Amyloidosis of the AL type

Amyloidosis of the AL Type

Clinical, Morphologic and Biochemical Aspects of the

Response to Therapy with Alkylating Agents and Prednisone

]OEL N. BLJXBAI JM. M.D. MARGARET E. HCJRLEY. M.D.

JOSEPfI (:HIJBA, Ph.D.

‘I’HE,(~DORE SPIRO, M.D.*

Nt:rv York, New York

From the Medical Service, Manhattan Veterans

Administration Hospital, and the Departments of Medicine and Pathology, New York Univer- sity Medical Center, New York, New York. ‘l’hcsc studies were partially supported by the Medical Research Service of the Veterans Ad- ministration. Requests for reprints should be iitltlressed to Dr. Joel Buxbaum, Research Service 15lC. Manhattan Veterans Administration 1 Iospital, First Avenue at 24th Street, New York, New York 10010. Manuscript accepted May 2, I 979.

* Pwscnt address: Service d’Hematologie, Institut Jules Bordct, 1. rue Heger-Bordet, 1000 Hrrlxcllcs. Belgique.

A patient with nephrotic syndrome was found to have amyioid deposition in the kidneys, skin and gastrointestinal tract. His ierum and urine contained monocionai X light chains. In the serum, the light chains were found as tetramers and dimers, whereas the urinary form was primarily dimer. His inarrow contained 16 per cent plasma ceils at the time of diagnosis. He was treated with the aiikyiating agents meiphaian and cyciophosphamide plus prednisone. He ex- hibited remarkable clinical improvement during the period of therapy concomitant with the eventual disappearance ofmonocionai light chain production, marked reduction in proteinuria, increased normal serum protein production and documented reduction in tissue amyioid deposition.

Biosynthetic studies were carried out with the patient’s bone marrow ceils obtained before, during and after cytotpxic therapy. Initially, X-chain tetramers were noted in cytopiasmic extracts and secretions of the marrow ceils in short-term tissue cuiture.‘Reduction and aikyiation of the molecules precipitabie with anti-light chain antiserums revealed light chain monomer and a smaller moie’cuie of approximate!y 10 to 12,OOO’daitons in size. Experiments carried out after the completion of therapy revealed no detectribie excess light chain synthesis. Similar studies in three other patients with AL fibrii deposition: indicated that ail synthesized light chain dime& but none produced larger polymers either intra- or extraceiiuiariy. A smaller molecule was identified in one of the three.

These observations support the previously suggested association of the occurrence of high molecular weight light chain polymers in the serum with AL amyibid deposition. In addition, the clinical improvement noted in conjunction with cytotoxic therapy raises the possibility of the existence of subsets of patients with AL deposition who will respond to these agents.

The substance histologically recognized as amyioid is chemically heterogeneous. At least two major chemical entities are recognized as amyloid [I]. One, found primarily in the AA type of amyloid, cons&s of fibrils formed from a protein with a molecular weight of approximately 8,500 daltons which shares antigenicity and chemical structure with an acute phase protein of human serum, SAA [Z-J].

In the second, or AL, type of amyioid, the major constituent of the fibril is composed of polymerized immunogiobuiin light chains or their fragments [5-Y]. The AL species is found in what was previously called primary amyloidosis and those cases associated with plasma.cell dy-

November 1979 The American Journal of Medicine Volume 67 667

AMYLOIDOSIS OF THE AL TYPE-BUXBAUM ET AL.

Figure 1. The renal biopsy specimen was stained with ‘Congo Red. Homogeneous deposits can be seen both in the mesangial regions of the glomeruli and the interstitial spaces. They showed green birefringence under polarized light microscopy and were metachromatic when other sections were stained with crystal violet.

scrasias. The pathway by which intact light chains are dine therapy. At the time of the present admission, his com- processed to amyloid fibrils in vivo has not yet been plaints were swelling of his lower extremities and increasing established.

. . . fatlgablhty.

