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Pathology (August 2011) 43(5), pp. 495–522

C O R R E S P O N D E N C E

Adenomatoid tumour of the adrenal gland in apatient with germline SDHD mutation: a casereport and review of the literature

Sir,Adenomatoid tumour, a benign neoplasm, is seen most com-monly in the male and female genital tract including epididy-mis, uterus and fallopian tubes.1,2 Adenomatoid tumours of theadrenal gland are rare and often an incidental finding. In somecases, clinical suspicion of phaeochromocytoma leads to resec-tion of the adrenal gland. Thus far, the tumour has not beendescribed in association with syndromic or genetic conditions.We report one case of adrenal adenomatoid tumour in a patientwith a personal and family history of succinate dehydrogenasecomplex subunit D (SDHD) mutation and carotid bodytumours.

The patient was a 24-year-old man who was referred for anadrenal mass. He had an unremarkable medical history. Hisfather was found to have cervical paragangliomas and upongenetic testing a germline P81L mutation in SDHD wasdetected. Site-specific predictive genetic testing confirmedthe presence of the family-specific germline mutation in SDHDin the patient. The patient did not report any blood pressureproblems or other symptoms worrisome for phaeochromocy-toma. His biochemical workup showed a 24 h urine norepi-nephrine of 59 mg (normal 15–100), epinephrine 4 mg (normal2–24), dopamine 249 mg, (normal 52–480), metanephrine180 mg (normal 25–222), and normal potassium 309 mmol(normal 40–412). His creatinine collection was 1.8 mg/dL andpotassium was 3.9 mmol/L. Computed tomography (CT) of hischest, abdomen and pelvis showed no intrathoracic pathologyand a 30� 20 mm mass, originating from the left adrenal gland.Magnetic resonance imaging (MRI) of the abdomen around thesame time showed a heterogeneous left adrenal mass, measuring30� 20� 25 mm. Additionally MRI of the neck showed bilat-eral carotid body masses measuring 25� 26 mm on the left and22� 15 mm on the right. The patient elected to undergo adre-nalectomy, after which he was going to consider removal of thecarotid body masses. After adrenalectomy, the patient wasinstructed to return in 6 months for a metanephrine level.

The specimen received in pathology measured 77� 35�20 mm and weighed 14.65 g. A portion of unremarkable adrenalgland was stretched over a 36� 33� 18 mm tumour mass thatseemed to arise from the adrenal cortex. On gross examination,the tumour was a solitary well-circumscribed solid mass with ahomogenous grey-yellow bulging cut surface. There were a fewsmall foci of haemorrhage, but no necrosis was noted (Fig. 1).

On microscopic examination, the tumour was composed ofvariably sized anastomosing tubules and channels (Fig. 2A)lined by bland cuboidal to epithelioid cells, some of whichcontained intracellular vacuoles (Fig. 2B). The tumour cellshad low nuclear/cytoplasmic ratios, with no appreciablemitotic activity or nuclear pleomorphism. Scattered aggregatesof mature lymphocytes were seen throughout the lesion.The tumour cells surrounded the adrenal cortical or medullarytissue, imparting an appearance of an infiltrative growthpattern. Focal involvement of adrenal capsule and extensioninto periadrenal fat was noted.

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By immunoperoxidase staining, the tumour was diffuselypositive for cytokeratin AE1/3 (Fig. 2C), calretinin (Fig. 2D)and WT-1 (Fig. 2E), focally positive for S-100 protein, andnegative for synaptophysin, chromogranin, CD31 and CD34,supporting the diagnosis of adenomatoid tumour. CD31 andCD34 immunostain underlined the presence of vessels.

We performed immunohistochemistry for SDHB (1:250;Sigma-Aldrich, USA) on the adenomatoid tumour and founda weak diffuse cytoplasmic blush (Fig. 3A), lacking definitegranularity. The adjacent adrenal gland (positive control)showed granular cytoplasmic staining in a mitochondrialpattern (Fig. 3A, arrows, and 3B).

Since clinical testing revealed a germline SDHD P81Lmutation, we sought to determine if a second somatic ‘hit’could have occurred in the tumour. PCR-based direct Sangersequencing of genomic DNA extracted from the paraffinembedded, formalin fixed adenomatoid tumour was performedper routine of Neumann et al.3 Sequencing of the promoter andall four exons (and flanking intronic regions) of the SDHDgene revealed only the germline heterozygous missensemutation P81L (c.242C>T) (Fig. 4). We then sought todetermine if somatic deletion of the remaining wildtypeallele occurred. Because the tumour was microdissectedand contained 100% tumour, we were able to validly inspectthe sequencing chromatogram which revealed two clearheterozygote peaks, i.e., no deletions were present (Fig. 4).

Approximately 34 other adrenal adenomatoid tumourshave been reported in the literature.1,4,5 These tumours areusually asymptomatic and found incidentally on radiology orat autopsy.1,5 Adenomatoid tumours have been described inother sites such as heart, intestinal mesentery, omentum,uterus, pancreas and pleura. The tumour has been reportedin association with AIDS and disseminated coccidioido-mycosis, micronodular adrenal cortical hyperplasia and in avascular cyst of an adrenal gland, but has never been describedin association with Carney’s complex.6 The reported meanage is 41 years (range 31–64) with a male predominance foradrenal adenomatoid tumours,1 differing slightly from ourpatient who is a younger (24-year-old) male. While otherstudies reported symptoms associated with the tumour suchas syncope and hypertension, in the majority of cases thetumour is non-functional.1,6 Histogenesis is thought to berelated to entrapment of primitive mesenchymal cells associ-ated with the mullerian tract in the adrenal or to the dislodge-ment of mesothelial inclusions.1,6,7

Histologically, four patterns have been described: adenoidal,angiomatoid, cystic, and solid, and more then one pattern canbe seen in a specimen. The most common pattern consists ofvariably sized tubules and fenestrated channels arranged invariably fibrous stroma. The tubules are lined with cells rangingfrom epithelioid to flattened, endothelial-like cells. Often signetring-like cells are seen. Lymphoid nodules are often present.4,5

Adrenal adenomatoid tumours display an immunophenotypesimilar to that of the genital tract.2,5,7,8 The tumours arestrongly reactive for calretinin, cytokeratin AE1/3, CAM5.2, CK5/6, WT-1, D2–40 and caldesmon. Calretinin, D2–40and WT-1 have shown a higher sensitivity then CK5/6 andcaldesmon.2

The differential diagnosis can be broad and includes: primaryadrenal tumours (adrenocortical tumour, phaeochromocytomas),

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Fig. 1 Well-circumscribed adrenal adenomatoid tumour with solid grey-yellow bulging cut surface.

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lymphangioma, angiosarcoma, primary as well as metastaticadenocarcinoma, malignant mesothelioma, and adrenal cyst.Lymphangioma is the most common mimic of adrenal angio-matoid tumour. Lymphangioma is positive for endothelial

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Fig. 2 (A) Adrenal adenomatoid tumour composed of variably sized anastomosing tubuintracellular vacuoles. Tumour cells show positive immunostaining for (C) cytokerati

Fig. 3 (A) Adenomatoid tumour showing a diffuse cytoplasmic blush (weak diffuse scortical tissue (A, arrows, B).

markers and negative for cytokeratin and mesothelial markersand the opposite is true of adrenal adenomatoid tumour.1

Epithelioid angiosarcomas can present as complex cystic-solidneoplasms with invasive growth of solid sheets and nests of cellswith epithelioid features and vascular spaces. Cytological atypiaand mitosis are common findings. These tumours are veryrare and the cells are positive for endothelial markers (CD31,CD34, and factor VIII).5,6 An adrenal cyst can be considered inthe differential diagnosis when cystic structures are prominent.If signet ring cells seem to be prominent, metastatic carcinomaneeds to be ruled out.6 Multicystic mesotheliomas maydemonstrate focal areas of adenomatoid-like change whichoccur in the peritoneum of young to middle-aged women.5

The genes for various familial tumour syndromes associatedwith phaeochromocytoma have been identified, such asthe proto-oncogene RET [associated with multiple endocrineneoplasia type 2 (MEN 2)], the tumour suppressor gene VHL

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les and channels lined by bland cuboidal to epithelioid cells, (B) some containingn AE1/3, (D) calretinin and (E) WT-1.

taining) in contrast to the true granular cytoplasmic staining of adjacent adrenal

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Fig. 4 Sequencing chromatograms of SDHD mutation. (A) Wild-typesequence in the codon 81 region compared to (B) heterozygous P81L SDHDmutation identified in the tumour-derived DNA. Note that this reflects thegermline mutation in tumour tissue. Because the DNA was extracted from aregion with 100% tumour (without normal tissue), the clear heterozygote patternshowing the germline mutated allele and the wildtype allele excludes a somaticdeletion (loss of heterozygosity).

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(associated with von Hippel-Lindau disease) and NF1(associated with type 1 neurofibromatosis). A recently ident-ified heritable form of paraganglioma-phaeochromocytomacaused by germline mutations in the genes encoding succinatedehydrogenase (SDH) complex has been described. A broadspectrum of germline mutations of the genes encodingvarious subunits SDH (SDHB, SDHC, and SDHD) have beendiscovered (reviewed by Eng 2010).9 Patients carryingthe mutations associated with familial head and neck para-ganglioma usually need to be followed for development ofphaeochromocytoma. It has been shown that 20–30% ofapparently non-syndromic phaeochromocytoma cases harbourgermline mutation.10,11 Clinical parameters such as age<45 years, multiple phaeochromocytoma, extra-adrenallocation, and previous head and neck paraganglioma canpredict for mutation carriers and prioritise gene testing inindividuals without a personal or family history of head andneck paraganglioma to reduce costs.10

Hereditary head and neck paragangliomas (HNP) are nearlyexclusively associated with germline mutations of at least threesuccinate dehydrogenase subunit genes (SDHx).11 Neumannand colleagues recently analysed clinical parameters aspotential predictors for finding germline mutations in HNPand found that family history, previous phaeochromocytoma,multiple HNP, age�40 years and male gender were predictorsfor an SDHx mutation. Our patient carried three of the red flags,family history, male and young age and was found to have agermline SDHD mutation by predictive testing. Interestingly,the adenomatoid tumour did not undergo loss or mutation ofthe remaining wild-type allele. SDHD is maternally imprintedwhich means that the allele inherited from this patient’s motherwould be silenced throughout the germline. So in effect, therealready exist two germline ‘hits,’ and a somatic mutation or lossof the remaining wild-type allele would not be necessary.

Recently, van Nederveen and colleagues proposed thatnegative immunostaining for SDHB is highly specific forphaeochromocytomas and paragangliomas associated withSDHB, SDHC, or SDHD gene mutations.12 All tumours witha germline SDH mutation (36 SDHB, 5 SDHC and 61 SDHD)were negative for SDHB immunohistochemistry. In four SDH-mutated tumours, the authors reported a weak and diffusecytoplasmic SDHB immunoreactivity in the tumour cells.Similar to our case, the staining pattern was clearly distinctfrom the strong speckled pattern present in the normal cells ofthe intratumoural fibrovascular network. Since independenttumour samples with the same mutation were clearly negative

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for SDHB immunoreactivity, the weak cytoplasmic staining inthe tumour cells was considered to be a non-specific back-ground artefact and scored as negative.12 Previous studies haveshown that immunohistochemistry for SDHB can be used totriage genetic testing.12,13 Gill and colleagues13 have found thatcompletely absent staining is more commonly found withSDHB mutations, whereas weak diffuse staining, as in ourcase, often occurs with SDHD mutation.

While there are reports of increased risk of renal tumours,14

thyroid tumours, and gastrointestinal stromal tumours (GISTs),there are no reports indicating the association of adenomatoidtumour with mutations of the SDHB and SDHD genes.15

Mutation analysis for the SDHD gene was performed in thecurrent adrenal gland tumour and a heterozygous germlinemutation was detected, confirming that the adenomatoid tumourreported in our patient was a SDHD mutated tumour. Germlinemutations affecting encoding subunits of SHDx may predisposeindividuals to different tumours in different organs and othermutation types or other as yet unidentified phaeochromocytomapredisposing genes might exist. Adenomatoid tumour ofthe adrenal gland is a rare tumour and has never beenpreviously reported in association with phaeochromocytomaand/or HNP. The current case report should inspire furtherinterest in this unusual tumour, especially in the setting ofSDHx mutations.

Abberly Lott Limbach*Ying Ni{Jie Huang*Charis Eng{Cristina Magi-Galluzzi*

*Pathology and Laboratory Medicine Institute, Department ofPathology, Cleveland Clinic, and {Genomic Medicine Institute,Cleveland Clinic Lerner Research Institute, Cleveland, Ohio,USA

Contact Dr C. Magi-Galluzzi.E-mail: magic@ccf.org

1. Bisceglia M, Carosi I, Scillitani A, Pasquinelli G. Cystic lymphangioma-like adenomatoid tumor of the adrenal gland: Case presentation and reviewof the literature. Adv Anat Pathol 2009; 16: 424–32.

2. Sangoi AR, McKenney JK, Schwartz EJ, Rouse RV, Longacre TA.Adenomatoid tumors of the female and male genital tracts: a clinicopatho-logical and immunohistochemical study of 44 cases. Mod Pathol 2009; 22:1228–35.

3. Neumann HP, Pawlu C, Peczkowska M, et al. Distinct clinical featuresof paraganglioma syndromes associated with SDHB and SDHD genemutations. JAMA 2004; 292: 943–51.

4. El-Daly H, Rao P, Palazzo F, Gudi M. A rare entity of an unusual site:adenomatoid tumour of the adrenal gland: a case report and review of theliterature. Patholog Res Int 2010; 2010: 702472.

5. Raaf HN, Grant LD, Santoscoy C, Levin HS, Abdul-Karim FW.Adenomatoid tumor of the adrenal gland: a report of four new casesand a review of the literature. Mod Pathol 1996; 9: 1046–51.

6. Chung-Park M, Yang JT, McHenry CR, Khiyami A. Adenomatoid tumorof the adrenal gland with micronodular adrenal cortical hyperplasia.Hum Pathol 2003; 34: 818–21.

7. Delahunt B, Eble JN, King D, Bethwaite PB, Nacey JN, Thornton A.Immunohistochemical evidence for mesothelial origin of paratesticularadenomatoid tumour. Histopathology 2000; 36: 109–15.

8. Nogales FF, Isaac MA, Hardisson D, et al. Adenomatoid tumors of theuterus: an analysis of 60 cases. Int J Gynecol Pathol 2002; 21: 34–40.

9. Eng C. Mendelian genetics of rare—and not so rare—cancers. Ann N YAcad Sci 2010; 1214: 70–82.

10. Erlic Z, Rybicki L, Peczkowska M, et al. Clinical predictors and algorithmfor the genetic diagnosis of pheochromocytoma patients. Clin Cancer Res2009; 15: 6378–85.

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11. Neumann HP, Erlic Z, Boedeker CC, et al. Clinical predictors forgermline mutations in head and neck paraganglioma patients: costreduction strategy in genetic diagnostic process as fall-out. Cancer Res2009; 69: 3650–6.

12. van Nederveen FH, Gaal J, Favier J, et al. An immunohistochemicalprocedure to detect patients with paraganglioma and phaeochromocytomawith germline SDHB, SDHC, or SDHD gene mutations: a retrospective andprospective analysis. Lancet Oncol 2009; 10: 764–71.

13. Gill AJ, Benn DE, Chou A, et al. Immunohistochemistry for SDHB triagesgenetic testing of SDHB, SDHC, and SDHD in paraganglioma-pheochro-mocytoma syndromes. Hum Pathol 2010; 41: 805–14.

14. Ricketts C, Woodward ER, Killick P, et al. Germline SDHB mutations andfamilial renal cell carcinoma. J Natl Cancer Inst 2008; 100: 1260–2.

15. Ricketts CJ, Forman JR, Rattenberry E, et al. Tumor risks and genotype-phenotype-proteotype analysis in 358 patients with germline mutations inSDHB and SDHD. Hum Mutat 2010; 31: 41–51.

DOI: 10.1097/PAT.0b013e3283486bb9

Microglandular adenosis with transition tobreast carcinoma: a series of three cases

Sir,Microglandular adenosis (MGA) is an uncommon benignproliferative glandular lesion of the breast and is generallyperceived as one with an indolent clinical course. It may bedetected incidentally on screening, as a mammographicdistortion, or as a clinically palpable mass or thickening.1–3

This lesion, especially when seen on core biopsy, may give riseto diagnostic difficulties in view of an infiltrative growthpattern and lack of myoepithelial layer.3,4 Although considereda benign lesion, several studies have described that carcinomais seen concurrently in up to 27% of patients with MGA.5–7

Whilst these findings may be a chance association, cases havebeen described in the literature describing MGA with atypicalMGA and carcinoma in some cases. The spectrum of lesionsshare a common immunohistochemical profile and molecularcharacteristics, suggesting that MGA is a candidate precursorlesion for certain types of carcinoma.

