Immunoglobulin Variable Heavy Chain Research Articles

Vitamin D insufficiency and prognosis in chronic lymphocytic leukemia

AbstractVitamin D insufficiency is common globally and low levels are linked to higher cancer incidence. Although vitamin D insufficiency is related to inferior prognosis in some cancers, no data exist for chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL). We evaluated the relationship of 25(OH)D serum levels with time-to-treatment (TTT) and overall survival (OS) in newly diagnosed CLL patients participating in a prospective cohort study (discovery cohort) and a separate cohort of previously untreated patients participating in an observational study (confirmation cohort). Of 390 CLL patients in the discovery cohort, 119 (30.5%) were 25(OH)D insufficient. After a median follow-up of 3 years, TTT (hazard ratio[HR] = 1.66; P = .005) and OS (HR = 2.39; P = .01) were shorter for 25(OH)D-insufficient patients. In the validation cohort, 61 of 153 patients (39.9%) were 25(OH)D insufficient. After a median follow-up of 9.9 years, TTT (HR = 1.59; P = .05) and OS (HR 1.63; P = .06) were again shorter for 25(OH)D-insufficient patients. On pooled multivariable analysis of patients in both cohorts adjusting for age, sex, Rai stage, CD38 status, ZAP-70 status, immunoglobulin heavy chain variable (IGHV) gene mutation status, CD49d status, and cytogenetic abnormalities assessed by interphase fluorescent in situ hybridization testing, 25(OH)D insufficiency remained an independent predictor of TTT (HR = 1.47; P = .008), although the association with OS was not significant (HR = 1.47; P = .07). Vitamin D insufficiency is associated with inferior TTT and OS in CLL patients. Whether normalizing vitamin D levels in deficient CLL patients would improve outcome merits clinical testing.

Segmental duplication as one of the driving forces underlying the diversity of the human immunoglobulin heavy chain variable gene region

BackgroundSegmental duplication and deletion were implicated for a region containing the human immunoglobulin heavy chain variable (IGHV) gene segments, 1.9III/hv3005 (possible allelic variants of IGHV3-30) and hv3019b9 (a possible allelic variant of IGHV3-33). However, very little is known about the ranges of the duplication and the polymorphic region. This is mainly because of the difficulty associated with distinguishing between allelic and paralogous sequences in the IGHV region containing extensive repetitive sequences. Inability to separate the two parental haploid genomes in the subjects is another serious barrier. To address these issues, unique DNA sequence tags evenly distributed within and flanking the duplicated region implicated by the previous studies were selected. The selected tags in single sperm from six unrelated healthy donors were amplified by multiplex PCR followed by microarray detection. In this way, individual haplotypes of different parental origins in the sperm donors could be analyzed separately and precisely. The identified polymorphic region was further analyzed at the nucleotide sequence level using sequences from the three human genomic sequence assemblies in the database.ResultsA large polymorphic region was identified using the selected sequence tags. Four of the 12 haplotypes were shown to contain consecutively undetectable tags spanning in a variable range. Detailed analysis of sequences from the genomic sequence assemblies revealed two large duplicate sequence blocks of 24,696 bp and 24,387 bp, respectively, and an incomplete copy of 961 bp in this region. It contains up to 13 IGHV gene segments depending on haplotypes. A polymorphic region was found to be located within the duplicated blocks. The variants of this polymorphism unusually diverged at the nucleotide sequence level and in IGHV gene segment number, composition and organization, indicating a limited selection pressure in general. However, the divergence level within the gene segments is significantly different from that in the intergenic regions indicating that these regions may have been subject to different selection pressures and that the IGHV gene segments in this region are functionally important.ConclusionsNon-reciprocal genetic rearrangements associated with large duplicate sequence blocks could substantially contribute to the IGHV region diversity. Since the resulting polymorphisms may affect the number, composition and organization of the gene segments in this region, it may have significant impact on the function of the IGHV gene segment repertoire, antibody diversity, and therefore, the immune system. Because one of the gene segments, 3-30 (1.9III), is associated with autoimmune diseases, it could be of diagnostic significance to learn about the variants in the haplotypes by using the multiplex haplotype analysis system used in the present study with DNA sequence tags specific for the variants of all gene segments in this region.

Telomeres and prognosis in patients with chronic lymphocytic leukaemia

In the present study, telomere length, telomerase activity, the mutation load of immunoglobulin variable heavy chain (IGHV) genes, and established prognostic factors were investigated in 78 patients with chronic lymphocytic leukaemia (CLL) to determine the impact of telomere biology on the pathogenesis of CLL. Telomere length was measured by an automated multi-colour flow-FISH, and an age-independent delta telomere length (ΔTL) was calculated. CLL with unmutated IGHV genes was associated with shorter telomeres (p=0.002). Furthermore, we observed a linear correlation between the frequency of IGHV gene mutations and elongation of telomeres (r=0.509, p<0.001). With respect to prognosis, a threshold ΔTL of -4.2kb was the best predictor for progression-free and overall survival. ΔTL was not significantly altered over time or with therapy. The correlation between the mutational load in IGHV genes and the ΔTL in CLL might reflect the initial telomere length of the putative cell of origin (pre- versus post-germinal center B cells). In conclusion, the ΔTL is a reliable prognostic marker for patients with CLL. Short telomeres and high telomerase activity as occurs in some patients with CLL with a worse prognosis might be an ideal target for treatment with telomerase inhibitors.

