- Email: [email protected]
IGHV Unmutated Status Influences Outcome More Than IGHV1-69 Gene Usage Per Se in Patients With Chronic Lymphocytic Leukemia
Brief Communication
IGHV Unmutated Status Influences Outcome More Than IGHV1-69 Gene Usage Per Se in Patients With Chronic Lymphocytic Leukemia Ester M. Orlandi, Silvia Zibellini, Cristiana Pascutto, Cristina Picone, Ilaria Giardini, Lara Pochintesta, Mario Lazzarino Abstract In this study, IGHV1-69 gene usage was detected in 46 out of 379 cases (12%) of chronic lymphocytic leukemia (CLL). In comparison with patients using alternative immunoglobulin heavy-chain variable (IGHV) genes, patients with IgHV1-69 CLLs more often presented at advanced stage, lacked somatic hypermutation (unmutated cases, 87% vs. 35%; P = .00001), and expressed unfavorable biologic characteristics. In 12 patients (26%), common amino acid motifs within the heavychain third complementarity-determining region were identified, allowing assignment to previously reported stereotyped subsets. In our study, treatment-free survival of patients with unmutated IGVH1-69 did not differ significantly from that of patients expressing unmutated alternative IGHV genes. As such, IGHV1-69 gene usage per se did not seem to be predictive of progressive disease, progression being primarily related to the unmutated IGHV profile. Clinical Lymphoma & Myeloma, Vol. 9, No. 5, 390-393, 2009; DOI: 10.3816/CLM.2009.n.076 Keywords: CD38, Binet stage, Gene rearrangement, HCDR3, ZAP70
Introduction
Patients and Methods
Chronic lymphocytic leukemia (CLL) cells express a restricted heavy-chain variable (HV) gene repertoire, and at least 27% of patients with CLL share closely homologous (stereotyped) B-cell antigen receptors (BCRs),1 suggesting common antigenic reactivity. Among immunoglobulin (IG)HV–expressed genes, IGHV1-69 is particularly intriguing: it is rearranged in a significant proportion of patients with CLL (10%-20%) and carries very few somatic mutations, and in several reported cases, stereotyped heavy-chain third complementarity-determining regions (HCDR3s) have been detected, with stereotypy showing clinical implications.1 On the contrary, IGVH1-69 gene usage and stereotypy are very uncommon among patients with CLL-like monoclonal B-cell lymphocytosis.2 In the current study, we evaluate the clinical and biologic characteristics and outcome in 46 patients with IGHV1-69 CLL in comparison with patients with non–IGHV1-69, to examine the clinical relevance of IGHV1-69 gene expression as a marker of prognosis.
From 1991 to September 2008, 391 patients with CLL were fully characterized for biologic prognostic parameters. The study was performed on peripheral blood (PB) or bone marrow (BM) samples collected after informed consent for routine diagnostic and followup procedures at diagnosis in 75% of cases and during follow-up, but before any treatment in the remaining cases. The diagnosis of CLL was according to National Cancer Institute-Working Group (NCI-WG) 1996 criteria and clinical stage according to Binet.
Clinic of Hematology, Fondazione IRCCS Policlinico San Matteo, University of Pavia, Italy Submitted: Jan 1, 2009; Revised: Feb 18, 2009; Accepted: Mar 2, 2009 Address for correspondence: Ester M. Orlandi, MD, Clinic of Hematology, Fondazione IRCCS Policlinico San Matteo, Viale Camillo Golgi, 19 27100 Pavia, Italy Fax: 39-0382-502250; e-mail: [email protected]
Analysis of Ig Gene Sequences Only immunoglobulin heavy chains were analyzed. Total RNA was extracted from PB or BM mononuclear cells; IGHV gene rearrangements were amplified using 6 family-specific HV leader primers together with a unique primer complementary to a constant region of cμ chain or with a HJ consensus primer. Polymerase chain reaction products were sequenced directly (automated ABI 3100 DNA sequencer using BigDye chemistry), and the sequences were analyzed using IMGT database and tools (ImMunoGeneTics; http://www.imgt.com). Sequences with homology to the nearest germline gene ≥ 98% were considered unmutated. The length of HCDR3 was computed according to IMGT.
Immunophenotype ZAP70 expression was investigated by flow cytometry using a
This summary may include the discussion of investigational and/or unlabeled uses of drugs and/or devices that may not be approved by the FDA. Electronic forwarding or copying is a violation of US and International Copyright Laws. Authorization to photocopy items for internal or personal use, or the internal or personal use of specific clients, is granted by CIG Media Group, LP, ISSN #1557-9190, provided the appropriate fee is paid directly to Copyright Clearance Center, 222 Rosewood Drive, Danvers, MA 01923 USA. www.copyright.com 978-750-8400.