Case 1 was a 49-year-old woman who presented with a self-detected lump in the upper outer quadrant of her left breast. Shehad no significant medical history and had no family history ofbreast or ovarian cancer. She had two children and her first fullterm pregnancy was at the age of 23. She had early menopauseat the age of 46 and had not taken any hormonal replacementtherapy. Physical examination revealed a 2.5 cm ill-definedlesion at 2 o’clock in the left breast. Mammogram and ultra-sound detected a 2.5 cm spiculated lesion in the left breast at 2o’clock, 5 cm from the nipple. Core biopsy showed neoplasticsheets and poorly formed glands in keeping with invasiveductal carcinoma. No in situ component was seen. Oestrogenand progesterone receptors were negative in the tumour. Thepatient underwent a wide local excision and sentinel nodebiopsy. Macroscopic examination showed a 25� 20� 18 mmovoid grey white hard lesion in the excision specimen.

Case 2 was a 58-year-old post-menopausal woman whounderwent a right breast wide local excision and level 3 axillarydissection for a 14 mm grade 1 invasive ductal carcinoma of nospecial type 7 years prior to this presentation. At the time, therewas metastatic carcinoma in one of 10 axillary lymph nodes,and clear surgical margins of the carcinoma were achieved. She

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was commenced on tamoxifen and underwent radiotherapy.Tamoxifen was subsequently ceased and replaced with ana-strazole (Arimidex). She suffered radionecrosis of her rightbreast and presented 7 years after her initial surgery for a righttotal mastectomy utilising a left breast flap and left reductionmammoplasty. Intraoperatively, a 3 cm mass was detected inthe left breast at 9 o’clock and a frozen section diagnosis ofinvasive carcinoma was made. A wide local excision of the leftbreast mass was performed. Macroscopic examination showeda 28� 16� 12 mm hard white nodule in the excision specimen.

Case 3 was a 67-year-old post-menopausal woman who wasrecalled for a 10 mm suspicious mass on the left breast at3 o’clock, 6 cm from the nipple, detected at routine mammo-graphy screening. She had no family history of breast cancerand had never taken hormone replacement therapy. Physicalexamination showed a 10 mm area of thickening over thecorresponding area, and ultrasound showed a radiologicallymalignant mass at the same location. Core biopsy wasperformed and subsequent excision was carried out. Macro-scopic examination of the excision specimen showed nodiscrete lesion.

Histologically, all three cases displayed a spectrum ofchanges ranging from uncomplicated MGA or atypicalMGA to in situ and invasive carcinoma.

Sections from Case 1 showed an invasive ductal carcinomaof no special type, Elston and Ellis grade 3 (tubule 3/3, nucleargrade 3/3, mitoses 3/3, giving a total score of 9/9). It displayedinfiltrating irregular tongues and nests of hyperchromaticpleomorphic cells in a desmoplastic stroma. At the peripheryof the invasive carcinoma, there was ductal carcinoma in situ,characterised by solid proliferation of tumour cells withintermediate to high grade nuclei, merging with glandsdisplaying MGA and atypical MGA covering an area of60 mm in maximum extent (Fig. 1A).

Histologically, uncomplicated MGA was identified bysmall round glandular structures with open lumen distributedrandomly in fibrocollagenous stroma. The glands were lined bya single layer of vacuolated cells with rounded nuclei andlacking a myoepithelial layer. Eosinophilic material was seen inthe lumen of the glands (Fig. 1B). This was intermixed withglands showing complex and irregular outlines, with back-to-back arrangement, mild to moderate nuclear pleomorphism,hyperchromasia, coarse chromatin and high nuclear tocytoplasmic ratios. There were diminished intraluminaleosinophilic secretions compared with the MGA (Fig. 1C).The morphology was consistent with atypical MGA.

All components of the lesion expressed AE1/AE3(cytoplasmic) and S-100 (cytoplasmic and nuclear) diffusely.It was weakly positive for c-kit and negative for CK5/6.Collagen type IV highlighted the basement membrane aroundMGA, atypical MGA (Fig. 1D) and ductal carcinoma in situ(DCIS). Invasive carcinoma was oestrogen and progesteronereceptors negative, which corresponded to that seen in theprevious core biopsy. No evidence of Her-2 gene amplificationwas seen using silver in situ hybridisation (SISH). There wasan increased level of Ki-67 labelling index as lesions pro-gressed from uncomplicated MGA of less than 1% (Fig. 1E),to atypical MGA of 3% (Fig. 1F), carcinoma in situ of 20%(Fig. 1G) and invasive carcinoma of 80% (Fig. 1H).

The uncomplicated MGA component in Case 1 shared somecommon morphological and immunohistochemical featureswhich raised the differential diagnosis of secretory carcinoma,including the presence of vacuolated cytoplasm. Secretory

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Fig. 1 Case 1. (A) Invasive ductal carcinoma (right) immediately adjacent to small round glands lined by hyperchromatic cells on a fibrous stroma (left) (H&E).(B) Microglandular adenosis showed small mostly rounded glands with bland ovoid cells which displayed vacuolated cytoplasm and luminal eosinophilic secretions(H&E). (C) Atypical microglandular adenosis with some back-to-back arrangement of glands displayed moderate nuclear pleomorphism and decreased eosinophilicsecretions (H&E). (D) Collagen type IV highlighted the basement membrane around atypical microglandular adenosis glands. (E) Ki-67 labelling index in uncomplicatedMGA of less than 1%. (F) Ki-67 labelling index in atypical MGA of 3%. (G) Ki-67 labelling index in DCIS of 20%. (H) Ki-67 labelling index in invasive carcinomaof 80%.

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carcinoma differs in other morphological aspects; thecytoplasm in secretory carcinoma cells generally appear morebubbly and granular, and their luminal secretions are betterdescribed as amphophilic and paler in colour, compared to thehyaline eosinophilia of those in MGA. Furthermore, the archi-tecture of the current case was purely of single round glands.This is in contrast to secretory carcinoma, which generallydisplays a more compact growth pattern with solid sheets andmicrocystic patterns admixed with its tubular component.

Sections of the left breast lesion from Case 2 showedinfiltrating cribriform and solid nests of cells displaying

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hyperchromatic enlarged nuclei, conspicuous nucleoli and smallamount of cytoplasm, with some nests displaying cylindroma-like microcystic spaces filled with basophilic material (Fig. 2A).These findings were consistent with a grade 2 adenoid cysticcarcinoma (tubules 2/3, nuclei 2/3, mitoses 2/3, giving a totalscore of 6/9). Oestrogen and progesterone receptors werenegative. No evidence of Her-2 gene amplification was seenusing SISH. The carcinoma was intimately intermixed witha second component, which showed a florid glandularproliferation with complex irregular glands lined by cytologi-cally atypical cells, and diminished intraluminal eosinophilic

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Fig. 2 Case 2. (A) Adenoid cystic carcinoma, characterised by cribriform and solid nests of cells with cylindromatous microcystic spaces (left), intimately admixed withatypical microglandular adenosis (right) (H&E). (B) Atypical microglandular adenosis displayed irregular complex glands with pleomorphic cells and decreasedeosinophilic secretions (H&E). (C) Ki-67 labelling index of greater than 80% in carcinoma (lower left) compared to 5% in adjacent atypical MGA (upper right).

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secretion (Fig. 2A,B). Keratin 14 (basal keratin) showedpositive staining in the adenoid cystic carcinoma component,but failed to stain the component of florid glandularproliferation. Furthermore, the carcinoma cells showed aKi-67 labelling index of 80% compared with 5% in the secondcomponent (Fig. 2C), supporting the concurrent diagnosis ofatypical microgladular adenosis over tubular variant of adenoidcystic carcinoma.

The initial core biopsy from Case 3 showed ductules lined bycolumnar cells with cytological atypia with luminal colloid-likeeosinophilic secretion and a complete absence of myoepithelialcells on H&E (Fig. 3A) and p63 staining (Fig. 3B). The featuresfavoured atypical MGA. Focal small tubules also showedfeatures suggestive of invasive carcinoma; however, due tothe small amount of tissue available, this was difficult toconfirm and excision was recommended for complete histo-logical assessment.

Subsequent excision showed a continuous spectrum ofatypical epithelial proliferative changes ranging fromatypical MGA to in situ and invasive carcinoma (Fig. 3C).The atypical MGA was characterised by an infiltrative butfocally circumscribed and lobulocentric proliferation of irre-gular tubular structures, some showing a branched architectureand containing periodic acid-Schiff positive and diastaseresistant eosinophilic secretion. The tubules were lined bycolumnar cells with crowded large ovoid nuclei with finechromatin and prominent nucleoli. Collagen type IV high-lighted the basement membranes around these tubules(Fig. 3D) and the lining cells stained positively with S-100.Adjacent to this were areas of DCIS, showing solid andcribriform groups of crowded atypical basaloid cells displayingintermediate to high grade nuclei. The in situ componentalso stained diffusely with S-100. Immediately adjacent wererounded clusters of atypical basaloid cells showing focalinfiltration into the adipose tissue with an absent myoepitheliallayer measuring approximately 1 mm in maximum dimension(Fig. 3E). Keratin 14 highlighted the cells in this group,but failed to stain any other component in the excision(Fig. 3F). A diagnosis of invasive carcinoma was favoured,with features of a solid variant of adenoid cystic carcinoma.There was absent staining with oestrogen and progesteronereceptors. Her-2 SISH showed no evidence of amplification.DCIS showed a 30% Ki-67 labelling index, whilst atypicalMGA showed a 5% labelling index (Fig. 3G). The small area ofinvasive carcinoma in Case 3 was cut out in subsequentsections and Ki-67 was unable to be performed on this com-ponent.

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MGA is an uncommon benign lesion arising in the breast,which may be detected as a localised, ill-defined density onmammography, with isolated cases reported as ‘suspiciousfor carcinoma’. Clinically, it may manifest as a palpable massor thickening.1–3 Histologically, it is characterised by ahaphazard distribution of small, uniform round glands linedby a single layer of bland, flat to cuboidal epithelial cells withrounded nuclei and abundant vacuolated to eosinophilic cyto-plasm. The glands may be clustered on a collagenous back-ground, but no sclerosis or compression of the glands is usuallyseen. The glands have a rounded lumen, which frequentlycontains periodic acid-Schiff (PAS) positive eosinophilicsecretion. The glands are devoid of myoepithelial cells,but are surrounded by a basement membrane, which stainsfor laminin and collagen IV. Electron microscopy shows amultilayered basement membrane surrounding the tubules.8

The epithelial cells stain positively with cytokeratin, S-100protein, epithelial membrane antigen (EMA), and negativelyfor oestrogen and progesterone receptors.1,2,8

MGA is benign in its uncomplicated form, however, therehave been rare reports describing its transition to atypicalMGA, in situ carcinoma and invasive carcinoma.1,2,5,9 AtypicalMGA has features of uncomplicated MGA but shows a greaterarchitectural complexity with back-to-back and interconnectedbudding glands. The glands are more closely opposed withdiminished intraluminal eosinophilic secretion. They arelined by cytologically atypical cells with mild to moderatenuclear pleomorphism, vesicular nuclei and prominentnucleoli. Mitoses may be seen. The glands, like uncomplicatedMGA, lack a myoepithelial layer but are surrounded by abasement membrane.1,2,4,5,8

Areas showing features of atypical MGA but with solidexpansile proliferation obliterating the lumen and/or severecytological atypia without the presence of desmoplasiaare considered carcinoma in situ arising in atypical MGA.It tends to retain the underlying alveolar growth pattern ofthe adenosis.10 Meanwhile, invasive carcinoma arising fromMGA (MGACA) is usually high grade and is characterised byinfiltrating nests/glands of malignant cells with a surround-ing desmoplastic stromal reaction.1,2,4,5 Different patternshave been observed, including duct-forming, clear cell,sarcomatoid, metaplastic, basal-like, acinic-like and adenoidcystic patterns.1,5 Invasive carcinoma differs from the othercomponents in the MGA spectrum by demonstrating a loss ofthe basement membrane.10

MGA, atypical MGA and carcinoma arising in MGAhave common immunohistochemical profiles. They are ‘triple

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Fig. 3 Case 3. (A) The core biopsies from Case 3 showed irregular glands with hyperchromatic atypical cells with no obvious myoepithelial layer (H&E). (B) p63showed no staining of myoepithelial cells in the core biopsies. (C) Sections of the excision in Case 3 showed atypical microglandular adenosis (right) and ductalcarcinoma in situ (left) (H&E). (D) Basement membrane around glands displaying atypical MGA stained positively with collagen type IV. (E) Within the area of atypicalMGA were rounded clusters of basaloid cells showing focal infiltration into the adipose tissue and an absence of myoepithelial layer (H&E). (F) Keratin 14 highlighted thecells in the group without any staining of other surrounding components. (G) Case 3 showed a Ki-67 labelling index of 30% in DCIS (right) compared to 5% in adjacentatypical MGA (left).

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negative’; that is, negative for oestrogen receptor (ER),progesterone receptor (PR) and Her-2. They stain positivelywith pancytokeratin (AE1/AE3), S-100, CK8/18 and epidermalgrowth factor receptor (EGFR). They are negative for CK5/6,and show focal staining with c-kit. Laminin and Type IVcollagen stain the basement membrane around most cases ofMGA, atypical MGA and carcinoma in situ. Myoepithelialmarkers such as p63 and smooth muscle actin (SMA) arenegative due to the lack of myoepithelial cells.1

Occasionally, the various components within the MGAspectrum may seem to merge imperceptibly together withno clear boundaries, and this is compounded by the fact thatthere are often overlapping morphological and immunohisto-

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chemical features. Ki-67 and p53 have been used in severalstudies as an adjunct tool in mapping the transition fromuncomplicated MGA to carcinoma.1,2,5 They generally showan increased level of positivity as lesions progress fromuncomplicated MGA to atypical MGA, carcinoma in situand invasive carcinoma, reflecting the increasing level ofcellular proliferation and malignant potential. A useful guideprovided by Khalifeh indicated that uncomplicated MGAgenerally show a Ki-67 labelling index of less than 3%, atypicalMGA show a labelling index of 5–10%, and in situ andinvasive carcinoma show an index greater than 30%.1

MGA is known for its resemblance to several benign entities,including sclerosing adenosis and apocrine adenosis,3,11 as well

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502 CORRESPONDENCE Pathology (2011), 43(5), August

as malignant diseases including tubular carcinoma.3,4 MGAwithout atypia is a benign mammary disease, and multiplestudies have emphasised the importance of clearly differentiat-ing MGA from tubular carcinoma, given that they share ahaphazard distribution of glands and an absence of myoe-pithelial cells.3,4,11 This is achieved by close microscopicexamination, as tubular carcinoma have angulated or ovalglands lined by columnar cells with apical snouts, and arrangedin an invasive pattern in a desmoplastic background.3,4

Immunohistochemistry is also a helpful adjunct in their differ-entiation, as tubular carcinoma, unlike MGA, is almost alwaysER and PR positive, S-100 negative and EMA positive.4

Other differential diagnoses such as sclerosing and apocrineadenosis are distinguished by the presence of p63 andSMA positive myoepithelial cells surrounding the cuboidalor apocrine epithelial cells. Morphologically, the glands insclerosing adenosis are compressed and attenuated, retaininga lobulocentric configuration.3,8 Apocrine adenosis displaystall columnar cells with eosinophilic granular cytoplasm and adistinct basal lamina. It is characterised by the presence of thespecific apocrine marker GCDFP-15, which is non-reactive inMGA. The absence of EMA in MGA also makes it uniqueamong other benign glandular proliferations in the breast.11

Although MGA was first described in 1968 by McDivittet al.,12 and further described in 1983 in three articles as abenign proliferative glandular disease of the breast with nomalignant potential,3,13,14 most of the literature in recent yearson MGA has focused on various case reports and smallseries of patients with carcinoma arising in association withMGA.1,5,6,7,9,10 Although MGA still remains under ‘benignepithelial proliferations’ in the current WHO classification,8

it has become an increasingly controversial entity in regardsto its biology and malignant potential.

