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B-cell chronic lymphocytic leukemia (B-CLL) accounts for the majority of CLLs in the United States and Europe.1 B-CLL occurs predominantly in elderly adults ...
Hematopathology / BIOLOGIC FEATURES IN FAMILIAL B-CLL

Familial B-Cell Chronic Lymphocytic Leukemia Analysis of Cytogenetic Abnormalities, Immunophenotypic Profiles, and Immunoglobulin Heavy Chain Gene Usage Patricia Aoun, MD, MPH,1 Guimei Zhou, MD,1 Wing C. Chan, MD,1 Cynthia Page, MT,1 Kellie Neth, MT,1 Diane Pickering, MS,2 Warren Sanger, PhD,2 Brigid Quinn-Laquer, MS,3 Patrice Watson, PhD,3 Jane F. Lynch, BSN,3 Henry T. Lynch, MD,3 and Dennis D. Weisenburger, MD1 Key Words: Chronic lymphocytic leukemia; CLL; Fluorescence in situ hybridization; FISH; Cytogenetics; Immunophenotyping; Variable region genes DOI: 10.1309/PFTPLL4HCK2D1ERK

Abstract B-cell chronic lymphocytic leukemia (B-CLL) is a heterogeneous disease that may exhibit familial clustering. We examined the cytogenetic, immunophenotypic, and VH gene usage characteristics of a family with B-CLL affecting 7 members in 3 generations. Interphase fluorescence in situ hybridization studies identified an acquired deletion of chromosome 13q14 in the leukemic cells of 6 affected members, accompanied by deletion 14q32 or trisomy 12 in 2 cases. VH gene analysis demonstrated clonal rearrangements of the VH3 gene family in 5 cases and of VH2 genes in 1 case. All 6 cases were mutated in VH2 or VH3. Two cases had a second VH1 family gene rearrangement that was unmutated. Flow cytometry performed on 5 cases showed the typical B-CLL immunophenotype; all were CD38–, but 3 expressed ZAP-70. Our findings support previous observations that familial and sporadic B-CLL cases are biologically similar and suggest that familial clusters will be useful for studying pathogenetic events in B-CLL.

B-cell chronic lymphocytic leukemia (B-CLL) accounts for the majority of CLLs in the United States and Europe.1 B-CLL occurs predominantly in elderly adults and is typically indolent but can have a highly variable clinical course. Numerous factors have been evaluated for prognostic effect, and known prognostic factors include clinical stage, atypical morphologic features, immunophenotypic characteristics such as CD38 and ZAP-70 expression, mutation status of the variable region of the immunoglobulin heavy chain gene (VH), and various chromosomal abnormalities.2-11 In recent years, VH mutation status and gene usage have emerged as important independent prognostic factors in B-CLL. Preferential use of the VH1, VH3, and VH4 gene families by B-CLL cells has been demonstrated, suggesting that chronic antigen stimulation may have a pathogenetic role.3,12-17 In B-CLL, the most frequently expressed genes are VH1-69, VH3-7, VH3-21, VH4-34, and VH4-39. Somatic mutations are found more frequently in cases expressing VH4-34 or VH3 family genes, whereas the VH1-69 and VH4-39 genes typically lack mutations.3,12,13,16 The use of VH3-21 is associated with a poor prognosis, regardless of mutation status.14,17,18 Recent studies also suggest that there are biologic relationships between VH mutation status and other prognostic factors. For example, somatic VH mutations are frequently observed in cases with del13q14 but are rare in cases with trisomy 12 and are associated with the lack of expression of CD38 and ZAP-70.6-8,10,19,20 Discordant ZAP-70 expression and VH mutation status may be related to the presence of high-risk genetic abnormalities such as deletions of 11q and 17p and expression of VH3-21.17,18,21 Familial clusters of B-CLL that are morphologically and immunophenotypically similar to sporadic cases have been reported, supporting a role for hereditary predisposition in a small proportion of cases.22-35 However, only a limited number Am J Clin Pathol 2007;127:31-38