In the present report we describe the clinical and laboratory findings in a patient with amyloid fibril deposition and bone marrow plasmacytosis who lacked evidence of any predisposing inflammatory disease. His serum contained monoclonal X light chains both as dimers and higher polymers, whereas his urine contained only X dimers. He was initially treated with melphalan and prednisone and, later, cyclophosphamide with remarkable symptomatic improvement and disappearance of the marrow plasma cells and of the serum and urine monoclonal,light chains. The X chains in his serum were succeeded by the appearance of a small amount of an immunoglobulin G (IgG) K M-protein which then appeared to fade into the background of his increasing normal immunoglobulin levels.

His blood pressure was 110/70 mm Hg. There was marked c&ma of the feet, legs and abdominal wall. The liver and spleen were not palpable.

Using techniques previously employed to examine the events involved in immunoglobulin biosynthesis in human and murine myeloma cells, we were able to analyze the cytoplasmic and secreted molecules produced by the patient’s bone marrow and to compare them with those synthesized by marrow cells obtained from patients with and without amyloidosis.

The complete blood count, serum glutamic oxaloacetic transaminase, lactic dehydrogenase, alkaline phosphatase, prothrombin time and bilirubin were normal. The calcium level was low (7.5 mg/dl) and the albumin level was 1.3 g/dl. Serum cholesterol was 720 mg/dl and the partial thrombo- plastin time was 33.2 seconds over 27.3 control. An x-ray series of the upper gastrointestinal tract and an oral cholecystogram disclosed no abnormalities. The patient’s urine contained 1 to 1.4 g protein/24 hours on several determinations. No cells or casts were seen. An intravenous pyelogram was within normal limits as were creatinine clearance and blood urea nitrogen. A renal biopsy revealed deposits of amyloid in the mesangium of the glomeruli. in the walls of blood vessels and in the in- terstitium [Figure 11. Electron microscopy and special stains confirmed the diagnosis of amyloidosis.

CASE REPORT

A 52 year old black man was admitted to the Manhattan Vet- erans Administration Hospital because of increasing edema. His past history was noteworthy for a diagnosis of thyrotoxi- cosis for which he was treated by subtotal thyroidectomy five years prior to admission. Histologic examination of the excised glancl revealed hyperplasia with involution secondary to io-

Over the ensuing 12 months, the patient’s clinical status deteriorated (Figure 2). He became overtly nephrotic, excreting from 10 to 30 g of protein daily. He was anemic, hypoalbumi- nemic, hypertriglyceridemic and hypercholesterolemic. His creatinine clearance diminished. His electrocardiogram showed low voltage. Immunoelectrophoreses of serum and urine revealed free X light chains in both fluids. Normal serum immunoglobulin levels were reduced. A bone marrow aspirate contained 16 per cent plasma cells but no clusters or abnormal cells. No amyloid was noted in the aspirate or in a subsequent biopsy specimen.

The patient was given melphalan, 20 mg/day, for four days and prednisone, 75 mg/day. for seven days. Shortly after in-

868 November 1979 The American Journal of Medicine Volume 67

,lMYI.OIIK)SIS OF ‘IlIE AI, ‘I‘YPE -HI IUAtlM E?‘ AI..

Figure 2. Case 1. The bottom panel shows the sequential changes in the patient’s immunoglobulin levels during the period of observation. His IgG (0 - - - l ), IgA (0 e---*0) and IgM (0-O) levels were below normal prior to the initiation of therapy. The IgG and IgM levels rose to normal; the IgA level remained the same. The increase in serum albumin is readily apparent. The middle panel shows the rapid increase and gradual decline in the patient’s degree of proteinuria. When the top and middle panels are compared, it is clear that the massive proteinuria preceded the diminution in creatinine clearance. Although the creatinine clearance and proteinuria have improved markedly, both are still abnormal. The therapeutic regimen is noted by the bars at the top of the figure. The initial square ( n ) represents a course of melphalan therapy, 20 mg/day, for four days and 75 mg of prednisone daily for seven days. The next bar (B n ) represents a period during which the patient received maintenance melphalan (2 mg/day). During the subsequent period (O), he received 2 and 4 mg of melphalan on alternate days. He was then maintained on a monthly regimen of melphalan and prednisone (0 0). Subsequently, he was given cyclophosphamide, 50 mg twice daily, during the period indicated by the last bar (a@).

stitution of this therapy, he suffered a femoral embolus. After a sncccssful cndartercctomy, he was given anticoagulants and the administration of mclphalan was stopped. He experienced an episode of retroperitoneal bleeding for which he required transfusion. Purpuric skin lesions also developed. Biopsies rc:vcalcd a nonspecific vasculitis and scattered dermal deposits of amyloid. A perorally obtained gastric biopsy specimen tlcmonstrated substantial amounts of amyloid in the gastric mucosa and submucosa. The patient began to suffer severe orthostatic hypertension which responded to sympathomimetic therapy. His nephrotic syndrome worsened, and cardiomegaly ~~2s noted.

