of mammography, the incidence of ductal carcinoma in situ (DCIS), a precursor ofbreast carcinoma, appears to be increasing. Traditionally DCIS was treated by mastectomy, butchanging trends in the treatment of breast cancer have raised the question of whether DCIS canbe further classified to find a treatment that is not too radical or noxious for a single patient (Sil-verstein, 1997).

This thesis was first designed in 1991 to study and compare multiple tumour-related characteris-tics as prognostic factors in patients treated uniformly for axillary node-negative breast cancer. Aseries of women had all undergone breast-conserving surgery and post-operative radiotherapy,but none had received adjuvant hormonal or chemotherapy.

We had a specific interest in the novel extracellular matrix (ECM) glycoprotein tenascin-C (Tn-C). The distinct, spatially and temporally restricted, expression of Tn-C had been characterizedduring mammary development (Inaguma� �� , 1988), during wound healing (Mackie� �� ,1988b) and in normal, hyperplastic and neoplastic breast tissue (Howeedy� �� , 1990). Tn-Cwas considered to be a product of fibroblasts and to have a role in epithelial-mesenchymal inter-actions (Sakakura��, 1991). Our aim was to quantify the expression of Tn-C in early breastcarcinomas and to study its relationship to other histopathological and prognostic parameters andfinally to disease outcome. We consistently observed a strong Tn-C immunoreactivity around theearly invasive nests in areas of the intraductal component. This led us to extend the study toductal carcinomas in situ.

Review of the literature10

($-'$/��$�#'�$(��($

"��Breast cancer is the leading type of cancer in women in the western world, constituting 20-25%of all cancer cases in women and causing 15-20% of all cancer deaths, or 2-5% of deaths fromall causes. The life time cumulative risk of developing breast cancer is now 10% (Blichert-Toft�� , 1997). In Finland, the 5-year relative survival rate has improved with time, being 79%among patients diagnosed in 1985-89 (Pukkala �� 1997).

Breast cancers are derived from the epithelial cells that line the lobular unit of the terminal duct.Cancer cells that remain within the basement membrane and the draining duct are classified as insitu or non-invasive. An invasive breast cancer is one in which there is dissemination of cancercells outside the basement membrane of the ducts and lobules into the surrounding adjacentnormal tissue. Both in situ and invasive cancers have characteristic patterns by which they can beclassified (Sainsbury��, 1994).

Intraductal carcinoma (DCIS)Intraductal carcinoma, �� or ductal carcinoma in situ (DCIS) is consid-ered a true precursor of invasive carcinomas. It is often noticed in a mammogram as microcalci-fications, and its incidence has risen now accounting for 15-20% of breast cancer cases com-pared with about 5% before the advent of cancer screening programmes (Elston and Ellis, 1998).

DCIS is classified into subtypes on a morphological basis. These subtypes include micropapil-lary, cribriform, comedo, solid and mixed types (Rosen, 1997). New classifications based onnuclear grade and the presence or absence of central necrosis have been presented for clinicalpurposes. (Silverstein et al., 1995, 1996; Silverstein, 1997, 1998; Elston and Ellis, 1998). Hol-land’s group has shown that DCIS is nearly always a unicentric process involving a single ductsystem. This emphasizes the importance of adequate excision and evaluation of excision marginsto cure the patient, leaving no risk of local recurrence or progression to systemic disease (Elstonand Ellis, 1998).

DCIS is called microinvasive when there are one or more clearly separate foci of infiltration ofnon-specialized interlobular stroma, none of which measures more than 1 mm (Elston and Ellis,1998). Areas of DCIS, an intraductal component, can often be observed inside an invasive carci-noma. When this intraductal component comprises 25% or more of the tumour area encompassedby the infiltrating tumour and when DCIS is present in the surrounding tissue also, it is called anextensive intraductal component (EIC) (Schnitt��, 1987).

Lobular carcinoma in situ (LCIS)LCIS is a proliferative lesion of small cells within breast lobules that is occasionally found inbreast biopsies. LCIS has been found to indicate an increased risk of about 10% of any particulartype of breast cancer that may develop in either breast within the next 10-15 years (Elston andEllis, 1998).

Review of the literature 11

Invasive ductal carcinoma (not otherwise specified)Invasive ductal breast carcinoma accounts for 41-75% of the published series. It is the mostcommon histological type of breast cancer. Its morphological features and cell size vary consid-erably and grading systems have been adopted to classify subtypes (Elston and Ellis, 1998).Grading was originally presented by Bloom and Richardson (Bloom and Richardson, 1957). It isbased on three parameters: tubule formation, nuclear pleomorphism and mitotic count, each ofwhich is scored on a scale of 1 to 3, and the total count is divided into grade 1 (3-5 points), grade2 (6-7 points) and grade 3 (8-9 points). Elston and Ellis have added a quantification for tubuleformation to make grading more standardized (Elston and Ellis, 1998). Though the assessment ofgrades is subjective and the methodology is not uniform, grading provides important prognosticinformation at all stages of ductal breast carcinoma (Roberti, 1997).

Invasive lobular carcinomaInvasive lobular carcinoma represents up to 15% of all breast cancers. The cancer cells are typi-cally small and the growth pattern is infiltrative (Elston and Ellis, 1998). Lobular carcinoma isalmost always steroid-receptor positive (Sastre-Garau��, 1996) and is associated with bettersurvival than invasive ductal carcinoma (Toikkanen��, 1997).

Lobular carcinoma is often difficult to detect either clinically or radiologically owing to its in-sidious growth pattern. It is often multicentric and may be bilateral (Simpson and Wilkinson,1998). However, it is considered suitable for breast-conserving treatment (Holland��, 1995;Sastre-Garau ��, 1996).

Other special types of breast carcinomaMedullary carcinoma is found in up to 10% of symptomatic breast cancers. Invasive cribriformcarcinoma, mucinous carcinoma and invasive papillary carcinoma all account for 1-4% of breastcarcinomas. Tubular carcinoma was previously considered rare, but in cases detected by mam-mography it is now found in 9 to 19% of all carcinomas. A common feature of these breast can-cer types is that at diagnosis they are often axillary node-negative and have a good or excellentprognosis (Elston and Ellis, 1998).

��8��

Breast-conserving surgeryThere are three main surgical options for invasive breast cancer: breast-conserving surgery, in-cluding axillary dissection with subsequent radiotherapy, total mastectomy with �� axillarydissection or total mastectomy with �� axillary dissection followed by immediate or de-layed reconstruction (Blichert-Toft ��, 1997).

As long ago as the 1930’s, Mustakallio was a pioneer in breast-conserving surgery (Rissanen,1969; Mustakallio, 1972). However, breast-conserving surgery was not widely accepted until themid 1980’s, when Fisher and Veronesi published the results of two large prospective randomizedtrials comparing breast-conserving surgery with radical surgery (Fisher��, 1985; Veronesi��, 1986). Later reports made breast-conserving surgery an established treatment modality forbreast cancer (Fisher��, 1989; Veronesi��, 1989; Blichert-Toft��, 1992; van Dongen��, 1992).

12 Review of the literature

Nowadays it is the treatment of choice for women with a tumour limited to one quadrant and notconsisting of extensive intraductal growth. An additional requirement is that a good cosmeticresult is achievable (Osteen, 1994). Young age, tumour size, the presence of a carcinoma at theresection margin and the presence of EIC in the tumour are associated with a higher risk of localrecurrence after breast-conserving surgery (Schnitt ��, 1987; Locker��, 1989; Boyages��, 1990; Holland��, 1990b; Borger��, 1994; Gage��, 1996).

Small tumour size, low grade and old age have been proposed as selection criteria to treat a sub-group of patients with surgery alone without postoperative radiotherapy (Clark� �� , 1992;Veronesi� �� , 1993; Liljegren� �� , 1994). However, a prospective clinical trial had to beclosed prematurely because the local recurrence rate rose to 16% in the group that did not re-ceive radiotherapy (Schnitt� �� , 1996). Adjuvant radiotherapy significantly improves localcontrol (Clark ��, 1992; Veronesi ��, 1993; Liljegren ��, 1994; EBCTCG, 1995; Fisher��, 1995a) and, for the present, is considered an essential adjunct to breast-saving surgery forall patients (Morrow��, 1995).

No significant difference in mortality was detected between the three groups of women treatedfor stage I and II breast cancer with either mastectomy, breast-conserving surgery and axillarydissection combined with postoperative radiotherapy, or breast-conserving surgery and axillarydissection without radiotherapy (Fisher �� , 1985; EBCTCG, 1995; Fisher �� , 1995a).Therefore, local recurrence was considered a fairly benign event although usually leading even-tually to breast loss. However, recent data report increased mortality among women with ipsilat-eral breast cancer recurrence (Whelan��, 1994; Kemperman��, 1995, Fortin ��, 1999).In women with an early local recurrence, breast cancer seems to be an aggressive disease withpoor prognosis, whether treated by mastectomy or by breast-conserving surgery (Tienhoven� ��, 1999). These results emphasize the importance of local control.

Radiotherapy after breast conserving surgeryA meta-analysis of 36 randomized studies revealed that adjuvant radiotherapy after breast con-servation reduces the rate of local recurrences to 6.7%, whereas with surgery alone the rate is19.6% (Fisher ��, 1995a). Radiation therapy also carries a small but important survival benefitfor women treated for axillary lymph-node-negative breast cancer with breast-conserving surgery(Levitt��, 1996). Post-operative radiotherapy is not administered after mastectomy for T1N0breast cancer (Finnish Breast Cancer Group, 1999).

Radiotherapy is usually administered on the breast at a dose of 50 Gy in 25 fractions during aperiod of 5 weeks. The patient has to commit herself to visit the clinic 5 days a week for 5weeks, which entails costs and may not be possible for some women, who therefore choosemastectomy rather than breast conservation.

The risk of a secondary sarcoma as a consequence of radiotherapy is fairly small, about nine per100 000 person-years (Taghian� �� , 1991). Common complications after breast-conservingtreatment include breast skin fibrosis in 19/82 patients (23%), breast oedema in 13 (16%), armoedema in 10 (12%), and pigmentation changes, teleangiectasiae or skin dimpling in 9 (11%)(Hunt��, 1998).

Adjuvant chemotherapyAdjuvant polychemotherapy targeted to micrometastatic disease with CMF (cyclophosphamide,methotrexate, fluorouracil) was shown to be effective as early as 1977 (Bonadonna��, 1977).


Adjuvant chemotherapy decreases recurrences and improves survival in women with axillarynode-positive cancer but also in node-negative patients (EBCTCG, 1992). The absolute benefitof adjuvant cytotoxic therapy on 10-year survival of women treated for node-negative breastcancer was 7%, and 11% for those with node-postive disease. The reduction in recurrences was35% in women under 50 years of age at randomization and 20% in women aged 50-69(EBCTCG, 1998a).

Adjuvant systemic therapy also reduces the rate of local recurrences after breast saving surgeryand radiotherapy (Haffty��, 1991). The local recurrence rate at 12 years was 5% in womenwho had received adjuvant systemic therapy compared with 10% in women treated with localsurgery and postoperative radiotherapy without chemotherapy (Fisher �� , 1995a). Adjuvantchemotherapy reduced the 10-year rate of development of new primary breast tumours from 6%to 1% (Arriagada and Rutqvist, 1991).

The adjuvant chemotherapy regimen with CMF, which is the treatment most widely used, causesfew toxic effects compared with other cytotoxic agents. Of 360 women treated, 18% had vomit-ing (grade 2 or more), 40% complete hair loss, 1,1% were admitted to hospital for febrile neu-tropenic episodes and one patient developed congestive heart failure (Levine��, 1998).

Adjuvant hormonal therapyPostmenopausal women have long been known to benefit from adjuvant tamoxifen treatment. Arecent report of the updated overview of 55 randomized trials revealed that all premenopausalwomen with oestrogen receptor-positive tumours also benefit from tamoxifen, and this effect isadditional to the effect of adjuvant chemotherapy (EBCTCG, 1998b). For trials of adjuvant tam-oxifen for 1 year, 2 years, and 5 years, the proportional reductions in recurrence produced among30 000 women during 10-year follow-up were 21%, 29% and 47%. The corresponding propor-tional mortality reductions were 21%, 17% and 26%, respectively. The proportional reductionsin mortality were similar for women with node-positive and node-negative diseases (EBCTCG,1998b).

The antitumour effects of tamoxifen are thought to be due to its anti-oestrogenic activity, medi-ated by competitive inhibition of oestrogen binding to oestrogen receptors. This effect is mostbeneficial in women whose tumours contain oestrogen receptors, i.e. ER (and/or PR) is a strongpredictive factor for anti-oestrogen treatment (Osborne, 1998).

The oestrogenic properties of tamoxifen are considered to account for its beneficial effects inlowering serum cholesterol and low-density lipoproteins, which are thought to reduce the rate ofatherosclerosis. Tamoxifen increases bone density in postmenopausal women, but rates of bonefractures in different studies are inconsistent (Osborne, 1998). The incidence of endometrialcancer was approximately doubled after 1 and 2 years of adjuvant tamoxifen treatment, andquadrupled after 5 years of treatment. However, the absolute incidence of contralateral breastcancer diminished more than the incidence of endometrial cancer increased (EBCTCG, 1998b).

The acute side effects of tamoxifen include menopausal symptoms such as hot flushes, atrophicvaginitis and irregular menses. Thromboembolic phenomena, often superficial thrombophlebitis,are reported in fewer than 1% of patients treated with adjuvant tamoxifen (Osborne, 1998).

��%'�At present, there is no consensus as to the best treatment of in-situ lesions. In DCIS a local exci-sion is not appropriate for extensive lesions (Blichert-Toft ��, 1997). Prognostic classifica-


tions have been developed and proposed for treatment selection (Holland��, 1990a; Silver-stein��, 1995, 1996; Silverstein, 1997, 1998). Patients suitable for breast-conserving surgeryalone are those who have low-grade lesions with little or no necrosis and in whom the surgicalmargins are clear. If a patient with risk factors for local recurrence chooses breast conservationover mastectomy, radiation added to surgery is recommended (Boyages��, 1999). Radiother-apy reduces local recurrences in DCIS (Fisher� �� , 1995b; Marks and Prosnitz, 1997). Pro-spective multicentre trials are in progress to search for treatment criteria to find out who shouldbe treated with resection only, who needs post-operative radiotherapy and who should undergomastectomy (Elston and Ellis, 1998). Axillary dissection is not indicated for women with pureDCIS (Silverstein��, 1991; Blichert-Toft ��, 1997).

*��Prognostic factors are used to predict the clinical course of breast cancer at the time of primarytreatment. They help to select the appropriate treatment for individual patients, to allow compari-sons of treatments between groups of patients at similar risk of recurrence or death, and to im-prove our understanding of breast cancer, which may permit the development of new strategiesor treatments. They can be broadly classified into chronological and biological factors. Chrono-logical factors are indicators of how long the cancer has been present; they relate to the stage ofthe disease at presentation. Biological factors relate to the intrinsic or potential behaviour of thetumour. In addition, patient related factors like age at diagnosis may be a risk factor (Miller��, 1994).

There are three major reasons for the use of prognostic factors. The first is to identify patientswhose prognosis is so good that adjuvant therapy after local surgery would not be ‘cost-beneficial’. The second is to identify patients whose prognosis is so poor that a more aggressiveadjuvant approach would be warranted. The third is to identify patients likely to be responsive orresistant to particular types of therapy. (Clark, 1994) The factors that are predictive of sensitivityto adjuvant treatment are called predictive factors (Gasparini��, 1993).

McGuire has presented guidelines for the evaluation of a biological prognostic factor. The factorstudied should possess a clear biological significance and the hypothesis to be tested should bedefined. Next, the investigator should state whether the study is a pilot study, a definitive studyor a confirmatory study. One should not try to draw conclusive clinical implications from theresults of a pilot study. The pilot study can generate estimates of the magnitude of the contribu-tion of a given factor, the extent of correlation of that factor with other prognostic factors, thedistribution of that factor in the population, the length of follow up and the number of recur-rences and deaths. These estimates are useful for a final, definitive study, for which the patientpopulation must be large enough, unbiased and appropriate, e.g. consisting entirely of node-negative patients, if the pilot study shows effect on node-negative patients. The laboratory meth-odology has to be validated, and assignment of cut-off values to dichotomize the final assayresult as yes/no, high/low, etc., for clinical purposes is often needed. And, finally, a confirmatorystudy is needed to test the factor’s reproducibility (McGuire, 1991).

There are many inconsistencies between the results of studies measuring the same prognosticfactor. This may be due to the use of different antibodies, different definitions for scoring ordifferent methods of tissue preparation (fresh/frozen vs. fixed) (Ravdin and Chamness, 1995).Storage of paraffin slides has been shown to diminish immunoreactivity (Jacobs� �� , 1996).Lack of consistency among the cut-off values chosen by laboratories also affects the results.


Instead of using “optimal” cut-off points to categorize populations as low and high risk groups,Altman et al. recommend the use of multiple prespecified cut-off points, so as not to lose infor-mation in the statistical analysis (Altman��, 1994). Cantor and Shuster emphasize that, beforecategorization, a continuous variable has to be shown to be prognostic, e.g. by Cox regression,and they then propose subdivision by effect size (hazard ratio) as a superior strategy to avoidbiases (Cantor and Shuster, 1994). Recently, artificial neural networks have been developed incomputer systems to improve the accuracy of cancer survival prediction (Burke��, 1998).

In prognostic studies, different end-points are usually encountered. Disease-free survival (DFS)and relapse-free survival (RFS) refer to the time between the original treatment and the diagnosisof disease recurrence. Local relapse-free survival (LRFS) and metastasis-free survival (MFS)specify these different events and are more accurate, when studying patients treated with breast-conserving surgery. Overall survival (OS) has death as the end-point.

Tumour sizeA fairly safe patient group to treat with surgery only are women, whose primary tumour is 10 mmor less in diameter. In this group, only 1/47 patients died during the 5-year follow-up. In node-negative patients whose tumours were 11 to 20 mm, the 5-year survival rate was 96%, whereas inthe node-positive group it was 62% (Joensuu and Toikkanen, 1991). In a node-negative smalltumour (<10 mm) population, the 10-year relapse-free survival was 85% for women 50 years orolder compared with 75% for women younger than 50 years (Fentiman��, 1996). During a20-year follow-up of node-negative patients, the relapse-free survival in tumours 10 mm or lesswas 86% compared with 69% in tumours 11-20 mm (Rosen��, 1989).

However, a good prognosis is related not only to tumour size, but rather more to axillary lymphnode status. The 5-year survival rate in women with node-negative tumours less than 2 cm indiameter was 96.3%, in those with 2-5 cm tumours 89.4% and in those with tumours larger than5 cm 82.2%, while the corresponding rates for women with 1-3 positive lymph nodes were87.4%, 79.9% and 73.0%, and the rates for those with more than 4 positive lymph nodes were66.0%, 58.7% and 45.5% (Carter��, 1989).

Besides tumour size, histology and grade, the possible vascular invasion is frequently included inthe pathology report, making no distinction between lymphatic and blood vessel invasion (Elstonand Ellis, 1998). Vascular invasion was significantly associated with survival in multivariateanalysis of 1704 consecutive invasive T1 and T2 breast carcinomas. It was also a predictor oflocal recurrence in patients treated by mastectomy or by wide local excision (Pinder��, 1994).

DNA flow cytometryThe method of assessing DNA content (ploidy) in paraffin-embedded tissues was presented byHedley (Hedley��, 1983). The normal chromosomal DNA content is diploid in post-mitoticcells (2 sets of 23 individual chromosomes in man) and this is expressed as a DNA index of 1(DI=1). When the DI differs from one, the tumour is aneuploid. Flow cytometry also providesinformation on the proliferative activity of a tumour in terms of the S-phase fraction (SPF). SPFis the fraction of cells with a DNA content corresponding to DNA-synthesizing cells.

In breast cancer, aneuploidy is linked with an adverse prognosis (Eskelinen� �� , 1989).Theprognostic values of DI and SPF are increased by combining the two parameters (Kallioniemi��, 1988; Joensuu��, 1990). Flow cytometry performed on 395 node-negative tumours dis-tinguished a low-risk diploid group with a 5-year disease-free survival of 88% and a high-risk


aneuploid group with 74% disease-free survival. Additionally, patients with diploid tumours andlow SPF had a 90% probability of disease-free survival, as compared with 70% in those withdiploid tumours and high SPF (Clark��, 1989). A similar observation of high SPF in diploidtumours was made in another study on breast cancer (Romero��, 1996) and in a study on softtissue sarcomas (Huuhtanen� �� , 1996). Discrepancies between some studies may be due tointerlaboratory differences (Kallioniemi��, 1990) or to differing cut-off values for SPF (Alt-man ��, 1994).

Ki-67 proliferation antigenThe Ki-67 proliferation antigen is an alternative to SPF for evaluating the proliferation activityof tumours (Isola��, 1990; Gasparini��, 1992). Ki-67 is detected with immunohistochem-istry that requires only one 5-µm-thin slice of tissue and saves material for other diagnostic andprognostic purposes. This is an advantage in small tumours. In addition, the Ki-67 antigen can bestained in cytological specimens and touch imprints (Schwarting, 1993). The Ki-67 antibodydetects a non-histone protein that is present in the cell nucleus in all phases of the cell cycleexcept G0 and early G1. It was first described in 1983 (Gerdes��, 1983).