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of studies have compared the biologic features of sporadic and familial cases. For example, few studies have systematically examined familial clusters of B-CLL using fluorescence in situ hybridization (FISH) to demonstrate recurrent cytogenetic abnormalities commonly seen in sporadic B-CLL.36-39 Likewise, preliminary studies have suggested that overexpression of CD38 and ZAP-70 occurs at similar rates33,40,41 and that there is preferential usage of the VH1, VH3, and VH4 gene families.28,42-44 The frequency at which specific genes are used is more difficult to ascertain owing to the limited number of cases studied. We have previously described 3 generations of a family in which the father and all 4 male offspring, including twin brothers, were affected by B-CLL.32 Conventional and molecular cytogenetic analysis of peripheral blood samples demonstrated the presence of an acquired del13q14 in the leukemic cells of all 4 affected offspring, without constitutional cytogenetic abnormalities.

In the present study, we used conventional and FISH cytogenetics and immunophenotypic studies to determine if additional asymptomatic family members were affected in this family. We also extended our previous FISH cytogenetic studies with additional probes and determined the mutational status, VH gene usage, and ZAP-70 expression of the B-CLL cells from affected family members to correlate these findings with each other and with the FISH cytogenetic results.

Materials and Methods Patients and Samples A family in which the father and all 4 male children were affected by B-CLL has been previously described.32 For the present study, additional family members were identified in 3 generations, for a total of 7 affected people ❚Figure 1❚. The 1

2

I ~d.60 ~d.70

1

2

3

d.70

d.83

d.64

4

5

II

1

2

CLL 77 d.87

d.82

CSU d.67

~d.60s

6

7

d.47 d.87 ~d.70 Co d.75

Pro 80 d.89

3

4

5

9 2

8

III

1

2

CLL 56 69

CLL 54 69

3

4

5 3 54 55 58

IV

1

2

3

CLL 51 Pro 73 80 4

5

CLL 39 73 6

V 36

42

39 CLL 34 53 36

49

7 2 40 54

6

7

~d.20 54

~d.70s

8

~d.50s

10 ~80

11 ~80

12

13

14

~80s

9

68 CLL 58 69

8 2 38 48

Proband Male 1 Female 2 Individual number Multiple primary cancers by pathology 47

CLL 53 55

33 47 Co 45 47

Unaffected Current age Cancer by pathology Age at diagnosis Current age

3

3

Number of unaffected progeny (both sexes) Identical twins

d.54

d.86

Cancer by family history Age of death

❚Figure 1❚ Pedigree of a family in which multiple members of 3 generations have been affected by B-cell chronic lymphocytic leukemia (B-CLL). The pattern is consistent with an autosomal dominant genetic predisposition to B-CLL. The decreasing age of onset across generations suggests anticipation. Co, colon cancer; CSU, cancer site unknown; Pro, prostate cancer. (Pedigree drawn by Tami Richardson-Nelson, BGS.)