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combination of 6 monoclonal antibodies (CD3-PE, CD56-PE, CD19Per-CPC5.5, CD5-APC, CD45-PECy7, and ZAP70 Alexa Fluor 488, Caltag Labs.; fixation and permeabilization kit: FIX & PERM, Caltag Labs.). ZAP70 expression was evaluated on T and natural killer cells and on pathologic CD5+/CD19+ lymphocytes. Samples with ≥ 20% ZAP70 B-CLL cells were considered positive. CD38 expression was determined using anti–CD38-PE (Becton Dickinson Labs), and the percentage of CD38 cells was measured in the CD19+/CD5+ fraction. Cases with ≥ 30% CD38 B-CLL cells were considered positive.
Interphase Fluorescence In Situ Hybridization Separate hybridization reactions were performed using probes for ATM (11q23.3), 13q14 (locus D13S319), p53 (17p13.1), and a centromeric probe (D12Z3) for chromosome 12 (Vysis, Inc.; Downers Grove, IL). At least 200 interphase nuclei were evaluated in each reaction.
Table 1 Clinical and Biologic Characteristics in 46 Patients With IGHV1-69 and 333 Patients With Non–IGHV1-69 Characteristic
P Value
IgHV1-69
Non–IGHV1-69
58 (35-81)
59 (27-85)
NS
35/11
192/141
.025
Stage A
33
286
–
Stage B
11
34
–
Stage C
2
13
–
Median Age, Years (Range) Male/Female Disease Stage, n
Stages B + C, n (%)
13 (28)
47 (14)
.02
Unmutated, n (%)
40 (87)
118 (35)
.00001
ZAP70 Positive, n (%)
18 (39)
70 (21)
.01
CD38 Positive, n (%)
12 (26)
46 (14)
.05
FISH, n
Statistical Analysis
Del(13q14)
10
149
–
Continuous variables are described in terms of median value and range. Categorical variables are summarized with count and relative frequency (%) of each category. Comparisons were performed using the Fisher exact test (2-tailed) for qualitative variables, and the nonparametric Mann-Whitney test was used for continuous variables. As to the outcome, we adopted treatment-free survival (TFS) as the clinical endpoint, being a reliable surrogate marker for disease progression. Treatment-free survival was defined as the time between date of diagnosis and the date of initiation of first treatment or date of last follow-up at which patients were known to be untreated. Treatment-free survival was estimated using the Kaplan-Meier method, and the Gehan-Wilcoxon test was applied to compare TFS between groups.
+12
11
25
–
Del(11q23)
5
28
–
Results In 12 of 391 patients, IGHV rearrangement was not assessable; 379 cases were evaluable. The most common HV family was HV3 (n = 196), followed by HV1 (n = 92) and HV4 (n = 80). IGHV1-69 was rearranged in 46 patients (12%). In Table 1, clinical and biologic characteristics of these patients are compared with 333 non–IGHV1-69 patients. We did not observe any association of IGHV1-69 with advanced age, whereas we did record a significant male predominance (P = .025). Advanced-stage disease (stage B or C) was more frequent in the IGHV1-69 subset. In the IGHV1-69 group, 40 patients (87%) did not have mutations, with 100% homology to germline in 38, whereas 6 patients had mutation, with homology < 96%. Conversely, in the non–IGHV1-69 group, 118 patients (35%) were not mutated (P < .00001). Unfavorable chromosome abnormalities, ie, deletion of chromosome 11q (del[11q]) and del(17p), as well as ZAP70 and CD38 expression were significantly more frequent in the IGHV1-69 cohort. Because in this group, negative prognostic parameters were exclusively associated with the unmutated status, we compared patients with unmutated IGHV1-69 with unmutated patients using alternative IGHV genes and did not observe any significant difference (ZAP70 expression: 18/40 vs. 50/118; CD38 expression: 12/40 vs. 30/118; unfavorable chromosomal aberrations: 14/40 vs. 31/118). Serology for hepatits
Del(17p)
9
15
–
No abnormalities
11
116
–
Unfavorable, n (%)
14 (30); All unmutated
43 (13); 31 Unmutated
.0037
Treatment, n Stages A + B Only
30/44
126/320
.0005
Treatment, n Unmutated Stages A + B Only
27/38
62/110
.16
Abbreviations: Del = deletion; FISH = fluorescence in situ hybridization; NS = nonsignificant