(Ihcmotherapy was resumed with the patient receiving 2 mg

of mclphalan orally daily. Several months later, the dose was changed to alternate daily doses of 2 and 4 mg. Bone marrow examination at this time revealed fewer than 5 per cent plasma cells. Again, no amyloid was seen in the bone marrow biopsy spccimcn.

During this period and over the next 18 months, the patient demonstrated gradual improvement. The administration of melphalan was continued except when the white blood cell and platelet counts decreased and the patient experienced an episode of herpes zoster. When the hematnpoietic system re- covered and the infection resolved, the patient was given monthly courses of melphalan. 16 mg/day. for four days and prednisone. 75 mg/day. for seven days.



Figure 3. Representative immunoelectrophoretic patterns from serum obtained from the patient at various times after diagnosis. A demonstrates the seagull type of precipitate obtained with anti-X antiserum at the time of diagnosis (arrow a). A normal serum is shown for comparison (N). Neither of the arcs correspond to material precipitable with y. cy, p or 6 antiserum. B shows a similar precipitate one year later. An anti-y precipitate is also shown. The serum proteins are quite similar. C represents serum obtained 10 months later during which the patient had received melphalan therapy. The x precipitate has lost its biphasic nature. The amounts of normal immunoglobulins have increased and there is a suggestion of an IgG K protein of restricted mobility (arrow b). D shows serum obtained a year later. During the preceding year, the patient received melphalan and prednisone monthly, then cyclophosphamide. There is a suggestion of a seagull deformity with both the y and K antiserum (arrow c). E shows the serum pattern six months later after the cessation of cyclophosphamide therapy. The 7, K and h arcs now appear to reflect the normal population of IgG molecules bearing both K and x determinants. The additional precipitin arc seen cathodally with the anti-y serum represents an artifact produced by interactions between the two antiserums diffusing from the parallel troughs.

Patient

Anti -A

N

Anti-Y

Patient

Anti-a

Patient

Anti - K.

Anti-Y

Patient

Anti-2

Anti -f( Anti -Y

Patient

Anti -2

Anti - /(

Anti-Y

Patient

Anti-3

The patient continued to do well with decreasing proteinuria, improving renal function and increasing normal immunoglobulin levels. Serial serum electrophoreses and immunoclectrophoreses revealed disappearance of the X chains but the appearance of a small amount of an apparently monoclonal IgG K protein (Figure 3). Repeat bone marrow examination showed no increase-in plasma cells or lymphocytes. However, because of the new protein, the administration of melphalan was discontinued and the administration of cyclophosphamide, 50 mg/twice daily, was begun. Repeat skin biopsy demonstrated no amyloid substance in the same location which previously had ample amyloid infiltration. The upper x-ray series of the gastrointestinal tract was repeated with marked improvement noted in gastric distensibility and motility: however, gastric biopsy was again positive for amyloid.

After six months, the cyclophosphamide therapy was discontinued. Subsequently, hypertension developed (blood pressure 180/110 mm Hg), which was controlled with pro- pranolol, furosemide and hydralazine. The patient continued to show proteinuria and mild renal insufficiency with creatinine concentrations ranging from 2 to 3 mg/dl.

MATERIALS AND METHODS

Serum and urine electrophoreses were carried out on cellulose acetate strips. Urine was concentrated 25- to 50-fold by either vacuum dialysis or membrane ultrafiltration. Serum electrophoresis was performed according to the micro method of Scheidegger using rabbit antiserum against human heavy and light chains prepared in our own laboratory or in the laboratory of Dr. E. C. Franklin [8]. The antiserums were raised against either intact isolated myeloma proteins or free light chains isolated from the urine of patients with Bence Jones proteinuria. Antiserums which reacted with either light chain class were elicited by immunization of rabbits with purified F(ab) fragments prepared from pooled human immunoglobulin. One antiserum raised against free X chains was obtained commercially (Behring, Lot No. 2225C].