Most of the cases described illustrated a transition tocarcinoma by morphological assessment and supported byimmunohistochemistry. The vast majority of cases showtransition with the concurrent presence of irregular shapedglands, increasing cytological atypia and mitotic figures(defined as atypical MGA) merging imperceptibly withareas of invasive carcinoma.1 The proliferative activity onmorphology mirrors that seen on Ki-67 and p53 staining, withincreasing labelling index seen as the lesion progresses fromMGA to carcinoma.1,2,5 Furthermore, Acs et al. believe thataltered myoepithelial cells form the major neoplastic elementin cases showing both atypical MGA and adenoid cysticcarcinoma as they stained positively with myoepithelial cellmarkers: S-100, smooth muscle actin, and vimentin.15 Otherssupport the premalignant potential of the lesion on the basis ofthe common immunohistochemical profile across the entirelesional spectrum including the carcinoma component.1

In addition, two recent studies investigated the molecularcharacteristics of eighteen cases displaying a spectrum ofchanges ranging from MGA to carcinoma using high resolutioncomparative genomic hybridisation (CGH).2,16 Both studiesshowed highly similar genetic profiles when comparing MGA,atypical MGA and carcinoma arising in MGA, with accumu-lation of additional genetic aberrations as cases ‘progressed’from MGA to MGACA. They also noted that the morphology,immunohistochemical profile and molecular characteristicsacross the MGA spectrum were similar to BRCA1-associated,triple-negative, basal-like breast carcinoma. It was concludedthat there was strong molecular evidence that all componentswithin the MGA spectrum were clonally related, and supported

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the hypothesis that MGA is a non-obligate precursor fordevelopment of intraductal and invasive carcinoma.2,16

Although the evidence points towards the premalignant poten-tial of MGA, the vast majority of MGA are reported in isolationwithout any proliferative changes. Hence, it is unlikely that it isan indolent invasive disease, but rather a precursor lesion withpotential to develop carcinoma in some cases. However, at thepresent time, there is no morphological, immunohistochemicalor molecular distinguishing feature that allows the accurateprediction of those cases of ‘pure’ MGA that will proceed tomalignancy. As mentioned by Shin et al.,2 the challenge for thefuture will be to study the molecular features of ‘pure’ MGAprospectively with clinical follow-up in order to identify mole-cular markers of risk for progression to cancer.

Given the evidence supporting their association withcarcinoma, current recommended management for bothMGA and atypical MGA is complete surgical excision withclinical follow-up. Recurrences have been observed afterincomplete excision.5,9 Carcinoma arising in MGA is treatedas per other types of breast carcinoma, including the achieve-ment of clear surgical excision margins, sentinel node biopsyand/or axillary clearance, as well as adjuvant chemotherapy andradiotherapy if required. Factors such as tumour stage and otherclinicopathological features must be considered.5,9

In summary, MGA, atypical MGA and carcinoma arising inMGA show overlapping morphological, immunohistochemicaland molecular features, and should be considered as part ofa progressive spectrum of changes. The presence of anycomponent should prompt a search of other possible com-ponents by the diagnostic pathologist and clinician so thatappropriate treatment can be implemented.

Acknowledgements: The authors wish to express appre-ciation to Dr Duncan Mcleod, Dr Kirsten Hills, Dr StuartAdams, Ms Jane Milliken and staff of the Department ofAnatomical Pathology at ICPMR, Westmead Hospital andNepean Hospital.

Lisa LinNirmala Pathmanathan

Department of Tissue Pathology, Westmead Hospital,Westmead, NSW, Australia

Contact Dr L. Lin.E-mail: lisalin10@gmail.com

1. Khalifeh IM, Albarracin C, Diaz LK, et al. Clinical, histopathologic, andimmunohistochemical features of microglandular adenosis and transitioninto in situ and invasive carcinoma. Am J Surg Pathol 2008; 32: 544–52.

2. Shin SJ, Simpson PT, Da Silva L, et al. Molecular evidence for progressionof microglandular adenosis (MGA) to invasive carcinoma. Am J SurgPathol 2009; 33: 496–504.

3. Rosen PP. Microglandular adenosis. A benign lesion simulating invasivemammary carcinoma. Am J Surg Pathol 1983; 7: 137–44.

4. Resetkova E, Flanders DJ, Rosen PP. Ten-year follow-up of mammarycarcinoma arising in microglandular adenosis treated with breast conser-vation. Arch Pathol Lab Med 2003; 127: 77–80.

5. Koenig C, Dadmanesh F, Bratthauer GL, Tavassoli FA. Carcinoma arisingin microglandular adenosis: an immunohistochemical analysis of 20intraepithelial and invasive neoplasms. Int J Surg Pathol 2000; 8: 313–5.

6. James BA, Milicent LC, Rosen PP. Carcinoma of the breast arising inmicroglandular adenosis. Am J Clin Pathol 1993; 100: 507–13.

7. Popper HH, Gallagher JV, Ralph G, Lenard PD, Tavassoli FA. Breastcarcinoma arising in microglandular adenosis: a tumour expressing S-100immunoreactivity. Report of five cases. Breast J 1996; 2: 154–9.

8. Tavassoli FA, Devilee P, editors. The WHO Classification of Tumours of theBreast and Female Genital Organs. Lyon: IARC Press, 2003; 82.

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9. Rosenblum MK, Purrazzella R, Rosen PP. Is microglandular adenosis aprecancerous disease? A study of carcinoma arising therein. Am J SurgPathol 1986; 10: 237–45.

10. ShuiR,YangW.Invasivebreastcarcinomaarising inmicroglandularadenosis:a case report and review of the literature. Breast J 2009; 15: 653–6.

11. Eusebi V, Betts CM, Millis RR, Bussolati G, Azzopardi JG. Microglandularadenosis, apocrine adenosis, and tubular carcinoma of the breast.An immunohistochemical comparison. Am J Surg Pathol 1993; 17: 99–109.

12. McDivitt RW, Stewart FW, Berg JW. Tumors of the Breast. Atlas of TumorPathology. Series 2, Fascicle 2. Washington DC: Armed Forces Institute ofPathology, 1968; 91.

13. Clement PB, Azzopardi JG. Microglandular adenosis of the breast – alesion simulating tubular carcinoma. Histopathology 1983; 7: 169–80.

14. Tavassoli FA, Norris HJ. Microglandular adenosis of the breast: a clin-icopathologic study of 11 cases with ultrastructural observations. Am J SurgPathol 1983; 7: 731–7.

15. Acs G, Simpson JF, Bleiweiss IJ, et al. Microglandular adenosis withtransition into adenoid cystic carcinoma of the breast. Am J Surg Pathol2003; 27: 1052–60.

16. Geyer FC, Kushner YB, Lambros MB, et al. Microglandular adenosisof microglandular adenoma? A molecular genetic analysis of a case asso-ciated with atypia and invasive carcinoma. Histopathology 2009; 55: 732–43.

DOI: 10.1097/PAT.0b013e32834864f4

Solitary juvenile xanthogranuloma in the lungof a young adult

Sir,Juvenile xanthogranuloma is a non-Langerhans cell histiocy-tosis seen most commonly in childhood and adolescence.It typically presents as a cutaneous tumour with rapid growthand spontaneous regression within months or years. Juvenilexanthogranuloma of extracutaneous sites are rare, and pose adiagnostic dilemma if there is no prior history of cutaneouscomponent. Only a handful of case reports are present in theliterature describing solitary extracutaneous juvenile xantho-granuloma.1–3 We report an interesting and extremely rare caseof juvenile xanthogranuloma in the lung without other currentor previously excised cutaneous or extracutaneous lesions in ayoung adult; to the best of our knowledge this is the first suchcase report.

A 23-year-old female presented to the hospital emergencydepartment for intermittent epigastric pain. She had a historyof polycystic ovarian syndrome and was an occasionalsmoker, smoking one to two packs per year for 3 years. Shehad no significant family history and did not take any regularmedication. Physical examination revealed moderate epigastrictenderness, and chest examination was unremarkable.Preliminary interpretation of the chest X-ray in the emergencydepartment showed normal cardiomediastinal contours and nogas under the diaphragm to suggest perforation of abdominalviscus. A trial of antacid and proton pump inhibitor wasimplemented for a provisional diagnosis of dyspepsia. Hersymptoms improved and she was discharged for follow-upwith her general practitioner.

She was recalled for further investigation as formalradiological examination of her chest X-ray showed a 3 cmrounded retrocardiac opacity with well-defined smoothmargins (Fig. 1A). Computed tomography (CT) showed a2.5 cm slightly lobulated uniform mass in the inferior portionof the left hilum just posterior to the left ventricle (Fig. 1B). Themass contained no calcification and did not display any contrast

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enhancement. No mediastinal adenopathy or other abnormalitywas identified. Further questioning revealed no respiratoryor systemic symptoms. Routine blood tests did not show anyabnormality. FDG positron emission tomography (PET) scanshowed an intensely metabolically active (peak SUV 25) softtissue mass in the left lower lobe corresponding to that seen inthe chest X-ray and CT scan (Fig. 1C). There were no otherlesions or areas with increased FDG uptake.

Fine needle aspiration biopsy of the lung mass wasperformed. The smears were moderately cellular and containedfrequent spindle shaped cells arranged in small fragmentsand individually, displaying ovoid nuclei, distinct nucleoliand a moderate amount of wispy cytoplasm (Fig. 1D,E). Noprominent cytological atypia or mitotic figures were identified.Cell block showed a small fragment of tissue with similarappearing spindled cells and scattered lymphocytes on astromal matrix. The cells were vimentin and Bcl-2 positive,TTF-1, keratin 7, CD34 and CD99 negative. S-100 showed anequivocal result. The overall findings were that of a spindle cellneoplasm, and differential diagnoses including solitary fibroustumour, sclerosing haemangioma and schwannoma weresuggested. However, definite classification was difficult, andbiopsy for histological assessment was recommended.

Since the diagnosis was still uncertain and the PET scandemonstrated a hot lesion, a left lower lobectomy wasperformed and showed a 27 mm, well circumscribed, firm,pale tan mass with a cream and partially haemorrhagic rimin the hilar region (Fig. 2A). The adjacent lung appearedunremarkable. Microscopic examination showed a circum-scribed lesion comprised of spindle cells arranged in intersect-ing fascicles and in a coarse storiform pattern, and in areasaccompanied by collagen fibres (Fig. 2B,C). The cells wereovoid to spindled with hyperchromatic nuclei displayingminimal to mild nuclear atypia and showing eosinophiliccytoplasm (Fig. 2D). Focally, these were merged with cellsdisplaying foamy cytoplasm (Fig. 2E). Scattered small lympho-cytes were seen admixed within the spindle cells. A solitaryosteoclast-like giant cell was identified, however no Toutoncell was present. Scattered mitotic figures were seen (5 per10 high power fields). Focal areas of haemorrhage andhaemosiderin deposition were also noted, possibly related toprevious FNA biopsy. The differential diagnosis at the timeincluded inflammatory myofibroblastic tumour, primary intra-pulmonary meningioma and follicular dendritic cell tumour.Immunoperoxidase staining showed strong staining withleukocyte common antigen (LCA), CD68, CD163, CD4 andfactor XIIIa (Fig. 3A–D). Vimentin also stained positively.There was patchy positivity with epithelial membrane antigen(EMA). Broad spectrum cytokeratin AE1/AE3, S100, SMA,CD99, and follicular dendritic cell markers (including CD21,CD23 and CD35) were negative. CD34 highlighted bloodvessels within the lesion but no staining of the spindled cellswas seen. Electron microscopy findings were non-contributory.

The overall findings were that of a spindle cell tumour, withmorphology and immunoperoxidase supporting the diagnosisof spindle cell variant of juvenile xanthogranuloma.

Given the diagnosis of juvenile xanthogranuloma, furtherquestioning revealed no other current or previously excisedcutaneous or extracutaneous lesions. Blood tests and haema-tological examination was normal.

Juvenile xanthogranuloma (JXG) is a well-recognisedmember of the non-Langerhans’ cell group of histiocyticproliferative disorders, with dermal dendrocytes considered

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Fig. 1 (A) AP chest X-ray shows a well-defined rounded retrocardiac opacity in the left lung. (B) Computed tomography of the chest with IV contrast shows a non-enhancing mass just posterior to the left ventricle. (C) FDG PET scan shows an intensely metabolically active soft tissue mass in the left chest. (D) Smear shows fragmentsof spindled shaped cells (Giemsa). (E) High power field shows spindled shaped cells with oval nuclei, distinct nucleoli and moderate amount of cytoplasm (Papanicolaou).

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to be the principal cell type in this entity.4 The first case wasdescribed as ‘congenital xanthoma multiplex’ by Adamson in1905,5 and the term ‘juvenile xanthogranuloma’ was proposedby Helwig and Hackney in 1954.6 It is a disease primarily foundin infants, children and adolescents, and is associated with avariety of medical conditions including neurofibromatosis,Niemann-Pick disease, urticaria pigmentosa and juvenile mye-lomonocytic leukaemia (JMML).7,8 Pathogenesis is largelyunknown, however the disease is believed to be a reactiveresponse to an unknown stimuli.9,10

Characteristically, it presents as solitary or multiple red-brown cutaneous papulonodules on the head and neck, upperpart of the trunk and proximal aspects of the limbs. Two-thirdsof all cases develop within the first six to nine months of life,and spontaneous involution is usually observed within monthsor years.11

Extracutaneous manifestations of JXG occur in approxi-mately 5% of cases,10 including involvement of mucosal

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surfaces, central nervous system, eye, liver, spleen, lung,lymph node and bone marrow.1,2,7 Although these extracuta-neous lesions are of benign nature, significant problems mayarise due to pressure effects and possible clinical misdiagnosisas malignant entities.

The first adult onset JXG case was reported in 1963.12

Approximately 15% of JXG occur in adults, with the majorityof the cases presenting as solitary cutaneous lesions in the headand neck.10 They usually occur in the second to third decades,however patients in their 60s and 70s have been known to beaffected. Only a handful of adult onset extracutaneous caseshave been reported in the literature, involving the eyes, skeletalmuscle, breast and gingiva, with most of these cases showingconcomitant cutaneous lesions.9,10,13

Both juvenile and adult forms of disease show a similarspectrum of histological features, displaying a mixture ofdifferent mononuclear and multinucleated cell types asdescribed in Zelger et al.’s overview of non-Langerhans cell

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Fig. 2 (A) Macroscopic examination shows a well-circumscribed pale tan mass with a cream and partially haemorrhagic rim. The adjacent lung appears unremarkable.(B) Low power field shows spindle cells arranged in fascicles and coarse storiform pattern (H&E). (C) Medium power field shows fascicles of spindled cells withintervening collagen fibres and scattered lymphocytes (H&E). (D) High power field shows ovoid to spindled cells with minimal to mild nuclear atypia and scatteredlymphocytes (H&E). (E) Focal infiltration of cells with foamy cytoplasm identified (H&E).

CORRESPONDENCE 505

histiocytoses.14 Spindle cell variant, characterised by spindle-shaped histiocytes arranged in a storiform pattern, is seen morecommonly in adult JXG in favour of vacuolated, xanthoma-tised, scalloped and oncocytic types, and characteristic Toutoncells may be sparse or absent in extracuatenous sites.2,7,10,11

Regardless, it is believed that the histological variation of JXGlesions is a time-dependent phenomenon rather than age orsite dependent, and mirrors the life cycle of the lesion witheventual capacity for self-healing or spontaneous regression.13

Fortunately for diagnostic purposes, JXG cells show a consist-ent immunoreactivity for vimentin, CD68, factor XIIIa andCD163, and are negative for S-100, CD1a and langerin.2,7

Electron microscopy shows bland fibrohistiocytic and/orxanthoma cells without Birbeck granules. Non-specificintracytoplasmic findings such as dense bodies, worm-likebodies, and popcorn bodies may be found.2,15

Spindle cell lesions of the lung raise several possibledifferential diagnoses. These include spindle cell carcinoma,inflammatory myofibroblastic tumour, primary intrapulmonary

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meningioma, follicular dendritic cell tumour, solitary fibroustumour and monophasic synovial sarcoma. Spindle cell carci-noma typically displays infiltrative margins and is usuallyassociated with more significant nuclear atypia, mitotic activityand/or necrosis. Furthermore, the absence of broad spectrumcytokeratin immunoreactivity makes this diagnosis unlikely.Follicular dendritic cell tumour, characterised by spindled toovoid cells forming fascicles, storiform arrays, whorls, diffusesheets or nodules, typically shows a more vesicular nucleiprofile,7 and the complete lack of staining with folliculardendritic markers (CD21, CD23, CD35) in this case excludesthis diagnosis. Monophasic synovial sarcoma often displaysa prominent haemangiopericytomatous vascular pattern,cytokeratin and CD99 positivity, all of which are lacking inour case. Our case also lacks the ‘patternless’ architecture,branching haemangiopericytoma-like vessels, and CD34 andCD99 positivity, distinctive in solitary fibrous tumour.16

Inflammatory myofibroblastic tumour, which tends to occurin the lung of a young adult, is a benign neoplasm with

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Fig. 3 (A–D) Histiocyte markers (A) LCA, (B) CD68, (C) CD163 and (D) factor XIIIa show positive staining of the spindle cells.