C virus was evaluated in 206 and was positive in 3 of 26 patients and in 10 of 180 patients in the 2 groups, respectively (P = .4). We were able to determine both IGHD and IGHJ gene usage in all patients with IGHV1-69: the most frequent HD genes were HD3-3 (n = 13), HD2-2 (n = 6), and HD3-10 (n = 5); HJ6 (n = 24) and HJ4 (n = 13) accounted for 78% of HJ genes. The combination HD3-3 with HJ6 was detected in 10 patients. HCDR3 median length was 22 amino acids for unmutated patients (range, 16-30 amino acids) and 15 for patients with mutations (range, 14-16 amino acids). We performed alignment analysis of the HCDR3 sequences and comparison with previously identified stereotyped subsets.1 In 12 patients (26%, all unmutated), common amino acid motifs within the HCDR3 were identified, allowing assignment to reported subsets (Table 2). Nine of these patients shared IGHV1-69 in combination with HD3-3 and HJ6, using alternative HD reading frames. Regarding TFS, we analyzed the impact of IGHV1-69 gene usage in 364 patients with stage A and B disease, as stage C patients were treated at diagnosis. The accepted indications to start treatment were based on the NCI-WG 1996 criteria during the study interval. At a median follow-up for both groups of 42 months, 30 of 44 patients (68%) and 126 of 320 patients (39%) received treatment (P = .0005), and TFS was shorter for patients with IGHV1-69 (median TFS, 29 months and 58 months, respectively;
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IGHV1-69 Gene Usage and Prognosis in CLL Table 2 IGHV1-69 Cases Showing HCDR3 Similarity to Published Stereotyped Subsets UPN
HD (RF)
HJ
Homology, %
HCDR3
HCDR3 Length, Amino Acids
Subset1
1
2-2*02 (3)
6*02
100
CAR ELPDIVVVPAAISRYYGMDV W
22
3
2
2-2*02 (3)
6*02
100
CAT PGQDWDIVVVPAAISYYYYGMDV W
25
3
3
3-16*02 (2)
3*02
100
CAR GGVYDYIWGSYRANDAFDI W
21
6
4
3-3*01 (2)
6*02
100
CAS GSPPYDFWSGYYPNYYYYGMDV W
24
7
5
3-3*01 (2)
6*02
100
CAR AGEGDFWSGYYPSNYYYGMDV W
23
7
6
3-3*01 (2)
6*02
100
CAR DCYDFWSGYRSLYYYYYGMDV W
23
7
7
3-3*01 (2)
6*02
100
CAR GTPNYDFWSGYSASYYYYYGMDV W
25
7
8
3-3*01 (2)
6*02
98.6
CAR VERPYYDFWSDYAVYYYYYGMDV W
24
7
9
3-3*01 (3)
6*02
100
CAR RGITIFGVVIYYYYGMDV W
20
9
10
3-3*01 (3)
6*02
100
CAR APTGEITIFGVADYYYYGMDV W
23
9
11
3-3*01 (3)
6*02
100
CAR DLGDLTIFGVVITNYYYYYGMDV W
25
9
12
3-3*01 (3)
6*02
100
CAG RKGDTIFGVVIHQYYYYYGMDV W
25
9
Abbreviations: RF = reading frame; HD = heavy-chain diversity region; HJ = heavy-chain join region; UPN = unique patient number
Figure 1 Treatment-Free Survival in 44 Patients With IgHV1-69 Versus 320 Patients With Non–IgHV1-69 A
100
44 IgHV1-69 Patients 320 non–IgHV1-69 Patients
Cumulative Probability of TFS
90 80 70 60 50 40 30 20 10
Discussion
0 0
24
48
72
96
120
144
168
192
216
240
Time, Months
B
100
44 IgHV1-69 Patients 320 non–IgHV1-69 Patients
Cumulative Probability of TFS
90 80 70 60 50 40 30 20 10 0 0
24
48
72
96
120
144
168
192
216
240
Time, Months A. TFS in 38 patients with UM IgHV1-69 vs. 110 patients with UM non–IgHV1-69 B. Only Binet stage A and B patients were included. Abbreviations: TFS = treatment-free survival; UM = unmutated
392
P = .0006; Figure 1A). When only unmutated patients were included in the analysis, these differences lost statistical significance (Table 1; P = .16; Figure 1B, P = .2338), although median TFS was somewhat shorter for IGHV1-69 unmutated patients (26 months and 37 months, respectively). As for patients with mutated IGHV1-69, 3 of 6 required treatment at 2 months, 32 months, and 41 months, respectively, from diagnosis. The small number of cases precludes any definitive conclusion on outcome associated with the mutated profile. Comparing unmutated HV1-69 patients with unmutated patients belonging to HV1 family not expressing 1-69 gene, to HV3 family, and to HV4 family, no significant differences were noted in terms of proportions of patients requiring treatment and TFS. In the IGHV1-69 group, stereotypy did not influence need of therapy, as 8 of 12 cases assigned to stereotyped subsets required treatment compared with 23 of 34 in the heterogeneous set (P = .7).