Ascending column chromatography was performed using columns packed with Sephadexm G-200, Sepharose 4B or Bio-Gel A 0.5. All columns were equilibrated with and the samples chromatographed in 0.3 M sodium chloride. Serum immunogiobulin levels were determined by the Mancini technique utilizing commercially available calibrated anti- immunoglobulin impregnated agar plates obtained from Kallestad Laboratories [g].

Bone marrow cells were obtained at the time of diagnostic aspiration [lo]. The samples were collected in heparinized syringes. The cells were washed, counted and incubated with radioactive amino acids. Aliquots were harvested after various times of incubation, washed and lysed with the nonionic de- tergent Nonidet P-40 (NP-40). Samples of supernatant medium containing cell secretions and cytoplasmic lysates were immunologically precipitated with antiserums specific for various immunoglobulin chains. The immunologic precipitates were separated by centrifugation, washed in a buffered salt solution and dissolved in sodium dodecyl sulfate (NaDodS04) prior to analytical electrophoresis either on cylindrical 5 per cent NaDodSOJ-containing acrylamide gels or on NaDodS04 slab gels with acrylamide concentrations ranging from 5 to 17.5 per


cent [ll]. Some samples were reduced with 0.15 M 2-mer-

captoethannl and alkylated with 0.2 M iodoacctamide prior to electrophoresis to determine the chain composition of the immunologically precipitated material [IZ].

RESULTS

Serum Proteins. Immunoelectrophoresis of the patient’s serum showed a’seagull type of precipitate when developed with anti-)\ serum. Neither of the limbs of the precipitate was precipitable with any of the anti-heavy chain serums (7, (Y, p and 61, indicating that the abnormal protein was comprised entirely of X light chains. Serial immunoclectrophoreses revealed the disappearance of the abnormal X chains and the subsequent appearance of the IgG K protein (Figure 3). Serial serum immunoglobulin levels are noted in Table I.

The initial cellulose acetate strip electrophoresis of the urine revealed the characteristic pattern seen in nephrotic urine. Immunoelectrophoresis of the urine demonstrated a protein which was precipitable with anti-X but not with antiserum directed toward any other immunoglobulin. When the serum and urine were immunoelectrophoresed on the same slide with the same anti-)\ serum, one of the serum proteins corresponded electrophoretically to that seen in the urine (Figure 4A and 4FJ. Since both serum proteins were composed exclusively of X chains and only one appeared in the urine, it was likely that one of the serum proteins was a high molecular weight X-chain polymer. Gel filtration showed the ehltion of the higher polymer at a position consistent with a molecular weight of 100,000 to 150,000 daltons (Figure 4B). The next eluted pool contained both the larger and smaller polymers (Figure 4c and 4D). Fractions eluting later contained only the smaller polymer which migrated identically with the urine protein.

Bone marrow cells obtained from the patient were studied on three occasions. Incorporation of radioactive amino acids into total protein [i.e., TCA precipitable) and immunoglobulins (anti-immunoglobulin precipitable) was linear over the 4 to 6 hour course of the experiments. The amount of incorporation of radioactivity into protein and its distribution between secreted and intracellular proteins varied with the number of plasma cells in the bone marrow sample.

The olcctropherograms of immunologically precipitated cvtoplasmic material obtained in experiment 1 are shown”in Figure 5. After 1 hour of incubation, the major cytoplasmic protein could be precipitated with the anti-F(ab) serum. It had a molecular weight of 55,000 daltons and was not precipitable with an antiserum specific for y heavy chains. At z hours of incubation, the 55,000 dnlton molecule was still prominent, but additional discrete peaks were also visible. Reduction and alkylation of these antilight chain precipitates followed by electrophoresis revealed two peaks of radioactivity. One had an estimated molecular weight of 25 to 27,000

TABLE I Serum lmmunoglobulin Levels (mg/dl) in Case 1

Date W

2174 305 5175 205 9175 280 11175 740 1176 810

9176 620 2177 1,640 a177 1,200 12177 1,200

12178 1,450

NOTE: ND = not determined.