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diagnosis of B-CLL was confirmed by review of the morphologic, immunophenotypic, cytogenetic, and/or clinical materials. A detailed hematologic and solid tumor family history was obtained from the proband (Figure 1, IV:4). Additional family members were contacted, and consent was obtained from each family member entering the study. Peripheral blood, skin biopsy, and buccal mucosa cytology samples were obtained for analysis from 6 affected and 9 unaffected family members. This study was reviewed and approved by the institutional review board at the Creighton University School of Medicine, Omaha, NE. Cytogenetic Studies Peripheral blood samples from 15 family members (6 affected and 9 unaffected) were studied by conventional and/or molecular cytogenetic techniques by FISH. Additional FISH studies were also performed on buccal mucosal samples from 2 affected members with multiple FISH abnormalities to determine whether these were constitutional or acquired. For conventional cytogenetic studies, blood samples were cultured with and without mitogen stimulation for 24 and 72 hours using standard techniques, and 20 G-banded metaphases were analyzed from each sample. Interphase FISH studies using probes to chromosomes 12, 13q14/13q34, and 14q32; the ATM locus on 11q23; and the p53 locus on 17p13.1 (all from Vysis, Downers Grove, IL) with hybridization on a HyBrite instrument (Vysis) were performed. At least 100 nuclei were examined for each probe. The false-positive and false-negative cutoff values used for these probes were those established by the Human Genetics Laboratory, University of Nebraska Medical Center in clinical studies of more than 500 analyses with these probes. Flow Cytometric Immunophenotyping Peripheral blood samples were obtained for immunophenotyping from 5 affected (Figure 1, IV:1, IV:2, IV:4, IV:9, and V:4) and 7 unaffected (Figure 1, V:1, V:2, V:3, V:6, V:7, V:7, V:8) family members. For flow cytometry studies, peripheral blood mononuclear cells (PBMCs) were isolated from EDTAanticoagulated peripheral blood by density gradient centrifugation (Accu-Prep, Accurate Chemical, Westbury, NY) and hypotonic lysis. PBMCs were then washed with phosphatebuffered saline and resuspended in RPMI 1640 (Life Technologies, Grand Island, NY) supplemented with 1 mol/L of Hepes buffer, L-glutamine, penicillin, streptomycin, and 10% fetal calf serum for immediate analysis or in Cell Culture Freezing Media with 10% dimethyl sulfoxide (GIBCO BRL/Invitrogen, Rockville, MD) for storage in liquid nitrogen. Isolated PBMCs were then labeled with antibodies to CD2, CD3, CD4, CD5, CD7, CD8, CD10, CD11c, CD16, CD19, CD20, CD23, CD24, CD25, CD38, CD45, CD56, CD103, and HLA-DR for multicolor flow cytometry studies

(Beckman Coulter EPICS XL or FC500 flow cytometer, Beckman Coulter, Miami, FL). B-CLL populations were further studied for cytoplasmic ZAP-70 expression using fresh or thawed sample aliquots, FIX AND PERM Cell Permeabilization reagents (Caltag Laboratories, Burlingame, CA), and 2 different ZAP-70 antibody clones (clone 1E7.2, Caltag Laboratories; clone 2F3.2, Upstate Laboratories, Charlottesville, VA). ZAP-70 expression by B-CLL cells was determined using a 20% positive cutoff and a T-cell threshold, as previously described.8 VH Gene Usage and Mutation Analysis VH gene usage and mutation analysis was performed on PBMCs from 6 affected family members (Figure 1, IV:1-4, IV:9, and V:4). DNA was isolated from fresh or frozen PBMCs after standard Proteinase K digestion, followed by phenol-chloroform extraction. Total cellular RNA was isolated from fresh PBMCs by Trizol reagent (Invitrogen, Carlsbad, CA). After DNase I (Promega, Madison, WI) treatment, 1 µg of total RNA was reverse transcribed to complementary DNA with random hexamers using Superscript II reverse transcriptase (Invitrogen). Rearranged VH genes were then amplified from extracted DNA by polymerase chain reaction (PCR) using VH family–specific primers to framework 1 (FR1) sequences and a consensus sequence at the 3' end of the J region (JH external) in a 50-µL volume, using a Biometra thermocycler (Biometra, Kent, England). Primer sequences are shown in ❚Table 1❚. The PCR reaction contained a final concentration of 1× PCR buffer with 0.2 µmol/L of each primer, 1.5 mmol/L of magnesium chloride (for VH1, VH3, and VH4) or 2.0 mmol/L of magnesium chloride (for VH2, VH5, and VH6), 0.2 mmol/L of each deoxynucleoside triphosphate, and 2.0 U of Taq DNA polymerase (Promega). Thirty-five cycles of PCR were performed using the following cycling conditions after an initial incubation at 94°C for 5 minutes: denaturing at 94°C for 40 seconds, annealing at 65°C for 40 seconds, and extension at 72°C for 1 minute. In cases in which 2 rearranged VH genes were identified, reverse transcriptase–PCR was also performed for these samples using the same PCR conditions except for an initial denaturing step of 2 minutes instead of 5 minutes. For these reactions, the leader primers of the VH1 and VH3 families replaced the FR1 primer. The PCR products were analyzed on 10% Precast TBE Gel (Bio-Rad, Hercules, CA) and visualized by ethidium bromide staining. The bands were excised, and the DNA was extracted by freeze thawing the gel slice (1 gel slice in 200 µL of sterile water) 3 times using a –70°C freezer. To prepare for sequencing, 2 µL of the extracted DNA was reamplified using the VH family–specific primer and M13RJH nested primers for 25 cycles. The PCR product was purified using the QIAquick Gel Extraction Kit (Qiagen, Santa Clarita, CA) and sent Am J Clin Pathol 2007;127:31-38