506 CORRESPONDENCE Pathology (2011), 43(5), August

histological features resembling spindled cell variants ofjuvenile xanthogranuloma, displaying a mixture of spindlecells arranged in fascicles or storiform pattern with minimalcytological atypia and admixed with lymphocytes, plasmacells and histiocytes.17 Both show common immunoreactivityincluding vimentin and selected histiocytic markers, howeverthe presence of diffuse strong staining with LCA and allhistiocytic markers together with a negative smooth muscleactin make this diagnosis unlikely. Similarly, the diffusehistiocytic marker positivity makes meningioma unlikely,taking note that epithelial membrane antigen may be positivein occasional cases of JXG.

All cases of pulmonary JXG in the literature have beenrecorded in children to date.1–3,18–20 All but one of these casesshow coexisting cutaneous or other visceral lesions,3 assistingin the diagnosis of pulmonary JXG. This made our casechallenging, given the rarity of pulmonary JXG and the lackof other JXG lesions in our patient.

In summary, this example of solitary pulmonary spindlecell juvenile xanthogranuloma arising in a young adult is, toour knowledge, the first reported case of extracutaneousmanifestion of adult-type juvenile xanthogranuloma in thelung. Juvenile xanthogranuloma should be considered in thedifferential diagnosis of spindle cell lesion encountered inany organ system and performing the appropriate immuno-peroxidase panel to exclude other conditions is the key todiagnosis.

Acknowledgements: The authors wish to express apprecia-tion to Dr John K. C. Chan for providing his expert opinion onthis case.

Lisa Lin*Elizabeth L. Salisburyz§jj

right © Royal College of pathologists of Australasia

Ian Gardiner{Winny Varikatt*§

*Department of Tissue Pathology, ICPMR, {Department ofRespiratory Medicine, Westmead Hospital, zDepartmentof Anatomical Pathology, Prince of Wales Hospital,§University of Western Sydney, and jjUniversity of NewSouth Wales, Sydney, NSW, Australia

Contact Dr L. Lin.E-mail: lisalin10@gmail.com

1. Freyer DR, Kennedy R, Bostrom BC, Kohut G, Dehner LP. Juvenilexanthogranuloma: forms of systemic disease and their clinical implications.J Pediatr 1996; 129: 227–37.

2. Dehner LP. Juvenile xanthogranuloma in the first two decades of life. Aclinicopathologic study of 174 cases with cutaneous and extracutaneousmanifestations. Am J Surg Pathol 2003; 27: 579–93.

3. Snijders D, Stenghele C, Monciotti C, et al. Case for diagnosis: 4-month-old infant with increasing cough, hemoptysis, and anemia. Pediatr Pulm2007; 42: 844–6.

4. Favara BE, Feller AC, Pauli M, et al. Contemporary classification ofhistiocytic disorders. Med Pediatr Oncol 1997; 29: 157–66.

5. Adamson N. Congenital xanthoma multiplex in a child. Br J Dermatol1905; 17: 222–3.

6. Helwig EB, Hackney VC. Juvenile xanthogranuloma (nevo-xantho-endothelioma). Am J Pathol 1954; 30: 625.

7. Swerdlow SH, Campo E, Harris NL, et al., editors. World HealthOrganization Classification of Tumours of Haematopoietic and LymphoidTissues. 4th ed. Lyon: IARC Press, 2008; 363–7.

8. Iwuagwu FC, Rigby HS, Payne F, Reid CD. Juvenile xanthogranulomavariant: a clinicopathological case report and review of the literature. Br JPlast Surg 1999; 52: 591–6.

9. Consolaro A, Sant’Ana E, Lawall MA, Consolaro MF, Bacchi CE. Gingivaljuvenile xanthogranuloma in an adult patient: case report with immuno-histochemical analysis and literature review. Oral Surg Oral Med OralPathol Oral Radiol Endod 2009; 107: 246–52.

10. Shin SJ, Scamman W, Gopalan A, Rosen PP. Mammary presentation of adult-type ‘juvenile’ xanthogranuloma. Am J Surg Pathol 2005; 29: 827–31.

11. Weedon D. Skin Pathology. 2nd ed. Edinburgh: Churchill Livingstone,2002; 1067–70.

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Fig. 1 Axial CT demonstrating large nasopharyngeal mass.

Fig. 2 Typical histomorphology of the extraskeletal myxoid chondrosarcomawith cords of malignant cells in a myxoid background (H&E).

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12. Gartmann H, Tritsch H. Klein-und großknotiges naevoxanthoendotheliom.Klin Exp Dermatol 1963; 215: 409–21.

13. Kuo FY, Eng HL, Chen SH, Huang HY. Intramuscular juvenile xantho-granuloma in an adult. A case report with immunohistochemical study.Arch Pathol Lab Med 2005; 129: e31–4.

14. Zelger BW, Sidoroff A, Orchard G, Cerio R. Non-Langerhans cell histio-cytosis: a new unifying concept. Am J Dermatopathol 1996; 18: 490–504.

15. Hsi ED. Hematopathology: A Volume in Foundations in DiagnosticPathology Series. Edinburgh: Churchill Livingstone, 2006; 535–9.

16. Fletcher CD, Unni KK, Mertens F. World Health Organization Classifica-tion of Tumours. Pathology and Genetics of Tumours of Soft Tissue andBone. Lyon: IARC Press, 2002; 86–90.

17. Travis WD, Brambilla E, Muller-Hermelink HK, Harris CC, editors. WorldHealth Organization Classification of Tumours. Pathology and Genetics ofTumours of Lung, Pleura, Thymus and Heart. 3rd ed. Lyon: IARC Press,2004; 105–6.

18. Bakir B, Unuvar E, Terzibasioglu E, Guven K. Atypical lung involvementin a patient with systemic juvenile xanthogranuloma. Pediatr Radiol 2007;37: 325–7.

19. Gupta AK, Bhargava S. Juvenile xanthogranuloma with pulmonary lesions.Pediatr Radiol 1988; 18: 70.

20. Garcia-Pena P, Mariscal A, Abellan C, Zuasnabar A, Lucaya J. Juvenile xan-thogranuloma with extracutaneous lesions. Pediatr Radiol 1992; 22: 377–8.

DOI: 10.1097/PAT.0b013e328348900d

Extraskeletal myxoid chondrosarcoma ofthe nasopharynx

Sir,Extraskeletal myxoid chondrosarcoma (EMCS) is a veryrare sarcoma with malignant chondroblast-like cells seen ina background of myxoid matrix.1 Although the name impliesorigin from the cartilage, there is no convincing evidence ofchondroid lineage of the malignant cells. EMCS was recog-nised by Stout and Verner in 1953 and defined as a distinctclinicopathological entity by Enzinger and Shiraki in 1972.2,3

It is a monoclonal neoplasm with either t(9;17) or t(9;22)translocations.4 The common sites of origin of these tumoursinclude extremities, especially legs.1,5

A 50-year-old male presented with nasal congestion anddull headache. Evaluation with computed tomography (CT)scan, magnetic resonance imagaing (MRI) and positronemission tomography (PET) scan was performed to reveal alarge, 7 cm in greatest dimension, expansile, destructive massin the nasal cavity, nasopharynx, bilateral ethmoid air cells,bilateral maxillary and right sphenoid sinuses (Fig. 1). Itdemonstrated avid contrast uptake in PET scan, suggestiveof a high grade tumour. The possibility of metastases fromanother location was ruled out based on the CT staging results.Ethmoid, frontal, maxillary and sphenoid sinusotomies werecarried out, along with tissue resection and mass debulking.Microscopic examination showed densely cellular tumour,consisting of cells with moderately pleomorphic, hyperchro-matic nuclei with prominent nucleoli. The cells were arrangedin ribbons, trabeculae and small nests present in a myxoidbackground (Fig. 2). Numerous mitotic figures were seen.The cytoplasm was scant and densely eosinophilic. Some ofthe tumour cells had rhabdoid appearance with eccentricallyplaced nuclei (Fig. 3). Additionally, extensive areas of necrosiswere present.

A panel of immunohistochemistry was performed andincluded vimentin, S-100, myogenin, Myo-D1, wide spectrumcytokeratin, CD30, ALK-1, CD45, EMA, synaptophysin

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and chromogranin. The tumour cells showed positivity withvimentin only.

Unfortunately no fresh tissue was collected for cytogeneticanalysis; therefore reverse-transcription polymerase chainreaction (RT-PCR) analysis on paraffin tissue for EWS-CHNfusion gene transcript was performed and was negative. RT-PCR against other transcripts found in EMCS was notperformed due to unavailability of the test at the laboratory.The material of the case was sent for consultation to MayoClinic, Rochester, USA, where the diagnosis was confirmed.

Chemotherapy was not given, considering the fact thatEMCS is typically resistant to that type of therapy. Four weeksafter the resection the tumour recurred and assumed a similarsize to the original pre-surgery dimensions. The patient startedto lose vision. He then received immediate radiotherapystarting with 4 fractions, with 800 cGy delivered to thesinonasal cavity and the right orbit utilising AP and two lateralfields and 3-dimensional conformal technique, 6-MV photons.Then this was followed by additional TomoTherapy(TomoTherapy, USA) with a dose of 4600 cGy delivered in23 fractions utilising 6-MV photons. The patient completed hisentire course of radiation treatment without any significantcomplaints. He reported significant improvement of his

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Fig. 3 Rhabdoid morphology with eccentrically placed nuclei (H&E).

508 CORRESPONDENCE Pathology (2011), 43(5), August

vision (almost back to the vision status prior to the radiation)and nasal congestion was resolved at the end of the radiationtreatment. However, this remarkable improvement wastemporary and following pulmonary and brain metastasesthe patient succumbed to the disease approximately 2 yearsafter the presentation.

Only five cases of extraskeletal myxoid chondrosarcoma(EMC) have been reported in the region of the head to date.The case reports include two tumours in the nasal cavity,1,6 onein the infratemporal fossa,7 one in the cerebellopontine angleof the brain8 and one in the maxillary antrum.9 Other rare sitesof origin besides the head include the supraclavicular region,10

intra-abdominal wall and female genitalia.5

EMCS usually occurs after the age of 30 years and ismore common in males. The thigh is the most commonlocation. The aetiological factors are unknown1 and the histo-genesis of the tumour is debatable. It may originate fromprimitive cartilage forming mesenchyme. One of the otherpossible sources of origin is synovial intimal cells, becausethe tumour most commonly arises in proximity to tendons andligaments.10,11 However, there has not been any substantiatedproof of these theories.

The clinical behaviour of this tumour may be indolentor aggressive, depending on the grade.12 Local recurrence,distal metastases or both may be present during the courseof the disease. Distal metastases have been recorded in lungs,soft tissues, bones, regional lymph nodes, subcutis, brain, bonesand testis.10,11 Recurrence and metastases after long intervalsare also known to occur.1

The patient presented in this report had several factorsthat unfavourably affected the course of this disease. Theseincluded age of the patient (> 50 years), male sex, location,relatively large size of the tumour, high grade, cellularity,mitoses, and atypical cytological features. The atypical featuresthat may be found in a high grade EMCS are rhabdoidphenotype and anaplasia.12

Microscopic examination of EMCS reveals two grades oftumours: low and high grade. The majority of tumours areconsidered low grade and are hypocellular, possess abundantmyxoid matrix, mild nuclear pleomorphism and rare mitoses.The high grade tumours are very cellular, growing predomi-nantly in diffuse solid sheets and largely devoid of myxoidmatrix. They have areas of necrosis, and areas of dedifferentia-tion consisting of rhabdoid morphology or high grade spindlecell sarcoma component.

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Immunohistochemical analysis reveals consistent expressionof vimentin. Variable expression of epithelial membrane antigen,neurofilament8 and synaptophysin has been reported in variousstudies.12 S-100 staining is very rare and is typically focal.

The differential diagnosis includes chondromyxoid fibroma,myxoid chondrosarcoma, chordoma, parachordoma, mixedtumours of salivary gland, and myxoid liposarcoma.5,7

The only similarity with chondromyxoid fibroma (CMF)is lobular growth pattern and myxoid stroma. CMF is acytologically bland tumour with stellate or spindle-shaped cellsin a myxoid background. Immunohistochemical stains are nothelpful in separating EMCS from CMF.

Separating EMCS from myxoid variant of chondrosarcomamay present a challenge on H&E; however, the former willusually have areas of recognisable chondrocytes and lacunae.It will stain with S-100 diffusely in the majority of the cases,while only rare cases of EMCS will stain with that marker andthe stain will highlight only rare cells.

Mixed salivary gland tumours may have areas resemblingEMCS, but these benign tumours are easily recognised bythe presence of variable epithelial and stromal componentselsewhere.

Myxoid liposarcomas have only some similarities withEMCS, mainly prominent myxoid background and blandcytology, but these tumours usually have small prominentlipoblasts and a rich ‘chicken wire-like’ vascular network.

This rare case highlights problems in establishing the diag-nosis of EMCS and the prognostic importance of unfavourablefeatures of this sarcoma such as location, rhabdoid phenotype,patient age and size of the tumour. Despite these factors,surgery with radiation, followed by chemotherapy, maycontribute significantly, although temporarily, to the qualityof life of patients with EMCS of the head.

Amarpreet Bhalla*Vladimir Osipov{

*Pathology Department, Danbury Hospital, Danbury, CT,USA; {Labtests, Department of Anatomical Pathology, MtWellington, Auckland, New Zealand

Contact Dr V. Osipov.E-mail: Vladimir.osipov@labtests.co.nz

1. Ceylan K, Kizilkaya Z, Yavanoglu A. Extraskeletal myxoid chondrosar-coma of the nasal cavity. Eur Arch Otorhinolaryngol 2006; 263: 1044–7.

2. Stout AP, Verner EW. Chondrosarcoma of the extraskeletal soft tissues.Cancer 1953; 6: 581–90.

3. Enzinger FM, Shiraki M. Extraskeletal myxoid chondrosarcoma.Hum Pathol 1972; 3: 421–35.

4. Meis-Kindblom JM, Bergh P, Gunterberg B, Kindblom LG. Extraskeletalmyxoid chondrosarcoma: a reappraisal of its morphologic spectrumand prognostic factors based on 117 cases. Am J Surg Pathol 1999; 23:636–50.

5. Santa Cruz MR, Proctor L, Thomas DB, Gehrig PA. Extraskeletal myxoidchondrosarcoma: A report of gynecologic case. Gynecol Oncol 2005; 98:498–501.

6. Liu Shindo M, Rice DH, Sherrod AE. Extraskeletal myxoid chondrosarcomaof the head and neck: a case report. Otolaryngol Head Neck Surg 1989; 101:485–8.

7. Acero J, Escrig M, Gimeno M, Montenegro T, Navarro-Vila C.Extraskeletal myxoid chondrosarcoma of the infratemporal fossa: a casereport. Int J Oral Maxillofac Surg 2003; 32: 342–5.

8. O’Brien J, Thornton J, Cawley D, et al. Extraskeletal myxoid chondro-sarcoma of the cerebellopontine angle presenting during pregnancy. Br JNeurosurg 2008; 22: 429–32.

9. Jawad J, Lang J, Leader M, Keane T. Extraskeletal myxoid chondrosarcomaof the maxillary sinus. J Laryngol Otol 1991; 105: 676–7.

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Fig. 1 Mammography, showing an infiltrating tumour mass, is suspicious forinvasive carcinoma.

Fig. 2 In some areas a spindle cell proliferation with sclero-elastotic cuffsaround individual open tubules and ducts are observed (H&E).

CORRESPONDENCE 509

10. Antonescu CR, Argani P, Erlandson RA, Healey JH, Ladanyi M, Huvos AG.Skeletal and extraskeletal myxoid chondrosarcoma. A comparative,clinicopathologic, ultrastructural and molecular study. Cancer 1998; 83:1504–21.

11. Goh Y-W, Spagnolo DV, Platten M, et al. Extraskeletal myxoid chondro-sarcoma: a light microscopic, Immunohistochemical and ultrastructural andimmunoultrastructural study indicating neuroendocrine differentiation.Histopathology 2001; 39: 514–24.

12. Oliveira AM, Sebo TJ, McGrory JE, Gaffey TA, Rock MG, Nascimento AG.Extraskeletal myxoid chondrosarcoma: A clinicopathologic, immunohistoc-hemical and ploidy analysis of 23 cases. Mod Pathol 2000; 13: 900–8.