IN @A w 30 59 ND 48 36 0

147 62 0

150 70 0 160 65 0

90 63 0

390 '70 0

120 78 0 155 a3 0

165 105 0

L

+

Patient Serum Unfractionated

B fi Fraction III

C :J L, _ IV

D f > V

E VI

F Patient Urine

Figure 4. A shows an immunoelectrophoretic pattern of the patient’s whole serum developed with anti-)\ serum. Panels B, C, D and E show the patterns obtained when his serum was chromatographed on Sephadex G-200, fractions pooled, concentrated by pervaporation, electrophoresed and precipitated with anti-)\ serum. F shows the pattern obtained when the patient’s urine was immunoelectrophoresed and developed with anti-h. Proteins with molecular weights of 120 to 140,000 daltons are in (B), those of 95 to 115,000 daltons in (C), those of 70 to 90,000 daltons in (D) and less than 70,000 daltons in (E). The largest x proteins comprise the anodal portion of the seagull, whereas the smaller proteins are cathodal and correspond in mobility to the x dimers found in the urine. Similar results were obtained when the serum was chromatographed on other media which separate proteins by molecular size.



claltons, the second 12,500 daltons (Figure 6). No radioactivity was found in the regions of the gel which would have contained heavy chains if they were present. After 6 hours of incubation, the anti-light chain precipitates contained major peaks of 97,000 and 55,000 daltons with shoulders of 69,000 and 150,000 daltons (Figure 5). The gel pattern after reduction and alkylation was similar to that of the %hour antilight chain precip-

_ 60Min

200 -

200 - - 120 Min.

a w a

cn 500 t 3601 din.

30 40

-\___ - I --4--_

-50 60 70 00 FRACTION NUMBER

Figure 5: Cytoplasm was prepared by NP-40 lysis from the bone marrow cell samples obtained after various period$ of incubation. The lysates were precipjtated with an antilight chain antiserum. The specific precipitates were dissolved in NaDodS04, boiled and applied to 5 per cent cylindrical acryfamide gels. Each 14G-labelled precipitate was co- electrophoresed with 3H-labelled secreted material prepared from the murine .myeloma tumor MPC-11. The marker preparation co,nta/ned HsL2 (molecular weight 150,000 daltons) ,migrating at fractton 27, H (55,000‘daltons),migratiAg at fraction 38 and L chains (23,500 daltons) mjgr’ating.at fractions 45 to 50 (- - -).

Figure 6. Electropherogram of the reduced and alkylate d anti-light chain precipitate (-) shown in Figure 5, top panel. The positions of the reduced and alkylated MPC-11 heavy and light chains are also shown (- - -).

__ FRACTION NUMBER

itate. These data indicated that the major cytoplasmic protein consisted primarily of polymerized light chains disulfide-linked to a smaller molecule and that, with time, the size of the intracellular polymers appeared to increase.

Attempts to extract cells labelled for 6 hours using the salt and water procedure of Pras et al. [13] yielded an electraphoretically heterodispersed collection of molecules which had no predominant polypeptide species.

When material secreted by the cells over the same 6-hour period was analyzed in a similar manner, two major peaks were precipitable with the anti-light chain antiserums (Figure 7, lower panel). These had molecular weights of 55,000 and 90,000 daltons. They were not precipitable with an antiserum specific for K light chains nor with any of the anti-heavy chain serums, hence, like the intracellular material, they were composed exclusively of X-chain-containing molecules. A third peak was also precipitable with the anti-light chain serum. In contrast to the dominant molecules, it was largely precipitable with a specific anti-K antiserum and comigrated with the murine IgG marker. By both size and antigenic criteria, it appeared to represent the normal intact IgG molecules synthesized by the normal plasma cells in the marrow sample and was unrelated to the X-polymers.