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In this study, relative expression of HV families and the frequency of IGHV1-69 gene usage in our series (12%) were consistent with those observed in large CLL cohorts with a different geographical composition.1,3 At our institution, the usage of the IGHV1-69 gene was uncommon in patients with non-Hodgkin lymphoma (NHL): IGHV1-69 gene was demonstrated on PB/BM cells in 3 among 59 patients with splenic marginal zone NHL (5%)4 and in 10 of 156 patients with NHL (6%) with different histology in whom IGHV-D-J rearrangement analysis was carried out (personal data). We confirm the predominance of male sex5 and unmutated status,1,6,7 along with high frequency of negative prognostic features such as unfavorable genetic abnormalities and ZAP70 and CD38 expression. In particular, this association seems to be related to the predominance of the unmutated profile in the IGHV1-69 cohort. Preferential association with certain HD and HJ genes encoding relatively long HCDR3 regions was observed, and in 12 cases (26%), distinct amino acid motifs could be identified, determining marked homology to stereotyped HCDR3 subsets.1 Specific HV gene rearrangements have been associated with an independent prognostic role. In this respect, patients with
Ester M. Orlandi et al IGHV3-21 CLL commonly show highly homologous HCDR3s and experience poor outcome regardless of the mutational status.8 As for the clinical relevance of IGHV1-69 gene usage, some uncertainty remains. Although IGHV1-69 expression has been associated with an unfavorable clinical course and a distinct biology of the HV1-69 subset among unmutated CLLs has been suggested,9 IGHV1-69 usage did not influence overall survival in a cohort of 25 unmutated cases,5 whereas the mutated profile appeared to correlate with indolent disease in another published series.7 Moreover, a relationship was found between the type of stereotyped HCDR3 and outcome, with different clinical course between subsets.1 In our study of a large cohort of patients with CLL, TFS of patients with unmutated IGVH1-69 did not differ significantly from that of unmutated patients expressing alternative IGHV genes. As such, IGHV1-69 gene usage per se does not turn out to be predictive of progressive disease. Poor prognosis in IGHV1-69 CLL seems related to the high proportion of unmutated patients without mutations, who are more likely to express adverse biologic characteristics and to display differential responses to BCR signaling.10
Acknowledgments Ester M. Orlandi designed the study, collected and analyzed clinical data, and drafted the article. Silvia Zibellini performed IGHV analysis. Cristina Pascutto performed statistical analysis. Cristina Picone performed immunophenotyping. Ilaria Giardini performed fluorescence in situ hybridization analysis. Lara Pochintesta col-
lected and analyzed clinical data. All authors revised the manuscript critically and gave final approval of the version to be submitted.
Disclosures The authors have no relevant relationships to disclose.
References 1. Stamatopoulos K, Belessi C, Moreno C, et al. Over 20% of patients with chronic lymphocytic leucemia carry stereotyped receptors. Pathogenetic implications and clinical correlations. Blood 2007; 109:259-70. 2. Dagklis A, Fazi C, Sala C, et al. The immunoglobulin gene repertoire of low-count CLL like MBL is different from CLL: diagnostic implications for clinical monitoring. Blood 2009; 114:26-32. 3. Murray F, Darzentas N, Hadzidimitriou A, et al. Stereotyped pattern of somatic mutation in subsets of patients with chronic lymphocytic leukaemia: implications for the role of antigene selection in leukemogenesis. Blood 2008; 111:1524-33. 4. Zibellini S, Arcaini L, Passamonti F, et al. Splenic marginal zone B-cell lymphoma: clinical clustering of immunoglobulin heavy chain repertoires. Blood 2008; 112: (Abstract 1775). 5. Walewska R, Majid A, Davis Z, et al. Male preponderance in chronic lymphocytic leukemia utilizing IGHV 1-69. Leukemia 2007; 21:2537-8. 6. Panovska-Stavridis I, Ivanovski M, Siljanovski N, et al. Chronic lymphocytic leukaemia patients with a V1-69 gene rearrangement do not have inferior survival with respect to patients that express other unmutated V(H) genes. Leuk Res 2007; 31:245-8. 7. Galligan L, Catherwood MA, Matthews C, et al. Mutated IgHV1-69 gene usage represents a distinct subgroup associated with indolent disease in chronic lymphocytic leukemia. Leuk Lymphoma 2008; 49:763-8. 8. Thorsélius M, Kröber A, Murray F, et al. Strikingly homologous immunoglobulin gene rearrangements and poor outcome in VH3-21-using chronic lymphocytic leukemia patients independent of geographic origin and mutational status. Blood 2006; 107:2889-94. 9. Kienle D, Benner A, Kröber A, et al. Distinct gene expression patterns in chronic lymphocytic leukemia defined by usage of specific VH genes. Blood 2006; 107:2090-3. 10. Guarini A, Chiaretti S, Tavolaro S, et al. BCR ligation induced by IgM stimulation results in gene expression and functional changes only in IgV H unmutated chronic lymphocytic leukemia (CLL) cells. Blood 2008; 112:782-92.