DOI: 10.1097/PAT.0b013e3283488fec

Low grade periductal stromal sarcoma withmarked sclero-elastosis

Sir,Low grade periductal stromal sarcoma (PDSS) is a rare biphasiclesion characterised by a neoplastic spindle cell proliferationlocalised around mammary ducts/tubules that retain openlumens in absence of leaf-like processes typically seen inphyllodes tumours.1 Although the term PDSS has been usedas a synonym for phyllodes tumour,2 it should be betterrestricted to those lesions that appear to arise from theperiductal/perilobular stroma instead of intralobular stroma.3

Burga and Tavassoli1 first defined the basic histologicalfeatures of PDSS: (i) sarcomatous proliferation of spindle-shaped cells with variable cytological atypia, around individualopen tubules and ducts devoid of a phyllodes pattern; (ii) oneor often multiple nodules that may have coalesced or may beseparated by adipose tissue; (iii) �3 mitoses � 10 highpower fields (HPF) in the area with the greatest mitotic activity;(iv) infiltration into surrounding mammary fibroadipose tissue.PDSS has a tendency for local recurrence when incompletelyexcised, with a metastatic potential, at least in thosecases harbouring more aggressive sarcomatous component.1

Complete excision with uninvolved margins is the recom-mended treatment.1

We report the first case of PDSS exhibiting extensive sclero-elastosis that obscured more than 80% of the underlyingtumour, raising differential diagnostic problems. A 84-year-old woman presented a slightly painful right breast lesion. Noprevious history of breast carcinoma was present. Physicalexamination revealed an immobile mass located in theupper outer quadrant of her right breast. No axillary lympha-denopathy was observed. Mammography revealed an infiltra-tive, poorly circumscribed tumour mass with calcifications,measuring 2 cm in its greatest diameter, located in the upperouter quadrant of her right breast (Fig. 1). An invasivecarcinoma was suspected and the patient underwentlumpectomy without axillary lymphadenectomy. The surgicalspecimen consisted of a tumour mass with infiltrating borders,firm in consistency and whitish in colour. Histologically,the tumour mass was surprisingly composed of a diffusesclero-elastosis approximately accounting for more than 80%of the entire lesion (Fig. 2). In some areas multiple noduleswere seen, consisting of a proliferation of spindle-shaped cellsforming cuffs around individual open tubules and ducts (Fig. 3).Some of these nodules were coalescing and infiltrating intothe surrounding fibro-adipose tissue. Neoplastic cells exhibited

right © Royal College of pathologists of Australasia

a mild to moderate nuclear pleomorphism and were embeddedin a variably sclero-elastotic stroma (Fig. 3). Mitotic activityranged from 1 up to 3 mitoses per 10 HPF. Even after ameticulous search of the whole tumour tissue, papillae orleaf-like structures typically observed in phyllodes tumourswere not observed. The resection margins were tumour-free.Sclero-elastotic changes were not observed in the surroundingbreast parenchyma. Immunohistochemically, tumour cells werepositive only to vimentin and CD34. No immunostainingwas obtained with a whole panel of cytokeratins (AE1/AE3,CKMNF, CAM 5.2, 34bE12, CK5/6, CK7, CK19), EMA, a-smooth muscle actin, desmin, S-100 protein, CD117 and p63.Proliferative index, assessed by Ki-67 staining, was low

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Fig. 3 High magnification showing a proliferation of atypical spindle-shapedcells around ducts. Neoplastic cells are infiltrating the surrounding breastparenchyma (H&E).

510 CORRESPONDENCE

(<3%). Based on morphological and immunohistochemicalfeatures, the diagnosis of ‘low grade PDSS with extensivesclero-elastosis’ was made. The patient underwent additionalradiotherapy and 8 months after surgery she is well with noevidence of recurrence.

Stromal elastosis, defined as dense aggregations of elasticfibres, may be found in nearly half of all women over the ageof 50 years with no breast disease,4 as well as in some casesof fibroadenoma, fibrocystic disease and in radial scar as acharacteristic stromal component.5 Additionally, sclero-elastosiscan be variably encountered in the stroma surrounding bothin situ and invasive ductal carcinoma.5 Differential diagnosismainly revolves around low-grade sarcomatoid/metaplasticcarcinoma exhibiting a fibromatosis/nodular fasciitis-likepattern and myofibroblastoma.6,7 The former can be easilyexcluded with the use of immunohistochemistry for lowmolecular weight keratins which identify epithelial foci withspindle morphology.6 Unlike our case, myofibroblastoma istypically a spindle cell tumour with well circumscribed borders,which expresses desmin and a-smooth muscle actin.7 Addition-ally, most cases show immunoreactivity for CD34, Bcl-2protein, CD99 and CD10.7

The present case highlights the possibility that PDSScan exhibit such diffuse sclero-elastosis that the underlyingmalignant cells may be easily overlooked. As previouslypostulated for breast carcinomas cells, it is likely that sclero-elastosis may merely represent a reactive change of theindigenous mammary stromal cells in response to unknownstimuli.8 With regard to our case, the absence of similar stromalchanges in the perilesional breast parenchyma of this 84-year-oldwoman seems to exclude an age-related phenomenon. Apartfrom these pathogenetic considerations, the present case stronglysuggests that pathologists dealing with a diffuse sclero-elastosisof the breast should carefully examine the whole lesion to rule outan underlying malignant tumour, including PDSS.

Rosario CaltabianoGaetano MagroGiada Maria VecchioSalvatore Lanzafame

Department G. F. Ingrassia, Division of Anatomic Pathology,University of Catania, Italy

right © Royal College of pathologists of Australasia

Contact Dr R. Caltabiano.E-mail: rosario.caltabiano@unict.it

1. Burga AM, Tavassoli FA. Periductal stromal tumor: a rare lesion with low-grade sarcomatous behavior. Am J Surg Pathol 2003; 27: 343–8.

2. Rosen PP. Breast Pathology. 3rd ed. Philadelphia: Lippincott Williams andWilkins, 2009; 202.

3. Tavassoli FA, Devilee P, editors. World Health Organization Classification ofTumours. Pathology and Genetics of Tumours of the Breast and FemaleGenital Organs. Lyon: IARC Press, 2003.

4. Farahmand S, Cowan DF. Elastosis in the normal aging breast. Ahistopathologic study of 140 cases. Arch Pathol Lab Med 1991; 115:1241–6.

5. Parfrey NA, Doyle CT. Elastosis in benign and malignant breast disease.Hum Pathol 1985; 16: 674–6.

6. Kurian KM, Al-Nafussi A. Sarcomatoid/metaplastic carcinoma of thebreast: a clinicopathological study of 12 cases. Histopathology 2002; 40:58–64.

7. Magro G. Mammary myofibroblastoma: a tumor with a wide morphologicspectrum. Arch Pathol Lab Med 2008; 132: 1813–20.

8. Tamura S, Enjoji M. Elastosis in neoplastic and non-neoplastic tissuesfrom patients with mammary carcinoma. Acta Pathol Jpn 1988; 38:1537–46.

DOI: 10.1097/PAT.0b013e328348a764

Expressional and mutational analysis ofPHLDA3 gene in common human cancers

Sir,Alterations of tumour suppressor gene p53 and protooncogeneAKT play critical roles in cancer development.1,2 Pleckstrinhomology-like domain family A member 3 (PHLDA3) is a p53-responsive gene and is required for p53-dependent apoptosis.3

PHLDA3 competes with AKT for binding membranephospholipids, thereby inhibiting AKT translocation to thecellular membrane and activation.3 Functionally, PHLDA3suppresses anchorage-dependent cell growth.3 At tissue level,PHLDA3 locus is frequently lost and PHLDA3 protein down-regulated in human lung endocrine tumours.3 Together, thesedata suggest that PHLDA3 may be a tumour suppressor geneand inactivation of PHLDA3 might contribute to tumorigenesis.Somatic mutation is a main mechanism by which functions oftumour suppressor genes are inactivated in human cancers.1,2

Somatic mutation of PHLDA3 has been analysed in a smallnumber (n¼ 12) of lung endocrine tumours, and there wasno PHLDA3 mutation.3 Because PHLDA3 is related to p53and AKT that are important in many types of human cancers,it is possible that PHLDA3 mutation might occur in othercancers.

In this study, we analysed somatic mutations of PHLDA3gene in methacarn-fixed tissues of 45 prostate carcinomas, 159non-small cell lung cancers (70 squamous cell carcinomas and89 adenocarcinomas), 37 gastric carcinomas, 42 colorectalcarcinomas, 43 breast carcinomas, and non-fixed bonemarrow aspirates of 45 leukaemias (30 acute myelogenousand 15 acute lymphoblastic leukemias) by polymerase chainreaction (PCR) and single-strand conformation polymorphism(SSCP) assay. In solid tumours, malignant cells and normalcells were selectively procured from haematoxylin and eosin(H&E) stained slides using a 30G1/2 hypodermic needle

Pathology (2011), 43(5), August

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Fig. 2 Visualisation of PHLDA3 expression in prostate cancer tissues byimmunohistochemistry. A prostate cancer shows strong PHLDA3 immunostain-ing in normal prostate cells (N), whereas surrounding prostate cancer cells showweak or negative PHLDA3 immunostaining.

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affixed to a micromanipulator, as described previously.4 Geno-mic DNA each from tumour cells and normal cells from thesame patients were amplified with three primer pairs coveringthe entire coding region of human PHLDA3 gene. Radioisotope([32P]dCTP) was incorporated into PCR products for detectionby autoradiography. Direct DNA sequencing was performed inthe cancers with mobility shifts in the SSCP. With immuno-histochemistry, we analysed tissue expression of PHLDA3protein in prostate cancrcinoma tissues (n¼ 107) usingDako Real EnVision System (Dako, Denmark) and a rabbitpolyclonal antibody against human PHLDA3 (dilution 1:50;Assay Biotechnology, USA). Other procedures for mutationand immunohistochemistry were as described in our previousreports.4,5

SSCP analysis detected aberrantly migrating bands ofPHLDA3 in one lung adenocarcinoma compared to wild-typebands from its normal DNA. There was no aberrantlymigrating band of PHLDA3 in the other cancer types. How-ever, the mutation was a silent mutation [c.9G–>A(p.Ala3Ala)] (Fig. 1). Positive immunostaining for PHLDA3was observed in 83 (78%) of the 107 prostate cancers, whilenegative immunostaining was observed in 24 (22%) (Fig. 2).Normal prostate glandular cells were positive for PHLDA3immunostaining in 101 (94%) of the 107 cases. Statistically,there was a significant difference in the PHLDA3 immuno-staining between normal tissue and carcinoma of the prostate(Fisher’s exact test, p< 0.001). However, there was nosignificant association between the immunostaining andvarious pathological parameters in the cancer patients(age, tumour size, vascular invasion, Gleason grade, and stage)(x2 test, p> 0.05).

The aim of this study was to determine whether somaticmutation of PHLDA3 is present in common solid cancers. In the

right © Royal College of pathologists of Australasia

Fig. 1 Mutation of PHLDA3 gene. Direct DNA sequencing of exon 3 ofPHLDA3 gene shows a silent mutation in a lung adenocarcinoma.

present study, we detected only one silent mutation in thecancers, indicating that somatic mutation of PHLDA3 is rarein common human cancer types analysed in the presentstudy. Another aim of our study was to determine whetherPHLDA3 expression is altered in cancers. We found thatPHLDA3 expression was lost in 22% of prostate cancers.These results indicate that PHLDA3 are altered in prostatecancers by loss of expression. To our knowledge, this is the firstreport on PHLDA3 expression in human tissues besideslung endocrine cancers. Until now, alterations of PHLDA3expression have been studied only in prostate and lung cancers.For better understanding of the roles of PHLDA3 in cancerpathogenesis, analysis of PHLDA3 expression in other cancersis needed in future studies.

Acknowledgements: This study was supported by a grantfrom Ministry for Health, Welfare and Family Affairs(A100098).

Nam Jin YooYoo Ri KimSug Hyung Lee

Department of Pathology, College of Medicine, The CatholicUniversity of Korea, Seoul, Korea

Contact Dr S. H. Lee.E-mail: suhulee@catholic.ac.kr

1. Oren M. Decision making by p53: life, death and cancer. Cell Death Differ2003; 10: 431–42.

2. Cully M, You H, Levine AJ, Mak TW. Beyond PTEN mutations: the PI3Kpathway as an integrator of multiple inputs during tumorigenesis. Nat RevCancer 2006; 6: 184–92.

3. Kawase T, Ohki R, Shibata T, et al. PH domain-only protein PHLDA3 is ap53-regulated repressor of Akt. Cell 2009; 136: 535–50.

4. Lee SH, Shin MS, Park WS, et al. Alterations of Fas (Apo-1/CD95) gene innon-small cell lung cancer. Oncogene 1999; 18: 3754–60.

5. Kim SS, Ahn CH, Kang MR, et al. Expression of CARD6, an NF-kappaBactivator, in gastric, colorectal and oesophageal cancers. Pathology 2010; 42:50–3.

DOI: 10.1097/PAT.0b013e3283489036

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512 CORRESPONDENCE Pathology (2011), 43(5), August

Pandemic (H1N1) 2009 influenza withneurological complications diagnosed usingspecific serology with the haemagglutinininhibition assay

Sir,Influenza is usually diagnosed using methods directly detectingvirus. Serology has proven useful in diagnosing pandemicinfluenza where direct detection methods are negative,1

particularly where complications occur later in clinical illness.We report three cases of severe neurological disease inAustralian children during the 2009 pandemic influenza seasonwith clinical influenza where direct detection methods werenegative or not available, and serology was used to confirmrecent infection with pandemic (H1N1) 2009 influenza A.

The clinical and laboratory features of the cases aresummarised in Table 1.

Case 1 was a 14-year-old boy who presented with an alteredconscious state and a 6-day history of fever and cough. He wasagitated with an inexpressive facies but able to walk normally.Electroencephalogram was consistent with encephalopathy,while magnetic resonance imaging (MRI) of the brain wasnormal. He was treated with oseltamivir and improved over thenext 4 days.

Case 2 was a 10-year-old girl who presented with lower limbweakness and urinary retention 1 week after a febrile illnesswith cough. She had patchy sensory changes in her lower limbsand was hyper-reflexic with up-going plantar reflexes. MRIrevealed transverse myelitis. She was commenced on methyl-prednisolone and made a gradual recovery.

Case 3 was an 8-year-old girl, who presented with febrilecomplex partial status epilepticus with no preceding respiratoryillness. She was treated with anticonvulsants, and required

right © Royal College of pathologists of Australasia

Table 1 Clinical and laboratory features of cases

Case 1

Age (years) / sex 14 / MaleNeurological complication EncephalopathyOnset of respiratory symptoms

prior to admission6 days

Contacts Siblings with ILISerological results

Interval between samples 4 daysInfluenza A CFT admission 128Influenza A CFT repeat >256H1 pandemic HI admission 8H1 pandemic HI repeat 512Mycoplasma serology IgM negative

Virological resultsNasal swab on admission Negative IFA for respiratory viruses*

and influenza. Negative influenzaA/H1N109 PCR

CSF resultsCSF white cell count (cells/mL) 3 mononuclear cellsCSF red cell count (cells/mL) 0CSF protein (g/L) 0.24CSF glucose (mmol/L) 3.2CSF bacterial culture NegativeCSF PCR HSV, enterovirus, VZV,

influenza A negativeTreatment Oseltamivir

* Respiratory syncytial virus; parainfluenza viruses 1, 2, 3; adenovirus; influenzaCFT, complement fixation titre; CSF, cerebrospinal fluid; HI, haemagglutinin inhIgM, immunoglobulin M; ILI, influenza-like illness; PCR, polymerase chain rea

intubation. She had no focal neurological findings andwas extubated promptly. At follow-up 2 weeks later, shehad made a complete recovery.

All of the patients were previously well, with no history ofprevious influenza or influenza vaccination. Two of the threehad negative nasopharyngeal swabs for influenza but all threehad diagnostic serology based upon a �4-fold rise in specificserological testing for pandemic (H1N1) 2009 influenza byhaemagglutinin inhibition assay (HI). The HI assay used thereference strain A/California/7/2009 as the assay antigen. Allpaired serum samples were tested in parallel at South EasternArea Laboratory Services. The HI assay detects serum anti-bodies to the viral haemagglutinin by measuring the inhibitionof virus-mediated agglutination of erythrocytes.2 As it is sub-type specific and distinguishes between seasonal and pandemicinfluenza, it has been widely used in epidemiological studies.2

All patients were also tested by HI for seroconversion toseasonal H1 (using strain A/Brisbane/59/2007-H1N1) andH3N2 (A/Brisbane/10/2007-H3N2) influenza. The HI assaythat was used was modified from established techniques3 usingpurified haemagglutinin prepared from reference strains andcontrol sera supplied by the World Health Organization (WHO)Collaborating Centre for Reference and Research on Influenza,Melbourne, Australia.