Experiment 2 was performed with cells obtained after the patient had been treated with melphalan and prednisone. There were fewer than 5 per cent plasma cells in the sample. The absolute quantity of immunoglobulin synthesized for secretion was considerably lower, although the rates of secretion were similar,

The electropherograms of antilight chain precipitates obtained from l-hour cytoplasm in the two experiments are compared in Figure 8. In experiment 2, light chain monomer (approximately 30,000 daltops], light chain dimer (approximately 56,000 daltons) and a split peak


____-

300 L ANTI-K

225

150

75

?

is 0 11

5

5 a 525 - ANTI-FAB

? 0

$450 -

0

O3?5 -

300 -

150 -

_*__--y’ ,

IO 20 30 40 50 60 70 80 9 FRACTION NUMBER

_

Figure 7. Aliquots of material secreted by cells labelled for 6 hours in experiment 1 were precipitated with either anti-K serum (top panel) or an anti-F (ab) serum which reacted with both K and x light chains and intact IgG (bottom panel) and electrophoresed as noted previously. The positions of the MPC-11 marker proteins are shown in the bottom panel (- - -). Direct precipitation with an anti-)\ serum could not be performed, but the molecular species of the X-containing material can be established by subtraction. The K-containing molecules comprise only the portion of the anti-F(ab) precipitate which comigrates with the HzL2 marker.

with estimated molecular weights of 105,000 and 88,000 daltons are all visible. Although immunoglobulin synthesis was markedly reduced as measured by the amount of radioactive amino acids incorporated into immunoglobulin, the qualitative representation of light chain polymers was similar to that seen in the first experiment.

The third study was performed approximately one year later when the patient was clinically improved after intermittent melphalan therapy. His marrow was normal, although his serum contained a small amount of an apparently monoclonal immunoglobulin of the IgG K type. The major radioactive protein in both cytoplasm and secretions was an apparently intact IgG molecule which yielded normal-sized heavy and light chains on reduction and alkylation (Figures 9 and llJ]. Neither free light chains nor light chain dimers were noted in the unreduced samples.

Similar experiments were carried out with marrow

cells from three other patients with AL, amyloid. The data are summarized in Table II. The cytopiasms of three patients (Cases 2, 3 and 4) contained light chain monomers and dimers in varying proportions without larger polymers (Figure 11). These findings corresponded to those noted in experiments performed with cells obtained from patients with L-chain myeloma without demonstrable amyloid deposition [IO]. In two of our cases (Cases 1 and 4). the antilight chain serum also precipitated molecules smaller than light chains. The smaller molecule was not detected in the unrc- duced sample in either case.

COMMENTS

Over the last decade, a number of investigators have come to perceive primary amylnidosis as a plasmacytic

f L 140 -

M-2

0’ - Exp 2 120- 1 E i 0

OL- _1_ --.L_~__-J

IO 20 30 40 50 60 70 80 90 FRACTION NUMBER

igure 8. Electropherograms of anti-F(ab) precipitated 60-minute-cytoplasmic lysates obtained in experiments 1 and 2 are compared. The positions of the MPC-11 H2L2 and L- chain markers are indicated by arrows. As noted in Figure 6, the major intracellular immunoglobulin-containing molecule in experiment 1 was the L2 dimer. In experiment 2, the monomer and dimer are equally prominent, and molecular species seen only as shoulders at this time in experiment 1 are more clearly defined.



Figure 9. Autoradiograph of a 10 per cent polyacrylamide slab gel of material immunologically precipitated from radioactive cytoplasm and secreted material obtained in the third experiment carried out with bone marrow cells from our patient (Case 1). The positions of the MPC-11 markers are noted. Channel 1 contains the cytoplasmic molecules precipitated by an anti-y serum; channel 2 contains those precipitated by the anti-F(ab) serum used in experiments 1 and 2. Channel 3 contains unpre- cipitated secretions, whereas channels 4 and 5 contain the same secretions precipitated with the anti--y and anti-F(ab) serums, respectively. The marrow -ells obtained at this time did not synthesize or secrete any free light chains of either class.

dyscrasia because of the frequently associated marrow plasmacytosis and the recent chemical evidence indicating that the fibrillar tissue deposits are composed primarily of monoclonal light chains or their fragments [14,15]. The feature responsible for the malignant out- come of the disease is the compromise of vital organ function secondary to deposition of the insoluble light chains. The realization that the AL type of amyloid was a plasma cell product led to a number of attempts to treat these patients with the same cytotoxic agents used in the treatment of overt myeloma.