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❚Table 1❚ Primers Used in the Polymerase Chain and Sequencing Reactions Primer Sequence* CCATGGACTGGACCTGGA CCATGGAGTTTGGGCTGAGCT ACCTGAGGAGACGGTGAC CCTCAGTGAAGGTTTCCTGCAAGG TCCTGCGCTGGTGAAACCCACACA GGTCCCTGAGACTCTCCTGTGCA TCGGAGACCCTGTCCCTCACCTGC GAAAAAGCCCGGGGAGTCTCTGA CCTGTGCCATCTCCGGGGACAGTG ACCAGGGTCCCTTGGCCCCA AGCGGATAACAATTTCACAC * All

Primer Name

Primer Use

VH1 leader VH3 leader JH external VH1FR1 VH2FR1 VH3FR1 VH4FR1 VH5FR1 VH6FR1 JH nested M13 reverse tag

PCR primer PCR primer PCR primer PCR and sequencing PCR and sequencing PCR and sequencing PCR and sequencing PCR and sequencing PCR and sequencing PCR primer Sequencing primer

sequences are written 5' to 3'.

directly for sequencing using the family-specific FR1 primer or M13-reverse primer for sequencing reactions.45 Sequencing runs were performed on a Beckman Coulter CEQ 2000 XL8 capillary DNA sequencer (Beckman Coulter) using dye-terminator chemistry. The sequences obtained from each sample were compared with previously published germline VH, DH, and JH segment sequences in the V Base sequence directory (http://www.mrc-cpe.cam.ac.uk/vbase), and the closest germline sequence was assigned. If 2% or more of the VH gene sequence differed from the germline sequence, the sample was classified as mutated.

only known female case (Figure 1, IV:9) is a cousin of the proband. The median age at diagnosis was 55 years (range, 34-77 years). The affected father was deceased at the time of the study. Absolute lymphocyte counts at the time of diagnosis were available for 4 of 7 affected people and ranged from 7.0 × 109/L (case IV:1) to 164.9 × 109/L (case III:1). Of the 6 living affected members, 5 had received treatment for B-CLL at the time of the study. The remaining affected family member was initially identified as a result of participation in the study and had not been treated. In addition to B-CLL, 1 of the 4 brothers (Figure 1, IV:3) also had prostate carcinoma. A detailed family history indicated other unaffected family members with prostate (Figure 1, III:7) or colon (Figure 1, III:6) carcinoma and 1 additional family member with a malignancy of unknown type (Figure 1, II:3).

Results

Cytogenetic Features The del13q14 was identified by interphase FISH in peripheral blood samples from all 6 affected family members alive at the time of the study and was the sole abnormality in 4 of 6 cases ❚Table 2❚. In 1 patient (IV:3), interphase FISH cytogenetic studies demonstrated deletion of both copies of

Clinical Features B-CLL was diagnosed clinically in 7 family members (Figure 1), including the affected father (Figure 1, III:1), his 4 sons (Figure 1, monozygotic twins IV:1 and IV:2, IV:3, and IV:4), and a son of 1 of the twin brothers (Figure 1, V:4). The ❚Table 2❚ Clinical and Laboratory Features of Family Members With B-CLL Case No.