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IGHV Unmutated Status Influences Outcome More Than IGHV1-69 Gene Usage Per Se in Patients With Chronic Lymphocytic Leukemia Ester M. Orlandi, Silvia Zibellini, Cristiana Pascutto, Cristina Picone, Ilaria Giardini, Lara Pochintesta, Mario Lazzarino Abstract In this study, IGHV1-69 gene usage was detected in 46 out of 379 cases (12%) of chronic lymphocytic leukemia (CLL). In comparison with patients using alternative immunoglobulin heavy-chain variable (IGHV) genes, patients with IgHV1-69 CLLs more often presented at advanced stage, lacked somatic hypermutation (unmutated cases, 87% vs. 35%; P = .00001), and expressed unfavorable biologic characteristics. In 12 patients (26%), common amino acid motifs within the heavychain third complementarity-determining region were identified, allowing assignment to previously reported stereotyped subsets. In our study, treatment-free survival of patients with unmutated IGVH1-69 did not differ significantly from that of patients expressing unmutated alternative IGHV genes. As such, IGHV1-69 gene usage per se did not seem to be predictive of progressive disease, progression being primarily related to the unmutated IGHV profile. Clinical Lymphoma & Myeloma, Vol. 9, No. 5, 390-393, 2009; DOI: 10.3816/CLM.2009.n.076 Keywords: CD38, Binet stage, Gene rearrangement, HCDR3, ZAP70
Introduction
Patients and Methods
Chronic lymphocytic leukemia (CLL) cells express a restricted heavy-chain variable (HV) gene repertoire, and at least 27% of patients with CLL share closely homologous (stereotyped) B-cell antigen receptors (BCRs),1 suggesting common antigenic reactivity. Among immunoglobulin (IG)HV–expressed genes, IGHV1-69 is particularly intriguing: it is rearranged in a significant proportion of patients with CLL (10%-20%) and carries very few somatic mutations, and in several reported cases, stereotyped heavy-chain third complementarity-determining regions (HCDR3s) have been detected, with stereotypy showing clinical implications.1 On the contrary, IGVH1-69 gene usage and stereotypy are very uncommon among patients with CLL-like monoclonal B-cell lymphocytosis.2 In the current study, we evaluate the clinical and biologic characteristics and outcome in 46 patients with IGHV1-69 CLL in comparison with patients with non–IGHV1-69, to examine the clinical relevance of IGHV1-69 gene expression as a marker of prognosis.
From 1991 to September 2008, 391 patients with CLL were fully characterized for biologic prognostic parameters. The study was performed on peripheral blood (PB) or bone marrow (BM) samples collected after informed consent for routine diagnostic and followup procedures at diagnosis in 75% of cases and during follow-up, but before any treatment in the remaining cases. The diagnosis of CLL was according to National Cancer Institute-Working Group (NCI-WG) 1996 criteria and clinical stage according to Binet.
Clinic of Hematology, Fondazione IRCCS Policlinico San Matteo, University of Pavia, Italy Submitted: Jan 1, 2009; Revised: Feb 18, 2009; Accepted: Mar 2, 2009 Address for correspondence: Ester M. Orlandi, MD, Clinic of Hematology, Fondazione IRCCS Policlinico San Matteo, Viale Camillo Golgi, 19 27100 Pavia, Italy Fax: 39-0382-502250; e-mail: [email protected]
Analysis of Ig Gene Sequences Only immunoglobulin heavy chains were analyzed. Total RNA was extracted from PB or BM mononuclear cells; IGHV gene rearrangements were amplified using 6 family-specific HV leader primers together with a unique primer complementary to a constant region of cμ chain or with a HJ consensus primer. Polymerase chain reaction products were sequenced directly (automated ABI 3100 DNA sequencer using BigDye chemistry), and the sequences were analyzed using IMGT database and tools (ImMunoGeneTics; http://www.imgt.com). Sequences with homology to the nearest germline gene ≥ 98% were considered unmutated. The length of HCDR3 was computed according to IMGT.
Immunophenotype ZAP70 expression was investigated by flow cytometry using a
This summary may include the discussion of investigational and/or unlabeled uses of drugs and/or devices that may not be approved by the FDA. Electronic forwarding or copying is a violation of US and International Copyright Laws. Authorization to photocopy items for internal or personal use, or the internal or personal use of specific clients, is granted by CIG Media Group, LP, ISSN #1557-9190, provided the appropriate fee is paid directly to Copyright Clearance Center, 222 Rosewood Drive, Danvers, MA 01923 USA. www.copyright.com 978-750-8400.