Case 1 demonstrated 4-fold rises in titres to both seasonalstrains but with lower rises than to the pandemic (H1N1) 2009influenza strain and these are likely to represent cross-reactingantibodies. Cases 2 and 3 did not demonstrate a 4-fold rise intitre to the seasonal strains. Complement fixation titre (CFT)testing for influenza A was also performed for all cases, withhigh titres suggesting recent infection but without the samediagnostic rise in titre.

Investigation of all three cases for alternative causes showedno other aetiology for the illness, other than that describedhere, and all made a good recovery. Case 3 did not have a

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Case 2 Case 3

10 / Female 8 / FemaleTransverse myelitis Status epilepticus7 days No respiratory symptoms

No known contacts Siblings with ILI

22 days 12 days>256 128256 128128 1281024 512IgM positive IgM negative

Negative IFA for respiratoryviruses* and influenza

Not done

1 PMN 1 mononuclear cell2 1890.54 0.294.1 2.7Negative NegativeHSV, enterovirus, VZV,

influenza A negativeHSV, enterovirus, VZV,

influenza A negativeCorticosteroids Anticonvulsants

A/B.ibition assay; HSV, herpes simplex virus; IFA, immunofluorescence assay;ction; PMN, polymorphonuclear leukocyte; VZV, varicella zoster virus.

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CORRESPONDENCE 513

nasopharyngeal sample collected for influenza testing as she hadno respiratory symptoms on presentation. Case 2 also hada positive Mycoplasma pneumoniae IgM antibody assay,with Mycoplasma complement fixation titres of 640 and 1280(elevated, but not demonstrating a 4-fold rise in titre). Thismay represent a false positive result or present an alternativeaetiology for her illness. Myelitis attributed to either Myco-plasma or influenza is rare but dual infections have beenreported.4,5

Encephalitis, seizures and transverse myelitis are knowncomplications of seasonal and pandemic influenza.6 Patientsmay present after viral shedding has ceased, with post-infectious inflammatory disease, as is likely in our cases, orin settings where nucleic-acid testing is not available. Whendirect virological tests are negative, additional serologicaltesting has been shown to enhance diagnostic yield forinfluenza in critically ill adults.1 This may be particularlyimportant for pandemic (H1N1) 2009 influenza, as reportssuggest increased rates of neurological disease followinginfection with this now commonly circulating strain.7 Influenzashould be considered as a cause of encephalitis or transversemyelitis in children, especially during the influenza season.If nucleic acid testing is negative, acute and convalescentserology for influenza and other pathogens should be usedas an additional diagnostic tool.

Acknowledgements: Dr Annie Bye, Department of Neuro-logy, Sydney Children’s Hospital for information on cases.

Brendan McMullan*{zGabriel Dabscheck*{Peter Robertson§Heather Johnston*William RawlinsonjjTony Walls*{�

*Sydney Children’s Hospital, Randwick, {School of Women’sand Children’s Health, UNSW, Sydney, zDepartment ofMicrobiology, St Vincent’s Hospital, Sydney, §Serology, andjjVirology, SEALS Pathology, Prince of Wales Hospital,Sydney, Australia; �Christchurch School of Medicine,University of Otago, Christchurch, New Zealand

Contact Dr B. McMullan.E-mail: brendanoch@yahoo.co.uk

1. Iwasenko JM, Cretikos M, Paterson DL, et al. Enhanced diagnosis ofpandemic (H1N1) 2009 influenza infection using molecular and serologicaltesting in intensive care unit patients with suspected influenza. Clin InfectDis 2010; 51: 70–2.

2. Noah DL, Hill H, Hines D, White EL, Wolff MC. Qualification of thehemagglutination inhibition assay in support of pandemic influenza vaccinelicensure. Clin Vaccine Immunol 2009; 16: 558–66.

3. McClelland LHR. The adsorption of influenza virus by red cells and a new invitro method of measuring antibodies for influenza virus. Can J PublicHealth 1941; 32: 530.

4. Tsiodras S, Kelesidis T, Kelesidis I, Voumbourakis K, Giamarellou H.Mycoplasma pneumoniae-associated myelitis: a comprehensive review.Eur J Neurol 2006; 13: 112–24.

5. Steininger C, Popow-Kraupp T, Laferl H, et al. Acute encephalopathyassociated with influenza A virus infection. Clin Infect Dis 2003; 36:567–74.

6. Centers for Disease Control and Prevention (CDC). Neurologiccomplications associated with novel influenza A (H1N1) virus infectionin children – Dallas, Texas, May 2009. MMWR Morb Mortal Wkly Rep 2009;58: 773–8.

right © Royal College of pathologists of Australasia

7. Ekstrand JJ, Herbener A, Rawlings J, et al. Heightened neurologic compli-cations in children with pandemic H1N1 influenza. Ann Neurol 2010; 68:762–6.

DOI: 10.1097/PAT.0b013e3283486b46

Coxiella burnetii culture-negativeendocarditis diagnosed by 16S rRNAsequencing of heart valve tissue

Sir,This report describes the clinical features of Coxiella burnetiiendocarditis and discusses the role of 16S rRNA sequencingin the diagnosis of culture-negative endocarditis. Polymerasechain reaction (PCR) is a useful method to help identifyfastidious organism in patients who have already receivedantibiotics and in whom blood cultures remain negative.Treatment options for Coxiella burnetii endocarditis are alsodiscussed.

A 54-year-old man presented with a 6 month historyof progressive dyspnoea on exertion, paroxysmal nocturnaldyspnoea, orthopnoea and 18 kg weight loss. He denied anyfever, chills, night sweats, or change in his chronic cough orsputum production. His medical history included 30 pack-yearsof smoking, but was otherwise unremarkable. His hobbiesincluded working on his farm, where he had exposure to cattle,horses, pigs, chickens, cats and dogs.

Physical examination revealed a cachectic gentlemanwho did not appear septic. His temperature was 35.58C, bloodpressure was 130/57 mmHg, pulse 85 beats per minute, andoxygen saturation 98%. A III/VI systolic ejection murmurand III/VI diastolic ejection murmur, both loudest at the leftlower sternal border at end-expiration, were auscultated. Awaterhammer pulse and Quincke’s sign were present (capillarynail bed pulsation, manifested by alternating reddeningand blanching of the nailbed with each heartbeat). A pistolshot sound was audible over the femoral artery. There were nostigmata of subacute bacterial endocarditis. Laboratory datarevealed a white blood cell count of 6.9 cells� 109/L (normalrange 3.0–10.5� 109/L), haemoglobin 127 g/L (115–155 g/L)and a troponin I of 0.18 mg/L (<0.04 mg/L). Electrocardiogramrevealed first degree block, with a PR interval of 232 milli-seconds. Chest X-ray was suggestive of congestive heartfailure. The patient was admitted to hospital and respondedwell to diuresis.

Transoesophageal echocardiogram revealed a bicuspid para-valvular aortic root abscess and pseudoaneurysm consistentwith infective endocarditis. There was 4þ aortic regurgitation.Ejection fraction was 65%. Four sets of blood cultures, drawn atdays 2, 3, 7 and 11 of admission, were all negative. Multipleblood cultures after prolonged incubation (21 days) as well assyphilis and Legionella serologies were also negative.

Serology (performed by the Public Health Laboratories,Ontario Agency for Health Protection and Promotion) forCoxiella burnetii subsequently revealed a phase II IgG of1:16 384, phase II IgM of 1:2048, phase I IgG of 1:32 768and a phase I IgM of 1:4096. Coxiella burnettii complementfixation test (CF) was positive at 1:512. Bartonella henselaeIgG immunofluorescence assay was elevated at 1:512,

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Fig. 1 Excised gross aortic valve tissue with extensive valve cusp destruction, irregular edges, and adherent grey-tan thrombus vegetation.

514 CORRESPONDENCE Pathology (2011), 43(5), August

suggestive of a likely cross-reacting antibody. At this point thepatient was started on oral doxycycline 100 mg twice daily andgentamicin 100 mg IV once daily. He underwent an aortic valvereplacement, aortic root replacement and coronaryartery bypass grafting day 18 after admission. Pathologicalexamination of valve tissue revealed degenerative changeswith nodular calcification and fibrosis with acute andchronic inflammatory cells (Fig. 1 and 2A). Warthin-Starrystain revealed coccobacilliary structures (Fig. 2B). Identifi-cation of the organism was performed by sequencing of the16S rRNA gene. DNA extracted from the aortic tissue wasamplified by PCR using universal primers targeting a 1000 bpsegment of the bacterial 16S rRNA gene as previouslydescribed.1 Amplified product was purified and sequenced.The partial 16S rRNA sequence was compared for homologyusing an online search engine (www.ncbi.nlm,nih.gov/).

right © Royal College of pathologists of Australasia

Fig. 2 (A) Haematoxylin-phloxine-saffron stained valve tissue demonstratingfibrous tissue (left) and chronic inflammation, chiefly lymphocytes, macro-phages and some plasma cells adjacent to fibrin. (B) Warthin-Starry stain of thevalve tissue demonstrating small coccobacillary structures in the tissue.

A greater than 99% match was obtained with the 16S rRNAgene sequence of C. burnetii Cbuk_Q154 (GenBank accessionCP001020.1)

After surgery the patient was switched to oral twice dailydoses of doxycycline 100 mg and hydroxychloroquine 200 mg.Throughout his admission he remained without signs ofinfection. He was discharged home with these antibiotics,the duration of which is expected to be a minimum of18 months. When seen at follow-up after 1 year, he continuedto remain well and without signs of infection.

Culture-negative infective endocarditis (IE) accounts for2.5–31% of all cases of IE.2 Coxiella burnetii is a fastidious,slow-growing organism often implicated in culture-negativeIE. While a major Duke criterion for diagnosis of IE is thepresence of bacteraemia, diagnosis is often delayed in patientswhose blood cultures remain negative. In one series, the meandelay in diagnosis of Coxiella species IE was 14 months.2

Delays in appropriate antibiotic administration may result inprogressive valve destruction, septic emboli and increasedmortality.2

Coxiella burnetii, the aetiological agent of Q-fever, isan intracellular Gram negative coccobacillus which possessesa life cycle that includes a desiccant resistant form and can existin two antigenic states: phase I, in which the organisms reactwith late convalescent guinea pig sera and are highly infectious,and phase II, whereby lack of gene expression results inavirulent organisms. Repeated passage of phase I virulentorganisms in embryonated chicken eggs leads to gradualconversion to phase II.3 Common animal reservoirs includecattle, sheep, goats and parturient cats, who shed desiccantresistant organisms in urine, faeces and birth products.Humans become infected with this pathogen by inhalation ofcontaminated aerosols or due to bites from ticks or otherarthropods. Clinical manifestations vary and may includea self-limited febrile illness lasting 2–14 days, a rapidlyprogressive atypical pneumonia syndrome, hepatitis, osteo-myelitis, meningitis and IE. IE accounts for 60–70% ofthe cases of chronic Q-fever and often occurs in the settingof pre-existing valvular disease and in immunosuppressedhosts. Patient contact with animal reservoirs is the maindiagnostic clue on history.3

Due to delays and difficulty with organism culture, serologyis the main diagnostic modality for Coxiella species. Acute Q-fever is diagnosed by a four-fold rise in antibody titre andan antibody response to phase II antigen. In contrast, chronic Q-fever is characterised by a higher response to phase I antigen,and is characterised by a complement fixation titre of >1:200or an IgG antibody titre of >1:800 by microimmuno-fluorescence.4 Serological cross-reactions are common, as seenin our case. In a study of 258 patients with Q-fever, 258 serumsamples were tested against Bartonella henselae and

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CORRESPONDENCE 515

Bartonella quintana antigens. More than 50% of chronic Q-fever patients had antibodies which reacted against Bartonellahenselae antigen to a significant level. However, as the levels ofspecific antibody in true cases of Bartonellosis are typicallyvery high (titres >1:1600), low-level cross-reactions betweenCoxiella burnetii antibodies and Bartonella hensenlae antigenin cases of Q-fever IE should not lead to misdiagnosis,providing serology testing is done for both agents.3 It is alsonoteworthy that, since the serological assay used between labsmay not be identical, titres may vary between laboratories.Furthermore, the titre used to define chronic Q-fever may alsodiffer between laboratories.

Given that bacterial DNA may persist during treatment ininfected valves for long periods, polymerase chainreaction (PCR) can be useful in identifying the causativeorganism.5 The PCR method is especially useful when thecausative organism is fastidious or when the specimen istaken during antimicrobial treatment.5 Kotilainen et al.examined heart valve tissue from 56 patients with broad-range PCR. Identification was performed by partial DNAsequencing of the 16S and 23S rDNA genes. This madepossible the final diagnosis of definite IE in 36 patients andpossible IE in two patients, while the diagnosis was rejected in18 patients. In four patients, all of whom received antibiotics,the PCR approach was the only method to yield the aetiologicaldiagnosis. The causative agent of IE was recognisable by PCRfrom resected valve tissue for up to 58 days after commence-ment of intravenous antibiotics, in contrast to bacterial culturesof valve tissue which became negative within only a few days.5

In another study of 177 heart valve samples, sensitivity,specificity, negative and positive predictive values for thisreal-time PCR method were 96%, 95.3%, 98.4% and 88.5%,respectively, when compared to results of blood culturestaken upon admission or prior to surgery. When organismsisolated by culture from resected valves were comparedwith blood, sensitivity, specificity, negative and positivepredictive values for PCR were 24.3%, 56.4%, 73.8% and12.8%, respectively.6

PCR would be best utilised as a supplement to blood andheart valve culture.6 It has been suggested that results ofthis technique be included as a new major Duke criterion forthe diagnosis of IE.5 Currently, PCR is predominantlyperformed on heart valve tissue of patients undergoing surgery,but theoretically it could also be performed on peripheralblood samples, including blood from patients who do notrequire surgery.6 Limitations of PCR negative results at thetime of surgery do not indicate that the patient has respondedwell to antimicrobials, and they cannot be used to monitortreatment response. PCR is also prone to false positives as PCRreagents may contain bacterial DNA, reducing its positivepredictive value.6

Treatment of Coxiella IE consists of antimicrobials andsurgery. A minimum of two or more drugs active againstCoxiella burnetii should be used for IE. Oral ciprofloxacin750 mg twice daily plus oral rifampin 300 mg once daily ororal hydroxychloroquine 600 mg once daily are suggestedregimens for IE.3,4 Twice daily doses of oral doxycyline100 mg and oral ciprofloxacin 750 mg is an alternative regi-men.4 In the present case, hydroxychloroquine and doxycylinewere chosen based on increasing evidence suggesting thatthis regimen should be first line treatment for Coxiella IE.4

All patients require long-term follow-up over several years dueto the possibility of late relapse. Antibody titres to phase I and II

right © Royal College of pathologists of Australasia

antigens should be measured with microimmunofluorescenceevery 3 months to ensure decline. When the antiphase I IgAis <1:200, therapy may be discontinued.4 Although mortalityfrom Q-fever was high in the 1970s, mortality has decreasedfrom 60% to 5% in more recent years,2 due to better diagnostictechniques.

In summary, Coxiella species are common causes of culture-negative IE. PCR is a useful method to help identify fastidiousorganisms and in patients who have already received antibioticsand whose blood cultures remain negative. It is also a substitutefor conventional heart valve cultures. A positive universalor specific PCR result for organism in heart valve tissuemay eventually be included as a major Duke criterion forIE. With appropriate and timely therapy, mortality from thesezoonotic illnesses is low.

Cecilia T. Costiniuk*Bing Wang{Daniel JohnstonezPeter Jessamine*{Baldwin Toye*{John P. Veinot{Marc Desjardins{

*Department of Medicine, Division of Infectious Diseases,{Department of Pathology and Laboratory Medicine,University of Ottawa, and zOttawa Hospital ResearchInstitute, University of Ottawa, Ottawa, ON, Canada

Contact Dr C. T. Costiniuk.E-mail: ccostiniuk@ottawahospital.on.ca

1. Chakrabarti P, Das BK, Kapil A. Application of 16S rDNA based seminestedPCR for diagnosis of acute bacterial meningitis. Indian J Med Res 2009; 129:182–8.

2. Houpikian P, Raoult D. Blood culture-negative endocarditis in a referencecenter: Etiologic diagnosis of 348 cases. Medicine 2005; 84: 162–73.

3. La Scola B, Raoult D. Serological cross-reactions between Bartonellaquintana, Bartonella henselae, and Coxiella burnetii. J Clin Microbiol1996; 34: 2270–4.