Age at Duration of Absolute LymphoDiagnosis (y) Disease (y) cytes (× 109/L)* Karyotype

FISH (% Nuclei)

VH Mutations

IV:1 IV:2 IV:3 IV:4

56 54 51 39

13 15 29 34

90.4 13.3 NA 202.0

46,XY 46,XY 46,XY NR

del13q14 (84) del13q14 (18); del14q32 (34) del13q14 (9); nullisomy 13q14 (68) del13q14 (95)

P P P P

IV:9

58

11

3.9

46,XX

del13q14 (39); +12 (57)

P

V:4

34

2

24.3

NA

del13q14 (90)

P

CD5

CD38

ZAP-70 (% Positive B-CLL Cells)

P P NA P, low density P, low density P

N N NA N

N (8) P (27) NA N (3)

N

P (97)

N

P (46)

B-CLL, B-cell chronic lymphocytic leukemia; FISH, fluorescence in situ hybridization; N, negative; NA, no sample available for testing; NR, no result owing to the lack of mitoses; P, positive. * Absolute lymphocyte count at the time of the study. The specimen for case IV:9 was obtained shortly after therapy. Values are given in Système International units; to convert to conventional units (/µL), divide by 0.001.

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chromosome 13q14 (nullisomy) in 68% of the nuclei and deletion of 1 copy of chromosome 13q14 in 9% of the nuclei. Conventional cytogenetic studies following 72-hour cultures with mitogen stimulation of this patient’s peripheral blood sample demonstrated a normal karyotype, and FISH studies for chromosome 13q14 performed on buccal mucosa were normal, confirming that del13q14 was an acquired cytogenetic abnormality. Repeated interphase FISH studies performed on a peripheral blood sample obtained 4 years later demonstrated nullisomy of 13q14 in 80% of nuclei, del13q14 in 17% of nuclei, and an additional deletion of the p53 locus at 17p13.1 in 73% of nuclei, consistent with cytogenetic progression. The del13q14 was accompanied by a deletion of 14q32 in 1 case (Figure 1, IV:2) and trisomy 12 (Figure 1, IV:9) in another. Interphase FISH studies for case IV:9 showed trisomy 12 and del13q14 abnormalities present simultaneously in the majority of nuclei. Conventional cytogenetic studies of peripheral blood samples from both of these patients yielded normal karyotypes. Interphase FISH studies for abnormalities of 13q14 and 14q32 performed on buccal mucosa cells from case IV:2 were negative for abnormalities of either region. Additional skin or buccal mucosa samples were not available for case IV:9. Conventional cytogenetic (5 cases) and/or interphase FISH (9 cases) studies performed on peripheral blood samples from 9 unaffected family members did not demonstrate any constitutional or acquired abnormalities. Immunophenotypic Features Immunophenotyping by flow cytometry was performed on peripheral blood samples from 5 of 6 living affected members and showed the typical CD5+, CD19+, dim CD20+, and CD23+ pattern of B-CLL, with low- or intermediate-density monotypic κ (2/5) or λ (3/5) light chain expression. All 5 cases were negative for CD38. However, 3 of the 5 were positive for ZAP-70 (Table 2). Of the 5 cases, 2 had low-density (dim) CD5 expression, and the other 3 showed moderately intense expression. Neither ZAP-70 expression nor intensity of CD5 expression correlated with mutational status.

Immunophenotyping by flow cytometry was also performed in 7 of the 9 clinically unaffected family members participating in the study. None of these family members had an absolute or relative lymphocytosis at the time of the study. No subclinical, CD5+, monoclonal B-cell populations were identified among these clinically unaffected family members. Immunoglobulin Gene Usage and Heavy Chain Variable Region Mutation Status For 6 affected members, material was available for VH mutation analysis. The mutation data are summarized in ❚Table 3❚. Sequencing of the PCR products demonstrated a single clonally rearranged VH gene in 4 of 6 cases. In the other 2 cases, 2 clonally rearranged VH genes were identified. The VH3 gene family was expressed in 5 of 6 cases, and VH2 was expressed in 1 case. In 3 of 5 cases using the VH3 family, the VH3-7 gene was rearranged. Both cases having 2 VH gene rearrangements used the VH3 and VH1 families. All 6 cases were classified as mutated in VH2 or VH3. In the 2 cases with 2 VH rearrangements, the VH1 family genes were unmutated. In both of these cases, expression of 2 in-frame rearrangements was confirmed by reverse transcriptase–PCR, suggesting the presence of 2 different clones in each sample. However, analysis of the phenotypic characteristics of the leukemic cells from these cases did not clearly identify 2 clonal populations, and the intensity of CD5 expression was uniform in both cases.