Initial results were not encouraging [16]. Nonetheless, sporadic successes have been reported using similar regimens [W-21]. The use of these agents assumes that they can eliminate the cell population producing the amyloid precursor, i.e., light chain. When that population is no longer present, the previously deposited tissue amyloid can be mobilized or resorbed [22]. A few cases

have been reported in which spontaneous disappearance of the Congo Red binding amyloid has been noted [23-281. However, all the reports have been of patients whose diseases were usually associated with the AA, rather than the AL, type protein, and the apparent re- sorption coincided with alleviation of the original inflammatory condition.

Cytotoxic therapy with melphalan entails risk to the patient [29-311. Our decision to use the melphalan- prednisone regimen was based on the extremely poor prognosis carried by our patient in the absence of therapeutic intervention. The patient showed signs of clinical improvement coincident with the disappearance of the X chains in serum and urine and the diminution in the amount of amyloid found in those organs acces- sible to biopsy. The persistent, although diminished, proteinuria indicates that there must be residual renal disease related to either the incomplete resolution of the


deposits or some other concomitant process. The response to melphalan therapy was also characterized by an increase in normal immunoglobulin levels as the monoclonal light chains disappeared. The increase in normal immunoglobulin frequently accompanies ef- fective chemotherapy of myeloma [32].

An additional phenomenon noted in our patient was the appearance of an apparently homogeneous IgG K protein in the strum. No other evidence for clinical myeloma was present. Melphalan has been demonstrated to cause variations in immunoglobulin synthesis by murine myeloma cells in culture [33,34]. In addition, the phenomenon of “light chain escape” has been described clinically [35]. The present case is unusual in that an HzLZ.protein was produced in a patient in whom only light chains were produced formerly after a therapy known to be mutagenic. Since the original protein was X and the new protein was K, it is unlikely that the new cell population represents a subclone of the previously X-producing plasma cells. The IgG K-producing cells may represent a new clone which has been transformed by melphalan and which may have less proliferative capacity than its predecessor. Two other cases have been described in which new immunoglobulin-pro- tlllcing clones have emerged in the course of therapy with alkylnting agents. However, in both cases the transition was from the production of one HzLz molecule to another [36,37].

The presence of light chain polymers in the serum has become recognized more frequently as more laboratories have gained access to techniques required to detect them [38-401. Some attempts have been made to relate the presence of polymers to the deposition of amyloid 1411, A number of patients with polymers in their serum did not have evidence of tissue amyloid, whereas others without polymers had tissue deposition. The size of the polymers was not noted in these patients. Several patients have been described in whom light chain tetra- mcrs were found in the serum. Careful evaluation of the patients, either clinically or at postmortem, has revealed amyloid in half of them.

H2L2 -

H -

L -

Figure 10. Autoradiographs of reduced and alkylate immunoprecipitates from the labelled cytoplasm and secretions obtained in the third experiment (see Figure 9) which have been electrophoresed on a slab gel. Channel 1 shows the anti-y precipitate (see Figure 9, channel 4) reduced to its component chains, whereas channel 2 shows a similarly treated anti-F(ab) precipitate (see Figure 9, channel 5). The positions of the markers are noted.

TABLE II Clinical and Laboratory Features of Patients Synthesizing Free x Chains

Case No. Diagnosis Clinical Serum Urine Cytoplasm Secretions

1 Primary amyloidosis (AL) Nephrotic, skin, Free h, h x,,x X polymer, X2 X4,X2 gastrointestinal, cardiac polymers + 12,500 vasomotor fragment

2 Multiple myeloma f Plasmacytomata, x2 A, h*,X x2 amyloid (AL) neuropathy,

hypoganimaglobulinemic 3 Primary amyloidosis (AL) Nephrotic, cardiac, liver, x2 A, x,,x A,

spleen 4 Primary amyloidosis (AL) Neuropathy x2 A:, X%X + x2

fragment 5 Multiple myeloma Frequent infections, renal 12 w x*,x

failure, no amyloid



A

H-

L-

L

-

1’ ; 3 .

*

Figure 11. Autoradiographs of unreduced (a) and reduced and alkylated (b) immunoprecipitates obtalned trom the cytoplasm of one of our patients (Case 4) and electrophoresed on polyacrylamide slab gels as noted previously. A shows labelled bands corresponding to light chain dimer (molecular weight 45,000 daltons) and light chain monomer. In B, channel 1 indicates that no radioactive material was precipitable from 2-hour labelled cytoplasm with an antiserum made against the acid-soluble component of human amyloid, whereas channels 2 and 3 show the results obtained when the same cytoplasm is precipitated with the anti-F(ab) serum, then reduced and alkylated. After this procedure, only light chain monomers were noted.