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combination of 6 monoclonal antibodies (CD3-PE, CD56-PE, CD19Per-CPC5.5, CD5-APC, CD45-PECy7, and ZAP70 Alexa Fluor 488, Caltag Labs.; fixation and permeabilization kit: FIX & PERM, Caltag Labs.). ZAP70 expression was evaluated on T and natural killer cells and on pathologic CD5+/CD19+ lymphocytes. Samples with ≥ 20% ZAP70 B-CLL cells were considered positive. CD38 expression was determined using anti–CD38-PE (Becton Dickinson Labs), and the percentage of CD38 cells was measured in the CD19+/CD5+ fraction. Cases with ≥ 30% CD38 B-CLL cells were considered positive.
Interphase Fluorescence In Situ Hybridization Separate hybridization reactions were performed using probes for ATM (11q23.3), 13q14 (locus D13S319), p53 (17p13.1), and a centromeric probe (D12Z3) for chromosome 12 (Vysis, Inc.; Downers Grove, IL). At least 200 interphase nuclei were evaluated in each reaction.
Table 1 Clinical and Biologic Characteristics in 46 Patients With IGHV1-69 and 333 Patients With Non–IGHV1-69 Characteristic
P Value
IgHV1-69
Non–IGHV1-69
58 (35-81)
59 (27-85)
NS
35/11
192/141
.025
Stage A
33
286
–
Stage B
11
34
–
Stage C
2
13
–
Median Age, Years (Range) Male/Female Disease Stage, n
Stages B + C, n (%)
13 (28)
47 (14)
.02
Unmutated, n (%)
40 (87)
118 (35)
.00001
ZAP70 Positive, n (%)
18 (39)
70 (21)
.01
CD38 Positive, n (%)
12 (26)
46 (14)
.05
FISH, n
Statistical Analysis
Del(13q14)
10
149
–
Continuous variables are described in terms of median value and range. Categorical variables are summarized with count and relative frequency (%) of each category. Comparisons were performed using the Fisher exact test (2-tailed) for qualitative variables, and the nonparametric Mann-Whitney test was used for continuous variables. As to the outcome, we adopted treatment-free survival (TFS) as the clinical endpoint, being a reliable surrogate marker for disease progression. Treatment-free survival was defined as the time between date of diagnosis and the date of initiation of first treatment or date of last follow-up at which patients were known to be untreated. Treatment-free survival was estimated using the Kaplan-Meier method, and the Gehan-Wilcoxon test was applied to compare TFS between groups.
+12
11
25
–
Del(11q23)
5
28
–
Results In 12 of 391 patients, IGHV rearrangement was not assessable; 379 cases were evaluable. The most common HV family was HV3 (n = 196), followed by HV1 (n = 92) and HV4 (n = 80). IGHV1-69 was rearranged in 46 patients (12%). In Table 1, clinical and biologic characteristics of these patients are compared with 333 non–IGHV1-69 patients. We did not observe any association of IGHV1-69 with advanced age, whereas we did record a significant male predominance (P = .025). Advanced-stage disease (stage B or C) was more frequent in the IGHV1-69 subset. In the IGHV1-69 group, 40 patients (87%) did not have mutations, with 100% homology to germline in 38, whereas 6 patients had mutation, with homology < 96%. Conversely, in the non–IGHV1-69 group, 118 patients (35%) were not mutated (P < .00001). Unfavorable chromosome abnormalities, ie, deletion of chromosome 11q (del[11q]) and del(17p), as well as ZAP70 and CD38 expression were significantly more frequent in the IGHV1-69 cohort. Because in this group, negative prognostic parameters were exclusively associated with the unmutated status, we compared patients with unmutated IGHV1-69 with unmutated patients using alternative IGHV genes and did not observe any significant difference (ZAP70 expression: 18/40 vs. 50/118; CD38 expression: 12/40 vs. 30/118; unfavorable chromosomal aberrations: 14/40 vs. 31/118). Serology for hepatits
Del(17p)
9
15
–
No abnormalities
11
116
–
Unfavorable, n (%)
14 (30); All unmutated
43 (13); 31 Unmutated
.0037
Treatment, n Stages A + B Only
30/44
126/320
.0005
Treatment, n Unmutated Stages A + B Only
27/38
62/110
.16
Abbreviations: Del = deletion; FISH = fluorescence in situ hybridization; NS = nonsignificant