The prognostic value of Ki-67 in breast cancer has been shown in several studies (Gasparini ��, 1992; Railo��, 1993; Veronese��, 1993; Haerslev��, 1996). The disease-free sur-vival in 212 T1 node-negative patients followed for 8 years was significantly poorer in Ki-67-positive tumours (cut-off 10% or more of tumour cells positive) (Railo��, 1997)

erbB-2 oncogene and proteinThe erbB-2 (neu / HER-2) gene belongs to a family of genes whose protein products are calledreceptor tyrosine kinases. These are transmembrane proteins that participate in extracellularsignal transduction by acting as receptors for a number of peptide growth factors (Sherbet andLakshmi, 1997). The erbB-2 gene is amplified and the protein can be observed by immunohisto-chemistry in about 20% of breast carcinomas (Callahan, 1989; Paik� �� , 1990). There is astrong correlation between gene amplification and protein overexpression for erbB-2 (Ciocca��, 1992).

The first study to show prognostic power for erbB-2 gene amplification in node-positive but notin node-negative breast cancer was published in 1987 (Slamon��, 1987). Since then, numer-ous studies have been performed on the prognostic usefulness of erbB-2, most of them showingerbB-2 to be a strong prognostic factor for overall survival in axillary-node-positive patients(Ravdin and Chamness, 1995). In 174 node-negative patients, erbB-2 overexpression shown byimmunohistochemistry correlated with overall survival in both univariate and multivariate analy-sis (Kallioniemi��, 1991). Ravdin collected results from 11 studies on the prognostic value oferbB-2 immunohistochemistry in node-negative breast cancer, each consisting of more than 100patients and with follow-ups lasting for more than 3 years. In five studies there was a positivecorrelation between erbB-2 expression and prognosis, whereas in six studies no correlation couldbe observed (Ravdin and Chamness, 1995). Recently, a significant association between erbB-2gene amplification and adverse prognosis in axillary node-negative breast carcinomas has beenshown with sufficient patient numbers and follow-up (Press��, 1997; Andrulis��, 1998).

Nevertheless, erbB-2 may have its use as a predictive marker. erbB-2 positive tumours may beresistant to adjuvant treatments with CMF or tamoxifen (Ravdin and Chamness, 1995) but sensi-tive to anthracyclines (Muss��, 1994).


p53 proteinThe normal p53 gene codes for a tumour suppressor protein, that is found only at low levelswhich are not detectable by immunohistochemistry. The function of p53 is to police the integrityof the genome by promoting apoptosis of cells with damaged DNA or to stop the cell from pro-ceeding into the DNA replication phase, thus allowing DNA repair. The p53 gene is mutated inhalf of all malignant tumours and in 40% of breast carcinomas. Mutation of the p53 gene leads tooverexpression of the protein, with a prolonged half-life, and loss of its tumour-suppressiveproperties (Levine��, 1994; Carson and Lois, 1995; Sherbet and Lakshmi, 1997).

p53 overexpression, detected by immunohistochemistry, has been shown to be a prognostic fac-tor for disease-free survival in node-negative breast cancer in several reports (Isola��, 1992;Thor��, 1992; Allred��, 1993; Silvestrini��, 1993; Gasparini��, 1994). However,Rosen did not find p53 to be a reliable prognostic factor in 440 node-negative breast cancerpatients followed for 10 years (Rosen��, 1995). The different storage times of sample slides(Jacobs ��, 1996) and different antibodies used (Jacuerner��, 1994) may explain some ofthe inconsistencies between studies using p53 immunohistochemistry.

Cathepsin-DCathepsin-D (cath-D) is an aspartic lysosomal proteinase that is active at acidic pH. Cath-D issuggested to have active functions in carcinomas either as a proteolytic enzyme or a mitogen(Westley and May, 1996). High concentrations of cath-D in breast cancer tissue extracts havebeen shown to correlate significantly with a poor prognosis in numerous studies (Thorpe��,1989; Duffy��, 1991; Kute��, 1991; Namer��, 1991; Duffy��, 1992; Pujol��,1993; Fernö��, 1994), also in patients with node-negative breast cancer (Tandon��, 1990;Granata��, 1991; Spyratos��, 1992).

Studies using immunohistochemistry to detect and quantify cath-D in breast carcinomas haveyielded somewhat controversial results. Henry found, quite unexpectedly, a correlation betweencath-D expression in malignant epithelial cells and a good prognosis (Henry� �� , 1990). Noassociation was found between the cath-D content in carcinoma cells and the prognosis in severalother studies using immunohistochemistry (Domagala��, 1992; Kandalaft��, 1993; Win-stanley��, 1993; Armas��, 1994; Castiglioni��, 1994; Ravdin��, 1994; Alo��,1996). However, cath-D expression in the carcinoma cells (n=95/262) in node-negative patientswas significantly correlated with shortened disease-free survival and overall survival during an 8-year follow up (Isola��, 1993). Another study correlated intense cath-D staining in carcinomacells with an adverse prognosis in node-positive but not in node-negative patients (Aaltonen��, 1995). The stromal fibroblasts and macrophages may also stain for cath-D and an associationbetween this stromal expression (but not cancer cell expression) and a high risk of disease recur-rence has been shown in three studies (Tetû��, 1993; Joensuu��, 1995; O’Donoghue��, 1995). A recent meta-analysis finds a relationship between high cath-D values and less fa-vourable prognoses in node-negative breast carcinoma (Ferrandina��, 1997).

uPA and PAI-1Urokinase plasminogen activator (uPA) is a serine protease which activates zymogen forms ofother proteases such as plasmin and gelatinase A (also called MMP-2 or type IV collagenase)and these both degrade type IV collagen (Liotta��, 1981; Dano��, 1985; Keski-Oja��,1992), one of the main constituents of the basement membranes. Type-1 plasminogen activatorinhibitor (PAI-1) is a specific inhibitor of uPA. uPA and PAI-1 are both overexpressed in breast


cancer tissue compared with the normal breast and benign breast conditions (Foucré��, 1991;Reilly��, 1992; Costantini��, 1996).

Several studies have demonstrated that increasing amounts or activity of uPA and PAI-1 areassociated with aggressive disease and a poor prognosis in breast cancer (Duffy� �� , 1988;Jänicke��, 1989; Foekens��, 1992; Spyratos ��, 1992; Grondal-Hansen� ��, 1993;Jänicke��, 1993; Bouchet��, 1994; Foekens��, 1994; Grondal-Hansen��, 1997).However, few prognostic studies using immunohistochemistry have been published. Overexpres-sion of uPA in Dukes’ B colorectal carcinomas was associated with a poor prognosis (Mulcahy��, 1994), as was the overexpression of either uPA or PAI-1 in renal carcinomas (Hofmann��, 1996). A study of breast cancer noted a non-significant association between immunohisto-chemically detected high levels of uPA and prognosis, but a significant association between cath-D (both stromal and carcinoma cell expression) and disease-free survival (Göhring��, 1996).

Therapeutic implications of prognostic factorsAn axillary lymph node metastasis during surgery is an indication for adjuvant treatment. Fornode-negative patients, several prognostic indices or risk groups have been presented. However,the recent update of the American Society of Clinical Oncology guidelines on use of tissue tu-mour markers in the management of patients with breast cancer recommended the measurementof only ER and PR (ASCO, 1998).

The recent European risk categorization into low, intermediate and high risk groups is based ontumour size, steroid receptor status, grade and age. Low-risk patients are 35 years or older andhave a grade-1 steroid receptor-positive tumour that is one centimeter or smaller. High-risk pa-tients have at least one of the listed factor: tumour larger than 2 cm, grade 2-3, steroid receptor-negative tumour or age younger than 35. Adjuvant chemotherapy is recommended for all high-risk patients except for steroid-receptor-positive elderly women, who are offered tamoxifenalone. Adjuvant tamoxifen treatment (for 2 years) is recommended for all those women whosecancer is either ER- or PR-positive, and the disease carries an intermediate or high risk of recur-rence (Goldhirsch��, 1998).

Measurement of erbB-2 expression for prediction of response to anthracycline-containing che-motherapy and for resistance to treatment with tamoxifen or CMF is considered to require furtherprospective verification, because currently available information is derived exclusively fromretrospective studies (Goldhirsch ��, 1998). Nevertheless, the evidence that tumours with highlevels of erbB-2 protein respond better to doxorubicin is mounting and erbB-2 is coming intoclinical use in many centres. There is still a lack of standards, and a commonly approved meth-odology for erbB-2 detection is needed (McNeil, 1999).

In spite of the vast quantity of scientific data on cancer biology, disappointingly few tumourmarkers are useful in treatment decisions. Many of these tumour markers are prognostic, but theydo not bring additional information to the classical prognostic factors, i.e. axillary nodal status(N), tumour size (T), tumour grade, steroid receptor status (ER and/or PR) and patient age. Still,a few tumour-related markers are predictive of a treatment response. Some tumour markers mayalso be useful in cancer diagnostics and surveillance. In addition, prognostic studies illustrate theimpact of a marker on tumour character and behaviour. Ultimately, a biological tumour markercan be the target of new therapies: for example, erbB-2 is the target of the anti-HER2/neu anti-body, which has been successfully used in combination with cytotoxic cisplatin therapy againstmetastatic breast cancer (Pegram��, 1998).


$%1�� "1�� Tissues are made up of cells and an extracellular matrix (ECM), which is composed of an intri-cate network of macromolecules. These macromolecules are secreted locally by cells of the tis-sue, often by fibroblasts. The ECM constituents can be divided into glycosaminoglycans (whencovalently linked with proteins, proteoglycans), and fibrous proteins of two functional types:mainly structural (e.g. collagen and elastin) and mainly adhesive (e.g. fibronectin and laminin).Fibronectin promotes the attachment of fibroblasts and various other cells to the matrix in con-nective tissues, while laminin promotes the attachment of epithelial cells to the basement mem-brane (BM). Epithelial cells bind to each other mainly by transmembrane proteins, cadherins.The cell-to-matrix adhesions are mediated by integrins, also transmembrane proteins of the cells.Fibronectin and laminin are both important for cell attachment, cell migration, differentiation andmorphogenesis (Alberts��, 1994). The ECM not only functions as a structural support, butalso plays an active role in differentiation (Hay, 1993), tissue repair (Yamada and Clark, 1996)and cancer invasion (Ruoslahti, 1992).

In epithelial tissues, the ECM consists of the BM, which is situated next to the epithelium, andthe loose connective tissue that lies beneath the BM. The BM is largely synthesized by the cellsthat rest on it; it consists of type IV collagen, the large heparan sulfate proteoglycan perlecan andthe glycoproteins laminin and entactin. (Alberts ��, 1994)

Malignant epithelial neoplasia are divided into in situ and invasive carcinomas by the morpho-logical distinction that in carcinomas in situ the BM seems continuous in light microscopy(Rosen, 1997). Invasion of the ECM can be resolved into four steps: detachment of the tumourcells from each other, attachment of the tumour cells to ECM components, degradation of theECM and migration of the tumour cells (Kumar� ��, 1997). During invasion, the BM is de-graded by cascades of proteolytic enzymes (Mignatti and Rifkin, 1993) but cancer cells may alsodeposit or induce stromal deposition of BM material (Bosman, 1994; Tani��, 1997).

Tumours of epithelial origin, i.e. carcinomas, can be divided into two discrete but interdependentcompartments, the malignant cells themselves and the stroma. The tumour stroma differs fromnormal connective tissue and resembles the aggregations of mesenchyme observed during mor-phogenesis (Birchmeier and Birchmeier, 1993) or the granulation tissue that forms during woundhealing (Dvorak, 1986). ECM remodelling takes place in all these three situations (Koukoulis��, 1993).

The complex cell-matrix and cell-cell interactions of the normal breast are slightly altered inhyperplastic processes and benign neoplasms whereas profound changes occur in carcinomas(Koukoulis��, 1991b; Koukoulis ��, 1993). Concomitantly, tenascin-C (Tn-C) immunore-activity which is noted in the periepithelial stromal regions of adult resting and aging breast ductsand acini, is moderately enhanced in physiological conditions such as foetal growth and gesta-tion, as well as hyperplasias, dysplasias and benign tumours, whereas it is markedly enhanced inintraductal and infiltrating carcinomas (Howeedy ��, 1990).

��9%Tenascin (Tn-C) is a very large multidomain ECM glycoprotein which is expressed in areas ofepithelial-mesenchymal interaction in various tissues during embryogenesis, reparative processesand neoplasia (Shrestha and Mori, 1997). The protein was first described as long ago as in 1975(Yamada��, 1975). Almost 10 years later several independent groups rediscovered tenascin(Sakakura ��, 1991). It was called glioma mesenchymal extracellular matrix antigen (GMEM)(Bourdon� �� , 1983), myotendinous antigen (Chiquet and Fambrough, 1984a; Chiquet and


Fambrough, 1984b), hexabrachion protein (Erickson and Inglesias, 1984), J1 glycoprotein(Kruse��, 1985) and cytotactin (Grumet��, 1985).

The name tenascin was proposed by Ruth Chiquet-Ehrismann and co-workers in 1986. It is acombination of two Latin verbs “tenere” to (hold) and “nasci” (to be born), which provided theroots for the English words “tendon” and “nascent”. These describe its location as a myotendi-nous antigen in tendon development, and its developmental expression (Chiquet-Ehrismann� ��, 1986).

The first member of the tenascin family is named tenascin-C (Tn-C, C for cytotactin), since othertenascins have also been described (Erickson, 1993; Vollmer, 1997; Kusakabe and Sakakura,1998). Tn-R (earlier restrictin (Rathjen� �� , 1991) or J1-160/180 (Pesheva� �� , 1989)) isrestricted to the central nervous system and is most prominent in development (Carnemolla��,1996). Tn-X , the largest tenascin glycoprotein, has been found in foetal tissues (Bristow��,1993; Burch��, 1995). Tn-X has an essential role in connective tissue and its deficiency isassociated with the Ehlers-Danlos syndrome (Burch��, 1997). Recently, a new family mem-ber, Tn-Y has been cloned. It is secreted by differentiated fibroblasts in the connective tissue ofmuscles and differs from other tenascins in its domain structure (Hagios��, 1996).

Tn-C glycoproteins from various species have been isolated and characterized and similarlycDNAs from various species have been cloned and sequenced. High levels of Tn-C have beendetected in numerous developing tissues and organs, as well as in all tumours and precancerouslesions examined so far (Vollmer, 1997). Its typical expression is spatially restricted to interfacesof epithelia and mesenchyme and temporally to processes of active stromal remodelling (Natali��, 1991; Shrestha and Mori, 1997).

Structure, functions and regulation of Tn-CThe Tn-C molecule is a hexabrachion, a six-armed glycoprotein (Erickson and Inglesias, 1984).Each of the six monomers consists of four structural domains: a hydrophobic N-terminal domaininvolved in formation of the hexamer, epidermal growth factor (EGF)-like repeats, fibronectintype III-like repeats and a C-terminal fibrinogen-like domain. Tn-C cDNAs from different animalspecies differ in the number of fibronectin type III-like repeats. For human Tn-C, up to foursplice variants with different numbers of fibronectin type III-like repeats have been detected(Vollmer, 1997). The major form of human Tn-C is composed of the N-terminal domain, 14 ½EGF-like repeats, 15 fibronectin type III-like repeats and the C-terminal fibrinogen-like domain.The molecular weights of Tn-C monomers range from 190 kDa to 320 kDa (Kusakabe and Sa-kakura, 1998). The full length human Tn-C cDNA corresponds to a monomer protein of 2203amino acids and the molecular weight of the hexamer in human Tn-C is 1900 kDa (Vollmer,1997). In humans, the Tn-C gene was demonstrated to be located on chromosome 9, bands q32-q34 (Gulcher��, 1990; Rocchi��, 1991).

The first property of Tn-C to be discovered was haemagglutination, suggesting a role in celladhesion (Yamada ��, 1975; Sakakura ��, 1991). However, the distribution of Tn-C seemsto follow the pathways of neural crest cell migration (Mackie��, 1988a) and migrating fibro-blasts (Halfter� �� , 1990), suggesting mitogenic and anti-adhesive functions for Tn-C. Anti-adhesive and growth-promoting functions have been shown in several cell culture studies (Chi-quet-Ehrismann ��, 1986, 1988, 1989; Kawakatsu��, 1992; Shrestha��, 1996). Tn-Cmay also have an effect on the immune system (Ruegg��, 1989; Hemesath��, 1994).

The active properties of the Tn-C molecule depend on the functionally distinct domains. A cell-and a heparin-binding domain have been identified on each hexabrachion arm and these are


situated in the fibronectin type III-like repeats and the fibrinogen-like terminal knob (Vollmer,1997). The EGF-like domains have been implicated as potential mitogenic structures (Engel,1989), but the fibronectin type III-like domains have also been associated with mitogenic activity(End� �� , 1992). The transiently expressed transmembrane proteoglycan syndecan bindsgrowth factors and Tn-C and its expression correlates with that of Tn-C and proliferation duringdevelopment (Vainio� �� , 1989; Salmivirta� �� , 1991; Thesleff� �� , 1996). Epidermalgrowth factor-dependent proliferation has been shown to depend on Tn-C (Jones��, 1997).

Earlier studies suggested that Tn-C expression was uniquely restricted to mesenchymal-derivedcells, mainly fibroblasts (Chiquet-Ehrismann, 1990). However, the epithelial origin of Tn-C wasshown using in situ hybridization in the early chicken lung bud, where tenascin mRNA wasfound to be produced exclusively by the epithelium at the sites of active growth of bronchialtubes (Koch��, 1991). Several studies have shown both normal and malignant epithelial cellsto be sources of Tn-C (Prieto��, 1990; Herlyn��, 1991; Onda��, 1991; Kawakatsu ��, 1992; Hiraiwa��, 1993; Ibrahim��, 1993; Linnala��, 1993; Lightner��, 1994;Ishihara��, 1995; Lohi��, 1995; Aukhil��, 1996; Tuominen��, 1997; Yoshida��, 1997).

Tn-C in the tissue may be heterogeneous in both structure and function. Tumour cell lines seemto produce the larger Tn-C isoform (250kD), a fragment of which has cell detachment activity.The epithelial Tn-C has been suggested to enable the cancer cell to move easily. In contrast, thestromal Tn-C produced by fibroblasts has been suggested to inhibit tumour growth by creatingcell boundaries, inhibiting cell migration and regulating cancer cell growth (Sakakura and Kusa-kabe, 1994). An alternatively spliced segment of the Tn-C molecule has been shown to down-regulate the integrity of focal adhesion sites (Murphy-Ullrich��, 1991). The larger isoformsof Tn-C are often associated with rapidly proliferating and migrating cells (Borsi� ��, 1992)and with malignant cells (Borsi��, 1996). The smallest Tn-C mRNA splice variant was foundin all ovarian tumours, while the larger splice variants were predominantly limited to malignanttumours (Wilson� �� , 1996). The larger mRNA isoform, including an alternatively splicedsequence of Tn-C, was found in lung cancer but not in normal lung tissues (Kusagawa� �� ,1998).

The possible functions of Tn-C have been studied in cell cultures and by transferring cells ortissues to knock-out mice. Tn-C-deficient knock-out mice were cloned and they turned out todevelop normally (Saga��, 1992). Although the presence of Tn-C may not be a prerequisitefor normal development, this does not mean that Tn-C has no functions (Sakakura and Kusakabe,1994). Mice, in which other genes have been deleted, e.g. TGF-β1, src or p53 did not developmajor disturbances in their phenotype (Vollmer, 1997). Nevertheless, knock-out mice haveproved to be important research tools generally (Majzoub and Muglia, 1996) and in Tn-C re-search (Lohi ��, 1995; Kusakabe and Sakakura, 1998).

In a cancer cell transplantation experiment, GLMT1 mouse mammary tumour cells that do notproduce Tn-C under culture conditions were transplanted into Tn-C knock-out mice and intowild type mice (Kusakabe and Sakakura, 1998). Tumours in the wild type mice grew faster andexpressed Tn-C strongly in the host dermal connective tissue and the growing tumour nests,whereas there was no Tn-C expression in the knock-out mice. It seems obvious that the expres-sion of Tn-C was a response to a factor produced by the wild type mouse connective tissue(Kusakabe and Sakakura, 1998). Such a factor had been reported earlier (Hiraiwa ��, 1993).The expression of Tn-C can be induced in fibroblasts but also in tumour cell lines by growthfactors, especially TGF-β (transforming growth factor-β) (Pearson� ��, 1988), cytokines andother soluble factors (Vollmer, 1997).


Tn-C in embryogenesisInteractions between mesenchyme and epithelium are known to play important roles in suchdevelopmental events as determination, growth, morphogenesis and functional differentiation inmany organs (Saxen��, 1975). These interactions are mediated by ECM substances like fi-bronectin and laminin, which participate in cell attachment, cell migration, differentiation andmorphogenesis (Ruoslahti��, 1985).

Tn-C was first found as a myotendinous antigen early in chick limb morphogenesis at sites con-necting developing muscle fibres, tendons and bone (Chiquet and Fambrough, 1984a). It wasthen suggested to play an active role in connective tissue development by modulating the inter-actions between fibronectin and cells (Mackie��, 1987a). Tn-C is also expressed during thedevelopment of the central nervous system and peripheral nerves (Grumet ��, 1985; Steindler��, 1989). The migrating neural crest cells are themselves a major source of Tn-C and, simi-larly, subpopulations of proliferating and migrating glia produce Tn-C (Tucker��, 1994). Tn-C expression shows similar site specificity and a similar temporal appearance (Crossin� �� ,1986; Mackie ��, 1988a) to epithelial tissues.