Construction and molecular characterization of mouse single-chain variable fragment antibodies against Burkholderia mallei and Burkholderia pseudomallei

We have selected two lipopolysaccharide (LPS) specific Burkholderia mallei mouse monoclonal antibodies (mAbs) and four anti-capsular B. pseudomallei-specific mAbs to generate mouse single-chain variable fragment (scFv) antibodies. This selection was made through extensive in vitro and in vivo assay from our library of mAbs against B. mallei and B. pseudomallei. We initially generated the mouse immunoglobulin variable heavy chain (VH) and light chain (VL) regions from each of these six selected mAbs using a phage display scFv technology. We determined the coding sequences of the VH and VL regions and successfully constructed two B. mallei-specific scFv phage antibodies consisting of two different VH (VH1 and VH2) and one Vλ1 families. Four scFvs constructed against B. pseudomallei had two VH (VH1 and VH6) and two VL (Vκ4/5 and Vκ21) families. All of six scFv antibodies constructed demonstrated good binding activity without any rounds of biopanning against B. mallei (M5D and M18F were 0.425 and 0.480 at OD405nm) and B. pseudomallei (P1E7, P2I67, P7C6, and P7F4 were 0.523, 0.859, 0.775, and 0.449 at OD405nm) by ELISA, respectively. A comparison of the immunoglobulin gene segments revealed that the gene sequences in complementarity-determining regions (CDRs) of three out of four B. pseudomallei-specific scFvs are highly conserved. We determined that the two B. mallei-specific scFvs have different CDRs in the VH, but the amino acid sequences of CDRs in the VL are conserved. This high sequence homology found in CDRs of VH or VL of these mAbs contributes to our better understanding and determination of binding to the specific antigenic epitope(s). The scFv phage display technology may be a valuable tool to develop and engineer mAbs with improved antigen-binding affinity.

Research on the Characteristics of IGHV Gene In Chronic Lymphocytic Leukemia of China.

AbstractAbstract 4609 ObjectiveThe usage, mutation status and prognostic impact of immunoglobulin heavy chain variable (IGHV) gene in Chinese patients with chronic lymphocytic leukemia (CLL) is unclear. We set out to define the characteristics of IGHV gene and its relevance to clinical and biological parameter in our patients. MethodsIGHV gene mutations were detected by multiplex PCR in 202 Chinese CLL patients and the purified PCR amplification products were sequenced. IGHV somatic hypermutation status and gene usage were analyzed by IMGT/V-QUEST software. The association analysis between IGHV somatic mutation status and the clinical and biological features, including Binet staging, immunophenotype, cytogenetic aberrant, were also emphasized in this study. ResultsThe results showed that 129 patients had mutated (M) IGHV, and the remaining 73 patients had unmutated (UM) IGHV according to the cutoff value of accordance rate 98%. The most frequent VH gene family was found to be VH3 (47.5%), followed by VH4 (34.7%), VH1 (11.4%), VH2 (2.5%), VH5(1.5%), VH7(1.5%) and VH6(0.9%) gene families, which was similar to other Asian populations. The overall survival (OS) time of UM IGHV group was significant shorter than M IGHV group (P=0.025). Significance was found in the expression of CD38 and ZAP-70 between patients with and without IGHV mutations (P<0.0001 and P=0.015, respectively). Binet staging was significantly different with IGHV mutation status (P<0.001). “Unmutated” sequences had significantly longer heavy chain complementarity-determining region 3 (HCDR3). Seven of these patients used VH1-69, which was similar to other Asia countries, but in striking contrast to those in Western countries, where VH1-69 was one of the most frequently used genes. FISH was performed in 117 cases, del(11q22) was considered as high risk factors, and 10(10/42, 23.8%) cases with UM IGHV gene. On the other hand, there were 7(7/75, 9.3%) cases with M IGHV gene (P=0.033). No significance was found in del(17p),del(13q),del(6q),add(12),IGH translocation between IGHV mutation and unmuatioan patients. A total of five stereotyped BCR were identified, IGHV3-21/IGHD3-9/IGHJ6, IGHV4-34/IGHD2-15/IGHJ6, IGHV1-3/IGHD6-19/IGHJ4, IGHV4-59/IGHD3-22/IGHJ6 and IGHV4-39/IGHD6-13/IGHJ5. ConclusionsThe usage of IGHV gene families indicates significant difference in Chinese CLL patients compared with Western patients, suggesting involvement of ethnic and/or environmental factors in CLL disease initiation. In the development course of CLL, BCR play an important role in the immunological recognition and selection. There are intimate relationships between mutation status of IGHV gene and prognosis. The usage of IGHV provides enlightment for the occurrence mechanism of CLL. Disclosures:No relevant conflicts of interest to declare.

Analysis of Stereotyped IGHV Distribution In a Series of 1133 Chronic Lymphocytic Leukemia Patients: The Experience of a Multicenter Italian Study Group