4. Marrie TJ. Coxiella burnetii (Q fever). In: Yu VL, Weber R, Raoult D,editors. Antimicrobial Therapy and Vaccines. Volume I: Microbes. 2nd ed.New York: NY Apple Trees Production, updated November 2007. http://www.antimicrobe.org/r08.asp.

5. Kotilainen P, Heiro M, Jalava J, et al. Aetiological diagnosis of infectiveendocarditis by direct amplification of rRNA genes from surgically removedvalve tissue: An 11-year experience in a Finnish teaching hospital. Ann Med2006; 38: 263–73.

6. Marin M, Munoz P, Sanchez M, et al. Molecular diagnosis of infectiveendocarditis by real-time broad-range polymerase chain reaction (PCR)and sequencing directly from heart valve tissue. Medicine 2007; 86:195–202.

DOI: 10.1097/PAT.0b013e3283486b77

Reversible bone marrow necrosis afterall-trans retinoic acid induction therapy foracute promyelocytic leukaemia

Sir,Bone marrow necrosis (BMN) is a unique clinicopathologicalentity distinct from avascular bone necrosis and bone marrow

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Fig. 1 Bone marrow disrruption of normal architecture and substitution ofhaematopoietic tissue and fat spaces by an extense necrotic material (PAS).

Fig. 2 Unstructured purple material and altered, shadowy cells with indistinctoutlines (PAS).

516 CORRESPONDENCE Pathology (2011), 43(5), August

aplasia. The histological features of BMN are disruption of thenormal marrow architecture, necrosis of myeloid tissue andmedullary stroma, and loss of fat spaces. Unlike avascularnecrosis, in BMN the spicular architecture is preserved, andunlike aplastic anaemia, the reticular structure is destroyed.BMN seems to be an extremely rare disease, at least in livingpatients, but in autopsy reports it is encountered more com-monly.1

Almost all the patients reported in the literature had anunderlying disease associated with BMN. Malignancy is themost common underlying disease and it is seen in 90% ofthe cases. Haematological malignancies are seen in 60% ofthe cases and acute leukaemia is the leading cause.1,2 Othermalignant causes of BMN are lymphomas, chronic myelopro-liferative disorders, and solid tumours. Main non-malignantcauses of BMN are infection, drugs, sickle cell disease, anddiffuse intravascular coagulation.2

We report the development of bone marrow necrosisfollowing induction treatment with all-trans retinoic acid(ATRA) in a patient with acute promyelocytic leukaemia(APL).

A 25-year-old man was admitted with cutaneousbleeding and haematuria, in the absence of significant medicalhistory. Haemogram at this moment revealed: leukocytes4.9� 109/L (atypical promyelocytes 8%), haemoglobin109 g/L and platelets 31� 109/L. Bone marrow aspirateconfirmed the clinical suspect of APL, including 93% ofatypical promyelocytes, the presence of t(15;17) on karyotype,and the existence of PML/RARa rearrangement (21%)on fluorescence in situ hybridisation (FISH). Coagulationtesting showed incoagulable prothrombin and activatedpartial thromboplastin times, fibrinogen 136 mg/dL, andD-dimer 1276 ng/mL. After correction of the haemostaticalterations with fresh frozen plasma transfusion, and theadministration of fibrinogen concentrate and antifibrinolytics,standard induction chemotherapy with ATRA (45 mg/m2/day), plus idarubicin (12 mg/m2 days 2, 4, 6 and 8) wasstarted.

On day þ1 of chemotherapy the patient presented a haema-toma of the psoas muscle which required percutaneousembolisation by angiotherapeutic techniques. On day þ13,after administration of parenteral piperacilin-tazobactam fora febrile episode, and concomitantly with a picture offever and bloody diarrhoea, a pseudomembranous cholitiswas diagnosed. Clostridium difficile infection was succesfullytreated with oral vancomycin. On day þ30 the haemogramshowed that WBC count and haemoglobin level were unex-pectedly low (leukocytes 1.1� 109/L, neutrophils 0.6� 109/L,haemoglobin 84 g/L), and a bone marrow examination wasindicated. By marrow aspiration, a watery dark brown fluid wasobtained and microscopy showed an amorphous extracellulareosinophilic proteinaceous material surrounding necrotic cells.A bone marrow biopsy was performed that showed disruptionof normal architecture with an eosinophilic silhouette ofbone marrow and loss of fat spaces, necrosis of myeloid tissueand medullary stroma, affecting more than 50% of the bonecylinder (Fig. 1 and 2). Some areas of haematopoietic preservedcells were located, forming islets, mainly of erythroid appear-ance (Fig. 3). A diagnosis of extensive bone marrow necrosiswas established.

We believed that the most likely cause for this marrownecrosis was the induction treatment with ATRA. Amongthe remaining drugs that were administered to our patient,

right © Royal College of pathologists of Australasia

no other is recognised to be associated with this complication.ATRA was then suspended and blood count returned tonormal gradually. After 1 month, blood counts were as follows:leukocytes 6.2� 109/L (neutrophils 3.6� 109/L), haemoglobin117 g/L, and platelets 285� 109/L. Seven weeks after thesuspension of ATRA, the haemogram was completely normaland a bone marrow aspiration showed signs of haematopoieticregeneration and absence of PML/RARa rearrangement byFISH.

Consolidation chemotherapy scheme with ATRA andidarubicin (three cycles) was administered and then mainten-ance treatment with ATRA, 6-mercaptopurine and methro-texate was continued for 2 years. No significant adverseevents occurred and currently, more than 2 years after thebone marrow necrosis episode, the patient continues in com-plete remission.

APL is a disease occasionally associated with BMN.Although BMN may develop before the diagnosis of APL isestablished,3 or at relapse,4 most of the reported cases occurredafter induction therapy with ATRA5,6 or with arsenic trioxide,especially when it is given at high doses.7,8

Introduction of ATRA has been the major breakthroughin the treatment of APL and it has dramatically improvedthe prognosis of this disease. It is recognised as themolecular targeted therapy directed at the chimeric protein(PML-RARa) generated by the specific chromosomaltranslocation in this disease [t(15;17)]. ATRA induces

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Fig. 3 Inmunohistochemical stain showing scarce residual areas of haemato-poietic preserved cells (CD43).

degradation of PML-RARa and activates normal RARa andPML, resulting in differentiation of APL cells. However,several adverse effects have been reported to occur afterclinical application of ATRA, especially ATRA syndrome.9

This syndrome is associated with the massive increaseof differentiated neutrophils that secrete inflammatory cyto-kines.

The mechanism of BMN is poorly understood, but failure ofthe microcirculation must be considered as the critical event.1,2

In ATRA associated cases it is believed that massivedifferentiation of leukaemic cells produces a dramatic increasein the oxygen demand, which cannot be supplied by the marrowcirculation. This situation leads to a hypoxaemic state thatfinally induces the necrosis. Probably, the higher the percentageof marrow leukaemic cells, the greater the possibility of thisadverse effect. Concomitant cytotoxic treatment is usuallyadministered to APL patients to avoid ATRA syndrome andprobably this fact makes BMN less frequent in this disease.BMN has been reported in APL patients treated with arsenictrioxide where the antileukaemic mechanism seems alsoto be the stimulation to cell differentiation, supporting thishypothesis.

When the microvascular failure can be stopped, therepair reaction starts and repopulation of the necrotic bonemarrow with normal haematopoietic tissue is the rule,1,2 asoccurred in our patient. We stopped treatment with ATRA, andafter 7 weeks bone marrow was recovered achieving completeremission. We could then continue treatment including ATRAwithout any other complication.

Our patient presented with a very high percentage ofatypical promyelocytes in the bone marrow (93%) at diagnosis.Perhaps if we had started ATRA treatment at reduced doses(as when WBC count is >10� 109/L for avoiding ATRAsyndrome) the risk of developing BMN would have beenmuch lower.

S. Lakhwani*J. M. Raya*G. Gonzalez-Brito*H. Alvarez-Arguelles{M. L. Brito*L. Hernandez-Nieto*

Departments of *Hematology, and {Pathology, HospitalUniversitario de Canarias, La Laguna, Spain

right © Royal College of pathologists of Australasia

Contact Dr S. Lakhwani.E-mail: sunillakhwani@hotmail.com

1. Janssens AM, Offner FC, Van Hove WZ. Bone marrow necrosis. Cancer2000; 88: 1769–80.

2. Paydas S, Ergin M, Baslamisli F, et al. Bone marrow necrosis: Clinico-pathologic analysis of 20 cases and review of the literature. Am J Hematol2002; 70: 300–5.

3. Wisecarver J, Harrington D. Bone marrow necrosis obscuring thediagnosis of acute promyelocytic leukemia. Hematol Pathol 1988; 2: 51–4.

4. Chim CS, Lam CC, Wong KF, Man C, Kam S, Kwong YL. Atypical blastsand bone marrow necrosis associated with near-triploid relapse of acutepromyelocytic leukemia after arsenic trioxide treatment. Hum Pathol 2002;33: 849–51.

5. Limentani SA, Pretell JO, Potter D, et al. Bone marrow necrosis in twopatients with acute promyelocytic leukemia during treatment with all-transretinoic acid. Am J Hematol 1994; 47: 50–5.

6. Cull GM, Eikelboom JW, Cannell PK. Exacerbation of coagulopathy withconcurrent bone marrow necrosis, hepatic and renal dysfunction secondaryto all-trans retinoic acid therapy for acute promyelocytic leukemia. HematolOncol 1997; 15: 13–7.

7. Ishitsuka K, Shirahashi A, Iwao Y, et al. Bone marrow necrosis in a patientwith acute promyelocytic leukemia during re-induction therapy with arsenictrioxide. Eur J Haematol 2004; 72: 280–4.

8. Zhou J, Meng R, Sui XH, Yang BF. Repeated arsenic trioxide intravenousinfusion causes focal bone marrow necrosis in two acute promyelocyticleukemia patients. Chin Med Sci J 2004; 19: 281.

9. Hatake K, Uwai M, Ohtsuki T, et al. Rare but important adverse effects of all-trans retinoic acid in acute promyelocytic leukemia and their management.Int J Hematol 1997; 66: 13–9.

DOI: 10.1097/PAT.0b013e3283489087

Extramedullary megakaryoblastic leukaemiawith massive generalised infiltration

Sir,Acute megakaryoblastic leukaemia is a subtype of acutemyeloid leukaemia (AML) that is classified as M7-AML inthe French-American-British (FAB) classification1 and in thecategory of AML not otherwise specified (NOS) in the WorldHealth Organization (WHO) classification.2 According toboth classification systems, the diagnosis requires 20% blasts,of which 50% are of megakaryocytic lineage. Positive immuno-staining for at least one of the platelet-derived antigens such asfactor VIII, glycoproteins (GP) IIb/IIIa (CD41), IIIa (CD61), Ib(CD42), IIIb (CD36) and/or CD31 is required.3 Here wepresent an autopsy case of massive organ infiltration of acutemegakaryoblastic leukaemia to anatomical sites that have notpreviously been reported.

The case is that of a 74-year-old woman with a historyof polycythaemia vera (PV) diagnosed in 1989, treated with6-mercaptopurine, hydroxyurea and interferon. In 2004 shepresented with fatigue, dyspnoea and purpura. Examination ofher blood revealed haemoglobin of 8.6 g/dL, white blood cell(WBC) count of 17� 109/L, platelet count of 7000� 109/L,lactate dehydrogenase (LDH) of 4670 U/L (normal range 100–238 U/L) and 25% leukaemic blasts in peripheral blood. Herbone marrow aspirate showed 40% blasts by visual inspection.Immunohistochemistry showed the blast cells to be positivefor CD34 (a marker of early haemopoietic progenitor cells)and negative for myeloperoxidase (MPO). A predominantpopulation of the leukaemic blasts (>50%) showed morpho-logical evidence of megakaryoblastic differentiation with

CORRESPONDENCE 517

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Fig. 1 The bone marrow biopsy showed highly pleomorphic giant cells withmorphology compatible with megakaryoblasts.

518 CORRESPONDENCE Pathology (2011), 43(5), August

large indented nuclei and basophilic cytoplasm (Fig. 1 and 2A).These cells showed positive cytoplasmic staining for CD61 andCD31, compatible with megakaryocytic lineage. Therewas also a significant population of maturing dysplastic mega-karyocytes with non-lobated and hyper-lobatednuclei. Reticulin staining showed myelofibrosis grade 4(Bauermeister’s classification)4 (Fig. 2B). The diagnosis waspolycythaemia vera with transformation to acute megakaryo-blastic leukaemia (M7 AML).

The patient continued to deteriorate and died after 10 daysof hospitalisation due to respiratory failure. An autopsy wasperformed (Fig. 2C,D and Fig. 3). On macroscopic examinationthere was mild to moderate enlargement and an irregularmicronodular appearance of lungs, heart, liver, spleen andkidneys. Generalised lymphadenopathy was evident. Histologyshowed massive involvement of the gastrointestinal tract(stomach, small and large intestine and appendix), liver, spleen,kidneys, adrenal glands, omentum, lungs, heart, thyroid

right © Royal College of pathologists of Australasia

Fig. 2 (A) The bone marrow biopsy (H&E) was completely replaced by highly pleomadvanced myelofibrosis in the bone marrow. (C) Immunohistochemcal expression for C

and multiple lymph nodes by a predominant population ofdiscohesive pleomorphic giant cells, with multi-lobated nuclei(Fig. 3). These cells were admixed with medium size cellswith 1–2 prominent nuclei of vesicular appearance andprominent nucleoli, compatible with blasts. Multiple dysplasticmegakaryocytes were also identified. As for the bonemarrow, these cells were positive for CD34, CD61 andCD31 (Fig. 2C,D). Examination of the brain was notperformed. The diagnosis was acute megakaryoblastic leukae-mia (M7-AML) with extensive extramedullary involvement.Cytogenetic studies were not performed. Consequently,we were unable to confirm somatic mutations typical ofpolycythaemia vera, such as JAK2 V617F2 and the possibilityof clonal transformation to M7-AML.

Polycythaemia vera (PV) is a myeloproliferative neoplasm(MPN) that undergoes transformation to myelodysplasticsyndrome or acute myeloid leukaemia in up to 20% of thecases. In untreated patients, the transformation risk is only 2–3%, but long-term treatment, specifically with alkylatingagents, increases the incidence of clonal transformation.5

Progression from long-standing PV to M7-AML has beenpreviously described.5–7 In two different cases, megakaryo-blastic transformation from PV was accompanied by develop-ment of hypercalcaemia6 and disseminated intravascularcoagulation.7 Although somatic mutations might be present,no genetic abnormalities are yet specific for PV.

Acute megakaryoblastic leukaemia is a rare sub-typeof AML that comprises less than 5% of all AML cases andis frequently associated with antecedent haematologicaldisorders such as myelodysplasia or MPN.8 According to theMD Anderson Cancer Center’s series, adult patients with M7-AML had antecedent haematological disorder, including MPNin 60% as opposed to 39% of non-M7-AML patients.8

Regarding the WHO classification of AML, leukaemiais only assigned to the NOS group if it does not meet thecriteria for other AML categories. In our case, the prior history

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orphic cells compatible with myeloid blasts. (B) The reticulin staining showedD61 and (D) CD31 in liver, compatible with megakaryocytic-derived lineage.

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Fig. 3 Massive infiltration of M7-MLA to (A) stomach, (B) colon and (C) kidney. (A) Blast cells and dysplastic maturing megakaryocytes with the abundanteosinophilic cytoplasm.

CORRESPONDENCE 519

of cytotoxic therapy precludes the diagnosis of AML withmyelodysplasia-related change.2 Our case is also excludedfrom the category of therapy-related AML, due to transform-ation from MPN. As cytogenetic studies were not available, wecannot entirely exclude the category of AML with recurrentgenetic abnormalities. Specific entities in this group includemegakaryoblastic leukaemia with t(1;22)(p13;q13);RBM15-MKL1.2

Other differential diagnoses include acute panmyelosiswith myelofibrosis.9 Unlike megakaryoblastic leukaemia, acutepanmyelosis shows trilineage differentiation with a prolifer-ation of granulocyte, megakaryocyte and erythroid precursors.