The selective distribution of Tn-C around budding epithelia has been described in foetal hairfollicles, teeth and mammary glands (Chiquet-Ehrismann ��, 1986; Thesleff��, 1987). Theexpression of Tn-C in the developing epithelia of the urinary (Aufderheide� �� , 1987) andgastrointestinal tract (Aufderheide and Ekblom, 1988) has been described. Fetal prostatic tissueshows strong and diffuse Tn-C immunoreactivity around the developing glands (Ibrahim ��,1993). Koch showed that, during chicken bronchial morphogenesis, the advancing epithelial cellsare the only source of Tn-C that is expressed around the tips of branching bronchi and is down-regulated when differentiation is over (Koch ��, 1991). The expression of Tn-C is similarlytransient in developing bile ducts (Tereda and Nakanuma, 1994).

Tn-C appears in the mesenchyme of the developing mouse mammary gland at day 14 of gesta-tion in the dense mesenchyme immediately surrounding the epithelium, the mammary endbuds. Itis present around the epithelia while the buds start to form sprouts, but later, after day 17, whenthe epithelium continues to grow and ramifies to form a mammary gland tree with about 15-20branching ducts, Tn-C disappears (Inaguma ��, 1988; Sakakura ��, 1991). The epithelialcells of the murine mammary bud synthesize Tn-C on the 14th and 15th days of gestation and themesenchymal cells at the epithelial-mesenchymal border of the growing bud synthesize Tn-Cfrom the 14th day to the 17th day, as shown by in situ hybridization (Kalembeyi��, 1997).

Tn-C in reparative processesWound healing is a complex process which in many ways resembles developmental processesand malignant growth. In normal rat skin, Tn-C was sparsely distributed, predominantly in asso-ciation with BM. The expression of Tn-C in healing rat wounds was markedly increased at thewound edge at all levels of the skin. There was also particularly strong Tn-C staining at the der-mal-epidermal junction beneath the migrating, proliferating epidermis. Tn-C was presentthroughout the matrix of the granulation tissue, but was not detectable in the scar after woundcontraction was complete (Mackie �� , 1988b). Similar changes in Tn-C distribution werenoted in rat tongue mucosa in incisional, excisional and laser wounds (Luomanen and Virtanen,1992). In rabbit corneal wounds, Tn-C appeared in the corneal stroma in the wound area, thewound edge showing the most intense reactivity (Tervo��, 1989).

The source of Tn-C in wound healing seems to be the migrating epithelial cells (Aukhil ��,1996; Latijnhouwers��, 1997). Wounding leads to loss of laminin and type IV collagen in the


region of the wound and expression of fibronectin and Tn-C, and to changes in the integrins onthe migrating keratinocytes (Juhasz��, 1993; Clark��, 1996; Haapasalmi��, 1996). Anupregulation of Tn-C has been associated with hyperproliferative skin disorders (Schalkwijk��, 1991a; Schalkwijk��, 1991b; Steijlen��, 1994).

In Tn-C-deficient knock-out mice, no impairment of wound healing was noted (Forsberg��,1996). Nevertheless, in normal individuals Tn-C is upregulated in various epithelial-mesenchymal interactions. The expression of Tn-C has been used to estimate the phase of woundhealing. Tn-C shows the area of wounding in experimental studies (Juhasz ��, 1993; Christo-fidou-Solomidou� �� , 1997). In forensic medicine, Tn-C expression in a wound reveals thewound to be more than 2-3 days old (Betz, 1995). A time-dependent transient upregulation ofTn-C takes place in myocardial scars after infarction and suggests a role in the reparative processof myocardial scarring (Willems��, 1996). A strong Tn-C immunoreactivity can be seen inthe granulation tissue of benign gastric and pyloric ulcers (Tiitta��, 1994).

A continuous and strong Tn-C reactivity was shown at the epithelial-stromal interface in samplesof acute cystitis (Tiitta��, 1993) and in Crohn’s colitis, chronic diverticulitis, chronic ischae-mic colitis and ulcerative colitis (Riedl��, 1992). Tn-C staining is also increased in the BMregion of bronchial biopsies in asthmatics (Laitinen� �� , 1996; Laitinen� �� , 1997). Snuff,being an epithelial irritant, causes an increase in Tn-C reactivity beneath the oral mucosa (Luo-manen��, 1997). The Tn-C content has been suggested to be an instrument for assessing theactivity of stromal remodelling in diseases of the large bowel (Riedl��, 1998). The pattern ofTn-C distribution in inflammatory conditions resembles that of wound healing and may indicatethe degree of active ongoing remodelling and repair.

Tn-C in normal, dysplastic and neoplasic tissuesThe presence of Tn-C in carcinomas was first reported in carcinogen-induced rat mammary tu-mours. Tn-C was found in the stroma surrounding neoplastic epithelial cells of all 20 carcinomasstudied. However, Tn-C was not detectable in the normal adult rat mammary gland, even duringpregnancy (Chiquet-Ehrismann ��, 1986). In benign and malignant human breast tumours Tn-C immunoreactivity differed and Tn-C was regarded as a stromal marker of malignancy (Mackie��, 1987b; Inaguma ��, 1988).

Immunohistochemistry using the monoclonal antibody 100EB2 on human breast tissue revealedthat Tn-C could also be found in normal adult tissues (Howeedy ��, 1990). The distribution ofTn-C in normal as well as physiological and pathological hyperplastic breast tissue has been wellcharacterized (Ferguson��, 1990; Howeedy ��, 1990; Shoji��, 1992). In the adult rest-ing breast, a thin, focally discontinuous band of Tn-C immunoreactivity was found around theducts and acini. In the lactating breast a broad reactivity layer could be seen around the entireductal tree. In fibrocystic disease, continuous bands of varying thickness were constantly found,the most intense staining appearing in the stroma adjacent to the proliferating epithelial struc-tures (Howeedy ��, 1990). Tn-C expression has been shown to be regulated cyclically. Duringthe first week of the menstrual cycle, Tn-C was present as a thin bright discontinuous line. In thesecond week, Tn-C appeared as a broad, paler and more complete band, and by the third week,there was an overall increase in the amount of Tn-C, which reached a maximum during the fourthweek (Ferguson ��, 1990).

In all types of intraductal carcinomas of the human breast, Tn-C immunoreactivity was seen asprominent bands, which at times appeared to comprise irregularly concentric rings; the Tn-Cstaining contrasted with the delicate, attenuated and focally discontinuous line of laminin immu-


nostaining (Howeedy ��, 1990). Infiltrating ductal carcinomas showed strong and generallydiffuse Tn-C immunostaining around clusters of carcinoma cells and even around individual cells(Howeedy ��, 1990). The stromal Tn-C immunoreactivity in infiltrating lobular carcinomaswas strong (Howeedy ��, 1990), including some desmoplastic areas (Gould��, 1990). Inlobular carcinomas the most intense reactivity was noted in areas immediately adjacent to thetumours (Gould ��, 1990). Malignant epithelial cells are an important source of Tn-C in intra-ductal and invasive breast carcinomas (Lightner ��, 1994; Yoshida ��, 1997).

A thin rim-like immunoreactivity for Tn-C is seen next to the BM in various normal epithelialtissues. In hyperplastic and dysplastic processes it becomes thicker and in infiltrating carcinomasthere is usually strong Tn-C expression (Natali ��, 1991; Koukoulis� �� , 1991a; Shresthaand Mori, 1997). Tn-C has been found in all carcinomas studied so far (Vollmer, 1997).

In the normal human endometrium, weak periglandular Tn-C immunoreactivity was observed in50% of specimens in the proliferative phase but not in the secretory phase, and intense and dif-fuse staining was seen around endometrial carcinoma cells (Sasano��, 1993). Similar Tn-Cdistribution and additionally, an association between Tn-C and proliferation of endometrial car-cinoma cells has been reported (Yamanaka��, 1996).

In normal adult human prostatic tissue, only scant focal periglandular and stromal Tn-C immu-noreactivity could be seen, whereas periglandular staining with diffuse stromal extension wasnoted in all grades of adenocarcinoma (Ibrahim ��, 1993). In prostatic intraepithelial neopla-sia, a putative precursory lesion of prostate carcinoma, periepithelial Tn-C expression was low inlow-grade lesions, resembling the Tn-C expression in normal and hyperplastic prostatic tissue,and high in high-grade lesions (Xue��, 1998).

Tn-C staining of normal human lung tissue showed an intensely reactive subepithelial, bandlikezone, corresponding to the region of the BM, beneath the bronchial respiratory epithelium andalso by the BM of the alveolar epithelial cells. In all types of malignant lung tumours there wasan intense stromal Tn-C immunoreactivity (Soini� �� , 1993). Salivary gland tumours werefound to express Tn-C in the stroma, whereas in normal tissues Tn-C was restricted to the BMregion of the ductal epithelium (Soini��, 1992). An increase in Tn-C in colon carcinomas ascompared to normal colon mucosa has been reported (Riedl ��, 1992; Sakai��, 1993).

This change in the distribution of Tn-C in pre-neoplastic and neoplastic tissues has been dis-cussed in a recent thesis by Tiitta. She made a systematic examination of cervical and vulvarepithelia, mucosa of the urinary bladder and gastric and oral mucosae in various inflammatory,dysplastic and neoplastic conditions. She suggests that Tn-C may have a role in inflammatoryresponses and participate in the stromal tissue remodelling that occurs concomitantly with tu-mour invasion (Tiitta, 1995).

The first report of Tn-C in malignant neoplasia already drew attention to the strong stromal ex-pression in “mesenchyme, particularly in the areas apposing the tumor epithelium” (Chiquet-Ehrismann ��, 1986). Tn-C was described as a prominent part of the matrix that surroundsmammary tumours (Inaguma �� , 1988). In occasional larger ductal breast carcinomas, theperipheral regions were observed to be more reactive for Tn-C (Howeedy �� , 1990). Tn-Cexpression in basal cell carcinomas of the skin appeared to become more widespread and diffusein sclerosing and infiltrative areas (Stamp, 1989). In primary melanomas a higher degree of Tn-Cexpression was seen in lesions of greater dermal invasiveness (Natali� �� , 1990). In 22/36human endometrial carcinomas the immunoreactivity for Tn-C was intense, especially at thefront of muscle invasion, and also at deep invasion into muscles. Tn-C expression correlated withvascular invasion and metastasis (Doi��, 1996). In a recent study on the often recurring and


metastasizing adenoid cystic carcinoma of the salivary gland, Tn-C reactivity was located in thestroma and cytoplasm in invasive areas and this was correlated with tumour cell proliferation(Shintani��, 1997).

Tn-C and cancer prognosisFew prognostic studies have been performed on Tn-C. Every case of 20 colon carcinomas with-out lymphogenous metastasis showed strong Tn-C expression, whereas no expression was notedin 9/61 metastatic tumours and the remaining 52/61 had moderate expression. (Sugawara��,1991). Breast cancer patients whose tumours showed intense Tn-C staining in fibrotic stroma(59/82 patients) had a significantly better prognosis in univariate analysis with a 5-year follow-up (Shoji� ��, 1993). On the basis of these results, Tn-C has been proposed to be a stromalmarker for cancers with a better prognosis (Sakakura and Kusakabe, 1994). In addition, in arecent study of 60 colon cancer patients it was concluded, that patients with more Tn-C expres-sion had better long-term survival than patients with absent or weak Tn-C expression (Iskaros��, 1997).

A study of 32 infiltrating breast carcinomas found no correlation between Tn-C expression andprognostic factors such as nodal metastasis, tumour necrosis, invasion of blood vessels, ploidy orSPF. However, in cases with increased stromal inflammation, significantly higher reticular andperiepithelial tenascin expression was noted (Moch� �� , 1993). Iskaros and co-workers re-corded stromal Tn-C expression on a scale of 0-4. In 115 patients with ductal carcinoma fol-lowed up for 65 months, this score correlated significantly with grade but not with survival(Iskaros��, 1998). No association between Tn-C expression and the clinical disease behav-iour was found in a study of 103 salivary gland carcinomas (Kärjä��, 1995) and another studyof 120 gastric carcinomas (Ilunga and Iriyama, 1995). However, in primary tumours of meta-static oral squamous cell carcinoma, the expression of Tn-C was higher than in tumours of non-metastatic cases (Harada��, 1994).

A relatively large series of 210 patients with breast cancer followed for 5 years was studied withimmunohistochemistry and scored for both stromal and cytoplasmic expression. Patients with noTn-C expression either in stroma or inside the carcinoma cells had a good prognosis and tendedto be lymph-node negative and oestrogen receptor (ER) -positive. The 12 patients with the worstprognosis had tumours expressing Tn-C both in the cytoplasm of the carcinoma cells and in thestroma. These Tn-C-positive cells were often located at the margin of cancer nests in contactwith the underlying mesenchyme. Additionally, when nine fresh cancer tissues were examinedfor the expression of Tn-C mRNA with in situ hybridization and with immunohistochemistry, 4/9expressed Tn-C mRNA at the periphery of cancer nests and two of these four stained for Tn-C inimmunohistochemistry (Ishihara ��, 1995). The same group suggested that the Tn-C which isproduced by cancer cells leads to detachment and this Tn-C may promote cancer cell invasionand metastasis (Yoshida��, 1995).

The prognostic studies on Tn-C in carcinomas have come to somewhat inconsistent conclusions.During embryogenesis and wound healing, Tn-C is expressed transiently in areas of advancingepithelia, mostly by the epithelial cells themselves, and is then downregulated. The distributionof Tn-C coincides with that of invasive nests in the periphery of many carcinomas, resemblingthe distribution in differentiation and in repair. In desmoplastic tumour stroma, however, a strongimmunoreactivity is seen (Gould ��, 1990; Melis��, 1997) and this Tn-C seems to be pro-duced mainly by fibroblasts (Yoshida ��, 1997). The association between stromal Tn-C ex-pression and a favourable prognosis suggests that stromal Tn-C may, indeed, reflect an effective


host response. This study concentrates on early breast carcinomas and the possible connectionbetween Tn-C expression in areas of invasion and disease character.

Aims of the study 27

�'1��$��2

The main purpose of this study has been to find biological tumour-related prognostic discrimi-nants of women treated for early breast cancer to better optimize the treatment. The specific aimshave been:

• to study and compare multiple prognostic factors of axillary node-negative breast cancer.

• to study factors related to local recurrence after breast conserving surgery and radiotherapy.

• to quantify and examine the distribution of Tn-C in clinical series of DCIS and early breastcancer.

• to study the associations between Tn-C and other histopathological and prognostic factors ofbreast cancer.

• to find out whether a relation exists between Tn-C and disease outcome in patients withearly breast carcinomas.

28 Patiens and methods

*��'$&��&��1$��

*��

Study IThe 136 patients with 137 node-negative breast cancers were collected retrospectively from twosurgical clinics in Helsinki: the Fourth Department of Surgery of Helsinki University CentralHospital (HUCH) and Maria Hospital. The patients had all been treated with breast-conservingsurgery, axillary dissection and post-operative radiotherapy from 1985 to 1989. These patientschosen for breast preservation had presented pre-operatively with a clinically unifocal cT1N0 (2cm or less in diameter) tumour with no cancer involvement at the resection margin found at sur-gery. A clear resection margin was defined as 5 mm or more of normal tissue measured his-tologically. Patients with narrower margins were referred for re-resection or mastectomy. Mas-tectomized patients were excluded from the study. Radiotherapy, 25 times 2 Gy administeredduring 5 weeks to the breast area, was given at the Department of Oncology of HUCH. None ofthe patients had received adjuvant chemotherapy or hormonal therapy. The minimum follow-upwas 5 years.

The tumours were reviewed and classified according to Azzopardi (Azzopardi, 1979) , and theirgrade (Bloom and Richardson, 1957) and size were assessed. The tumours measured one to 26mm, median 15 mm, allowing the whole cross-section of the tumour to be examined in mostcases.

Studies II and IIIIn studies II and III a combined group of patients (original and additional patients) was used toincrease statistical power. The original group consisted of 143 women with 144 node-negativebreast cancers (one bilateral) treated with breast-saving surgery and axillary dissection at theMaria Hospital (55 patients) and the Fourth Department of Surgery of HUCH (88 patients) in1985-1989. The treatment criteria and treatment were identical to those of study I. The medianfollow-up time was 7.8 years, range 5.4-11.4 years.

All the additional patients had had a recurrence. In 1985-1990 there were 238 women with axil-lary node-negative breast cancer (not included in study I) who had been treated similarly andreferred to the Department of Oncology of HUCH for radiotherapy. Twenty-six (11%) of thesehad had a recurrence. The rates of both local relapse (15/238, 6%) and distant metastasis(16/238, 7%) at 5-year follow-up were comparable with those in the original patient group, inwhich 4% had local recurrences and 8% distant metastases (study I), suggesting an equal stan-dard of treatment. Paraffin-embedded specimens were available for 15 of the 26 tumours thatrecurred, seven local recurrences and 11 distant metastases, three patients having both. These 15patients formed the new series.

Sections of paraffin-embedded tissue specimens of the primary tumours were reviewed and clas-sified for histology (Azzopardi, 1979). The extent of the intraductal component (ductal carci-noma in situ, DCIS) was recorded. It was called extensive (EIC) if DCIS comprised 25% ormore of the tumour area encompassed by the infiltrating tumour and DCIS was also present inthe surrounding tissue (Schnitt ��, 1987). The grades of the ductal carcinomas were assessed(Bloom and Richardson, 1957). Tumour size was measured in the histological sections and var-

Patients and methods 29

ied from 1 to 26 mm. In most cases, the whole cross-section of the tumour was examined. Inprognostic studies with immunohistochemistry and flow cytometry, 6-11% of the samples couldnot be interpreted on account of technical failures or because there was too little malignant tissueleft. The histopathological and prognostic parameters were assessed by pathologists blinded tothe outcome. A median size of 13 mm was chosen to dichotomize the study group. The medianage at operation of these patients was 51.9 years. For analysis, the patients were divided into twogroups, 50 years or older and less than 50 years old.

Study IVThere were 133 patients, operated on for primary DCIS between 1974-1996 at the Fourth De-partment of Surgery, HUCH. The pathology reports and specimens of these 133 tumours werereviewed and the most representative samples were selected for Tn-C immunohistochemistry. In36 cases, either no paraffin block was available or there was no malignant tissue left in the paraf-fin block. A pre-operative core-needle biopsy had been taken from 16 tumours. These tumourswere excluded to avoid misinterpretation of any stromal expression of Tn-C related to trauma(Mackie ��, 1988b). The remaining 89 tumours were included.

The group of 44 patients with small (1-25 mm) axillary node-negative tumours that comprisedboth an invasive area and an intraductal component is a subgroup of study I.

The tumours were re-examined and classified for histological subtype (Rosen, 1997). Nucleargrade was classified as low (1), intermediate (2) or high (3) (Holland ��, 1990a). The largestdimension of the tumours was obtained from the pathology reports. The mean size was 15 mm(range 2-40 mm). In 26 tumours (31%) the pathologist had not reported the size of the tumourapparently because of widespread multifocality. The paraffin-embedded tumours were cut at 5µm for Tn-C immunohistochemistry, and the sections were stained with haematoxylin-eosin forhistological studies.

Steroid receptors and Ki-67 had been routinely determined in the laboratory of most of the tu-mours. ER was available in 64 (72%), PR in 63 (71%) and of Ki-67 antigen in 61 (69%) of thetumours.

1��

Tn-C immunohistochemistry (studies I-IV)For immunohistochemistry the sections were cut at 5 µm, deparaffinized and treated with pepsin(0.1% in 0.01 M HCl at 37 °C) for 30 min. They were incubated with MAb 143DB7 against Tn-C. The MAb 143DB7 reacts with the fibrinogen-like knob region of the protein and detects bothTn-C isoforms (Tiitta� �� , 1992). This was followed by alkaline phosphatase-coupled rabbitimmunoglobulins to mouse IgG (Dakopatts, Glostrup, Denmark). Some sections were treated bythe alkaline-phosphatase/anti-alkaline-phosphatase bridge method (Dakopatts) with identicalresults. After exposure to the conjugate the colour reaction was developed with naphthol AS-B1phosphate (Sigma, St. Louis, MO, USA). Endogenous alkaline phosphatase activity was abol-ished by incubation with 1 M levamisole (Sigma). The sections were then gently counterstainedwith haematoxylin. Sections incubated with an irrelevant MAb or with the conjugate only wereused as negative controls and gave no reaction.

30 Patients and methods

The extent and intensity of Tn-C expression was scored as -, +, ++ and +++, corresponding tonegative, weak, moderate, and strong immunoreactivity. Tn-C staining was scored for periductaland stromal expression.

In the combined groups in studies II and III, an invasion border of the tumour could be identi-fied in 121 of the 159 tumours. In the rest, the invasion border was not included in the specimensavailable. Tumours expressing Tn-C in the area of invasion with adjacent normal tissue clearlyvisible were called Tn-C positive.

Immunohistochemical analysis of p53 protein, Ki-67 antigen and erbB-2protein (study II)The monoclonal antibody to the p53 protein (clone DO-7) and polyclonal anti-human Ki-67 werepurchased from Dako (Glostrup, Denmark). The optimal working dilutions were determined byserial dilutions and were 1:300 for anti-p53 and 1:500 for anti-Ki-67. Mouse monoclonal anti-body E2-4001 raised against the intracytoplasmic domain of the human erbB-2 antigen was ob-tained from Molecular Oncology, Inc., Gaithersburg, MD, USA. The final concentration used inthe working solution was 6 µg/ml (dilution 1:20). The p53 and Ki-67 primary antibodies wereapplied overnight and erbB-2 antibody for 2 hours at room temperature in humidified chambers.