AbstractAbstract 2423Chronic lymphocytic leukemia (CLL) is characterized by an extremely variable clinical course. Mutational status of the immunoglobulin heavy-chain variable (IGHV) region defines two disease subsets with different prognosis. A fraction of CLL cases carries highly homologous B-cell receptors (BCR), i.e. characterized by non-random combinations of immunoglobulin heavy-chain variable (IGHV) genes and heavy-chain complementarity determining region-3 (HCDR3).We performed sequence analysis to characterize IGHV regions in a panel of 1133 CLL patients investigated by a multicenter Italian study group. A total of 1148 rearrangements were identified; the analysis of stereotyped subsets was performed based on previously reported criteria (Messmer et al, J Exp Med 2004; Stamatopuolos et al, Blood 2007). Specifically, we compared all our sequences with those found in three different publicly available data sets (Stamatopoulos et al, Blood 2007; Murray et al, Blood 2008 and Rossi et al, 2009 Clin Cancer Res). In addition, a pairwise alignment within all sequences was performed in order to discover novel potential subsets (HCDR3 identity > 60%).Based on the 2% cut-off used to discriminate between Mutated (M) and Unmutated (UM) cases, 777 sequences (67.59%) were classified as M, while 371 sequences (32.3%) as UM. The most represented IGHV genes within mutated cases were IGHV4-34 (104/118) and IGHV3-23 (85/96), whereas IGHV1-69 (97/112) was the most frequently used in the UM group. Interestingly, the IGHV3-21 gene, reported to be frequently expressed in CLL patients from Northern Europe, was present in only a small fraction of cases (24; 2.07%), confirming a previous finding reported by Ghia et al (Blood 2005) in a smaller panel. In our series, stereotyped HCDR3 sequences were found in 407/1148 (35.45%) patients, 177 of whom were M and 230 were UM cases. Overall, we observed that stereotyped sequences were significantly associated with UM IGHV status (Fisher's exact test, P<0.0001). Among the 407 stereotyped HCDR3 sequences, 345 belong to the clusters reported by Murray et al and 14 to those described by Rossi et al., 2009 Clin Cancer Res. The most frequent stereotyped subsets identified in our panel were #1 (35 cases), #7 (28 cases), #4 (24 cases), #3 and #9 (16 cases), #28 (13 cases), and #2 (12 cases), together with subsets #5, #8, #10, #12, #13, #16 and #22 (all ranging from 6 to 9 cases). Finally, we were able to identify by auto-matching analysis 48 sequences potentially specific for 23 novel putative stereotype subsets.In our series we identified 407/1148 (35.45%) stereotyped HCDR3 sequences. The percentage was higher than that reported by Stamatopoulos et al and Murray et al. This discrepancy may partially be due to the different approach used in our analysis, namely the matching to a general data set including all published stereotyped subsets instead of the auto-matching performed by those Authors. We demonstrated a significant association between IGHV status and stereotyped sequences and confirmed the finding that #1 is the most frequent subset identified so far. Finally, we were able to identify a series of 23 novel putative subsets that will require further confirmation. Disclosures:No relevant conflicts of interest to declare.

Incidence of Cytogenetic Abnormalities In Newly Diagnosed Binet Stage A CLL and Relationship with Prognostic Biomarkers and with Stereotyped B Cell Receptors: Updated Results on 344 Patients Included In the Prospective O-CLL1 GISL Study.

AbstractAbstract 4613 Background:Biologic risk factors such as immunoglobulin variable heavy chain (IgVH) gene mutation status and CD38 and ZAP-70 expression levels, along with genomic aberrations, have been integrated in clinical prognostic evaluation of CLL. Additionaly, CLL subsets expressing a certain stereotyped B-cell receptors have also been indicated to share biological and clinical features. Aims:We investigated, by FISH, the incidence of the major cytogenetic alterations (+12 and 13q14, 17p13, 11q23 deletions), their clinical implication and their relationship with prognostic biomarkers in 344 out of 384 Binet A CLLs enrolled in the prospective multicenter O-CLL1 GISL trial. Stereotypy subsets identification have been performed in 324 patients. Methods:Molecular markers characterization and FISH protocols were previously reported (Cutrona et al. Haematologica, 2008; Fabris et al. Genes Chromosomes Cancer, 2008), while stereotyped subsets were defined according to Stamatopoulos et al (Blood, 2007) and Murray et al (Blood, 2008). Results:At least one abnormality was found in 225/344 (65.4%) cases. The most frequent abnormality was del(13q14), detected in 173 CLLs (50.3%) followed by +12 (44/344;12.8%) (one case harboring 17p13 deletion), del(17p13) (9/344, 2.6%) and del(11q23) (18/344, 5.2%). 13q14 deletion was found as a sole abnormality in 155 (45%) patients; in the remaining cases, it was combined with +12 (3 pts) and 17p13 (4 pts) or 11q23 deletions (11 pts). The 13q deletion was found as a monoallelic deletion in 139/173 (80.3%); the presence of a biallelic deletion (> 20% of interphase nuclei) was found in the remaining 34 cases. No acquisition of new cytogenetic aberrations was evidenced among the 13 CLLs developing progressive disease (range, 6 to 32 months; median, 20 months); in only one case, the proportion of nuclei with 17p13 and 13q14 deletions increased from the time of diagnosis (from 33% to 92%). Biomarkers data were available in all of the patients. CD38 percentages (mean value ± sem) were 7.9±1.3, 15.1±1.9, 51.7±5.5, 22.0±7.8,40.8±13.2, 39.8±7.3 for del(13q14), normal karyotype, +12, del(11q23), del(17p13) and multiple alterations, respectively (p<0.0001). The percentages of IgVH mutations significantly correlated with cytogenetic alterations; namely, 5.7±0.2 for cases with del(13q14), 4.7±0.4 for normal karyotype, 2.3±0.5 for +12, 0.05±0.05 for del(11q23), 2.0±1.1 for del(17p13) and 1.0±0.4 for multiple alterations (p<0.0001). Similarly, a significant correlation was found for ZAP-70 expression: namely 32.9±1.6 for cases with del(13q14), 38.5±2.1 for normal karyotype, 46.4±3.6 for +12, 67.0±8.3 for del(11q22), 41.0±12.8 for del(17p13) and 50.7±5.4 multiple alterations (p<0.0001). Cytogenetic abnormalities were clustered in 3 risk groups [i.e. low del(13q14) and normal; intermediate (+12); and high risk 17p13 and 11q23 deletions] and correlated with a scoring system in which patients were stratified in 4 different groups according to the absence or presence of 1, 2 or 3 biomarkers (Morabito et al., Br. J. Haematol., 2009). Notably, 166/175 cases scoring 0, gathered in the low FISH group, whereas 21/26 high FISH risk cases clustered in scoring 2–3 (p<0.0001). A significantly higher risk of starting treatment was found in high vs. intermediate (p=0.024) and low FISH risk (p=0.001) CLLs. Finally, stereotyped IgVH sequences were found in 108/324 (33%). Unfavorable stereotyped subsets (#1, #2, #3, #7 and #9) were significantly more frequent in CLLs with poor-prognostic aberrations (p=0.0203; RR=3.589). Conclusions:Our data indicate that cytogenetic lesions predicting unfavorable prognosis show a relatively low incidence in newly diagnosed Binet stage A CLLs and are significantly associated with negative prognostic biomarkers predictive of disease progression. Our prospective study also confirms the prognostic value of risk FISH categories in predicting the time to the first treatment and revealed a higher rate of unfavorable stereotyped IgVH subsets in patients carrying poor-prognostic genomic aberrations. Finally, preliminary evidence in a limited number of cases indicates that the acquisition of new genetic abnormalities seem to be an infrequent event during disease progression. Disclosures:No relevant conflicts of interest to declare.