C virus was evaluated in 206 and was positive in 3 of 26 patients and in 10 of 180 patients in the 2 groups, respectively (P = .4). We were able to determine both IGHD and IGHJ gene usage in all patients with IGHV1-69: the most frequent HD genes were HD3-3 (n = 13), HD2-2 (n = 6), and HD3-10 (n = 5); HJ6 (n = 24) and HJ4 (n = 13) accounted for 78% of HJ genes. The combination HD3-3 with HJ6 was detected in 10 patients. HCDR3 median length was 22 amino acids for unmutated patients (range, 16-30 amino acids) and 15 for patients with mutations (range, 14-16 amino acids). We performed alignment analysis of the HCDR3 sequences and comparison with previously identified stereotyped subsets.1 In 12 patients (26%, all unmutated), common amino acid motifs within the HCDR3 were identified, allowing assignment to reported subsets (Table 2). Nine of these patients shared IGHV1-69 in combination with HD3-3 and HJ6, using alternative HD reading frames. Regarding TFS, we analyzed the impact of IGHV1-69 gene usage in 364 patients with stage A and B disease, as stage C patients were treated at diagnosis. The accepted indications to start treatment were based on the NCI-WG 1996 criteria during the study interval. At a median follow-up for both groups of 42 months, 30 of 44 patients (68%) and 126 of 320 patients (39%) received treatment (P = .0005), and TFS was shorter for patients with IGHV1-69 (median TFS, 29 months and 58 months, respectively;
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IGHV1-69 Gene Usage and Prognosis in CLL Table 2 IGHV1-69 Cases Showing HCDR3 Similarity to Published Stereotyped Subsets UPN
HD (RF)
HJ
Homology, %
HCDR3
HCDR3 Length, Amino Acids
Subset1
1
2-2*02 (3)
6*02
100
CAR ELPDIVVVPAAISRYYGMDV W
22
3
2
2-2*02 (3)
6*02
100
CAT PGQDWDIVVVPAAISYYYYGMDV W
25
3
3
3-16*02 (2)
3*02
100
CAR GGVYDYIWGSYRANDAFDI W
21
6
4
3-3*01 (2)
6*02
100
CAS GSPPYDFWSGYYPNYYYYGMDV W
24
7
5
3-3*01 (2)
6*02
100
CAR AGEGDFWSGYYPSNYYYGMDV W
23
7
6
3-3*01 (2)
6*02
100
CAR DCYDFWSGYRSLYYYYYGMDV W
23
7
7
3-3*01 (2)
6*02
100
CAR GTPNYDFWSGYSASYYYYYGMDV W
25
7
8
3-3*01 (2)
6*02
98.6
CAR VERPYYDFWSDYAVYYYYYGMDV W
24
7
9
3-3*01 (3)
6*02
100
CAR RGITIFGVVIYYYYGMDV W
20
9
10
3-3*01 (3)
6*02
100
CAR APTGEITIFGVADYYYYGMDV W
23
9
11
3-3*01 (3)
6*02
100
CAR DLGDLTIFGVVITNYYYYYGMDV W
25
9
12
3-3*01 (3)
6*02
100
CAG RKGDTIFGVVIHQYYYYYGMDV W
25
9
Abbreviations: RF = reading frame; HD = heavy-chain diversity region; HJ = heavy-chain join region; UPN = unique patient number
Figure 1 Treatment-Free Survival in 44 Patients With IgHV1-69 Versus 320 Patients With Non–IgHV1-69 A
100
44 IgHV1-69 Patients 320 non–IgHV1-69 Patients
Cumulative Probability of TFS
90 80 70 60 50 40 30 20 10
Discussion
0 0
24
48
72
96
120
144
168
192
216
240
Time, Months
B
100
44 IgHV1-69 Patients 320 non–IgHV1-69 Patients
Cumulative Probability of TFS
90 80 70 60 50 40 30 20 10 0 0
24
48
72
96
120
144
168
192
216
240
Time, Months A. TFS in 38 patients with UM IgHV1-69 vs. 110 patients with UM non–IgHV1-69 B. Only Binet stage A and B patients were included. Abbreviations: TFS = treatment-free survival; UM = unmutated
392
P = .0006; Figure 1A). When only unmutated patients were included in the analysis, these differences lost statistical significance (Table 1; P = .16; Figure 1B, P = .2338), although median TFS was somewhat shorter for IGHV1-69 unmutated patients (26 months and 37 months, respectively). As for patients with mutated IGHV1-69, 3 of 6 required treatment at 2 months, 32 months, and 41 months, respectively, from diagnosis. The small number of cases precludes any definitive conclusion on outcome associated with the mutated profile. Comparing unmutated HV1-69 patients with unmutated patients belonging to HV1 family not expressing 1-69 gene, to HV3 family, and to HV4 family, no significant differences were noted in terms of proportions of patients requiring treatment and TFS. In the IGHV1-69 group, stereotypy did not influence need of therapy, as 8 of 12 cases assigned to stereotyped subsets required treatment compared with 23 of 34 in the heterogeneous set (P = .7).