For immunostaining, 5-µm thick paraffin sections were cut, mounted on slides coated with 3-aminopropyl-triethoxy-silane (APES) (Sigma, St. Louis, MO, USA) and treated in a microwaveoven as described earlier (Victorzon��, 1996).

Immunohistochemistry was performed by the avidin-biotin complex (ABC) immunoperoxidasetechnique, applying a commercial Elite ABC Kit (Vectastain, Vector Laboratories, Burlingame,CA, USA), also earlier described (Victorzon ��, 1996).

The levels of immunoreactivity of the p53 and Ki-67 antigens were expressed as percentages ofthe positive cancer cell nuclei. Interpreting erbB-2 staining, all tumours with positive cell mem-branes were scored as positive. The p53 staining could be interpreted in 143/159 (90%), the Ki-67 staining in 141/159 (89%), and the erbB-2 staining in 142/144 (99%) of the tumours. Theanti-erbB-2 antibody E2-4001 was no longer available when the new material of 15 tumours wastested.

A cut-off value of 20% of positive nuclei was used for p53. For Ki-67, 5-15% positive expres-sion in the nuclei was called weak (+), 16-29% moderate (++), and 30% or more strongly posi-tive (+++). Because there was no difference in metastasis between the Ki-67 +, ++ and +++tumours, the classification was simplified by combining these classes into one Ki-67-positivecategory of 5% or more positive nuclei. Five percent was also the median of the Ki-67 immu-nostaining. The cut-off values for prognostic factors presented here were also used when ana-lyzing local recurrences after breast-saving surgery.

DNA flow cytometry (study II)A modification of the method of Hedley et al. was employed (Hedley ��, 1983). In brief, two50 µm-thick sections were treated with 10 mg/ml proteinase K (Sigma) for 30 min at room tem-perature. After filtration, the nuclei were treated with RNase (10 mg/ml) and stained with 25µg/ml ethidium bromide (Sigma) for at least 1 h. The DNA was determined by flow cytometry(FACScan, Becton Dickinson, Mountain View, CA, USA) using 15 mW excitation at 488 nm,and the total emission above 560 nm was recorded. As the staining intensity of the fixed nuclei


varies from one sample to another, no internal standard was added. The lowest peak was as-signed a DNA index (DI) value of 1.00 and the DI values of other peaks were calculated withthis as a reference. Therefore, any hypodiploid peaks were identified as diploid and the normaldiploid peak as hyperdiploid. The S-phase fraction (SPF) was calculated either using the Cellfitprogram of the FACScan flow cytometer or manually by a modified rectilinear method (Baisch�� , 1975). If the automatic and manual methods gave different results, the lower SPF waschosen. Usually the manual method gave a lower result, because it was applied only to thosetumours in which it was felt that the automatic method had given too high a value for SPF, e.g.when there was a skewness to the right of the G1 peak. If the sample contained less than 15% ofaneuploid cells, the SPF was not calculated. At least 10 000 nuclei from each specimen wereanalyzed. DNA ploidy could be determined in 150/159 (94%) and SPF in 147/159 of the tu-mours (92%). The median SPFs of the diploid (2.4%) and aneuploid tumours (8.4%) were usedas cut-off values.

Immunohistochemical analysis of cath-D , uPA and PAI-1 (study III)Formalin-fixed and paraffin-embedded tumour samples were cut into 5-µm-thick sections anddeparaffinized, and endogenous peroxidase was blocked for 30 minutes in 0.3% H2O2 in metha-nol. Alkaline phosphatase anti-alkaline phosphatase (APAAP kit system 40, cat. no. K0670,DAKO A/S, Denmark) and streptavidin-peroxidase (StreptAB complex/HRP kit, cat. no. K492,DAKO A/S, Denmark) methods were applied according to the manufacturer’s instructions. 3-amino-9-ethyl carbazole was used as the chromogen for peroxidase, Mayer’s haematoxylin wasused for nuclear staining and the sections were mounted with Aquamount (BDH Limited, Poole,UK). Mouse monoclonal antibodies against cath-D (clone 1C11, Triton Diagnostics, Alameda,CA., USA), uPA (American Diagnostica Inc., Greenwich, CT, USA) and PAI-1 (American Di-agnostica Inc., Greenwich, CT, USA) were applied at dilutions 1:20 (0.13 µg/ml, 1 h at roomtemperature), 1:75 (13 µg/ml, 1 h at 37°C) and 1:100 (10 µg/ml, 1 h at 37°C), respectively.Positive and negative controls were included in each set of stainings.

All tumours were evaluated for cath-D, uPA and PAI-1 expression by a pathologist unaware ofthe outcome of the case. A few samples in each immunohistochemistry set could not be inter-preted because of technical failure or lack of carcinoma tissue.

The expression of cath-D was typically granular and confined to the cytoplasm of carcinomacells, but also of stromal macrophages and fibroblasts. The cath-D-positive cells showed nopreference of location between the central parts and the periphery of the tumours. The scoring ofcytoplasmic cath-D immunoreactivity was assessed according to Isola and co-workers: tumourswith a clearly detectable level of 10% or more of strongly positive carcinoma cells were definedas positive for cytoplasmic reactivity (Isola ��, 1993). When 10% or more of the stromal cellswere strongly positive, the stromal expression was called positive (Tetû �� , 1993). Cath-Dexpression could be interpreted in 155 out of 159 tumours. There were 73 tumours (47%) show-ing immunoreactivity for cytoplasmic cath-D and 68 tumours (44%) for stromal cell expression;43 tumours (28%) expressed both.

Immunoreactivity for uPA and PAI-1 was seen in the vast majority of tumours. Expression wasseen in the cytoplasm of carcinoma cells and diffusely in the surrounding stroma, but also fre-quently in the cytoplasm of benign breast epithelia and stroma. For uPA and PAI-1, cytoplasmicand diffuse stromal immunoreactivities were classified visually, taking account of both the inten-sity and the proportion of cells or the area of the tumour stained. The categories were negative (-), and slightly (+), moderately (++) or strongly positive (+++). In some samples the stromal fi-broblasts showed a moderate to strong intensity, which seemed to be independent of the diffuse

32 Patients and methods

stromal immunoreactivity, and this was recorded separately (-/+). For purposes of analysis, thecategories of cytoplasmic and diffuse stromal uPA and PAI-1 reactions were combined to form alow-grade and a high-grade group for both cytoplasmic and diffuse stromal staining for uPA andPAI-1. Cytoplasmic uPA was missing in 9 tumours (6%) but diffuse stromal reactivity was notedin all tumours. PAI-1 was expressed in the cytoplasm in every tumour, but stromal staining wasnot found in 21 tumours (13%). Fibroblasts expressed uPA in 39 tumours (32%) and PAI-1 in 42(35%).

ER and PR immunohistochemistry (study IV)Until October 1995 the steroid receptors were stained in frozen sections. Frozen sections wereused for determinations of ER in 46 tumours and of PR in 45 tumours. The immunohistochemis-try was performed using the ERICA and PRICA kits according to the instructions of the manu-facturer (Abbott Laboratories, Chicago, IL, USA). The result was scored as weakly positive (+)when 10-40% of cells were stained, as moderately positive (++) when 40-70% were stained, andas strongly positive (+++) when >70% were stained. The staining intensity was not recorded.After October 1995 the staining was done on paraffin-embedded sections. The tissue arrivedfresh at the laboratory and the tumour specimen was immediately fixed in formaldehyde. Steroidreceptors were determined from paraffin sections of 18 tumours, using the monoclonal anti-human ER antibody (clone 1D5, Dako, Glostrup, Denmark) and PRICA kit for PR� The stainingmethod for PR has been described earlier (Boguslawski, 1994). ER was detected using the samemicrowave pretreatment as for PR but detection was done with a commercial ABC kit (Vectas-tain Elite, Vector Laboratories, Burlingame, CA, USA). The scoring was the same as for frozensections. For analysis, the steroid receptors were dichotomized to positive and negative groups.

Ki-67 immunohistochemistry (study IV)Ki-67 immunohistochemistry was performed using monoclonal Ki-67 antiserum (Dakopatts) onfrozen tissue in 21 tumours before February 1994 (for methods, see (Railo �� , 1993)) andthereafter, polyclonal Ki-67 antibody (Dako, Glostrup, Denmark) on paraffin-embedded tissue in40 tumours (for methods, see (Railo ��, 1997). The level of immunoreactivity was expressedas the proportion of Ki-67-positive nuclei. The intensity of the staining was not recorded. Thefrozen sections were scored as weakly positive (+) when 1-2% of the nuclei were positive, asmoderately positive (++) when 3-10% of the nuclei were positive and as strongly positive (+++)when >10% of nuclei were positive. For the paraffin-embedded tumour material, the corre-sponding proportions of Ki-67-positive nuclei were 5-15% (+), 15-30% (++) and >30% (+++).For analysis, the Ki-67-positive groups were combined.

Statistical analysisThe chi-square test, Fisher’s exact test and the Mann-Whitney U test were used to test for asso-ciations between variables (studies I-IV) and possible differences between patients with relapsesin the original and in the new patient groups (study II). The statistical significance of differencesin periductal and stromal Tn-C distribution between the intraductal component of invasive can-cers and pure DCIS was tested with the chi-square test (study IV). Metastasis-free survival(MFS) in the original patient group according to each prognostic parameter was estimated withthe Kaplan-Meier method. The log-rank test was used to show the statistical significance of dif-ferences in outcome between patients with and without each prognostic factor (studies I and II).The statistical significance of the effects of tumour grade and size on MFS was tested with theCox proportional hazard model, the variable to be tested being the only covariate (study I). The


statistical significance of differences in outcome between patients with or without a prognosticfactor were calculated in the overall patient group, using the Cox proportional hazard model tocompute the relative risks of disease recurrence. The statistical significance of the effect of thecontinuous variables on disease recurrence was also tested with the Cox proportional hazardmodel, the variable to be tested being the only covariate. The multivariate analysis for metastasiswas performed with the Cox proportional hazard model, entering the variables that were signifi-cant in the univariate analysis (studies II-III). Two-sided p-values smaller than 0.05 were consid-ered significant.

In the first study, the statistical power was relatively low because of the small number of recur-rences in the series with favourable prognostic characteristics. In order to increase the statisticalpower, the number of cases was increased in studies II and III. Since statistical power is almostexclusively dependent on the number of cases with events (recurrences, deaths) the power wouldnot have been increased by staining the vast number of cases without a recurrence. This some-what unusual statistical method may introduce a bias if the study group is used to estimate therisk of recurrence in a Tn-C-positive case, for example. For the estimation of Tn-C positivity inrecurrent versus non-recurrent cases and for estimation of the relative risk of recurrence in Tn-C-positive versus Tn-C-negative cases the study group was not biased.

Results34

($��#��

�� 8��9%�� :��;�� '<Of the 137 invasive tumours, 44 (32%) contained a component of intraductal carcinoma. In 3cases the available sections did not contain invasive areas, and therefore stromal Tn-C could bestudied in 134 cancers. Tn-C staining and its intensity (-,+,++,+++) were noted periductally andin the stroma, both in intraductal and invasive areas. In 122 tumours (89%) stromal Tn-C wasseen and in 40 (29%) periductal Tn-C was present. In 7 tumours (5%) Tn-C was not present,either stromally or periductally. Stromal Tn-C was present in the different histological types aswell as in ductal carcinomas of all grades. The different histological types of breast cancer andgrades of ductal carcinoma did not differ in their distribution of stromal Tn-C staining. Inflam-mation was noted in 96 tumours (70%), while lymphocytes were present in the Tn-C-positivearea in 27 tumours (20%). An invasion border could be seen in sections of 110 tumours and Tn-C immunoreactivity was present in the same area in 54 (49%).

During the 5-year follow-up, 6 local recurrences (4.4%) and 11 distant metastases (8.1%) oc-curred, 2 patients had both. None of the patients who relapsed had a Tn-C-negative tumour. Onehad no Tn in the stroma, but Tn was found periductally. Neither stromal Tn-C expression nor itsassociation with lymphocytes could predict a distant metastasis. Two cases of metastasis oc-curred in the group of 27 patients in whom no invasion border could be identified. However, Tn-C immunoreactivity was noted in the invasion border in 54 tumours, and in this group 8 distantmetastases developed during the follow-up period. One case of metastasis was noted in the groupof 56 tumours with no Tn-C in the invasion border. The difference in MFS (85% vs. 98%) be-tween patients with Tn-C-positive and -negative invasion borders was statistically significant(p=0.008). There was a statistically significant association of Tn-C in the invasion border withhistological grade (p=0.03) but not of Tn-C with tumour size (p=0.32). The prognostic impact ofTn-C in the invasion border (p=0.008) on MFS was stronger than that of tumour size (p=0.03)and grade (p=0.11).

��8�:��9%��8�� ;�� '9'''<The expression of Tn-C at the site of invasion was correlated with the grade (p=0.03), with ahigher proliferation rate as measured by the expression of the Ki-67 antigen (more than 5% ofpositive nuclei) (p=0.03) and with a high SPF (p=0.004). There was also an association withtumours not comprising an intraductal component (p=0.04). A Tn-C-positive invasion borderwas associated with cath-D expression both in the carcinoma (p=0.05) and the stromal cells(p=0.03) and with fibroblastic PAI-1 (p=0.05).

(�� ;�� ''�� '''<The median follow-up of patients in the original study group was 7.8 years (range 5.7-11.4years). Seven local relapses occurred (5%), 6 in the ipsilateral breast and one in the axilla. Inaddition, one patient had had an angiosarcoma in the treated breast. One mastectomy was per-formed because of a painful postradiation mastitis, and one mastectomy with immediate recon-struction because of a poor cosmetic result. One cancer developed in the contralateral breast. Adistant metastasis occurred in 14 women (10%), four of whom also had a local recurrence. The

Results 35

median time of the local recurrence was 2.9 years (range 1.3 - 6.9 years) and that of a distantmetastasis 3.6 years (range 0.7 - 8.2 years) in the overall patient group (original and additionalpatients combined).

*��;�� ''�� '''<In univariate analysis for metastases, size (HR=2.56, CI=1.13-5.94, p=0.02), SPF of diploidtumours (HR=3.16, CI=1.02-9.81, p=0.05), Ki-67 (HR=5.5, CI=1.6-18.7, p=0.006) and Tn-C inthe invasion border (HR=3.4, CI=1.1-10.4, p=0.03) were significant prognostic factors. In studyIII of proteolytic enzymes, cytoplasmic cath-D (HR=3.80, CI=1.50-9.57, p=0.005), stromal cellcath-D (HR=4.28, CI=1.70-10.78, p=0.002) and fibroblastic PAI-1 (HR=2.44, CI=1.06-5.61,p=0.04) were significant prognostic factors. No association was found between uPA and metas-tasis.

In multivariate analysis, Ki-67 was the strongest prognostic factor for metastasis, followed bystromal cath-D, Tn-C in the invasion border, cytoplasmic cath-D, fibroblastic PAI-1 and tumoursize, in this order.

*��;�� ''�� '''<Tn-C in the invasion border was the only statistically significant prognostic factor for local recur-rence in study II with a hazard ratio of 11.0, CI 1.4-85.1, p=0.022. In study III, stromal uPA(HR=7.24, CI=1.62-32.34, p=0.01) and fibroblastic PAI-1 (HR=4.3, CI=1.44-12.85, p=0.009)also showed prognostic power for local recurrence.

��9%�� %'��;�� '-<Tn-C was expressed periductally in 77 tumours (87%). It was weak in 23 (26%), moderate in 29(33%) and strong in 25 tumours (28%). In some samples, Tn-C was not present round all of theaffected ducts. In a few cases, periductal staining was also seen round benign hyperplastic ducts.When stromal Tn-C was encountered with microinvasion, it seemed to accompany the invadingcells. Typically, the stromal Tn-C was focal, often forming bridges between the ducts of DCIS.In two cases, stromal Tn-C was expressed in areas of benign fibrotic stroma. Stromal expressionwas absent in 67 tumours (75%), weak in nine (10%), moderate in nine (10%) and strong in four(5%).

Comedo or cribriform tumours were more frequently Tn-C-positive than DCIS of other his-tological types. Periductal Tn-C was associated with nuclear grade (p=0.04), comedo type(p=0.002), microinvasion (p=0.04), missing PR (p=0.008) and positive Ki-67 (p=0.04). StromalTn-C expression was associated with strong periductal staining (p=0.02) and microinvasion(p=0.005).

The distribution of Tn-C expression between pure DCIS (89 tumours) and the intraductal com-ponent areas of 44 small invasive breast carcinomas was compared. There was no difference inthe distribution of periductal staining, but stromal expression was present in 25% of the pureDCIS and in 82% of the intraductal components (p<0.0001).

Discussion36

�'�%��'�&

The first study of this thesis showed that there was an association between Tn-C expressed in theinvasion border of breast carcinomas and an increased risk of a distant metastasis. The intensityor extent of immunostaining for Tn-C in the tumour stroma did not relate to the disease progno-sis. Iskaros and co-workers have reported a similar result (Iskaros ��, 1998), whereas Shoji’sgroup found an association between stromal Tn-C and a favourable prognosis (Shoji �� ,1993). Two studies on colon carcinoma have found a similar association with better survival andstrong stromal Tn-C immunoreactivity (Sugawara ��, 1991; Iskaros ��, 1997). Stromal Tn-C expression seems to be produced partly by fibroblasts (Yoshida ��, 1997) and to be relatedto desmoplasia (Melis ��, 1997). In some tumours, stromal Tn-C may indicate a host-responseassociated with better survival. To the best of my knowledge, the present study is the first reportof a relationship between an adverse prognosis and Tn-C immunoreactivity in the invasion front.

In fact, an enhanced Tn-C expression in the invading tumour area has been reported in severalstudies (Stamp, 1989; Howeedy ��, 1990; Natali ��, 1990; Tiitta, 1995; Doi ��, 1996;Shintani ��, 1997). This restricted expression resembles that of Tn-C during differentiationand wound healing, where Tn-C coincides with advancing epithelium and seems to capture astage of active epithelial cell proliferation and remodelling of the ECM. The advancing cellsseem to produce the Tn-C (Koch ��, 1991; Aukhil ��, 1996; Yoshida ��, 1997). ThisTn-C immunoreactivity has been used as an indicator of the phase of these processes in woundhealing (Juhasz ��, 1993; Betz, 1995; Christofidou-Solomidou ��, 1997). The expression ofTn-C in invading areas of malignant tumours may illustrate an analogous active process of carci-noma cell proliferation and remodelling of the cancerous stroma.

The sites of Tn-C localization within tissues suggest a possible relationship with proliferativeevents, but it is not clear whether Tn-C serves as a mitogen in vivo, or simply parallels prolifera-tive events (Vollmer, 1997). In normal breast tissue, the highest proliferation found in the secondhalf of the menstrual cycle was accompanied by expression of Tn-C (Ferguson ��, 1990). Inthe present study, the expression of Tn-C in the invasion border of small breast carcinomas wasassociated with a high SPF and with the Ki-67 proliferation antigen (study II). High Ki-67 levelswere also associated with enhanced Tn-C expression round the ducts of DCIS (study IV). Tn-Chas also been shown to co-express with the proliferating cell nuclear antigen (PCNA) in endo-metrial hyperplasia and carcinoma (Yamanaka ��, 1996) and in adenoid cystic carcinoma ofsalivary glands (Shintani ��, 1997).

There was an association between Tn-C in the invasion border and cath-D expression in eitherstromal or carcinoma cells, and these three parameters all related to a higher risk of metastasis(study III). Tn-C was degraded by five different proteinases (Imai� �� , 1994). Plasmin acti-vated by uPA was also able to cleave Tn-C (Gundersen� ��, 1997). Kusagawa has proposedthat Tn-C degradation might be used as a marker for metastatic potential, since there was degra-dation of Tn-C into small fragments in six out of 13 lung cancers with lymph node metastases,but in only one out of 17 node-negative tumours (Kusagawa ��, 1998). Degradation of Tn-Cin the stroma of carcinomas may relate to increased proteolytic activity and a worse prognosis.This accords with reports of decreased stromal Tn-C expression and a poor prognosis (Sugawara��, 1991; Shoji ��, 1993; Iskaros ��, 1997).

The factors that were significantly prognostic for metastasis in univariate analysis in studies I-IIIinclude tumour size, high proliferation (Ki-67 in all tumours, SPF in diploid tumours), Tn-C inthe invasion border, cath-D in stromal and carcinoma cells and PAI-1 in stromal fibroblasts. No

Discussion 37

association was found between metastasis and erbB-2 or p53 expression. One explanation maybe that the patients were all treated with breast conserving surgery and had been selected on thebasis of small tumour size (cT1), whereas most other studies on node-negative breast cancerhave also included larger tumours. It is suggested that the mutations that lead to overexpressionof erbB-2 and p53 are not directly related to prognosis in the early phases of breast carcinoma.uPA and PAI-1 were widely expressed both diffusely in the stroma and in the cytoplasm of car-cinoma cells. However, we failed to show a consistent association between these factors anddisease outcome. This may be due to difficulties in quantification, which is based mainly on theimmunostaining intensity of these almost ubiquitous enzymes. An invasion border was not pres-ent in all the tumour blocks available, which may have diminished the prognostic value of Tn-Cin the invasion border, as compared with other prognostic factors (studies II and III).