Assessment of Minimal Residual Disease (MRD) In Relapsed CLL Patients Treated with Fludarabine and Cyclophosphamide with or without Rituximab (REACH)

AbstractAbstract 1390 Introduction:Levels of minimal residual disease (MRD) have been shown to correlate with PFS in previously untreated patients with CLL (CLL8, Boettcher et al. Leukemia, 2009). Patients who remain MRD positive after treatment have a higher risk of relapse. Eradication of MRD is therefore a desirable clinical endpoint of treatment. We were interested to assess this correlation in REACH, a randomized international clinical study in previously treated CLL patients, randomized 1:1 for treatment with rituximab, fludarabine and cyclophosphamide© R-FC (276 patients) or FC alone (276 patients); (Robak et al. JCO 2010). Methods:While MRD quantification by flow cytometry requires an identifiable stable phenotype and fresh blood samples, PCR based methods can be performed centrally on frozen samples. We have therefore developed a Realtime Quantitative (RQ) PCR method, using patient-specific IgVH (immunoglobulin variable heavy chain) gene rearrangements as targets. Briefly, genomic DNA was isolated from CD19 sorted B-cells. ASO (allele-specific oligonucleotide) primers were designed matching the hypervariable N-D-N region of the patient-specific leukemic clone and used with reverse consensus primers and hydrolysis probes annealing to the family-specific joining region of the IGH rearrangement (Brüggemann et al., Leukemia, 2004). Maximum sensitivity and quantitative range were defined for every RQ-PCR. Patients were categorized as molecular responders (MRD negative) if there was no detectable clonal IgH rearrangement, using a sensitivity cut-off of 1×10-4. Molecular response was assessed at the time of CR confirmation and 6 months later (if CR was maintained). Results:Among the 103 patients who achieved CR during the study, 86 patients had at least one MRD assessment in peripheral blood, 92 patients in bone marrow. Since many patients had a CR confirmation at different time points during the follow-up period, we initially analyzed the MRD levels only in patients who had achieved confirmed complete response at end of treatment +/−3 month (“EOT - period”).The rate of MRD negativity in blood (22 pts: 5(15) FC, 6(7)R-FC) at EOT was 33% for patients treated with FC, and 86% for patients treated with R-FC (p=0.06); In bone marrow at the EOT (61 patients: 5(27) FC, 20(34) R-FC) the rates were 19% and 59%, respectively (p= 0,02), indicating higher efficacy of the Rituximab containing regimen in eradication of residual disease; This is in line with the previously reported results using FACS analysis of MRD in the CLL8 trial; the differences in the detection rate in blood versus bone-marrow, suggest a higher sensitivity for detection of MRD in bone marrow.We therefore compared the levels of MRD negativity in samples from blood and bone marrow in patients where both samples were taken at the same time point. Results were concordant in 8/9 patients, one patient had a positive result in bone marrow with no detectable signal in blood. This supports the notion that assessment of MRD in bone marrow of CLL patients may be more sensitive than assessment in blood only. However, for a definitive statement larger sample size would be needed.We then correlated MRD status at EOT, regardless of treatment arm, with PFS: In line with previous reports, there was a clear trend to longer PFS in patients who had reached MRD negativity (median PFS not reached), while patients with residual disease had shorter PFS; however, due to small sample numbers, statistical significance could not be reached. We also analyzed the correlation of MRD negativity reached at any time during and after treatment with PFS, bearing in mind that this sample set is inherently biased, since patients with early progression will be lost from the analysis; the results are consistent with the EOT findings. Summary:ASO IgVH RQ-PCR is a powerful method to detect residual levels of disease in CLL patients with clinical complete response and undetectable MRD correlates with longer PFS. Among patients in REACH achieving clinical CR on either study arm, a higher percentage achieved MRD negativity on the R-FC arm, consistent with the increased efficacy shown for the Rituximab treatment arm by the REACH clinical data. Disclosures:Mundt:Roche: Employment. Smith:Roche: Employment. Lin:Genentech: Employment. Barrett:Roche: Employment. Hurst:Genentech: Employment. Geisler:Roche: Research Funding, Speakers Bureau. Hiddemann:Roche: Research Funding.

Analysis of Immunoglobulin Heavy Chain Variable Genes In Burkitt Lymphoma Uncovers An Antigen Role In the Pathogenesis of the Disease