Discussion The del13q14 is the most common genetic abnormality found in B-CLL when tested by FISH methods and is associated with a good prognosis when present as a sole abnormality.4,11,46 The presence of an acquired del13q14 in tumor cells from all members of this family affected by B-CLL, including identical twins, strongly suggests an inherited predisposition to developing B-CLL. The pattern of inheritance in this family seems to be autosomal dominant, and the identification of an affected cousin (IV:9) is consistent with incomplete

❚Table 3❚ Mutation Analysis in Family Members With B-Cell Chronic Lymphocytic Leukemia Case No. IV:1 IV:2 IV:3 IV:4 IV:9 V:4

VH Family

VH Gene

VH3 VH1 VH3 VH3 VH2 VH3 VH1 VH3

3-11 1-46 3-15 3-7 2-5 3-7 1-2 3-7

% Identity 96.0 100.0 97.0 95.0 95.0 96.0 99.5 93.0

DH Gene

JH Gene

D1-14 D3-22 D6-13 D4-17 D5-5 D6-13 D2-2 D3-22

JH4 JH4 JH4 JH3 JH3 JH6 JH4 JH3

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penetrance. Because the father and his 4 sons lived on a farm, an alternative explanation for this familial cluster would be a common environmental exposure. However, environmental risk factors have not been clearly identified in sporadic or familial B-CLL, although a small increase in risk of developing B-CLL has been associated with farming and has been attributed to exposure to certain pesticides (reviewed by Caporaso et al47). In this family, neither the offspring of one of the brothers, case V:4, nor the affected cousin, case IV:9, grew up on the same farm as the 4 brothers. This latter finding also supports the hypothesis that an inherited abnormality, rather than a common environmental exposure, had an important role in this familial disease cluster. To our knowledge, only 3 other studies have used FISH to systematically examine familial B-CLL cases for the presence of recurrent cytogenetic abnormalities commonly identified in sporadic cases. Summersgill et al36 identified a sibling pair with trisomy 12, and Espinet et al37 identified 2 brothers with different abnormal karyotypes by conventional studies but who shared del13q14 as shown by FISH studies. Mehes et al39 described 2 brothers with diploid Y chromosomes by FISH studies and Y chromosome gains by comparative genomic hybridization; 1 of the brothers was also positive for del13q14. Our findings, in conjunction with these reports, support the hypothesis that familial and sporadic B-CLL cases undergo similar genetic events during pathogenesis. A recent study of cytogenetic aberrations in sporadic cases of B-CLL found that in some patients, del13q14 was present in only a subset of purified CD5+CD19+ cells, suggesting that del13q14 is not the initiating oncogenic event.11 All of the affected family members in our study acquired the del13q14. Recent studies have suggested several candidate pathogenetic genes on 13q14.48,49 The predisposing gene in this family is an area of continuing study and may provide additional insights into the pathogenetic events in B-CLL. Anticipation, a phenomenon in which disease onset or increased severity of disease occurs at younger ages in successive generations, has been described in other familial B-CLL clusters, with a decrease in age of disease onset of approximately 16 to 20 years between generations.26,29,31 A similar phenomenon was observed in this family. The age of onset was 77 years in the first known affected person (Figure 1, III:1). The mean age of onset in the next generation (IV) decreased to 53 years. In the most recent generation (V), the sole affected member identified to date, case V:4, was 36 years old at diagnosis. However, it should be noted that this latter patient was diagnosed with B-CLL as a result of participation in the study and was initially clinically asymptomatic. Therefore, ascertainment bias cannot be completely excluded as an explanation for the earlier age of onset observed in this patient. Interphase FISH studies performed on peripheral blood samples from other members of generation V were all negative 36 36