Besides a higher risk of a distant metastasis, women with tumours expressing Tn-C in the inva-sion border also had a higher risk of local recurrence after breast-conserving surgery and post-operative radiotherapy (study II). Adjuvant treatment would probably have diminished this risk(Fisher ��, 1995a; EBCTCG, 1998b) Whether missing Tn-C identifies a subgroup of women,who could be treated safely with breast-conserving surgery without postoperative radiotherapyremains to be investigated. Few biological tumour markers have been presented for selectingpatients for breast conservation. Suggested predictive markers for the effect of post-operativeradiotherapy following breast conservation include elevated levels of p53 or GST-π (glutathioneS-transferase-π) proteins and absent or low levels of bcl-2 protein (Silvestrini��, 1997). Therate of local recurrences after breast conservation was higher in high-grade (Nixon��, 1996)and in ER-negative tumours (Yaghan� ��, 1998). EIC is a known risk factor for local recur-rence, but in the present study with small tumours and clean margins this was not the case. Indi-vidual tailoring of breast-conserving treatment is needed if we are to avoid overtreatment and toprevent local recurrences. According to recent data, an early local recurrence after breast-conserving treatment is associated with an equally poor prognosis as a local recurrence aftermastectomy (Tienhoven ��, 1999).

In DCIS, moderate to strong periductal Tn-C was related to microinvasion. Stromal Tn-C ex-pression was found in most intraductal component areas of small invasive breast carcinomas butonly in one tumour in four of pure DCIS (study IV). These results imply that the expression ofTn-C is related to invasion and Tn-C seems to be present from the very first phase of invasion. Ininvasive tumours, Tn-C in the invasion border was unrelated to tumour size (study I). Tn-C im-munostaining in the areas of invading cells may thus be an indicator of aggressive growth in allstages of early carcinomas. Archival material of small tumours was used and only one slide fromeach tumour was examined in this study. Whether Tn-C expression is present in all invasionborders of a three-dimensional, often spherical tumour, remains to be studied. Tn-C immu-nostaining was easy to interpret and immunohistochemistry is a preferable method in small tu-mours, because it saves tissue for diagnostics and other prognostic tests.

The associations between Tn-C in the invasion border and breast cancer prognosis (studies I andII), and between periductal Tn-C and the malignant characteristics of DCIS (high nuclear grade,comedo-type, Ki-67, missing PR and microinvasion) (study IV) suggest that tumours lacking Tn-C may be dormant. In the present study, those 40% of women with DCIS expressing weaklyperiductal Tn-C or none may have been cured with surgery alone. The 50% of women with earlybreast carcinomas whose tumours do not express Tn-C in the invasion border may not need ad-juvant hormonal therapy or chemotherapy. However, the results will have to be repeated in fu-ture studies with more patients before definite conclusions can be drawn.

38 Discussion

The expression of Tn-C in DCIS resembles the expression of Tn-C in prostatic intra-epithelialneoplasia (Ibrahim ��, 1993; Xue ��, 1998) and adenomas of the colon (Riedl ��, 1992).Similarities also exist in the distribution of Tn-C in various types of carcinoma (Shrestha andMori, 1997) and also in various types of mesenchymal tumours (Schnyder� �� , 1997). Theexpression of Tn-C in any dysplasia may indicate the earliest phase of invasion. In any invasivetumours, Tn-C immunoreactivity in areas of invasion may indicate an active phase of tumourgrowth with an increased tendency of the carcinoma cells to disseminate.

Conclusions 39

%�&%#��'�&�

In small axillary node-negative breast carcinomas, Tn-C was expressed in the invasion border inhalf the cases. This expression of Tn-C in the invasion border was a prognostic factor for bothlocal breast cancer recurrence and distant metastasis.

Tn-C in the invasion border was associated with grade, with a higher proliferation rate (Ki-67and SPF) and with cath-D expression in either carcinoma or stromal cells.

Other prognostic factors for metastasis were Ki-67, SPF in diploid tumours, tumour size, cath-Din carcinoma cells and stromal cells, and PAI-1 in stromal fibroblasts.

No consistent association was found between uPA or PAI-1 and prognosis. The reason may havebeen failure to quantify these enzymes immunohistochemically.

Moderate to strong Tn-C expression periductally in DCIS was related to invasion and to suchunfavourable characteristics as high nuclear grade, comedo type, Ki-67 expression and absentPR. Those 40% of DCIS cases with no Tn-C or only weak periductal expression may represent asubgroup of tumours with benign behaviour.

Summary40

��11�(2

Tenascin-C (Tn-C) is an extracellular matrix glycoprotein expressed transiently in areas ofepithelial-mesenchymal interaction during embryogenesis and wound healing. Strong immu-nostaining is often seen in the stroma of various carcinomas. The expression of Tn-C was studiedin 159 small axillary node-negative breast carcinomas. The patients had all been treated withbreast-conserving surgery and post-operative radiotherapy and followed up for a median of 8years. Stromal expression of Tn-C did not relate to prognosis, but the expression of Tn-C in theinvasion border was associated with metastasis and local recurrence. This expression in the inva-sion border was related to grade, proliferation (measured with Ki-67 antigen and S-phase frac-tion) and cathepsin-D. Tn-C immunostaining was also performed on 89 intraductal carcinomas(DCIS). A moderate to strong periductal Tn-C expression correlated with high nuclear-grade,comedo type, microinvasion, Ki-67 expression and absence of progesterone receptors. The ex-pression of Tn-C seems to be related to invasion. Its clinical use might be to indicate the veryearly phase of invasion in dysplasias. In invasive tumours, its expression at the invasion frontmay indicate a tumour ready to disseminate. In DCIS, tumours with no Tn-C or only weak peri-ductal expression might be cured with surgery alone. The small axillary node-negative invasivebreast carcinomas that did not express Tn-C in the invasion border had an excellent prognosis.Future studies are needed to find out whether this subgroup does not need adjuvant therapy.

Acknowledgements 41

�%7&�/#$�)$1$&��

This study was carried out at the Fourth Department of Surgery (the Surgical Hospital) and theDepartment of Oncology, Helsinki University Central Hospital during 1990-1999. I want toexpress my deep gratitude to Professor Juhani Ahonen, Head of the Fourth Department of Surge-ry and ”our Jussi” for the staff of the Surgical Hospital. He has offered not only facilities forresearch but also his support and good advice concerning the study. He is also acknowledged forimproving the training of surgeons in Finland. I also want to thank the former and present Pro-fessors of Oncology, Lars Holsti, Pentti Rissanen and Heikki Joensuu, for their positive attitudetowards my work. Professor Joensuu kindly contributed to the study design.

I have been lucky to have patient, inspiring and encouraging supervisors Docent Karl von Smit-ten, M.D., and Docent Ilkka Saario, M.D., who have tolerated me taking my time with the evolu-tion of this study. Ilkka Saario gave me the idea of studying the results of breast conserving sur-gery and was the first to teach me scientific thinking and writing. Karl von Smitten has beendeveloping the treatment of breast cancer patients and during the years has collected patient datafor clinical and scientific purposes at the Surgical Hospital. Unlike myself, he never seemed tobe disappointed with my not getting things done during this study.

The contribution of Professor Ismo Virtanen, Head of the Department of Anatomy and the Insti-tute of Biomedicine, University of Helsinki, has been of the utmost importance for the executionof this study. He has developed the antibody used in tenascin-C (Tn-C) immunohistochemistryand he and his co-workers have published the very basic observations about Tn-C in normal,hyperplastic and neoplastic breast tissue. During this study he placed the excellent laboratorystaff and facilities of the Department of Anatomy at my disposal. He also helped me to keep upwith the subject by providing me with dozens of publications about Tn-C. His prompt review ofall the manuscripts has been of great value.

Terttu Toivonen, M.D., has spent many hours looking at the microscope opposite me. Besidesteaching me breast pathology, she has amazed me by her vivid imagination, wit and knowledgeof medicine, biology, literature, music and social affairs. I never tired at those microscopy ses-sions.

Docent Stig Nordling, M.D., always had time to help me. He did a great work in interpreting theresults of flow-cytometry and p53 and Ki-67 immunohistochemistry. In addition, he turned out tobe a versatile consultant of computer programmes, data management, scientific writing and theEnglish language.

Professor Carl Blomqvist, M.D., taught me how to find my way at the Department of Oncologyand how to use statistical programs. His constructive criticism has been essential in preparing themanuscripts. Getting to know the ”oncological” way of thinking after many discussion with”Calle” has been useful not only in this study but also in my work as a surgeon.

Docent Caj Haglund, M.D., together with the expertise of Docent Kristina von Boguslawski,Ph.D., has been the promoter of many significant prognostic studies of tumour-related factors. Ihave been priviledged to collaborate with their group. Docent Heli Nevanlinna, Ph.D., broughtthe view of a geneticist to the study. Docent Veli-Matti Wasenius, Ph.D., was in charge of theimmunohistochemical analysis of proteolytic enzymes.

I am grateful to the reviewers of this thesis, Docent Kaija Holli, M.D., and Docent Ylermi Soini,M.D. for their constructive opinions and help in completing this manuscript.

42 Acknowledgements

Mrs Jean Margaret Perttunen, B.Sc., has revised the English language of this thesis. I have enjo-yed her accurate comments and sense of humour during the work.

Professor Eero Saksela, Head of the Department of Pathology, University of Helsinki, providedfavourable conditions and some good advice to complete this study.

At the beginning of this study, in the years 1989-1994, I was obtaining an important part of mysurgical training in Maria Hospital, Helsinki, where Heikki Tykkä, M.D., had started breast-conserving surgery in the mid-1980’s. I was impressed by the high quality of surgical treatmentof early breast cancer created by the multidisciplinary group of radiologists, surgeons and pat-hologists. The basic aims of this study arose from an idea of still improving the prognosis ofthese patients. Docent Vesa Perhoniemi, M.D., my chief in the Maria Hospital, showed interestin this study and sent me literature. I want to thank the former Heads of the Department of Sur-gery of Maria Hospital Docent Gustaf Fock, Docent Seppo Vainionpää and Jan Rusk for theirpositive attitude towards me.

When designing the study, I got valuable advice from Professor Kari Alitalo and Professor AnttiVaheri. My sincerest thanks are due to Docent Torsten Wahlström, M.D., who originally, bychance, suggested that I should study Tn-C. At that time I did not know anything about Tn-C and”Totte” Wahlström did not know me. Such unpredjudiced and unselfish communication is vitalin science.

Professor Seppo Sarna gave me some valuable advice concerning the statistical presentation.Professor Jorma Keski-Oja has been of help in revising some of the manuscripts and in our con-tinuous discussions on Tuesdays during short breaks of his dermatological and my surgical prac-tices at Diacor Reception Centre in Herttoniemi. I also want to thank Professor Peter Roberts andProfessor Krister Höckerstedt for their support and guidance.

The technical expertise of Ms Leena Pirjamo, Aili Takkinen, Marja-Leena Piironen, MonicaShoultz, Päivi Peltokangas, Aino Mäki, Kristiina Messina and Mr Reijo Karppinen has been ofgreat value.

To perform a study spread over several institutes and departments involves extra work. I havebeen lucky to have help from the following persons whenever I needed to find the right people,patient files, tumour samples, laboratory equipment or literature: Ms Hilkka Ahonen, Raija Lahti,Lea Hagman, Marjo Tukiainen, Leila Willström, Arja Tuokko, Anna-Leena Hästö, Irma Anders-son, Merja Stamm, Outi Rauanheimo, Margit Ahlskog, Seija Aro, Hannele Pitkänen Jaana Sopa-nen, Aini Hämäläinen and the staff of the patient archives of the Surgical Hospital.

I am grateful to my parents for teaching me how to study and work and never take anything forgranted. My mother Leena, M.Sc., took care that we children learnt English. My late fatherPentti Roukka, M.Sc., always trusted that I should manage to find my way, which still encoura-ges me.

Kaisa Roukka, M.Sc., a biologist and my aunt, has supported me in my life and studies for aslong as I can remember. With her husband Olli Halkka, Emeritus Professor of genetics, she hasbrought me more scientific writing than I was able to read throughout the years of this study.

I want to acknowledge the positive support of my parents-in-law, Oili and Antero Jahkola. Ante-ro, as a still working Emeritus Professor has represented an ideal of a disciplined scientist.

My dear husband and best friend Juha, M.Sc., helped me to create research files for this studyand also with the editing of this thesis. Our children Pyry, Lauri and Tuuli have grown quite

Acknowledgements 43

independent and capable of taking care of their duties during these years, whether because or inspite of their mother struggling with her thesis.

This study was financially supported by the Finnish Cancer Organization, the Finnish MedicalAssociation, the Finnish Breast Cancer Group and the Kurt and Doris Palander Foundation.

Thank you all for all. Alone I would not have achieved anything.

Helsinki June 1999

��

��

��

��!��"��

�� #�� !��

$�� %�&��'��'��()*+,

References44

($�$($&%$�

Aaltonen, M., Lipponen, P., Kosma, V.-M., Aaltomaa, S. and Syrjänen, K., Prognostic value ofcathepsin D expression in female breast cancer. ��, 15, 1033-1038 (1995).

Alberts, B., Bray, D., Lewis, J., Raff, M., Roberts, K. and Watson, J.D. (1994). Cells junctions,cell adhesion, and the extracellular matrix. In ��!��B. Alberts, D. Bray,J. Lewis, M. Raff, K. Roberts, & J. D. Watson (Ed.), (pp. 946-1010). New York, London: Gar-land Publishing, Inc.

Allred, D.C., Clark, G.M., Elledge, R., Fuqua, S.A.W., Brown, R.W., Chamness, G.C., Osborne,C.K. and McGuire, W.L., Association of p53 protein expression with tumour cell proliferationrate and clinical outcome in node-negative breast cancer. -� .�� , 85, 200-206(1993).

Alo, P.L., Visca, P., Marci, A., Mangoni, A., Botti, C. and Tondo, U.D., Expression of fatty acidsynthase (FAS) as a predictor of recurrence in stage I breast carcinoma patients. ��, 77,474-482 (1996).

Altman, D.G., Lausen, B. and Sauerbrei, W., Dangers of using “optimal” cutpoints in theevaluation of prognostic factors. -�.��, 86, 829-835 (1994).

Andrulis, I.L., Bull, S.B., Blackstein, M.E., Sutherland, D., Mak, C., Sidlofsky, S., Pritzker,K.P.H., Hartwick, R.W., Hanna, W., Lickley, L., Wilkinson, R., Qizilbash, A., Ambus, U., Lipa,M., Weizel, H., Katz, A., Baida, M., Mariz, S., Stoik, G., Dacamara, P., Strongitharm, D., Ged-die, W., McCready, D. for the Toronto Breast Cancer Study Group., neu/erbB-2 amplificationidentifies a poor-prognosis group of women with node-negative breast cancer. -��/��, 16,1340-1349 (1998).

Armas, O.A., Gerald, W.L., Lesser, M.L., Arroyo, C.D., Norton, L. and Rosen, P.P., Immuno-histochemical detection of cathepsin D in T2N0M0 breast carcinoma. � � -� 0�!� ��, 18,158-166 (1994).

Arriagada, R. and Rutqvist, L.E., Adjuvant chemotherapy in early breast cancer and incidence ofnew primary malignancies. 1��, 338, 535-538 (1991).

American Society of Clinical Oncology (ASCO)., 1997 update of recommendations for the useof tumour markers in breast and colorectal cancer. -��/��, 16, 793-795 (1998).

Aufderheide, E., Chiquet-Ehrismann, R. and Ekblom, P., Epithelial-mesenchymal interactions inthe developing kidney lead to expression of tenascin in the mesenchyme. -��2��, 105, 599-608 (1987).

Aufderheide, E. and Ekblom, P., Tenascin during gut development: appearance in the mesen-chyme, shift in molecular forms, and dependence on epithelial-mesenchymal interactions. -��2��, 107, 2341-2349 (1988).

Aukhil, I., Sahlberg, C. and Thesleff, I., Basal layer of epithelium expresses tenascin mRNAduring healing of incisional skin wounds. -��, 31, 105-112 (1996).

Azzopardi, J.G., Ahmed, A. and Millis R.R (1979). Problems in breast pathology. In �3�� !��4��((��Bennington, J.L. (Ed.), (pp. 240-257). London/Philadelphia: WBSaunders

References 45

Baisch, H., Göhde, W. and Linden, W.A., Analysis of PCP data to determine the fraction of cellsin various phases of cell cycle. ��5�4��2��, 2, 31-39 (1975).

Betz, P., Immunohistochemical parameters for the age estimation of human skin wounds. � �-6�� , 16, 203-209 (1995).

Birchmeier, C. and Birchmeier, W., Molecular aspects of mesenchymal-epithelial interactions.��4��2��, 9, 511-540 (1993).

Blichert-Toft, M., Rose, C. and Andersen, J.A., Danish randomized trial comparing breast con-servation with mastectomy: Six years of life table analysis. -�.��, Monogr 11, 19-25 (1992).

Blichert-Toft, M., Smola, M.G., Cataliotti, L. and O’Higgins, N., Principles and guidelines forsurgeons - management of symptomatic breast cancer. 5��-�0�!�/��, 23, 101-109 (1997).

Bloom, H.J.G. and Richardson, W.W., Histological grading and prognosis in breast cancer. Astudy of 1409 cases of which 359 have been followed for 15 years. 2�-��, 11, 359-377(1957).

Boguslawski, K.v., Immunohistochemical detection of progesterone receptors in paraffin sec-tions. A novel method using microwave oven pretreatment. �� , 102: 641-646, (1994).

Bonadonna, G., Rossi, A., Valagussa, P., Banfi, A. and Veronesi, U., The CMF program foroperable breast cancer with positive axillary nodes: updated analysis on the disease-free interval,site of relapse and drug tolerance. ��, 39 Suppl, 2904-2915 (1977).

Borger, J., Kemperman, H., Hart, A., Peterse, H., van Dongen, J. and Bartelink, H., Risk factorsin breast-conservation therapy. -��/��, 12, 653-60 (1994).

Borsi, L., Allemanni, G., Gaggero, B. and Zardi, L., Extracellular pH controls pre-mRNA alter-native splicing of tenascin-C in normal, but not malignantly transformed cells. ��-��, 66,632-635 (1996).

Borsi, L., Carnemolla, B., Nicola, G., Spina, B., Tanara, G. and Zardi, L., Expression of differ-ent tenascin isoforms in normal, hyperplastic and neoplastic human breast tissues. ��-��,52, 688-692 (1992).

Bosman, F.T., The borderline: basement membranes and the transition from premalignant tomalignant neoplasia. Review. ��7��, 29, 216-225 (1994).

Bouchet, C., Spyratos, F., Martin, P.M., Hacène, K., Gentile, A. and Oglobine, J., Prognosticvalue of urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitors PAI-1and PAI-2 in breast carcinomas. 2�-��, 69, 398-405 (1994).

Bourdon, M.A., Wikstrand, C.J., Furthmayer, H., Matthews, T.J. and Bigner, D.D., Human gli-oma-mesenchymal extracellular matrix antigen defined by monoclonal antibody. ��, 43,2796-2805 (1983).

Boyages, J., Delaney, G. and Taylor, R., Predictors of local recurrence after treatment of ductalcarcinoma in situ. ��, 85, 616-628 (1999).

Boyages, J., Recht, A., Connolly, J.L., Schnitt, S.J., Gelman, R., Kooy, H., Love, S., Osteen,R.T., Cady, B., Silver, B. and Harris, J.R., Early breast cancer: predictors of breast recurrencefor patients treated with conservative surgery and radiation therapy. ��/��, 19, 29-41(1990).

46 References

Bristow, J., Tee, M.K., Gitelman, S.E., Mellon, S.H. and Miller, W.L., Tenascin-X: a novelextracellular matrix protein encoded by the human XB gene overlapping p450c21B. -��2��,122, 265-278 (1993).

Burch, G.H., Bedolli, M.A., McDonough, S., Rosenthal, S.M. and Bristow, J., Embryonic ex-pression of tenascin-X suggests a role in limb, muscle, and heart development. "�4�"��, 203,491-504 (1995).

Burch, G.H., Gong, Y., Liu, W., Dettman, R.W., Curry, C.J., Smith, L., Miller, W.L. and Bris-tow, J., Tenascin-X deficiency is associated with Ehlers-Danlos syndrome. .��8��, 17, 104-108 (1997).

Burke, H.B., Hoang, A., Iglehart, J.D. and Marks, J.R., Predicting response to adjuvant andradiation therapy in patients with early stage breast carcinoma. ��, 82, 874-877 (1998).

Callahan, R., Genetic alterations in primary breast cancer. 2��, 13, 191-203 (1989).

Cantor, A.B. and Shuster, J.J., Re: Dangers of using “optimal” cutpoints in the evaluation ofprognostic factors. Correspondence. -�.��, 86, 1798-1799 (1994).

Carnemolla, B., Leprini, A., Borsi, L., Querze, G., Urbini, S. and Zardi, L., Human tenascin-R.Complete primary structure, pre-mRNA alternative splicing and gene localization on chromo-some 1q23-q24. -�2�� , 271, 8157-8160 (1996).

Carson, D.A. and Lois, A., Cancer progression and p53. Review. 1��, 346, 1009-1011(1995).

Carter, C.L., Allen, C. and Henson, D.E., Relation of tumor size, lymph node status, and survivalin 24,740 breast cancer cases. ��, 63, 181-187 (1989).

Castiglioni, T., Merino, M.J., Elsner, B., Lah, T.T., Sloane, B.F. and Emmert-Buck, M.R., Im-munohistochemical analysis of cathepsins D, B, and L in human breast cancer. 9� ��, 25,857-862 (1994).

Chiquet, M. and Fambrough, D.M., Chick myotendinous antigen. I. A monoclonal antibody as amarker for tendon and muscle morphogenesis. -��2��, 98, 1926-1936 (1984a).