AbstractAbstract 4135The analysis of the immunoglobulin heavy chain variable (IGHV) genes in B-cell derived tumors can yield relevant pathogenic information; it contributes to the definition of the normal cell counterpart and in some neoplasias allowed the identification of subsets of stereotyped B cell receptors associated to different clinical/phenotypic features and outcome. According to the WHO 2008 Classification, Burkitt lymphomas (BL) derive from either germinal center (GC) or post GC B cells. This study was aimed at a comprehensive investigation of the IGH genes in BL; for that, 27 samples of BL were studied, including both pediatric (n=15) and adult (n=12) cases from the 3 clinical variants of the disease, namely endemic (n=1), sporadic (n=22) and immunodeficiency-associated (n=4).All the samples analyzed harbored in frame IGHV-D-J rearrangements, the great majority (96.3%) being functionally productive. The comparison of the IGH genes usage in BL with the normal repertoire found in CD5 neg B cells detected no differences concerning IGHJ and IGHD usage. However, the usage of IGHV genes was significantly different from that of the normal B cell counterpart (p=0.21). Genes like IGHV3-21 and IGHV5-a were only found in BL samples, with a frequency of 11.1% and 3.7% respectively. Other genes were overrepresented in BL with respect to normal B cells, namely IGHV2-70 (7.4% vs 1.3%), IGHV3-30 (11.1% vs 5.2%), IGHV4-34 (7.4% vs 4.3%), IGHV4-39 (11.1% vs 5.2%) and IGHV4-59 (14.8% vs 9.1%). These results pointed to a biased used of certain IGHV genes by BL cells. No preferential V-D-J rearrangement was detected and only 3 samples had common HCDR3 features, although with different V-D-J rearrangements.IGHV mutational load was calculated as the percentage of germline identity (GI). Most of the BL samples (70.4%) harbored mutated IGHV genes (<98% GI), whereas borderline/minimally mutated IGHV genes (98-99.9% GI) were detected in a 22.2% of the samples. Only 2 out of 27 cases (7.4%) presented unmutated IGHV genes (100% GI). Interestingly, the pattern of the aminoacid (aa) substitutions introduced by the SHM process revealed a precise targeting: 81.5% of the cases showed the same aa replacements indicating that common antigens must be implicated.Intraclonal diversity (ID) was assessed by analysis of the subcloned nucleotide sequences. Only confirmed mutations, i.e.mutations observed more than once in the subclones from the BL case, were considered. Under this definition, 48.1% of BL cases had ongoing nucleotide mutations. Nevertheless, analysis of aa replacements introduced by unconfirmed nucleotide mutations showed that some aa substitutions were shared by other cases. Thus, considering that those unconfirmed mutations were confirmed by other case, these changes were acknowledged as real mutations, this raising the percentage of BL samples with ID to a 88.9%. Following this, it could be concluded that the majority of the cases presented ID, indicating that their normal counterpart is a centroblast experiencing the GC reaction.It has been previously shown that the IGHV3 subgroup harbors the Staphilococcal protein A (SpA) binding motif. In this sense, we found that a 44.4% of the BL cases used a gene from this subgroup. Interestingly, the SpA binding site was preserved in all of the IGHV3 aa sequences analysed. SpA is the prototypic B-cell superantigen and the fact that its binding motif was present in the main IGHV gene used by BL cells in this series suggest that superantigens could play a biological role in this disease.In conclusion, this work allowed the confirmation of the postulated normal counterpart for BL being a centroblast experiencing the GC reaction. More, the biased use of IGHV genes and the recurrent hypermutations that were detected suggest a role for common antigens in the selection of the clonogenic progenitors. Finally, the conserved SpA binding motif found in BL cases using IGHV3 genes indicates that superantigens may also be implicated. Disclosures:No relevant conflicts of interest to declare.

Predictors of Response to Salvage Therapy for Advanced Chronic Lymphocytic Leukemia (ECOG Protocol 2903)

AbstractAbstract 2463The treatment of relapsed/refractory B-Chronic Lymphocytic Leukemia (CLL) is difficult in terms of both inducing meaningful responses and being able to predict clinical outcome. In an attempt to address the latter aspect, we analyzed a battery of prognostic parameters obtained from a cohort of relapsed/refractory CLL patients entered onto ECOG trial E2903. This protocol included 2 steps: an induction phase (step 1) with pentostatin (4 mg/m2), cyclophosphamide (600 mg/m2), and rituximab (375 mg/m2) given every 3 weeks for 6 cycles and a consolidation phase (step 2) where complete responders (CR) were given 4 weeks of subcutaneous (sc) alemtuzumab (30 mg/m2, 3 times a week) or patients attaining partial response (PR), stable or progressive disease were given 18 weeks of sc alemtuzumab. Sixty-five eligible patients were enrolled; median age was 63 yr; male to female ratio of 4:1; median (range) number of prior therapies was 3 (1–7); and there were 16, 7, and 40 patients in Rai 0–1, Rai 2; and Rai 3–4 stages respectively. The median serum β-2 microglobulin was 4 (1–28). The overall response rate for step 1 in this cohort was 46% with 1 complete and 29 partial responses and 38.5% judged as having stable disease. Strict CR criteria, including CT imaging were utilized, demonstrating the infrequency of total response using CT scans. For traditional prognostic parameters, we found that age over 70 yr and high Rai risk stage (3–4) did not preclude a clinical response, but there was a significant difference between responders and non-responders in serum β-2 microglobulin levels (p=0.03). For molecular prognostic parameters, we found that the immunoglobulin variable heavy chain region (IGVH) mutational status did not preclude a lack of clinical response. However, for patients who were ZAP70 negative (−) or CD38 negative (−) these factors were positively associated with clinical response (p=0.046, 0.0005, respectively). The sensitivity in predicting a clinical response was 82% in ZAP70- and 73% in CD38-. In multivariable logistic regression analysis, only CD38 was significantly associated with response (Odds Ratio: 41, p=0.002). When patients were segregated into low (13q- or normal), intermediate (Trisomy 12) and high risk FISH (17p-,) there was no association with clinical outcome. In addition, if patients' CLL cells were unmutated and high risk FISH, there was no reduced clinical response. Of 65 patients included in this analysis, the 2-year overall survival (OS) was 55% (±7%) and the 2-year progression-free survival (PFS) is 25% (± 6.6%). The median follow-up time amongst survivors was 23 (range 6–60) months. The univariable analysis for PFS revealed that the IGVH mutated (2-yr PFS 53% for mutated vs. 18% for unmutated, p=0.037, Figure) and low risk FISH (2-yr PFS 42% for low risk vs. 23% for high risk, p=0.04) were significant. However, only IGVH mutated was significant (HR 0.33, p=0.027) for PFS in multivariable Cox model. In univariable analysis for OS, the combination of age <70 yr and β-2 microglobulin<2 upper limits [UL] was significant (2-yr OS: 68% vs. 39%, p=0.048), whereas only β-2 microglobulin was significant (HR 3.45 for >=2UL vs. <2UL, p=0.02) in multivariable Cox model. A total of 24 patients entered onto step 2 of the consolidation phase, receiving alemtuzumab; 17 patients who were responders from step 1 entered onto the 4 weeks of step 2; and 7 of the non-responders went onto the 18 weeks of step 2. The 2-year OS of these 24 patients was 62% from the date of consolidation phase: 61% for 17 responding patients to PCR and 60% for non responders to PCR (p=0.87). Current conclusions of this study are: (1) heavily pretreated high risk CLL patients may still have a respectable survival with the PCR combination; (2) β-2 microglobulin, ZAP70, and CD38 were important factors associated with response to PCR; (3) both IGVH mutated and FISH were prognostic markers associated with PFS; (4) addition of alemtuzumab may confer comparable survival rates for PCR non responders to the responders; (5) β-2 microglobulin was the only prognostic factor significant for OS. In total this study shows that both traditional and molecular markers can be used to predict clinical outcome for high risk relapsed/ refractory CLL patients treated with chemoimmunotherapy. We anticipate further valuable prognostic information to come from this still accruing study. [Display omitted] Disclosures:Rosen: Genentech: Speakers Bureau.