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for del13q14 at the time of study. Previous studies using flow cytometry for screening of larger cohorts have reported detection of subclinical clones with immunophenotypic features of B-CLL in 3.5% of the general population50,51 and in 14% to 18% of clinically unaffected relatives of patients with BCLL.33,40 These studies suggest that the risk of family members harboring subclinical disease may be as high as 7-fold.33 However, flow cytometric studies of peripheral blood samples performed in 7 of our clinically and cytogenetically unaffected generation V cases did not detect subclinical monoclonal Bcell populations. Our findings may be because we have tested only a small number (7) of the healthy members of this family. ZAP-70 expression was identified by flow cytometry in 3 cases. Preliminary studies have suggested that ZAP-70 overexpression in familial B-CLL cases occurs at rates similar to sporadic cases.41 ZAP-70 expression has been associated with unmutated VH genes and a worse prognosis.8-10,20,52-55 In 2 of the ZAP-70+ cases in our study, additional cytogenetic abnormalities (+12 and del14q32) were identified. In addition, the case with the highest proportion of B-CLL cells expressing ZAP-70 had 2 clonal VH rearrangements, one of which was unmutated. ZAP-70 expression was also detected in case V:4 with del13q14 as the sole abnormality and mutated VH genes, both factors generally associated with a good prognosis. Previous studies of sporadic cases, however, have also demonstrated that ZAP-70 expression and mutational status are discordant in a subset of cases. The relationship between specific cytogenetic abnormalities and ZAP-70 expression has not been extensively examined and merits additional study. Limited heterogeneity in VH family gene usage was present in this family. The most frequently used VH gene was VH3, and 3 of 5 family members with VH3 rearrangements used VH 3-7. Previous studies of familial B-CLL clusters have also reported limited heterogeneity in VH gene family usage.28,42-44 The VH3 gene family is expressed at the highest frequency in familial B-CLL 28,44 and in IgM-positive sporadic cases of BCLL.13 The VH3-7 gene, however, has not been specifically studied in B-CLL. Previous studies have also noted a high incidence of VH mutations in sporadic B-CLL cases with del13q14, 6,7 a low incidence in cases with trisomy 12,7 and a mixed pattern in rare cases with both chromosomal abnormalities.7 All of the affected members of this family with del13q14 had mutated VH3 or VH2 genes. In 2 affected members, a second clonal VH1 gene rearrangement was identified, and in each case, the second rearrangement was unmutated. Concurrent interphase FISH studies performed on the specimen from case IV:9 demonstrated +12 and del13q14. Both cytogenetic abnormalities were identified by FISH in most nuclei from this specimen, with only occasional nuclei observed to carry only +12 or del13q14. Although flow cytometry did not clearly demonstrate 2 clonal populations in either case, in each case, both VH rearrangements were © American Society for Clinical Pathology

Hematopathology / ORIGINAL ARTICLE

expressed in-frame. It is unlikely that a single B-cell clone would express 2 VH genes. More likely, 2 clones expressing different VH genes were present in each of these samples. For each of the 2 cases with 2 clonal rearrangements, both rearrangements were sequenced and shown to be unrelated (Table 3). The molecular cytogenetic abnormalities, pattern of immunoglobulin gene usage and mutation, and the immunophenotypic findings in this family are similar to those seen in sporadic B-CLL cases. Our findings support previous observations that familial cases of B-CLL demonstrate similarities to sporadic cases, and, therefore, familial clusters are likely to be useful for studying predisposing and initiating genetic events in B-CLL. From the Departments of 1Pathology and Microbiology and 2Pediatrics, University of Nebraska Medical Center; and 3Preventive Medicine and Public Health, Creighton University School of Medicine, Omaha. Supported in part by grant USPHS CA36727 from the National Cancer Institute, Department of Health and Human Services, Bethesda, MD. Address reprint requests to Dr Aoun: Dept of Pathology and Microbiology, University of Nebraska Medical Center, 983135 Nebraska Medical Center, Omaha, NE 68198-3135.

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