Chiquet, M. and Fambrough, D.M., Chick myotendinous antigen. II. A novel extracellular gly-coprotein complex consisting of large disulfide-linked subunits. -� �� 2��, 98, 1937-1946(1984b).

Chiquet-Ehrismann, R., What distinguishes tenascin from fibronectin? 6�052�-, 4, 2598-2604(1990).

Chiquet-Ehrismann, R., Kalla, P. and Pearson, C.A., Participation of tenascin and transforminggrowth factor-β in reciprocal epithelial-mesenchymal interactions of MCF7 cells and fibroblasts.��, 49, 4322-4325 (1989).

Chiquet-Ehrismann, R., Kalla, P., Pearson, C.A., Beck, K. and Chiquet, M., Tenascin interfereswith fibronectin action. ��, 53, 383-390 (1988).

Chiquet-Ehrismann, R., Mackie, E.J., Pearson, C.A. and Sakakura, T., An extracellular matrixprotein involved in tissue interactions during fetal development and oncogenesis. ��, 47, 131-139 (1986).

References 47

Christofidou-Solomidou, M., Bridges, M., Murphy, G.F., Albelda, S.M. and DeLisser, H.M.,Expression and function of endothelial cell α-v integrin receptors in wound-induced human an-giogenesis in human skin/SCID mice chimeras. � �-��, 151, 975-983 (1997).

Ciocca, D.R., Fujimura, F.K., Tandon, A.K., Clark, G.M., Mark, C., Lee-Chen, G.-J., Pounds,G.W., Vendely, P., Owens, M.A., Pandian, M.R. and McQuire, W.L., Correlation of HER-2/neuamplification with expression and with other prognostic factors in 1103 breast cancers. -�.��, 84, 1279-1282 (1992).

Clark, G.M., Do we really need prognostic factors for breast cancer? Review. 2��, 30, 117-126 (1994).

Clark, G.M., Dressler, L.G., Owens, M.A., Pounds, G., Oldaker, T. and McGuire, W.L., Predic-tion of relapse or survival in patients with node-negative breast cancer by DNA flow cytometry..�5�!��-� ��, 320, 627-33 (1989).

Clark, R.A.F., Ashcroft, G.S., Spencer, M.-J., Larjava, H. and Ferguson, M.W.J., Re-epithelialization of normal human excisional wounds is associated with a switch from αv β5 toαv β6 integrins. 2�-�"� ��, 135, 46-51 (1996).

Clark, R.M., McCulloch, P.B., Levine, M.N., Lipa, M., Wilkinson, R.H., Mahoney, L.J., Basrur,V.R., Nair, B.D., McDermot, R.S., Wong, C.S. and Corbett, P.J., Randomized clinical trial toassess the effectiveness of breast irradiation following lumpectomy and axillary dissection fornode-negative breast cancer. -�.��, 84, 683-689 (1992).

Costantini, V., Sidoni, A., Deveglia, R., Cazzato, O.A., Bellezza, G., Ferri, I., Bucciarelli, E. andNenci, G.G., Combined overexpression of urokinase, urokinase receptor, and plasminogen acti-vator inhibitor-1 is associated with breast cancer progression. ��, 77, 1079-1088 (1996).

Crossin, K.L., Hoffman, S., Grumet, M., Thiery, J.P. and Edelman, G.M., Site-restricted expres-sion of cytotactin during development of chick embryo. -��2��, 102, 1917-1930 (1986).

Dano, K., Andreasen, P.A., Grondal-Hansen, J., Kristensen, P., Nielsen, L.S. and Skriver, L.,Plasminogen activators, tissue degradation and cancer. ��4��, 44, 139-266 (1985).

Doi, D., Araki, T. and Asano, G., Immunohistochemical localization of tenascin, estrogen re-ceptor and transforming growth factor-β-1 in human endometrial carcinoma. 8�� /��4��, 41, 61-66 (1996).

Domagala, W., Striker, G., Szadowska, A., Dukowicz, A., Weber, K. and Osborn, M., CathepsinD in invasive ductal NOS breast carcinoma as defined by immunohistochemistry. � �-��,141, 1003-1012 (1992).

van Dongen, J.A., Bartelink, H. and Fentiman, I.S., Randomized clinical trial to assess the valueof breast-conserving therapy in stage I and II breast cancer. EORTC 10810 trial. -�.��, Monogr 11, 15-18 (1992).

Dowsett, M., Improved prognosis for biomarkers in breast cancer. Editorial. 1��, 351, 1753-1754 (1998).

Duffy, M.J., Brouillet, J.-P., Reilly, D., McDermott, E., O’Higgins, N., Fennelly, J.J., Maude-londe, T. and Rochefort, H., Cathepsin D concentration in breast cancer cytosols: correlationwith biochemical, histological, and clinical findings. �� , 37, 101-104 (1991).

48 References

Duffy, M.J., O’Grady, P., Devaney, D., O’Siorain, L., Fennelly, J.J. and Lijnen, H.J., Urokinase-plasminogen activator, a marker for aggressive breast carcinomas - preliminary report. ��,62, 531-533 (1988).

Duffy, M.J., Reilly, D., Brouillet, J.-P., McDermott, E.W.M., Faul, C., O’Higgins, N., Fennelly,J.J., Maudelonde, T. and Rochefort, H., Cathepsin D concentration in breast cancer cytosols:correlation with disease-free interval and overall survival. �� , 38, 2114-2116 (1992).

Dvorak, H.F., Tumors: wounds that do not heal. Similarities between tumor stroma generationand wound healing. Review. .�5�!��-� ��, 315, 1650-1659 (1986).

EBCTCG., Systemic treatment of early breast cancer by hormonal, cytotoxic, or immune ther-apy. 133 randomised trials involving 31,000 recurrences and 24,000 deaths among 75,000women. Early Breast Cancer Trialists’ Collaborative Group. 1��, 339, 1-15 and 71-85(1992).

EBCTCG., Effects of radiotherapy and surgery in early breast cancer. .�5�!��-� ��, 333, 1444-55 (1995).

EBCTCG., Polychemotherapy for early breast cancer: an overview of the randomized trials.1��, 352, 930-942 (1998a).

EBCTCG., Tamoxifen for early breast cancer: an overview of the randomized trials. 1��,351, 1451-1467 (1998b).

Elston, C.W. and Ellis, I.O. (1998). ��2�� '�0�� !� . Churchill Livingstone:Edinburgh, London, New York, Philadelphia, San Francisco, Sydney, Toronto.

End, P., Panayotou, G., Entwistle, A., Waterfield, M.D. and Chiquet, M., Tenascin: a modulatorof cell growth. 5��-�2�� , 209, 1041-51 (1992).

Engel, J., EGF-like domains in the extracellular matrix proteins: localized signals for growth anddifferentiation. 6520�1��, 251, 1-7 (1989).

Erickson, H.P., Tenascin-C, tenascin-R and tenascin-X: a family of talented proteins in search offunctions. Review. ��/��2��, 5, 869-876 (1993).

Erickson, H.P. and Inglesias, J.L., A six-armed oligomer isolated from cell surface fibronectinpreparations. .��, 311, 267-269 (1984).

Eskelinen, M.J., Pajarinen, P., Collan, Y., Pesonen, E., Alhava, E., Kettunen, K. and Nordling,S., Relationship between DNA ploidy and survival in patients with primary breast cancer. 2�-0�!, 76, 830-834 (1989).

Fentiman, I.S., Hyland, D., Chaudary, M.A. and Gregory, W.M., Prognosis of patients withbreast cancers up to 1 cm in diameter. 5��-��, 32A, 417-420 (1996).

Ferguson, J.E., Schor, A.M., Howell, A. and Ferguson, M.W., Tenascin distribution in the nor-mal human breast is altered during the menstrual cycle and in carcinoma. "��, 42,199-207 (1990).

Fernö, M., Baldetorp, B., Borg, Å., Brouillet, J.-P., Olsson, H., Rochefort, H., Sellberg, G.,Sigurdsson, H., Killander, D. for the South Sweden Breast Cancer Group., Cathepsin D, both aprognostic factor and a predictive factor for the effect of adjuvant tamoxifen in breast cancer.5��-��, 30A, 2042-2048 (1994).

References 49

Ferrandina, G., Scambia, G., Bardelli, F., Panici, P.B., Mancuso, S. and Messori, A., Relation-ship between cathepsin-D content and disease-free survival in node-negative breast cancer pa-tients: a meta-analysis. 2�-��, 76, 661-666 (1997).

Finnish Breast Cancer Group (1999) Rintasyövän valtakunnallinen diagnostiikka- ja hoitosuosi-tus. 0�� :�;�� ;�� Tampere: Department of Oncology, Tampere University Hos-pital.

Fisher, B., Anderson, S., Redmond, C.K., Wolmark, N., Wickerham, D.L. and Cronin, W.M.,Reanalysis and results after 12 years of follow-up in a randomized clinical trial comparing totalmastectomy with lumpectomy with or without irradiation in the treatment of breast cancer. .5�!��-� ��, 333, 1456-61 (1995a).

Fisher, B., Bauer, M., Margolese, R., Poisson, R., Pilch, Y., Redmond, C., Fisher, E.R., Wol-mark, N., Deutsch, M., Montague, E., Saffer, E., Wickerham, L., Lerner, H., Glass, A., Shibata,H., Deckers, P., Ketcham, A., Oishi, R. and Russel, I., Five-year results of a randomized clinicaltrial comparing total mastectomy and segmental mastectomy with or without radiation in thetreatment of breast cancer. .�5�!��-� ��, 312, 665-673 (1985).

Fisher, B., Redmond, C., Poisson, R., Margolese, R., Wolmark, N., Wickerham, L., Fisher, E.,Deutsch, M., Caplan, R., Pilch, Y., Glass, A., Shibata, H., Lerner, H., Terz, J. and Sidorovich,L., Eight-year results of a randomized clinical trial comparing total mastectomy and lumpectomywith or without irradiation in the treatment of breast cancer. .� 5�!�� -� ��, 320, 822-828(1989).

Fisher, E.R., Costantino, J., Fisher, B., Palekar, A.S., Redmond, C., Mamounas, E. and Investi-gators, for the National Surgical Adjuvant Breast and Bowel Project Collaborating Investiga-tors., Pathologic findings from the National Surgical Adjuvant Breast Project (NSABP) ProtocolB-17. Intraductal carcinoma (ductal carcinoma in situ). ��, 75, 1310-1319 (1995b).

Foekens, J.A., Schmitt, M., von Putten, W.L.J., Peters, H.A., Bontenbal, M., Jänicke, F. andKlijn, J.G.M., Prognostic value of urokinase-type plasminogen activator in 671 primary breastcancer patients. ��, 52, 6101-6105 (1992).

Foekens, J.A., Schmitt, M., Putten, von W.L.J., Peters, H.A., Kramer, M.D., Jänicke, F. andKlijn, J.G.M., Plasminogen activator inhibitor-1 and prognosis in primary breast cancer. -��/��, 12, 1648-1658 (1994).

Forsberg, E., Hirsch, E., Fröhlich, L., Meyer, M., Ekblom, P., Aszodi, A., Werner, S. and Fäss-ler, R., Skin wounds and severed nerves heal normally in mice lacking tenascin-C. ��.��0��<0�, 93, 6594-6599 (1996).

Fortin, A., Larochelle, M., Laverdiere, J., Lavertu, S. and Tremblay, D., Local failure is respon-sible for the decrease in survival for patients with breast cancer treated with conservative surgeryand postoperative radiotherapy. -��/�� 17, 101-109 (1999).

Foucré, D., Bouchet, C., Hacène, K., Pourreau-Schneider, N., Gentile, A., Martin, P.M., Des-places, A. and Oglobine, J., Relationship between cathepsin D, urokinase, and plasminogenactivator inhibitors in malignant vs benign breast tumours. 2�-��, 64, 926-932 (1991).

Gage, I., Schnitt, S.J., Nixon, A.J., Silver, B., Recht, A., Troyan, S.L., Eberlein, T., Love, S.M.,Gelman, R., Harris, J.R. and Connolly, J.L., Pathologic margin involvement and the risk of re-currence in patients treated with breast-conserving therapy. ��, 78, 1921-8 (1996).

50 References

Gasparini, G., Pozza, F., Bevilacqua, P., Meli, S., Boracchi, P., Reitano, M., Santini, G., Maru-bini, E. and Sainsbury, J.R.C., Growth fraction (Ki-67 antibody) determination in early-stagebreast carcinoma: histologic, clinical and prognostic correlations. ��2��, 1, 92-99 (1992).

Gasparini, G., Pozza, F. and Harris, A.L., Evaluating the potential usefulness of new prognosticand predictive indicators in node-negative breast cancer patients. -�.��, 85, 1206-1219 (1993).

Gasparini, G., Weidner, N., Bevilacqua, P., Maluta, S., Palma, P.D., Caffo, O., Barbareschi, M.,Boracci, P., Marubibi, E. and Pozza, F., Tumor microvessel density, p53 expression, tumor size,and peritumoral lymphatic vessel invasion are relevant prognostic markers in node-negativebreast carcinoma. -��/��, 12, 454-466 (1994).

Gerdes, J., Schwab, U., Lemke, H. and Stein, H., Production of a mouse monoclonal antibodyreactive with a human nuclear antigen associated with cell proliferation. ��-��, 31, 13-20(1983).

Goldhirsch, A., Glick, J.H., Gelber, R.D. and Senn, H.-J., Meeting highlights: International con-sensus panel on the treatment of primary breast cancer. -� .�� , 90, 1601-1608(1998).

Gould, V.E., Koukoulis, G.K. and Virtanen, I., Extracellular matrix proteins and their receptorsin the normal, hyperplastic and neoplastic breast. ��"��"�4, 32, 409-416 (1990).

Granata, G., Coradini, D., Cappelletti, V. and Fronzo, G.D., Prognostic relevance of cathepsin Dversus oestrogen receptors in node negative breast cancers. 5��-��, 27, 970-972 (1991).

Grondal-Hansen, J., Christensen, I.J., Rosenquist, C., Brunner, N., Mouridsen, H.T., Dano, K.and Blichert-Toft, M., High levels of urokinase-type plasminogen activator and its inhibitor PAI-1 in cytosolic extracts of breast carcinomas are associated with poor prognosis. ��, 53,2513-2521 (1993).

Grondal-Hansen, J., Hilsenbeck, S.G., Christensen, I.J., Clark, G.M., Osborne, C.K. and Brun-ner, N., Prognostic significance of PAI-1 and uPA in cytosolic extracts obtained from node-positive breast cancer patients. 2��, 43, 153-163 (1997).

Grumet, M., Hoffman, S., Crossin, K.L. and Edelman, G.M., Cytotactin, an extracellular matrixprotein of neural and non-neural tissues that mediates glia-neuron interaction. ��.��0��<0�, 82, 8075-8079 (1985).

Gulcher, J.R., Alexakos, M.J., LeBeau, M.M., Lemons, R.S. and Stefansson, K., Chromosomallocalization of the human hexabrachion (tenascin) gene and evidence for recent reduplicationwithin the gene. 8�� , 6, 616-622 (1990).

Gundersen, D., Chien, T.-T., Sordat, B., Mourali, F. and Ruegg, C., Plasmin-induced proteolysisof tenascin-C. Modulation by lymphocyte derived urokinase-type plasminogen activator andeffect on T-lymphocyte adhesion, activation, and cell clustering. -� � ��, 158, 1951-1060(1997).

Göhring, U.-J., Scharl, A., Thelen, U., Ahr, A. and Crombach, G., Comparative prognostic valueof cathepsin D and urokinase plasminogen activator, detected by immunohistochemistry, in pri-mary breast carcinoma. ��, 16, 1011-1018 (1996).

Haapasalmi, K., Zhang, K., Tonnesen, M., Olerud, J., Sheppard, D., Salo, T., Kramer, R., Clark,R.A.F., Uitto, V.-J. and Larjava, H., Keratinocytes in human wounds express αv β6 integrin. -��4��"� ��, 106, 42-48 (1996).

References 51

Haerslev, T., Jacobsen, G.K. and Zedeler, K., Correlation of growth fraction by Ki-67 and pro-liferating cell nuclear antigen (PCNA) immunohistochemistry with histopathological parametersand prognosis in primary breast carcinomas. 2��, 37, 101-103 (1996).

Haffty, B.G., Fischer, D., Rose, M., Beinfield, M. and McKhann, C., Prognostic factors for localrecurrence in the conservatively treated breast cancer patient: a cautious interpretation of thedata. -��/��, 9, 997-1003 (1991).

Hagios, C., Koch, M., Spring, J., Chiquet, M. and Chiquet-Ehrismann, R., Tenascin-Y: a proteinof novel domain structure is secreted by differentiated fibroblasts of muscle connective tissue. -��2��, 134, 1499-1512 (1996).

Halfter, W., Liverani, D., Vigny, M. and Monard, D., Deposition of extracellular matrix alongthe pathway of migrating fibroblasts. ��, 262, 467-481 (1990).

Harada, T., Shinohara, M., Nakamura, S. and Oka, M., An immunohistochemical study of theextracellular matrix in oral squamous cell carcinoma and its association with invasive and meta-static potential. =��#��4, 424, 257-266 (1994).

Hay, E.D., Extracellular matrix alters epithelial differentiation. Review. ��/��2��, 5,1029-1035 (1993).

Hedley, D.W., Friedlander, M.L., Taylor, I.W., Rugg, C.A. and Musgrove, E.A., Method foranalysis of cellular DNA content of paraffin-embedded pathological material using flow cy-tometry. -�9�� , 31, 1333-1335 (1983).

Hemesath, T.J., Marton, L.S. and Stefansson, K., Inhibition of T cell activation by the extracel-lular matrix protein tenascin. -�� , 152, 5199-5207 (1994).

Henry, J.A., McCarthy, A.L., Angus, B., Westley, B.R., May, F.E.B., Nicholson, S., Cairns, J.,Harris, A.L. and Horne, C.H.W., Prognostic significance of the estrogen-regulated protein, ca-thepsin D, in breast cancer. ��, 65, 265-271 (1990).

Herlyn, M., Graeven, U., Speicher, D., Sela, B.-A., Bennicelli, J.L., Kath, R. and Guerry, D.,Characterization of tenascin secreted by human melanoma cells. �� , 51, 4853-4858(1991).

Hiraiwa, N., Kida, H., Sakakura, T. and Kusakabe, M., Induction of tenascin in cancer cells byinteractions with embryonic mesenchyme mediated by a diffusible factor. -��0�, 104, 289-296 (1993).

Hofmann, R., Lehmer, A., Buresch, M., Hartung, R. and Ulm, K., Clinical relevance of uroki-nase plasminogen activator, its receptor, and its inhibitor in patients with renal cell carcinoma.��, 78, 487-492 (1996).

Holland, P.A., Shah, A., Howell, A., Baildam, A.D. and Bundred, N.J., Lobular carcinoma of thebreast can be managed by breast-conserving therapy. 2�-�0�!, 82, 1364-1366 (1995).

Holland, R., Connolly, J.L., Gelman, R., Mravunac, M., Hendriks, J.H.C.L., Verbeek, A.L.M.,Schnitt, S.J., Silver, B., Boyages, J. and Harris, J.R., The presence of an extensive intraductalcomponent following a limited excision correlates with prominent residual disease in the remain-der of the breast. -��/��, 8, 113-8 (1990b).

Holland, R., Hendriks, J.H.C.L., Verbeek, A.L.M., Mravunac, M. and Stekhoven, J.H.S., Extent,distribution, and mammographic/histological correlations of breast ductal carcinoma in situ.1��, 335, 519-522 (1990a).

52 References

Howeedy, A.A., Virtanen, I., Laitinen, L., Gould, N.S., Koukoulis, G.K. and Gould, V.E., Dif-ferential distribution of tenascin in the normal, hyperplastic, and neoplastic breast. 1�� 4��,63, 798-806 (1990).

Hunt, K.K., Singletary, S.E., Smith, T.L., Ross, M.I., Strom, E.A., McNeese, M.D. and Ames,F.C. (1998). Conservation surgery and radiation: The M. D. Anderson Cancer Center experiance.In ��2�� 4�� !� ��!�� !��K. I. Bland, & E.M. Copeland (Ed.), (pp. 1179-1182). Philadelphia: W.B.Saunders Company.

Huuhtanen, R., Wiklund, T., Blomqvist, C., Virolainen, M., Yi, P. and Tribukait, B., High S-phase fraction is an adverse prognostic sign in diploid soft-tissue sarcomas. ��, 77, 1815-22(1996).

Ibrahim, S.N., Lightner, V.A., Ventimiglia, J.B., Ibrahim, G.K., Walther, P.J., Bigner, D.D. andHumphrey, P.A., Tenascin expression in prostatic hyperplasia, intraepithelial neoplasia, andcarcinoma. 9� ��, 24, 982-989 (1993).

Ilunga, K. and Iriyama, K., Expression of tenascin in gastric carcinoma. 2�-�0�!, 82, 948-951(1995).

Imai, K., Kusakabe, M., Sakakura, T., Nakanishi, I. and Okada, Y., Susceptibility of tenascin todegradation by matrix metalloproteinases and serine proteinases. 6520� 1��, 352, 216-218(1994).

Inaguma, Y., Kusakabe, M., Mackie, E.J., Pearson, C.A., Chiquet-Ehrismann, R. and Sakakura,T., Epithelial induction of stromal tenascin in the mouse mammary gland: from embryogenesis tocarcinogenesis. "�4�2��, 128, 245-255 (1988).

Ishihara, A., Yoshida, T., Tamaki, H. and Sakakura, T., Tenascin expression in cancer cells andstroma of human breast cancer and its prognostic significance. �� , 1, 1035-41(1995).