Generation and validation of canine single chain variable fragment phage display libraries

Single chain variable region fragments (scFvs) are composed of an immunoglobulin (Ig) variable heavy (VH) and variable light (VL) chain joined by a flexible serine–glycine linker. They represent the smallest antibody fragments that maintain antigen specificity and they hold significant potential for therapeutic antigen targeting in vivo. Here we report on the design and validation of a series of degenerate primers that amplify the recombined variable regions of canine Ig heavy and light chain genes from lymphocyte cDNA. We show that these VH and VL amplicons can be randomly combined by a flexible linker using splicing by overlap extension PCR to form scFv constructs that can be expressed on the surface of M13 bacteriophage. To demonstrate that scFvs with specificity for previously encountered antigens are contained within these scFv phage display libraries we used simple panning procedures to isolate canine parvovirus (CPV) specific scFvs from a library made from the splenocytes of a dog immunized against CPV. These studies reveal the feasibility of this approach for generating diverse canine scFv libraries and pave the way toward future studies to isolate canine antigen-specific scFv of interest that may be tested as targeting agents for the treatment of infectious, inflammatory and neoplastic diseases in the dog.

Comparison between oral and intravenous fludarabine plus cyclophosphamide regime as front-line therapy in patients affected by chronic lymphocytic leukaemia: influence of biological parameters on the clinical outcome

The fludarabine plus cyclophosphamide (FC) regimen was reported to be superior to chlorambucil or fludarabine alone in terms of complete response (CR), overall response (OR) and progression-free survival (PFS) in previously untreated patients with chronic lymphocytic leukaemia (CLL). In the present study, we compared the efficacy and toxicity of FC administered through oral and intravenous route in 65 untreated patients affected by advanced CLL. No statistical differences were noticed between the two routes of administration in terms of OR, PFS, time to re-treatment (TTR) and overall survival (OS) of analysed patients. We also assessed the influence on the clinical outcome of the mutation status of the immunoglobulin variable region heavy chain (IgVH) gene, of the cytogenetic abnormalities and of the expression of ZAP70 and CD38 in patients' primary samples. Among the 58 evaluable patients, 31 (53%) achieved a CR and 18 (31%) a partial response. The median PFS was 35 months, median TTR was 42 months and median OS was not reached after 45 months (range, 1-161). A significantly lower OR rate was noticed in patients with high-risk cytogenetic abnormalities (del 17p, del 11q). In this study, high-risk cytogenetic abnormalities and unmutated IgVH genes were independent predictors of TTR. These results underline the importance of biological stratifications in front-line treatment of CLL patients. We confirm that FC is an effective regimen with mild toxicities; it could be recommended for patients with low-risk biological parameters who represent, in our experience, about 30% of the total.

Persistent infections and their relationship with selected oncologic and non-tumor pathologies

Our earlier studies of hepatitis C virus (HCV) infection rates among blood donors at the Kyiv Municipal Blood Center revealed a 3.45% HCV+ prevalence in these “healthy” hosts. In the study here, we analyzed HCV (as well as cytomegalovirus [CMV]) prevalence among Chernobyl nuclear power plant (NPP) accident sufferers—cleanup workers, local residents, NPP workers, and convalescent patients—who suffered acute radiation syndrome (ARS) as a result of the 1986 accident, and individuals who had not been exposed to ionizing radiation (IR). Serological analyses of antibodies against each pathogen (via enzyme-linked immunosorbent assay [ELISA]) revealed the highest HCV (i.e., 27.2%) and CMV (85.6%) prevalence in the convalescent hosts. Though the HCV prevalence (reflecting a current/past infection) among the cleanup workers (and other groups) was lower (i.e., 11–25%), viral presence was “associated” with a higher incidence of selected somatic diseases, for example, thyroiditis, goiter, hypertension, Type 1 diabetes, chronic hepatitis/gastritis, in the cleanup workers. A similar scenario with respect to CMV was also seen, i.e., lower prevalence rates [relative to in ARS patients] and “association” between CMV status and incidence of chronic gastritis, arthritis, and bronchitis, in the cleanup workers and IR-non-exposed controls. Further, irrespective of CMV status, there was a clear delineation between incidence rate(s) of each of the pathologies and whether or not the person was/was not exposed in 1986. We also investigated, due to a high incidence of chronic lymphocytic leukemia (CLL) among Chernobyl sufferers, if there was homology between immunoglobulins (Igs) generated by these transformed cells and known antiviral and antimicrobial Igs. Polymerase chain reaction (PCR) analyses of Ig heavy-chain variable (IgHV) genes in cells from CLL patients who were/were not exposed in 1986 revealed a significant homology of some IgHV genes with Igs directed against infectious agents. However, no differences were found between the sequences from IR-exposed and IR-non-exposed CLL patients. Based on the findings here, we conclude that a past/ongoing presence of certain viral infections (i.e., CMV and/or HCV) in a host can modify (aggravate) the clinical course of certain somatic (i.e., non-tumor) diseases and promote malignancies (i.e., CLL), and that each of these outcomes could be modulated as a result of that host’s past exposure to IR.