The overall survival for this subtype of acute myeloidleukaemia is poor, with a median survival of only 4–10 months.Treatment is based on administration of cytarabine andanthracycline or non-anthracycline based therapy. Bonemarrow transplantation, interferon, apopotosis inducingagents4 and stem cell transplantation have proven to increasesurvival.10

Extramedullary disease for M7-AML mainly involvesskin and central nervous system (CNS).3 In an Italian cohort(GIMEMA trials) of 3603 patients with AML, megakaryoblas-tic leukaemia comprised only 0.6% (n¼ 24) of all cases andonly 16% of these patients presented with extramedullaryinvolvement of brain and skin.3 In the Eastern CooperativeOncology Group (ECOG) trials, 1.1% of AML patients devel-oped M7-AML in 12-year period of time, but only two patientspresented with extramedullary involvement (spleen, liver andskin).4 Mediastinal involvement has also been described, withsites such as kidney only observed in children.11

CNS involvement can manifest as a spinal cord mass withcompression and paraplegia.12,13 In the case reported by Byrantet al., the autopsy revealed massive infiltration of M7-AMLto multiple organs: spleen, liver, lungs, mesentery, heart anduterine endometrium.12 Our case is unique in that infiltrationto the gastrointestinal tract (stomach, small and large intestineand appendix), kidneys, adrenal glands, and thyroid has notbeen previously reported.

Ana Cristina Vargas*Blake O’Brien{Maria Luisa MarquezzCarlos Ortiz-Hidalgo§Sunil R. Lakhani*{

right © Royal College of pathologists of Australasia

*The University of Queensland Centre for Clinical Research,{Pathology Queensland: The Royal Brisbane and Women’sHospital, Brisbane, Queensland, Australia; zCMNSXXI, IMSS,Mexico City, and §American British Cowdray Medical Center,Mexico City, Mexico

Contact Dr A. C. Vargas.E-mail: anacristina@uq.edu.au

1. Bennett JM, Catovsky D, Daniel MT, et al. Proposed revised criteriafor the classification of acute myeloid leukemia. A report of theFrench-American-British Cooperative Group. Ann Intern Med 1985;103: 620–5.

2. Swerdlow SH, Campo E, Harris NL. WHO Classification of Tumours ofHaematopoietic and Lymphoid Tissues. 4th ed. Lyon: IARC, 2008.

3. Pagano L, Pulsoni A, Vignetti M, et al. Acute megakaryoblastic leukemia:experience of GIMEMA trials. Leukemia 2002; 16: 1622–6.

4. Tallman MS, Neuberg D, Bennett JM, et al. Acute megakaryocyticleukemia: the Eastern Cooperative Oncology Group experience. Blood2000; 96: 2405–11.

5. Kurosawa M, Iwasaki H. Megakaryoblastic transformation of poly-cythemia vera with hypercalcemia. Ann Hematol 2002; 81: 668–71.

6. Stamatopoulos K, Yataganas X, Papadaki T, Paterakis G. Unusuallyprolonged survival of a case of acute megakaryoblastic leukemiasecondary to long-standing polycythemia vera. Leuk Res 2002; 26: 699–700.

7. Hino K, Sato S, Sakashita A, Tomoyasu S, Tsuruoka N, Koike T.Megakaryoblastic transformation associated with disseminated intravascu-lar coagulation in the course of polycythemia vera: a case report. RinshoKetsueki 1992; 33: 500–6.

8. Oki Y, Kantarjian HM, Zhou X, et al. Adult acute megakaryocyticleukemia: an analysis of 37 patients treated at MD Anderson CancerCenter. Blood 2006; 107: 880–4.

9. Orazi A, O’Malley DP, Jiang J, et al. Acute panmyelosis with myelofi-brosis: an entity distinct from acute megakaryoblastic leukemia. ModPathol 2005; 18: 603–14.

10. Garderet L, Labopin M, Gorin NC, et al. Hematopoietic stem cell trans-plantation for de novo acute megakaryocytic leukemia in first completeremission: a retrospective study of the European Group for Blood andMarrow Transplantation (EBMT). Blood 2005; 105: 405–9.

11. Dastugue N, Lafage-Pochitaloff M, Pages MP, et al. Cytogenetic profileof childhood and adult megakaryoblastic leukemia (M7): a study of theGroupe Francais de Cytogenetique Hematologique (GFCH). Blood 2002;100: 618–26.

12. Bryant BJ, Alperin JB, Elghetany MT. Paraplegia as the presentingmanifestation of extramedullary megakaryoblastic transformation of pre-viously undiagnosed chronic myelogenous leukemia. Am J Hematol 2007;82: 150–4.

13. Brahmanday GR, Gheorghe G, Jaiyesimi IA, et al. Primary mediastinalgerm cell tumor evolving into an extramedullary acute megakaryoblasticleukemia causing cord compression. J Clin Oncol 2008; 26: 4686–8.

DOI: 10.1097/PAT.0b013e32834864ba

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520 CORRESPONDENCE Pathology (2011), 43(5), August

Reversible bone marrow reticulin fibrosis as aside effect of romiplostim therapy for thetreatment of chronic refractoryimmune thrombocytopenia

Sir,Idiopathic thrombocytopenic purpura (ITP), also known asprimary immune thrombocytopenia is an acquired immunedisorder characterised by isolated thrombocytopenia (bloodplatelet count less than 100� 109/L), and the absence of anyobvious initiating/underlying cause of the thrombocytopenia.1

Initial treatment options include corticosteroids, intravenousimmunoglobulin (IVIG) and splenectomy, but up to 10% ofpatients are refractory to first-line therapies.2 Thrombopoiesisstimulating agents are a new class of drugs which have recentlybecome available for the management of this subgroup ofpatients. One such agent is romiplostim, a peptide body thatmimics endogenous thrombopoietin (TPO) by binding andactivating the human TPO receptor, resulting in increasedplatelet production. Whilst demonstrating good clinicalefficacy, the side effect of bone marrow fibrosis has beenobserved in a number of cases. Herein, we report a case whichpresented with increased bone marrow reticulin associated withromiplostim use, which was reversible upon drug withdrawal.

In 2008, a 69-year-old man was incidentally found to havethrombocytopenia during pre-operative work up (platelet count28� 109/L). A series of investigations did not identify anyunderlying cause for his thrombocytopenia. A bone marrowbiopsy was also performed, which did not show any dysplasticfeatures nor increased reticulin (WHO Grade 0) (Fig. 1A,B).The diagnosis of ITP was made. Over the next 9 months, he wastreated with steroids, IVIG and splenectomy, all of which failedto yield a sustained improvement of his platelet count. He waseventually commenced on romiplostim in late 2009 (Fig. 2).Due to ongoing haematuria, his romiplostim dose was titratedslowly up to the maximum recommended dose of 10 mg/kgto optimise platelet response. His platelet count peaked at63� 109/L at Week 16 (Fig. 2). However, it was also notedat this time that his surveillance full blood examination showedslowly worsening anaemia and a neutrophilia, with occasionalintermediate myeloid cells on blood film. A loss of plateletresponse followed despite the patient being on the maximumrecommended dose. A bone marrow biopsy was performed atweek 22 of therapy (Fig. 3A,B). This showed a hypercellularmarrow and plenty of megakaryocytes with occasionalclustering. Some megakaryocytes demonstrated unusual

right © Royal College of pathologists of Australasia

Fig. 1 Pre-treatment bone marrow trephine. (A) Normal cellularity with trilineage haemin uptake.

features such as hyper-lobation. In particular, reticulin uptakewas moderately increased with no collagen fibrosis detected(WHO Grade 1–2). Cytogenetics testing was not performeddue to insufficient sample obtained. Due to clinical necessity,the patient’s romiplostim was continued under close monitor-ing. By week 41 (Fig. 2), his thrombocytopenia worsened, eventhough his anaemia remained clinically insignificant. Further-more, his blood film showed mild tear drop poikilocytosis anda significantly increased number of nucleated red cells thatraised suspicion of transformation into myelofibrosis. Thepatient remained clinically asymptomatic without any hepato-megaly noted on examination. A repeat bone marrow biopsyperformed at week 43 of therapy (Fig. 3C,D) showed a mark-edly hypercellular marrow with myeloid hyperplasia and leftshifted granulopoiesis. Megakaryocytes were greatly increasedin numbers, with abnormal clustering into large groups. Theyshowed bizarre features such as emperipoleisis, hyperlobularand hyperchromatic nuclei, as well as irregular cytoplasmicedges. There was expansion in paratrabecular promyelocytepopulation but no increase in blast numbers and no associatedosteosclerosis. Recticulin uptake was markedly increased(WHO Grade 2) with no collagen fibrosis noted. Cytogeneticstesting was again unable to be performed due to insufficientsample obtained.

The patient’s romiplostim was subsequently ceased at week43. A recovery of haemoglobin and resolution of his neutro-philia was observed over the next 6 weeks. This, unfortunately,was accompanied by a relapse of his immune thrombocytope-nia (week 49, Fig. 2). A follow-up bone marrow biopsy wasperformed at week 53 (Fig. 4A,B). This showed notableimprovement both in terms of cellularity and megakaryocytemorphology, with reduced reticulin (WHO Grade1), althoughnot completely back to baseline. Due to ongoing severethrombocytopenia (<10� 109/L) and bleeding despite inter-mittent IVIG, romiplostim was recommenced at week 56 at amuch lower dose (3 mg/kg), with monthly monitoring of fullblood examination (FBE) indices and film. At a recent follow-up (week 60), the patient’s platelet count was 40� 109/L, withFBE showing a recurrence of mild neutrophilia but nosignificant anaemia or abnormal morphological features onblood film.

Reticulin is a normal component of the bone marrow and isdetectable by silver staining methods in 71–81% of healthysubjects.3 Increased bone marrow reticulin is associated with anumber of benign and malignant conditions (connective tissuedisorders, granulomatous disease, lymphoma) and is frequentlyreversible. On the contrary, collagen fibrosis, detectableby trichrome staining, is characteristically irreversible and is

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opoiesis and no dysplastic features (H&E). (B) Reticulin stain shows no increase

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associated with definite pathology such as myeloproliferativedisease or metastatic solid tumours. Although the patho-physiology of bone marrow fibrosis remains incompletelyunderstood, evidence suggests megakaryocytes play a centralrole. When stimulated, megakaryocytes activate growth factorsand cytokines which in turn promote synthesis of collagen bybone marrow fibroblasts.4 Bone marrow fibrosis (reticulin andcollagen) can be quantified using several different gradingschemes. In the modified Bauermeister scheme, the degreeof reticulin/collagen deposition is graded on a scale from 0 to 4.In a retrospective study looking at bone marrow of 40 ITPpatients not on thrombopoiesis stimulating agents using the

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Fig. 3 Bone marrow trephine during treatment. (A,B) Week 22. Hypercellular and mostain shows diffuse reticulin uptake with no collagen fibrosis. (C,D) Week 43. Markedmegakaryocytes (H&E). Reticulin stain shows dense reticulin uptake with coarse fibr

above scheme, 38% were found to have Grade 0 (no reticulinfibres); 50% were Grade 1 (occasional fine reticulin fibres);13% were Grade 2 (fine fibres throughout section); none wereGrade 3 (diffuse fibre network with scattered coarse fibres) orGrade 4 [as Grade 3 with areas of collagenisation (positivetrichrome stain)].5 In 2008, a simplified grading of myelofi-brosis was recommended by the fourth edition World HealthOrganisation Classification of Tumours of Haemopoietic andLymphoid Tissues. This semi-quantitative grading scheme isbased on the European consensus established in 2005 from anexpert panel, and consists of only four grades as opposed to fivein the modified Bauermeister scheme. Grade 0 is defined as

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derately increased number of megakaryocytes with clustering (H&E). Reticulinly hypercellular marrow with extensive clustering of abnormally hyperlobated

es. Collagen fibrosis is absent.

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Fig. 4 Bone marrow trephine following treatment cessation, week 53. (A) Normal number of megakaryocytes with no clustering or abnormal morphological features(H&E). (B) Reticulin stain shows patchy network of fine reticulin with only occasional intersections.

522 CORRESPONDENCE Pathology (2011), 43(5), August

scattered linear reticulin with no intersections (normal bonemarrow); Grade 1 fibrosis contains a loose network of reticulinwith many intersections, especially in perivascular areas; Grade2 fibrosis demonstrates diffuse and dense increase in reticulinwith extensive intersections, occasionally with focal bundles ofcollagen and/or focal ostersclerosis; Grade 3 fibrosis denotesdiffuse and dense increase in reticulin with extensive intersec-tions and coarse bundles of collagen, often associated withosteosclerosis.6,7 WHO grading was used to assess the degreeof marrow fibrosis in our case.

Bone marrow reticulin fibrosis is a known side effect ofromiplostim, with cases reported both during preclinical studiesand clinical trials. In a single arm, long-term safety and efficacystudy (n¼ 142), various degrees of increased bone marrowreticulin were observed in eight patients, which suggested anincidence of 5%. However its true incidence is unknown as pre-and post-treatment bone marrow biopsies were not routinelyperformed as part of the study protocol.8 Reticulin fibrosis isassociated with prior splenectomy, multiple prior ITP therapies,high romiplostim dose (greater than 5 mg/kg) and poor plateletresponse, all hallmarks of severe ITP. The patients showedno significant cytopenias on peripheral blood and nosplenomegaly (in those without splenectomy). There has beenno report of myeloid neoplasm to date. Follow-up serial bonemarrow examinations suggest that any increase in bone marrowreticulin during romiplostim treatment is probably reversible ondiscontinuation of treatment. It was observed in the studiesthat there was a two grade drop in reticulin where bone marrowbiopsies following treatment cessation were available foranalysis (n¼ 4). In particular, collagen deposition wasobserved in one case during therapy, but was not seen at thefollow-up marrow following discontinuation of treatment. Animprovement was observed between 8 and 12 weeks follwingromiplostim withdrawal.9 Nevertheless, it is difficult to drawdefinite conclusions regarding the natural history of romiplos-tim-induced bone marrow fibrosis from these data, owingto limited follow-up time and lack of post-treatment biopsyspecimens.

In the setting of using a relatively new drug with limited post-marketing data, our case supports the finding that some patientson romiplostim for treatment of chronic refractory ITPmay develop bone marrow fibrosis within a year of initiatingtherapy. Moreover, with serial bone marrow biopsiesperformed at key time-points pre- and post-treatment,we demonstrate that the fibrosis is at least partially reversibleon treatment cessation. Therefore, it is prudent that a baseline

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reticulin assessment as well as a uniform grading system beemployed when evaluating bone marrow specimens of ITPpatients on any thrombopoiesis stimulating agents. Wherepossible, it is recommended that the same pathologist shouldreview all of the subsequent bone marrow biopsies with referenceto prior specimens. Finally, all patients on romiplostim shouldhave a full blood examination and blood film at least monthly.There should be a low threshold to perform a bone marrow biopsyif there is any unexplained anaemia or neutrophilia, or if filmshows abnormal morphological features such as red cell poiki-locytosis, nucleated red cells or intermediate myeloid cells. Aloss of platelet response despite increasing romiplostim dose mayalso herald the presence of bone marrow fibrosis.

Teresa Leung*Julie Lokan{Paul Turner*Carole Smith*

Departments of *Haematology, and {Anatomical Pathology,Austin Health, Heidelberg, Victoria, Australia

Contact Dr T. Leung.E-mail: terezaleung@yahoo.com.au

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2. George JN. Management of patients with refractory immune thrombocyto-penic purpura. J Thromb Haemost 2006; 4: 1664–72.

3. Cuker A. Toxicities of the thrombopoietic growth factors. Semin Haematol2010; 47: 289–98.

4. Kimura A, Katoh O, Hyodo H, Kuramoto A. Transforming growth factor-beta regulates growth as well as collagen and fibronectin synthesis of humanmarrow fibroblasts. Br J Haematol 1989; 72: 486–91.

5. Mufti G, Hassaerjian R, Bain B, Kuter D, Dreiling L, Nichol J. Bone marrowreticulin in patient with immune thrombocytopenic pupura. Blood 2006; 108:Abstr 3982.

6. Swerdlow SH, Campo E, Harris NL, et al., editors. WHO Classification ofTumours of Haematopoietic and Lymphoid Tissues. 4th ed. Lyon: IARC,2008; 45–6.

7. Thiele J, Kvasnicka HM, Facchetti F, Franco V, van der Walt J, Orazi A.European consensus on grading bone marrow fibrosis and assessment ofcellularity. Haematologica 2005; 90: 1128–32.

8. Bussel JB, Kuter DJ, Pullarkat V, Lyons RM, Guo M, Nichol JL. Safety andefficacy of long-term treatment with romiplostim in thrombocytopenicpatients with chronic ITP. Blood 2009; 113: 2161–71.

9. Kuter DJ, Mufti GJ, Bain BJ, Hasserjian RP, Davis W, Rutstein M.Evaluation of bone marrow reticulin formation in chronic immune thrombo-cytopenia patients treated with romiplostim. Blood 2009; 114: 3748–56.

DOI: 10.1097/PAT.0b013e328348fecc

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