Iskaros, B.F., Hu, X., Sparano, J.A. and Fineberg, S.A., Tenascin pattern of expression and es-tablished prognostic factors in invasive breast carcinoma. -�0�!�/��, 68, 107-112 (1998).

Iskaros, B.F., Tanaka, K.E., Hu, X., Kadish, A.S. and Steinberg, J.B., Morphologic pattern oftenascin as a diagnostic biomarker in colon cancer. -�0�!�/��, 64, 98-101 (1997).

Isola, J., Visakorpi, T., Holli, K. and Kallioniemi, O.-P., Association of overexpression of tu-mour suppressor protein p53 with rapid cell proliferation and poor prognosis in node-negativebreast cancer patients. -�.��, 84, 1109-1114 (1992).

Isola, J., Weitz, S., Visakorpi, T., Holli, K., Shea, R., Khabbaz, N. and Kallioniemi, O.-P., Ca-thepsin D expression detected by immunohistochemistry has independent prognostic value inaxillary node-negative breast cancer. -��/��, 11, 36-43 (1993).

Isola, J.J., Helin, H.J., Helle, M.J. and Kallioniemi, O.-P., Evaluation of cell proliferation inbreast carcinoma. Comparison of Ki-67 immunohistochemical study, DNA flow cytometricanalysis, and mitotic count. ��, 65, 1180-1184 (1990).

Jacobs, T.M., Prioleau, J.E., Stillman, S.J. and Schnitt, S.J., Loss of tumour marker immu-nostaining intensity on stored paraffin slides of breast cancer. -�.��, 88, 1054-1059(1996).

References 53

Jacuerner, J., Moles, J.P., Penault-Lorca, F., Adelaine, J., Torrente, M., Vians, P., Birngaum, D.and Theillet, C., p53 immunohistochemical analysis in breast cancer with four monoclonal anti-bodies: comparison of staining and PCR-SSCP results. 2�-��, 69, 846-852 (1994).

Joensuu, H. and Toikkanen, S., Prognosis of breast cancer with small primary tumour (pT1).��/��, 30, 793-796 (1991).

Joensuu, H., Toikkanen, S. and Isola, J., Stromal cell cathepsin D expression and long-termsurvival in breast cancer. 2�-��, 71, 155-159 (1995).

Joensuu, H., Toikkanen, S. and Klemi, P.J., DNA index and S-phase fraction and their combina-tion as prognostic factors in operable ductal breast carcinoma. ��, 66, 331-340 (1990).

Johnson, K.A., Kramer, B.S. and Anderson, M.J. (1998). Adjuvant systemic therapy of breastcancer. In �� 2�� '� �� 4�� !� �� !�� !�� K. I.Bland, & E. M. Copeland (Ed.), (pp. 1197-1233). Philadelphia: W.B.Saunders Company.

Jones, P.L., Cowan, K.N. and Rabinovitch, M., Tenascin-C, proliferation and subendothelialfibronectin in progressive pulmonary vascular disease. � �-��, 150, 1349-1360 (1997).

Juhasz, I., Murphy, G.F., Yan, H.-C., Herlyn, M. and Albelda, S.M., Regulation of extracellularmatrix proteins and integrin cell substratum adhesion receptors on epithelium during cutaneoushuman wound healing in vivo. � �-��, 143, 1458-1469 (1993).

Jänicke, F., Schmitt, M., Pache, L., Ulm, K., Harbeck, N., Höfler, H. and Graeff, H., Urokinase(uPA) and its inhibitor PAI-1 are strong and independent prognostic factors in node-negativebreast cancer. 2��, 24, 195-208 (1993).

Jänicke, F., Schmitt, M., Ulm, K., Gössner, W. and Graeff, H., Urokinase-type plasminogenactivator antigen and early relapse in breast cancer. 1��, 334, 1049 (1989).

Kalembeyi, I., Yoshida, T., Iriyama, K. and Sakakura, T., Analysis of tenascin mRNA expres-sion in the murine mammary gland from embryogenesis to carcinogenesis: An in situ hybridiza-tion study. ��-�"�4�2��, 41, 569-573 (1997).

Kallioniemi, O.-P., Blanco, G., Alavaikko, M., Hietanen, T., Mattila, J., Lauslahti, K., Lehtinen,M. and Koivula, T., Improving the prognostic value of DNA flow cytometry in breast cancer bycombining DNA index and S-phase fraction. ��, 62, 2183-2190 (1988).

Kallioniemi, O.-P., Holli, K., Visakorpi, T., Koivula, T., Helin, H.H. and Isola, J.J., Associationof c-erbB-2 protein over-expression with high rate of cell proliferation, increased risk of visceralmetastasis and poor long-term survival in breast cancer. ��-��, 49, 650-655 (1991).

Kallioniemi, O.-P., Joensuu, H., Klemi, P. and Koivula, T., Inter-laboratory comparison of DNAflow cytometric results from paraffin-embedded breast carcinomas. 2��, 17,59-61 (1990).

Kandalaft, P.L., Chang, K.L., Ahn, C.W., Traweek, S.T., Mehta, P. and Battifora, H., Prognosticsignificance of immunohistochemical analysis of cathepsin D in low-stage breast cancer. ��,71, 2756-2763 (1993).

Kawakatsu, H., Shiurba, R., Obara, M., Hiraiwa, H., Kusakabe, M. and Sakakura, T., Humancarcinoma cells synthesize and secrete tenascin in vitro. -�� -� �� , 83, 1073-1080(1992).

54 References

Kemperman, H., Borger, J., Hart, A., Peterse, H., Bartelink, H. and van Dongen, J.v., Prognosticfactors for survival after breast conserving therapy for stage I and II breast cancer. The role oflocal recurrence. 5��-��, 31A, 690-698 (1995).

Keski-Oja, J., Lohi, J., Tuuttila, A., Tryggvason, K. and Vartio, T., Proteolytic processing of the72,000-Da type IV collagenase by urokinase plasminogen activator. 5>��, 202, 471-476(1992).

Koch, M., Wehrle-Haller, B., Baumgartner, S., Spring, J., Brubacher, D. and Chiquet, M.,Epithelial synthesis of tenascin at tips of growing bronchi and graded accumulation in basementmembrane and mesenchyme. 5>��, 194, 297-300 (1991).

Koukoulis, G.K., Gould, V.E., Bhattacharyya, A., Gould, J.E., Howeedy, A.A. and Virtanen, I.,Tenascin in normal, reactive, hyperplastic, and neoplastic tissues: Biologic and pathologic impli-cations. 9� ��, 22, 636-43 (1991a).

Koukoulis, G.K., Howeedy, A.A., Korhonen, M., Virtanen, I. and Gould, V.E., Distribution oftenascin, cellular fibronectins and integrins in normal, hyperplastic and neoplastic breast. Re-view. -�0�� , 25, 285-295 (1993).

Koukoulis, G.K., Virtanen, I., Korhonen, M., Laitinen, L., Quaranta, V. and Gould, V.E., Immu-nohistochemical localization of integrins in the normal, hyperplastic, and neoplastic breast. � �-��, 139, 787-799 (1991b).

Kruse, J., Keilhauer, G., Faissner, A., Timpl, R. and Schachner, M., The J1 glycoprotein - anovel nervous system cell adhesion molecule of the L2/HNK-1 family. .��, 316, 146-148(1985).

Kumar, V., Cotran, R.S. and Robbins, S.L. (1997). 2��!� . W.B.Saunders Company:Philadelphia.

Kusagawa, H., Onoda, K., Namikawa, S., Yada, I., Okada, A., Yoshida, T. and Sakakura, T.,Expression and degeneration of tenascin-C in human lung cancers. 2� -��, 77, 98-102(1998).

Kusakabe, M. and Sakakura, T. (1998). Extracellular matrix in tissue remodelling: tenascin-C asa modulator in cell-matrix interactions. In 5>�� >'��%� ��Y. Ninomiya, & e. al. (Ed.), (pp. 87-107). Tokyo/Basel: Japan Sci Soc Press/S Kar-ger.

Kute, T.E., Shao, Z.M., Sugg, N.K., Long, R.T., Rusell, G.B. and Case, L.D., Cathepsin D as aprognostic indicator for node-negative breast cancer patients using both immunoassays and en-zymatic assays. ��, 52, 5198-5203 (1991).

Kärjä, V., Syrjänen, K. and Syrjänen, S., Collagen IV and tenascin immunoreactivity as prog-nostic determinant in benign and malignant salivary gland tumours. ��/��!�� 0��,115, 569-575 (1995).

Laitinen, A., Altraja, A., Kämpe, M., Linden, M., Virtanen, I. and Laitinen, L.A., Tenascin isincreased in airway basement membrane of asthmatics and decreased by an inhaled steroid. � �-�� , 156, 951-958 (1997).

Laitinen, L.A., Laitinen, A., Altraja, A., Virtanen, I., Kämpe, M., Simonsson, B.G., Karlsson,S.E., Haakansson, L., Venge, P. and Sillastu, H., Bronchial biopsy findings in intermittent or“early” asthma. -��!�� , 98, S3-6 (1996).

References 55

Latijnhouwers, M., Bergers, M., Ponec, M., Dijkman, H. and Andriessen, M., Human epidermalkeratinocytes are a source of tenascin-C during wound healing. -��4��"� ��, 108, 776-783(1997).

Levine, A.J., Perry, M.E., Chang, A., Silver, A., Dittmer, D., Wu, M. and Welsh, D., The 1993Walter Hubert Lecture: The role of the p53 tumour-suppressor gene in tumorigenesis. 2�-��'��, 69, 409-416 (1994).

Levine, M.N., Bramwell, V.H., Pritchard, K.I., Norris, B.D., Shepherd, L.E., Abu-Zahra, H.,Findlay, B., Warr, D., Bowman, D., Myles, J., Arnold, A., Vandenberg, T., MacKenzie, R.,Robert, J., Ottaway, J., Burnell, M., Williams, C.K. and Tu, D., Randomized trial of intensivecyclophosphamide, epirubicin, and fluorouracil chemotherapy compared with cyclophospha-mide, methotrexate, and fluorouracil in premenopausal women with node-positive breast cancer.-��/��, 16, 2651-2658 (1998).

Levitt, S.H., Aeppli, D.M. and Nierengarten, M.E., The impact of radiation on early breast carci-noma survival. ��, 78, 1035-1042 (1996).

Lightner, V.A., Marks, J.R. and McCachren, S.S., Epithelial cells are an important source oftenascin in normal and malignant human breast tissue. 5>��, 210, 177-84 (1994).

Liljegren, G., Holmberg, L., Adami, H.-O., Westman, G., Graffman, S. and Bergh, J., Sectorresection with or without postoperative radiotherapy for stage I breast cancer: Five year resultsof a randomized trial. -�.��, 86, 717-722 (1994).

Linnala, A., Balza, E., Zardi, L. and Virtanen, I., Human amnion epithelial cells assemble tenas-cins and three fibronectin isoforms in the extracellular matrix. 6520�1��, 317, 74-78 (1993).

Liotta, L.A., Tryggvason, K., Garbisa, S., Robey, P.G. and Abe, S., Partial purification and char-acterization of a neutral protease which cleaves type IV collagen. 2�� , 20, 100-104(1981).

Locker, A.P., Ellis, I.O., Morgan, D.A.L., Elston, C.W., Mitchell, A. and Blamey, R.W., Factorsinfluencing local recurrence after excision and radiotherapy for primary breast cancer. 2�-�0�!,76, 890-894 (1989).

Lohi, J., Tani, T., Laitinen, L., Kangas, L., Lehto, V.-P. and Virtanen, I., Tenascin and fibronec-tin isoforms in human renal cell carcinomas, renal cell carcinoma cell lines and xenografts innude mice. ��-��, 63, 442-449 (1995).

Luomanen, M., Tiitta, O., Heikinheimo, K., Leimola-Virtanen, R., Heinaro, I. and Happonen, R.-P., Effect of snuff and smoking on tenascin expression in oral mucosa. -�/�� , 26,334-338 (1997).

Luomanen, M. and Virtanen, I., Distribution of tenascin in healing incision, excision and laserwounds. -�/�� , 22, 41-45 (1992).

Mackie, E.J., Chiquet-Ehrismann, R., Pearson, C.A., Inaguma, Y., Taya, K., Kawarada, Y. andSakakura, T., Tenascin is a stromal marker for epithelial malignancy in the mammary gland.��.��0��<0�, 84, 4621-4625 (1987b).

Mackie, E.J., Halfter, W. and Liverani, D., Induction of tenascin in healing wounds. -��2��,107, 2757-2767 (1988b).

56 References

Mackie, E.J., Thesleff, I. and Chiquet-Ehrismann, R., Tenascin is associated with chondrogenicand osteogenic differentiation in vivo and promotes chondrogenesis in vitro. -��2��, 105,2569-2579 (1987a).

Mackie, E.J., Tucker, R.P., Halfter, W., Chiquet-Ehrismann, R. and Epperlein, H.H., The distri-bution of tenascin coinsides with pathways of neural crest cell migration. "�4�� , 102,237-250 (1988a).

Majzoub, J.A. and Muglia, L.J., Molecular medicine - knockout mice. .�5�-� ��, 334, 904-907(1996).

Marks, L.B. and Prosnitz, L.R., Lumpectomy with and without radiation for early-stage breastcancer and DCIS. Review. /��!�, 11, 1361-1374 (1997).

McCormick, B., Selection criteria for breast conservation. ��, 74, (1994).

McGuire, W.L., Breast cancer prognostic factors: Evaluation guidelines. Editorial. -�.��, 83, 154-155 (1991).

McNeil, C., News: Using HER2 to choose chemotherapy in breast cancer: Is it ready for theclinic? -�.��, 91, 110-112 (1999).

Melis, M., Baiocchini, A., Soda, G. and Bosco, D., Tenascin expression in elastotic cuffs ofinvasive ductal carcinoma of the breast. ��, 193, 479-484 (1997).

Mignatti, P. and Rifkin, D.B., Biology and biochemistry of proteinases in tumor invasion (Re-view). ��4, 73, 161-195 (1993).

Miller, W.R., Ellis, I.O., Sainsbury, J.R.C. and Dixon, J.M., ABC of breast diseases. Prognosticfactors. 2� ��-, 309, 1573-1576 (1994).

Moch, H., Torhorst, J., Durmuller, U., Feichter, G.E., Sauter, G. and Gudat, F., Comparativeanalysis of the expression of tenascin and established prognostic factors in human breast cancer.��, 189, 510-514 (1993).

Morrow, M., Harris, J.R. and Schnitt, S.J., Local control following breast-conserving surgery forinvasive cancer: results of clinical trials. -�.��, 87, 1669-73 (1995).

Mulcahy, H.E., Duffy, M.J., Gibbons, D., McCarthy, P., Parfrey, N.A., O’Donoghue, D.P. andSheahan, K., Urokinase-type plasminogen activator and outcome in Dukes’ B colorectal cancer.1��, 344, 583-584 (1994).

Murphy-Ullrich, J.E., Lightner, V.A., Aukhil, I., Yan, Y.Z., Erickson, H.P. and Höök, M., Focaladhesion integrity is downregulated by alternatively spliced domain of human tenascin. -��2��, 115, 1127-1136 (1991).

Muss, H.B., Thor, A.D., Berry, D.A., Kute, T., Liu, E.T., Koerner, F., Cirrincione, C.T., Bud-man, D.R., Wood, W.C., Barcos, M. and Henderson, I.C., c-erbB-2 expression and response toadjuvant therapy in women with node-positive early breast cancer. .�5�!��-� ��, 330, 1260-1266 (1994).

Mustakallio, S., Conservative treatment of breast carcinoma - review of 25 years follow up. ��, 23, 110-116 (1972).

Namer, M., Ramaioli, A., Fontana, X., Etienne, M.-C., Héry, M., Jourlait, A., Milano, G., Fre-nay, M., Francois, E. and Lapalus, F., Prognostic value of total cathepsin D in breast tumors.2��, 19, 85-93 (1991).

References 57

Natali, P.G., Nicotra, M.R., Bartolazzi, A., Mottolese, M., Coscia, N., Bigotti, A. and Zardi, L.,Expression and production of tenascin in benign and malignant lesions of melanocyte lineage. ��-��, 46, 586-590 (1990).

Natali, P.G., Nicotra, M.R., Bigotti, A., Botti, C., Castellani, P., Risso, A.M. and Zardi, L.,Comparative analysis of the expression of the extracellular matrix protein tenascin in normalnormal human fetal, adult and tumor tissues. ��-��, 47, 811-816 (1991).

Nixon, A.J., Schnitt, S.J., Gelman, R., Gage, I., Bornstein, B., Hetelekidis, S., Recht, A., Silver,B., Harris, J.R. and Connolly, J.L., Relationship of tumor grade to other pathologic features andto treatment outcome of patients with early stage breast carcinoma treated with breast conservingtherapy. ��, 78, 1426-1431 (1996).

O’Donoghue, A.E.M.A., Poller, D.N., Bell, J.A., Galea, M.H., Elston, C.W., Blamey, R.W. andEllis, I.O., Cathepsin D in primary breast carcinoma: adverse prognosis is associated with ex-pression of cathepsin D in stromal cells. 2��, 33, 137-145 (1995).

Onda, H., Poulin, M.L., Tassava, R.A. and Chiu, I.-M., Characterization of a newt tenascincDNA during newt limb regeneration by in situ hybridization. "�4�2��, 148, 219-232 (1991).

Osborne, C.K., Tamoxifen in the treatment of breast cancer. .�5�!�� -� ��, 339, 1609-1618(1998).

Osteen, R., Selection of patients for breast conserving surgery. ��, 74, 366-371 (1994).

Paik, S., Hazan, R., Fisher, E.R., Sass, R.E., Fisher, B., Redmond, C., Schlessinger, J., Lippman,M.E. and King, C.R., Pathological findings from the National Surgical Adjuvant Breast andBowel Project: Prognostic significance of erbB-2 protein overexpression in primary breast can-cer. -��/��, 8, 103-112 (1990).

Pearson, C.A., Pearson, D., Shibahara, S., Hofsteenge, J. and Chiquet-Ehrismann, R., Tenascin:cDNA cloning and induction by TGF-β. 5 2/�-, 7, 2977-2982 (1988).

Pegram, M.D., Lipton, A., Hayes, D.F., Weber, B.L., Baselga, J.M., Tripathy, D., Baly, D.,Baughman, S.A., Twaddell, T., Glaspy, J.A. and Slamon, D.A., Phase II study of receptor-enhanced chemosensitivity using recombinant humanized anti-p185HER2/neu monoclonal anti-body plus cisplatin in patients with HER2/neu-overexpressing metastatic breast cancer refractoryto chemotherapy treatment. -��/��, 16, 2659-2671 (1998).

Pesheva, P., Spiess, E. and Schachner, M., J1-160 and J1-180 are oligodendrocyte-secretednonpermissive substrates for cell adhesion. -��2��, 109, 1765-1778 (1989).

Pinder, S.E., Ellis, I.O., Galea, M., O’Rourke, S., Blamey, R.W., and Elston C.W., Pathologicalprognostic factors in breast cancer. III. Vascular invasion: relationship with recurrence and sur-vival in a large study with long-term follow-up. 9��!�, 24, 41-47 (1994).

Powles, T.J., Adjuvant therapy for early breast cancer: a time to refine. Editorial. -�.��, 89, 1652-1654 (1997).

Press, M.F., Bernstein, L., Thomas, P.A., Meisner, L.F., Zhou, J.-Y., Ma, Y., Hung, G., Robin-son, R.A., Harris, C., El-Naggar, A., Slamon, D.J., Phillips, R.N., Ross, J.S., Wolman, S.R. andFlom, K.J., HER-2/neu gene amplification characterized by fluoresence in situ hybridization:Poor prognosis in node-negative breast carcinomas. -��/��, 15, 2894-2904 (1997).

58 References

Prieto, A.L., Jones, F.S., Cunningham, B.A., Crossin, K.L. and Edelman, G.M., Localizationduring development of alternatively spliced forms of cytotactin mRNA by in situ hybridization. -��2��, 111, 685-698 (1990).

Pujol, P., Maudelonde, T., Daures, J.-P., Rouanet, P., Brouillet, J.-P., Pujol, H. and Rochefort,H., A prospective study of the prognostic value of cathepsin D levels in breast cancer cytosol.��, 71, 2006-2012 (1993).

Pukkala, E., Sankila, R. and Vertio H (1997) Syöpä Suomessa. 0�� 0�:�;��3��'��3��?@�(p.49) Helsinki: Suomen Syöpärekisteri.

Railo, M., Lundin, J., Haglund, C., von Smitten, K., von Boguslawski, K. and Nordling, S., Ki-67, p53, ER-receptors, ploidy and s-phase as prognostic factors in T1 node negative breast can-cer. ��/��, 36, 369-374 (1997).

Railo, M., Nordling, S., von Boguslawski, K., Leivonen, M., Kyllönen, L. and von Smitten, K.,Prognostic value of Ki-67 immunolabelling in primary operable breast cancer. 2�-��, 68,579-583 (1993).

Rathjen, F.G., Wolff, J.M. and Chiquet-Ehrismann, R., Restrictin; a chick neural extracellularmatrix protein involved in cell attachment co-purifies with the cell recognition molecule F11."�4�� , 113, 151-164 (1991).

Ravdin, P.M. and Chamness, G.C., The c-erbB-2 proto-oncogene as a prognostic and predictivemarker in breast cancer: a paradigm for the development of other macromolecular markers - areview. 8��, 159, 19-27 (1995).