Mathematical analysis of antigen selection in somatically mutated immunoglobulin genes associated with autoimmunity

Affinity maturation is a process by which low-affinity antibodies are transformed into highly specific antibodies in germinal centres. This process occurs by hypermutation of immunoglobulin heavy chain variable (IgH V) region genes followed by selection for high-affinity variants. It has been proposed that statistical tests can identify affinity maturation and antigen selection by analysing the frequency of replacement and silent mutations in the complementarity determining regions (CDRs) that contact antigen and the framework regions (FRs) that encode structural integrity. In this study three different methods that have been proposed for detecting selection: the binomial test, the multinomial test and the focused binomial test, have been assessed for their reliability and ability to detect selection in human IgH V genes. We observe first that no statistical test is able to identify selection in the CDR antigen-binding sites, second that tests can reliably detect selection in the FR and third that antibodies from nasal biopsies from patients with Wegener's granulomatosis and pathogenic antibodies from systemic lupus erythematosus do not appear to be as stringently selected for structural integrity as other groups of functional sequences.

IGHV4-39 deletion polymorphism does not associate with risk or outcome of multiple sclerosis

The restricted use of immunoglobulin heavy chain variable (IGHV) family 4 gene segments by clonally expanded B cells in brain lesions and cerebrospinal fluid (CSF) of multiple sclerosis (MS) patients is well documented. Specifically, the overrepresentation of gene IGHV4-39 has been highlighted in multiple studies. To investigate the role of IGHV4-39 in MS, we screened 193 MS cases, representing the extremes of clinical outcome (benign and malignant), and 187 controls for a previously reported germline deletion polymorphism containing IGHV4-39. We did not reveal a genetic association linking this polymorphism to MS risk or progression.

High Expression of Lymphocyte-Activation Gene 3 ( LAG3) in Chronic Lymphocytic Leukemia Cells Is Associated with Unmutated Immunoglobulin Variable Heavy Chain Region ( IGHV) Gene and Reduced Treatment-Free Survival

Chronic lymphocytic leukemia (CLL) is characterized by a monoclonal expansion of mature B-lymphocytes. Mutational status of the immunoglobulin variable heavy chain region (IGHV) gene stratifies CLL patients into two prognostic groups. We performed microarray analysis of CLL cells using the Agilent platform to detect the most important gene expression differences regarding IGHV status in CLL cells. We analyzed a cohort of 118 CLL patients with different IGHV mutational status and completely characterized all described prognostic markers using expression microarrays and quantitative real-time RT-PCR (reverse transcription PCR). We detected lymphocyte-activation gene 3 (LAG3) as a novel prognostic marker: LAG3 high expression in CLL cells correlates with unmutated IGHV (P < 0.0001) and reduced treatment-free survival (P = 0.0087). Furthermore, quantitative real-time RT-PCR analysis identified a gene-set (LAG3, LPL, ZAP70) whose overexpression is assigned to unmutated IGHV with 90% specificity (P < 0.0001). Moreover, high expression of tested gene-set and unmutated IGHV equally correlated with reduced treatment-free survival (P = 7.7 * 10(-11) vs. P = 1.8 * 10(-11)). Our results suggest that IGHV status can be precisely assessed using the expression analysis of LAG3, LPL, and ZAP70 genes. Expression data of tested markers provides a similar statistical concordance with treatment-free survival as that of the IGHV status itself. Our findings contribute to the elucidation of CLL pathogenesis and provide novel prognostic markers for possible application in routine diagnostics.

Abstract 2125: Somatic hypermutations in hairy cell leukemia suggest a canonical process, except in patients with the poor-prognosis VH4-34 variant

Abstract Hairy cell leukemia (HCL) cells express an immunoglobulin (Ig) heavy chain variable (VH) gene with different degrees of somatic mutation. We recently reported that of 82 patients with HCL, 14 expressed VH4-34 and that these patients had a poorer outcome compared to the other 68 patients. Moreover, poor prognosis in the VH4-34 patients was independent of the known poor-prognosis variant of HCL, called HCLv. To confirm these findings, and to more extensively characterize the somatic mutations occurring in the non-VH4-34 patients, we studied mutations from these and an additional 32 patients. A total of 118 rearranged VH sequences from 114 HCL patients were analyzed. Four patients had 2 rearrangements each, none of them VH4-34. Seventeen (15%) of the 114 patients had VH4-34 rearrangements, comprising 10 (38%) of 26 HCLv and 7 (8%) of 88 classic HCL patients (p=0.0005). Sixteen (94%) of 17 VH4-34 rearrangements were unmutated (&amp;gt;98% homologous to germline), compared to 15 (15%) of the other 101 (p=2 × 10-10). Median homology to germline was 99.6% in VH4-34 vs 95.4% in the other rearrangements (p&amp;lt;0.0001). Previous clinical findings that VH4-34+ HCL is associated with high white blood cell count at diagnosis, inferior response and progression-free survival after initial cladribine therapy, and poor overall survival from diagnosis independent of HCLv status, were confirmed. Characterization of the locations of somatic VH mutations in lymphocytic leukemias is rare. One published study reported that in 172 chronic lymphocytic leukemia (CLL) rearrangements, mutations were concentrated in the complementarity determining (CDR) regions, with higher replacement/silent (R/S) mutation ratios for CDRs (4.60) vs framework (FR) regions (1.72) (Blood, 103:3490-3495, 2004). We found that in our 118 HCL rearrangements containing 1385 mutations, R/S was 2.40 overall, 4.29 for CDRs, and 1.83 for FRs, nearly identical to the reported CLL data. Other features reported in CLL, including mutations preferentially in RGYW hot spots and consisting predominantly of transitions rather than transversions, were also observed in HCL. These data suggest that like CLL, VH mutations in HCL result from canonical somatic hypermutation and select for stable antigen receptors, similar to the mutation process taking place in the normal germinal center. However, the origin of VH4-34+ HCL appears different and requires further study. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2125.