The role of covalent or noncovalent polymerization place when light chains are exposed to lysosomal en- in AL fibril formation is not clear. Amyloid fibrils can zymes extracted from human kidneys [46,47]. In both be formed in vitro by incubating intact amyloidogenic digestions, analysis of the products prior to fibril for- light chains with pepsin [42-451. As digestion progresses, mation has indicated that a high molecular weight an insoluble pellet forms which binds Congo Red and complex, composed of either intact or partially digested has the characteristic fibrillar structure of amyloid when light chains, is formed. This gradually disappears as examined electronmicroscopically. Similar events take digestion proceeds and the amount of insoluble material


increases. Hence, polymerization of either the intact or partially digested light chains may be critical in amyloid formation. The experiments with cells from three of our patients (Cases 2, 3 and 41, which demonstrated that, despite indisputable evidence of tissue amyloidosis, the marrow cells did not contain or secrete light chains in any polymeric form larger than dimers, indicate that intracellular polymerization is not obligatory in the I’ormation and deposition of AL proteins.

We were not able to isolate sufficient amounts of light chains from our patient (Case 1) for chemical analysis. On NaDodS04 gels, it and that of two other patients (Cases 4 and 5) appeared to be slightly larger than our murine K chain marker, having estimated molecular weights of 25,500 to 27,000 daltons; however, X chains are known to display anomalous electrophoretic be- havior in this system [48]. It is thus likely that the largest intracellular form was a tetramer which migrated more rapidly than would be predicted from its molecular weight, i.e., 100,000 versus 110,000 daltons, or a trimer attached to another peptide, i.e., 82,500 + 12,500 daltons. Although reduction and alkylation of the antilight chain precipitates yielded both the monomer and the

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2. Ixvin M. Franklin EC. Frantrione B. et al.: The amino acid scx~ucncc of a major noni;;7mnnoglobi*lin component of some amvloid fihrils. I Clin Invest 51: 2773. 1972.

3. (:orc!vic: Pd. Rosenthal ‘CJ. Franklin EC: Amyloid-related scrllm component (SAAJ-studies in acute infections, mcxlllllary thyroid carcinoma, and postsurgery. Clin Im- munol Immunopathol 6: 83, 1976.

4. Kos(>nthill CJ. Franklin EC, Frangione B, et al.: Isolation and I);lrti;til characterization of SAA-an amvloid-related ilrotein from human strum. J Immunol 116: i415, 1976.

5. Glenncr GG. Tcrrv W. Harada M. et al.: Amvloid fibril nro- teins: prook of homology with immunoglob;lin light ch;ins by sequence analyses. Scie’nce 17i: 1150, 1971.

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Putnam, FW, Whitley EJ Jr, Paul C, et al.: Amino acid se- cpxxx:c of a K Bence Jones protein from a case of primary amyloidosis. Biochemistry 12: 3763, 1973.

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. ” ”

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12,500~dalton peak, it was not clear if the smaller peptide carried light chain determinants or was a nonlight chain molecule which coprecipitatecl with the native light chain. If it was light-chain related. it was either a postsynthetic degradative product or a synthetic fragment which was rapidly bound to an intact light chain, since it did not appear to accumulate either in the cytoplasm or secretions of the labellcd marrow cells. The similar-sized molecules seen in some of the other experiments were not noted consistently enough to draw any firm conclusions concerning their role in amyloi- dogenesis.

ACKNOWLEDGMENT

We gratefully acknowledge the expert assistance of Dr. G. Sidhu in the interpretation of the pathologic material obtained from our patient. We are also grateful to Dr. E. C. Franklin for the generous gift of specific antiimmu- noglobulin heavy chain antiserums and to Dr. Franklin and Dr. R. S. Basch for their helpful discussions of the manuscript. We also appreciate the technical expertise of Ms. Frances Santiago-Schwarz.

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a78 November 1979 The American Journal of Medicine Volume 67

Amyloidosis of the AL type

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