Ravdin, P.M., Tandon, A.K., Allred, C., Clark, G.M., Fuqua, S.A.W., Hilsenbeck, S.H., Cham-ness, G.C. and Osborne, C.K., Cathepsin D by Western blotting and immunohistochemistry:failure to confirm correlations with prognosis in node-negative breast cancer. -��/��, 12,467-474 (1994).

Reilly, D., Christensen, L., Duch, M., Nolan, N., Duffy, M.J. and Andreasen, P.A., Type-1 plas-minogen activator inhibitor in human breast carcinomas. ��-��, 50, 208-214 (1992).

Riedl, S., Faissner, A., Schlag, P., Herbay, A.v., Koretz, K. and Möller, P., Altered content anddistribution of tenascin in colitis, colon adenoma, and colorectal carcinoma. 8��!�,103, 400-406 (1992).

Riedl, S., Kadmon, M., Tandara, A., Hinz, U., Möller, P., Herfarth, C. and Faissner, A., Mucosaltenascin C content in inflammatory and neoplastic diseases of the large bowel. "��'�� , 41, 86-92 (1998).

Rissanen, P.M., A comparison of conservative and radical surgery combined with radiotherapyin the treatment of stage I carcinoma of the breast. 2�-��, 42, 423-426 (1969).

Roberti, N.E., The role of histologic grading in the prognosis of patients with carcinoma of thebreast. Review. ��, 80, 1708-1716 (1997).

Rocchi, M., Archidiacono, N., Romeo, G., Saginati, M. and Zardi, L., Assignment of the genefor human tenascin in the region q32-q34 of chromosome 9. 9� �� 8��, 86, 621-622(1991).

Romero, H., Schneider, J., Burgos, J., Bilbao, J. and Rodriquez-Escudero, F.J., S-phase fractionidentifies high-risk subgroups among DNA-diploid breast cancer. 2��, 38,265-275 (1996).

References 59

Rosen, P.P. (1997). Intraductal carcinoma. In ��2��!��P. P. Rosen (Ed.), (pp.227-274). Philadelphia, Pennsylvania: Lippincott-Raven.

Rosen, P.P., Groshen, S., Saigo, P.E., Kinne, D.W. and Hellman, S., A long term follow-upstudy of survival in stage I (T1N0M0) and stage II (T1N1M0) breast carcinoma. -��/��, 7,355-366 (1989).

Rosen, P.P., Lesser, M.L., Arroyo, C.D., Cranor, M., Borgen, P. and Norton, L., p53 in node-negative breast carcinoma: an immunohistochemical study of epidemiologic risk factors, histo-logic features, and prognosis. -��/��, 13, 821-30 (1995).

Ruegg, C.R., Chiquet-Ehrismann, R. and Alkan, S.S., Tenascin, an extracellular matrix protein,exerts immunomodulatory activities. ��.��0�, 86, 7437-7441 (1989).

Ruoslahti, E., Control of cell motility and tumour invasion by extracellular matrix interactions.2�-��, 66, 239-242 (1992).

Ruoslahti, E., Hayman, E.G. and Pierschbacher, M.D., Extracellular matrices and cell adhesion.��, 5, 581-594 (1985).

Saga, Y., Yagi, T., Ikawa, Y., Sakakura, T. and Aizawa, S., Mice develop normally withouttenascin. 8��A�"�4�� , 6, 1821-1831 (1992).

Sainsbury, J.R.C., Anderson, T.J., Morgan, D.A.L. and Dixon, J.M., ABC of breast diseases:Breast cancer. 2� ��-, 309, 1150-1153 (1994).

Sakai, T., Kawakatsu, H., Hirota, N., Yokoyama, T., Sakakura, T. and Saito, M., Specific ex-pression of tenascin in human colonic neoplasms. 2�-��, 67, 1058-1064 (1993).

Sakakura, T., Ishihara, A. and Yatani, R. (1991). Tenascin in mammary gland development:from embryogenesis to carcinogenesis. In ��!�� 2�� D. R.Lippman ME (Ed.), (pp. 383-400). Boston/Dordrecht/London: Kluwer Academic Publishers.

Sakakura, T. and Kusakabe, M., Can tenascin be redundant in cancer development? ��"�4�.��, 2, 111-16 (1994).

Salmivirta, M., Elenius, K., Vainio, S., Hofer, U., Chiquet-Ehrismann, R., Thesleff, I. and Jal-kanen, M., Syndecan from embryonic tooth mesenchyme binds tenascin. -� 2�� , 266,7733-7739 (1991).

Sasano, H., Nagura, H., Watanabe, K., Ito, K., Tsuiki, A., Sato, S., Yajima, A., Kusakabe, M.and Sakakura, T., Tenascin expression in normal and abnormal human endometrium. ��, 6, 323-326 (1993).

Sastre-Garau, X., Jouve, M., Asselain, B., Vincent-Salomon, A., Beuzeboc, P., Dorval, T., Du-rand, J.-C., Fuorquet, A. and Pouillart, P., Infiltrating lobular carcinoma of the breast. Clini-copathologic analysis of 975 cases with reference to data on conservative therapy and metastaticpatterns. ��, 77, 113-120 (1996).

Saxen, L.N., Karkinen-Jääskeläinen, M., Lehtonen, E., Nordling, S. and Wartiovaara, J. (1975).Inductive tissue interactions. In �� !��4�� P.Poste, & G. L. Nicholson (Ed.), (pp. 331-407). Amsterdam: North-Holland.

Schalkwijk, J., Steijlen, P.M., van Vlijmen-Willems, I.M.J.J., Oosterling, B., Mackie, E.J. andVerstraeten, A.A., Tenascin expression in human dermis is related to epidermal proliferation. � -��, 139, 1143-1150 (1991b).

60 References

Schalkwijk, J., van Vlijmen, I., Oosterling, B., Perret, C., Koopman, R., van den Born, J. andMackie, E.J., Tenascin expression in hyperproliferative skin diseases. 2�-�"� ��, 124, 13-20 (1991a).

Schnitt, S.J., Connolly, J.L., Khettry, U., Mazoujian, G., Brenner, M., Silver, B., Recht, A., Bea-dle, G. and Harris, J.R., Pathologic findings on re-excision of the primary site in breast cancerpatients considered for treatment by primary radiation therapy. ��, 59, 675-81 (1987).

Schnitt, S.J., Hayman, J., Gelman, R., Eberlein, T.J., Love, S.M., Mayzel, K., Osteen, R.T.,Nixon, A.J., Pierce, S., Connolly, J.L., Cohen, P., Schneider, L., Silver, B., Recht, A. and Harris,J.R., A prospective study of conservative surgery alone in the treatment of selected patients withstage I breast cancer. ��, 77, 1094-100 (1996).

Schnyder, B., Semadeni, R.O., Fisher, R.W., Vaughan, L., Car, B.D., Heitz, P.U., Winterhalten,K.H. and Odermatt, B.F., Distribution pattern of tenascin-C in normal and mesenchymal tissues.��-��, 72, 217-224 (1997).

Schwarting, R., Little missed markers and Ki-67. Editorial. 1��4��, 68, 597-599 (1993).

Sherbet, G.V. and Lakshmi, M.S. (1997). ��!�� . Academic Press: London.

Shintani, S., Alcalde, R.E., Matsumura, T. and Terakado, N., Extracellular matrices expressionin invasion area of adenoid cystic carcinoma of salivary glands. ��1��, 116, 9-14 (1997).

Shoji, T., Kamiya, T., Tsubura, A., Hamada, Y., Hatano, T., Hioki, K. and Morii, S., Tenascinstaining positivity and the survival of patients with invasive breast carcinoma. -�0�!��, 55,295-297 (1993).

Shoji, T., Kamiya, T., Tsubura, A., Hatano, T., Sakakura, T., Yamamoto, M. and Morii, S., Im-munohistochemical staining patterns of tenascin in invasive breast carcinomas. =��#��4��9��, 421, 53-56 (1992).

Shrestha, P. and Mori, M. (1997). Tenascin: an extracellular matrix protein in cell growth, adhe-sion and cancer . Springer: Heidelberg.

Shrestha, P., Sumitomo, S., Lee, C.H., Nagahara, K., Kamegai, A., Yamanaka, T., Takeuchi, H.,Kusakabe, M. and Mori, M., Tenascin: growth and adhesion modulation - extracellular matrixdegrading function: an in vitro study. /��/��5��-��, 32B, 106-113 (1996).

Silverstein, M.J. (1997). "�� . Williams & Wilkins: Baltimore.

Silverstein, M.J., Ductal carcinoma in situ of the breast (clinical review). Br Med J, 317, 734-739 (1998).

Silverstein, M.J., Gierson, E.D., Colburn, W.J., Rosser, R.J., Waisman, J.R. and Gamagami, P.,Axillary lymphadenectomy for intraductal carcinoma of the breast. 0�!�8��/��, 172,211-214 (1991).

Silverstein, M.J., Lagios, M.D., Craig, P.H., Waisman, J.R., Lewinsky, B.S., Colburn, W.J. andPoller, D.N., A prognostic index for ductal carcinoma in situ of the breast. ��, 77, 2267-2274 (1996).

Silverstein, M.J., Poller, D.N., Waisman, J.R., Colburn, W.J., Barth, A., Gierson, E.D., Lewin-sky, B., Gamagami, P. and Slamon, D.J., Prognostic classification of breast ductal carcinoma-in-situ. 1��, 345, 1154-57 (1995).

References 61

Silvestrini, R., Benini, E., Daidone, M.G., Veneroni, S., Boracchi, P., Cappelletti, V., Fronzo,G.D. and Veronesi, U., p53 as an independent prognostic marker in lymph node-negative breastcancer patients. -�.��, 85, 965-70 (1993).

Silvestrini, R., Veneroni, S., Benini, E., Daidone, M.G., Luisi, A., Leutner, M., Maucione, A.,Kenda, R., Zucali, R. and Veronesi, U., Expression of p53, glutathione S-transferase-pii, andBcl-2 proteins and benefit from adjuvant radiotherapy in breast cancer. -�.��, 89,639-645 (1997).

Simpson, J.F. and Wilkinson, E.J. (1998). Malignant neoplasia of the breast: Infiltrating carci-nomas. In �� 2�� 4�� !� �� !�� !�� K. I.Bland, & E. M. Copeland (Ed.), (pp. 285-295). Philadelphia: W.B.Saunders.

Slamon, D.J., Clark, G.M., Wong, S.G., Levin, W.J., Ullrich, A. and McGuire, W.L., Humanbreast cancer: Correlation of relapse and survival with amplification of the Her-2/neu oncogene.0��, 235, 177-182 (1987).

Soini, Y., Pääkkö, P., Nuorva, K., Kamel, D., Linnala, A., Virtanen, I. and Lehto, V.-P., Tenas-cin immunoreactivity in lung tumors. � �-��, 100, 145-150 (1993).

Soini, Y., Pääkkö, P., Virtanen, I. and Lehto, V.-P., Tenascin in salivary gland tumours. ='��#��4��9��, 421, 217-222 (1992).

Spyratos, F., Martin, P.-M., Hacène, K., Romain, S., Andrieu, C., Ferrero-Pous, M., Deytieux,S., Doussal, V.L., Tubiana-Hulin, M. and Brunet, M., Multiparametric prognostic evaluation ofbiological factors in primary breast cancer. -�.��, 84, 1266-1272 (1992).

Stamp, G.W.H., Tenascin distribution in basal cell carcinomas. -��, 159, 225-229 (1989).

Steijlen, P.M., Maessen, E., Kresse, H., van Vlijmen, I.M.J.J., Verstraeten, A.A., Traupe, H. andSchalkwijk, J., Expression of tenascin, biglycan and decorin in disorders of keratinization. 2�-"� ��, 130, 564-568 (1994).

Steindler, D.A., Cooper, N.G.F., Faissner, A. and Schachner, M., Boundaries defined by adhe-sion molecules during development of the cerebral cortex: the J1/tenascin glycoprotein in themouse somatosensory cortical barrel field. "�4�2��, 131, 243-260 (1989).

Sugawara, I., Hirakoshi, J., Masunaga, A., Itoyama, S. and Sakakura, T., Reduced tenascin ex-pression in colonic carcinoma with lymphogenous metastasis. ��4�� , 11, 325-331(1991).

Taghian, A., Vathaire, F.d. and Terrier, P., Long-term risk of sarcoma following radiation treat-ment for breast cancer. ��-��/��2��, 21, 361-367 (1991).

Tandon, A.K., Clark, G.M., Chamness, G.C., Chirgwin, J.M. and McGuire, W.L., Cathepsin Dand prognosis in breast cancer. .�5�!��-� ��, 322, 297-302 (1990).

Tani, T., Lumme, A., Linnala, A., Kivilaakso, E., Kiviluoto, T., Burgeson, R.E., Kangas, L.,Leivo, I. and Virtanen, I., Pancreatic carcinomas deposit laminin-5, preferably adhere to laminin-5, and migrate on the newly deposited basement membrane. � � -� ��, 151, 1289-1302(1997).

Tereda, T. and Nakanuma, Y., Expression of tenascin, type IV collagen and laminin during hu-man intrahepatic bile duct development and in intrahepatic cholangiocarcinoma. 9��!�,25, 143-150 (1994).

62 References

Tervo, K., Tervo, T., van Setten, G.B., Tarkkanen, A. and Virtanen, I., Demonstration of tenas-cin-like immunoreactivity in rabbit corneal wounds. �� /�� , 67, 347-350(1989).

Tetû, B., Brisson, J., Côté, C., Brisson, S., Potvin, D. and Roberge, N., Prognostic significanceof cathepsin D expression in node-positive breast carcinoma: an immunohistochemical stydy. ��-��, 55, 429-435 (1993).

Thesleff, I., Mackie, E., Vainio, S. and Chiquet-Ehrismann, R., Changes in the distribution oftenascin during tooth development. "�4�� , 101, 289-296 (1987).

Thesleff, I., Vaahtokari, A., Vainio, S. and Jowett, A., Molecular mechanisms of cell and tissueinteractions during early tooth development. ��, 245, 151-161 (1996).

Thor, A.D., Moore, D.H., Edgerton, S.M., Kawasaki, E.S., Reihsaus, E., Lynch, H.T., Marcus,J.N., Schwartz, L., Chen, L.-C., Mayall, B.H. and Smith, H.S., Accumulation of p53 tumoursuppressor gene protein: An independent marker of prognosis in breast cancers. -�.��, 84, 845-855 (1992).

Thorpe, S.M., Rochefort, H., Garcia, M., Freiss, G., Christensen, I.J., Khalaf, S., Paolucci, F.,Pau, B., Rasmussen, B.B. and Rose, C., Association between high concentrations of M 52,000cathepsin D and poor prognosis in primary human breast cancer. ��, 49, 6008-6014(1989).

van Tienhoven, G., Voogd, A.C., Peterse, J.L., Nielsen, M., Andersen, K.W., Mignolet, F.,Sylvester, R., Fentiman, I.S., van der Schueren, E., van Zijl, K., Blichert-Toft, M., Bartelink, H.and van Dongen, J.A., Prognosis after treatment for loco-regional recurrence after mastectomy orbreast conserving therapy in two randomized trials (EORTC 10801 and DBCG-82TM). 5��-��, 35, 32-38 (1999).

Tiitta, O. (1995). Tenascin expression in preneoplastic and neoplastic lesions and inflammatoryconditions. University of Helsinki

Tiitta, O., Sipponen, P., Gould, V. and Virtanen, I., Tenascin expression in inflammatory, dys-plastic and neoplastic lesions of the human stomach. =��#��4, 425, 369-374 (1994).

Tiitta, O., Wahlström, T., Paavonen, J., Linnala, A., Sharma, S., Gould, V.E. and Virtanen, I.,Enhanced tenascin expression in cervical and vulvar koilocytotic lesions. � � -� ��, 141,907-13 (1992).

Tiitta, O., Wahlström, T., Virtanen, I. and Gould, V.E., Tenascin in inflammatory conditions andneoplasma of the urinary bladder. =��#��4�2��, 63, 283-287 (1993).

Toikkanen, S., Pylkkönen, L. and Joensuu, H., Invasive lobular carcinoma of the breast has bet-ter short- and long-term survival than invasive ductal carcinoma. 2�-��, 76, 1234-1240(1997).

Tucker, R.P., Brunso-Bechtold, J.K., Jenrath, D.A., Khan, N.A., Poss, P.M., Sweatt, A.J. andXu, Y., Cellular origins of tenascin in the developing nervous system. ��"�4�.��,2, 89-99 (1994).

Tuominen, H., Pöllänen, R. and Kallioinen, M., Multicellular origin of tenascin in skin tumors -an in situ hybridization study. -��, 24, 590-596 (1997).

References 63

Vainio, S., Jalkanen, M. and Thesleff, I., Syndecan and tenascin expression is induced byepithelial-mesenchymal interactions in embryonic tooth mesenchyme. -��2��, 108, 1945-1954 (1989).

Veronese, S.M., Gambacorta, M., Gottardi, O., Scanzi, F., Ferrari, M. and Lampertico, P., Pro-liferation index as a prognostic marker in breast cancer. ��, 71, 3926-3931 (1993).

Veronesi, U., Banfi, A. and Vecchio, M.D., Comparison of Halsted mastectomy with quadran-tectomy, axillary dissection, and radiotherapy in early breast cancer. Long-term results. 5�� -��/��, 22, 1085-1087 (1986).

Veronesi, U., Luini, A., Vecchio, M.D., Greco, M., Galimberti, V., Merson, M., Rilke, R., Sac-chini, V., Saccozzi, R., Savio, T., Zucali, R., Zurrida, S. and Salvadori, B., Radiotherapy afterbreast-preserving surgery in women with localized cancer of the breast. .� 5�!�� -� ��, 328,1587-91 (1993).

Veronesi, U., Salvadori, B., Luini, A., Banfi, A., Zucali, R., Vecchio, M.D., Saccozzi, R.,Beretta, E., Boracci, P., Farante, G., Galimberti, V., Mezzanotte, G., Sacchini, V., Tana, S. andMarubini, E., Conservative treatment of early breast cancer. Long term results of 1232 casestreated with quadrantectomy, axillary dissection, and radiotherapy. �� 0�!, 211, 250-259(1989).

Victorzon, M., Roberts, P.J., Haglund, C., von Boguslawsky, K. and Nordling, S., Ki-67 immu-noreactivity, ploidy and S-phase fraction as prognostic factors in patients with gastric carcinoma./��!�, 53, 182-191 (1996).

Vollmer, G., Biologic and oncologic implications of tenascin-C/hexabrachion proteins. ��4/��9� ��, 25, 187-210 (1997).

Westley, B.R. and May, F.E.B., Cathepsin D and breast cancer. 5�� -� ��, 32A, 15-24(1996).

Whelan, T., Clark, R., Roberts, R., Levine, M., Foster, G. and Investigators of the Ontario Clini-cal Oncological Group, Ipsilateral breast tumour recurrence postlumpectomy is predictive ofsubsequent mortality: Results from a randomized trial. ��-��/��2��, 30, 11-6(1994).

Willems, I.E.M.G., Arends, J.-W. and Daemen, M.J.A.P., Tenascin and fibronectin expression inhealing human myocardial scars. -��, 179, 321-325 (1996).

Wilson, K.E., Langdon, S.P., Lessells, A.M. and Miller, W.R., Expression of the extracellularmatrix protein tenascin in malignant and benign ovarian tumours. 2�-��, 74, 999-1004(1996).

Winstanley, J.H.R., Leinster, S.J., Cooke, T.G., Westley, B.R., Platt-Higgins, A.M. and Rudland,P.S., Prognostic significance of cathepsin-D in patients with breast cancer. 2� -� ��, 67,767-772 (1993).

Xue, Y., Smedts, F., Latijnhouwers, M.A., Ruijter, E.T., Aalders, T.W., de la Rosette,J.J.M.C.H., Debruyne, F.M.J. and Schalken, J.A., Tenascin-C expression in prostatic intraepi-thelial neoplasia (PIN): A marker of progression. ��, 18, 2679-2684 (1998).

Yaghan, R., Stanton, P.D., Robertson, K.W., Going, J.J., Murray, G.D. and McArdle, C.S., Oes-trogen receptor status predicts local recurrence following breast conservation surgery for earlybreast cancer. 5��-�0�!�/��, 24, 424-462 (1998).

64 References

Yamada, K.M. and Clark, R.A.F. (1996). Provisional matrix. In �� !��#��R. A. F. Clark (Ed.), (pp. 51-93). New York: Plenum Press.

Yamada, K.M., Yamada, S.S. and Pastan, I., The major cell surface glycoprotein of chick em-bryo fibroblasts is an agglutinin. ��.��0��<0�, 72, 3158-3162 (1975).

Yamanaka, M., Taga, M. and Minaguchi, H., Immunohistological localization of tenascin in thehuman endometrium. 8��/��4��, 41, 247-252 (1996).

Yoshida, T., Ishihara, A., Hirokawa, Y., Kusakabe, M. and Sakakura, T., Tenascin in breastcancer developement - is epithelial tenascin a marker for poor prognosis. Review. ��1��,90, 65-73 (1995).

Yoshida, T., Matsumoto, E.-I., Hanamura, N., Kalembeyi, I., Katsuta, K., Ishihara, A. and Saka-kura, T., Co-expression of tenascin and fibronectin in epithelial and stromal cells of benign le-sions and ductal carcinomas in the human breast. -��, 182, 421-428 (1997).

Tenascin C in early breast cancer - Helda - University of Helsinki

Documents

Transcript of Tenascin C in early breast cancer - Helda - University of Helsinki