Analysis of human immunoglobulin gene repertoire in NOD/scid IL2Rγ null mice engrafted with human cord blood derived hematopoietic stem cells (36.14)

Abstract The humanized NOD/scid IL2rg-/- (hNSG) and similar mouse models hold promise as testing platforms for preclinical studies. Here, we analyzed the human immunoglobulin (IG) variable heavy and light chain gene repertoire from sorted single B-cells of hNSG mice engrafted with cord blood derived hematopoietic stem cells. Unique IGHV (339) and IGLV (364, kappa and lambda) gene segments were derived from major B cell subsets identified in a largely normal B cell development pathway. However, the hNSG mice expressed elevated CD5+ mature B-1-like cells in the periphery. In mice and humans, B-1 cells are the major source of autoantibodies. Although the hNSG mice expressed a highly diverse repertoire comparable to humans, detailed genetic analysis of IG gene segments from the mature B cell subset revealed the presence of long IGH-CDR3 regions with positive charges—a hallmark for auto/polyreactivity. For functional tests, several in vitro expressed scFv-Fc IG candidates, derived from cloned heavy and light chain cognate pairs of single B cells, tested positive in an anti-nuclear antibody assay for auto/polyreactivity and/or bound to HIVgp120 in ELISA assays. The natural induction during HIV-1 infection of broadly neutralizing antibodies (BnAbs) which display autoantibody-like characteristics may be curtailed by the checkpoint control mechanisms during B cell ontogeny. We propose the hNSG mouse can be a model platform to test this hypothesis and interrogate how BnAbs are formed.

35 BCR-signaling profiles associated with prognosis and progression in B-CLL

Background. B-cell chronic lymphocytic leukemia (B-CLL) patients exhibit a variable clinical course. Several biological parameters have been shown to be associated with clinical outcome in CLL. Among them, the most reliable markers are represented by the absence of somatic mutations within the immunoglobulin variable heavy chain genes (IGHV), the expression of CD38 antigen, or the presence of the ZAP-70 tyrosine kinase. These parameters of poor clinical outcome are structurally and/or functionally linked to B-cell Receptor (BCR) expressed by CLL cells, thereby strengthening the hypothesis that antigenic stimulation mediated by the BCR represents a driving event in the onset and progression of the malignant B cells. Methods. We applied a “network level” analysis of BCR signaling by measuring single-cell profiles of phosphoprotein networks by flow cytometry. We evaluated the response to BCR engagement in primary cells isolated from 27 CLL patients by analyzing the phosphorylation states of 5 phosphoproteins on the route of BCR signaling, including p-Syk, p-NF-kappaB, p-Erk1/2, p-p38 and p-JNK. BCR was cross-linked by incubating cells with anti-IgM antibodies. Results. Unsupervised clustering analysis distinguished BCR response profiles of phosphoproteins that differentiated cases of CLL with mutated IGHV from those with unmutated IGHV (P=0.0003), cases with low levels of CD38 expression from those with high levels (P=0.0004) and cases with ZAP-70negative leukemic cells from cases that were ZAP-70-positive (P=0.001). Furthermore, the same BCR response profiles were also associated with time to progression (P=0.0014) and with overall survival (P=0.049), as assessed by Kaplan–Meier curves and the log-rank test. Independent survival analysis of time to progression via fitting Cox proportional hazards models comprising clinical covariates and/or BCR network response to modulation demonstrated that measuring modulated BCR network signaling can yield improved prognostic information compared to CD38 status alone (likelihood ratio test 5.8 for CD38 versus 10.6 for signaling) and enhance prognostic assessment using IGHV status (likelihood ratio test for IGHV = 14.8 versus for IGHV + signaling = 17.9).

Molecular and clinical features of chronic lymphocytic leukemia with stereotyped B-cell receptors in a Ukrainian cohort

A fraction of chronic lymphocytic leukemia (CLL) carries highly homologous B-cell receptors, characterized by non-random combinations of immunoglobulin heavy-chain variable (IGHV) genes and heavy-chain complementarity-determining region-3 (HCDR3), often associated with a restricted selection of IG(K/L)V light chains. We analyzed the features of CLL expressing homologous HCDR3 in a cohort of 264 Ukrainian patients by merging them with a recently published reference series of 1426 cases. This approach allowed us to identify 96/264 (36%) cases as expressing homologous HCDR3, subdivided into 47 subsets. Among these, 27 apparently novel subsets were identified, although most of them were composed of two sequences per subset (‘potential subsets’). CLL cases belonging to several stereotyped subsets showed HCDR3 homologies with various autoreactive clones. Our analysis identified molecular and clinical features of a Ukrainian cohort of patients with CLL.