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In this study, relative expression of HV families and the frequency of IGHV1-69 gene usage in our series (12%) were consistent with those observed in large CLL cohorts with a different geographical composition.1,3 At our institution, the usage of the IGHV1-69 gene was uncommon in patients with non-Hodgkin lymphoma (NHL): IGHV1-69 gene was demonstrated on PB/BM cells in 3 among 59 patients with splenic marginal zone NHL (5%)4 and in 10 of 156 patients with NHL (6%) with different histology in whom IGHV-D-J rearrangement analysis was carried out (personal data). We confirm the predominance of male sex5 and unmutated status,1,6,7 along with high frequency of negative prognostic features such as unfavorable genetic abnormalities and ZAP70 and CD38 expression. In particular, this association seems to be related to the predominance of the unmutated profile in the IGHV1-69 cohort. Preferential association with certain HD and HJ genes encoding relatively long HCDR3 regions was observed, and in 12 cases (26%), distinct amino acid motifs could be identified, determining marked homology to stereotyped HCDR3 subsets.1 Specific HV gene rearrangements have been associated with an independent prognostic role. In this respect, patients with
Ester M. Orlandi et al IGHV3-21 CLL commonly show highly homologous HCDR3s and experience poor outcome regardless of the mutational status.8 As for the clinical relevance of IGHV1-69 gene usage, some uncertainty remains. Although IGHV1-69 expression has been associated with an unfavorable clinical course and a distinct biology of the HV1-69 subset among unmutated CLLs has been suggested,9 IGHV1-69 usage did not influence overall survival in a cohort of 25 unmutated cases,5 whereas the mutated profile appeared to correlate with indolent disease in another published series.7 Moreover, a relationship was found between the type of stereotyped HCDR3 and outcome, with different clinical course between subsets.1 In our study of a large cohort of patients with CLL, TFS of patients with unmutated IGVH1-69 did not differ significantly from that of unmutated patients expressing alternative IGHV genes. As such, IGHV1-69 gene usage per se does not turn out to be predictive of progressive disease. Poor prognosis in IGHV1-69 CLL seems related to the high proportion of unmutated patients without mutations, who are more likely to express adverse biologic characteristics and to display differential responses to BCR signaling.10
Acknowledgments Ester M. Orlandi designed the study, collected and analyzed clinical data, and drafted the article. Silvia Zibellini performed IGHV analysis. Cristina Pascutto performed statistical analysis. Cristina Picone performed immunophenotyping. Ilaria Giardini performed fluorescence in situ hybridization analysis. Lara Pochintesta col-
lected and analyzed clinical data. All authors revised the manuscript critically and gave final approval of the version to be submitted.
Disclosures The authors have no relevant relationships to disclose.
References 1. Stamatopoulos K, Belessi C, Moreno C, et al. Over 20% of patients with chronic lymphocytic leucemia carry stereotyped receptors. Pathogenetic implications and clinical correlations. Blood 2007; 109:259-70. 2. Dagklis A, Fazi C, Sala C, et al. The immunoglobulin gene repertoire of low-count CLL like MBL is different from CLL: diagnostic implications for clinical monitoring. Blood 2009; 114:26-32. 3. Murray F, Darzentas N, Hadzidimitriou A, et al. Stereotyped pattern of somatic mutation in subsets of patients with chronic lymphocytic leukaemia: implications for the role of antigene selection in leukemogenesis. Blood 2008; 111:1524-33. 4. Zibellini S, Arcaini L, Passamonti F, et al. Splenic marginal zone B-cell lymphoma: clinical clustering of immunoglobulin heavy chain repertoires. Blood 2008; 112: (Abstract 1775). 5. Walewska R, Majid A, Davis Z, et al. Male preponderance in chronic lymphocytic leukemia utilizing IGHV 1-69. Leukemia 2007; 21:2537-8. 6. Panovska-Stavridis I, Ivanovski M, Siljanovski N, et al. Chronic lymphocytic leukaemia patients with a V1-69 gene rearrangement do not have inferior survival with respect to patients that express other unmutated V(H) genes. Leuk Res 2007; 31:245-8. 7. Galligan L, Catherwood MA, Matthews C, et al. Mutated IgHV1-69 gene usage represents a distinct subgroup associated with indolent disease in chronic lymphocytic leukemia. Leuk Lymphoma 2008; 49:763-8. 8. Thorsélius M, Kröber A, Murray F, et al. Strikingly homologous immunoglobulin gene rearrangements and poor outcome in VH3-21-using chronic lymphocytic leukemia patients independent of geographic origin and mutational status. Blood 2006; 107:2889-94. 9. Kienle D, Benner A, Kröber A, et al. Distinct gene expression patterns in chronic lymphocytic leukemia defined by usage of specific VH genes. Blood 2006; 107:2090-3. 10. Guarini A, Chiaretti S, Tavolaro S, et al. BCR ligation induced by IgM stimulation results in gene expression and functional changes only in IgV H unmutated chronic lymphocytic leukemia (CLL) cells. Blood 2008; 112:782-92.
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