Late Appearance of 14q32/IGH Translocation in Chronic Lumphocytic Leukemia

Abstract 2429 Up to 80% of Chronic Lymphocytic Leukemia (CLL) harbour clonal chromosome aberrations having important clinical implications (i.e. 13q deletion, +12, 11q/ATM and 17p/TP53 deletions). 14q32/IGH rearrangements were recently found in 6–19% of CLL patients and were associated with therapy-demanding disease and inferior outcome. Whereas evidence was provided that some of the classical aberrations, such as 11q-, 17p-, may appear late in CLL clinical history, no information is presently available concerning 14q32/IGH translocations. The aim of this study was i) to analyze the incidence of 14q32/IGH translocations occurring at clonal evolution in CLL, ii) to analyze the clinicobiologic significance of late-appearing 14q32/IGH translocations. One hundred-five CLL cases seen at our institution in a 10-year period were submitted to FISH analysis at diagnosis or before 1st line treatment as part of routine diagnostic workup. In 47 patients with indolent disease (untreated or treated with 1 line without relapse, group 1) FISH analysis was repeated after 48–96 months (median 72). In 58 relapsed patients who started 2nd line treatment (group 2), FISH was performed sequentially before administration of the 2nd line and before each subsequent line of therapy. These 105 patients fulfilled the following criteria: a) diagnosis of bona fide CLL based on morphology and immunophenotyping (CD5/CD19+, CD23+ as minimal requirement), b) clinical records available for review, c) successful FISH analysis at diagnosis and during follow-up. Those cases with t (11;14)(q13;q32)/CCND1-IGH or other 14q32/IGH translocations present at diagnosis were excluded from this study. Sequential FISH studies were performed in all patients on peripheral blood (PB) samples using commercially available probes for the identification of deletions at 13q14, 11q22/ATM, 17p13/TP53, of trisomy 12 and of 14q32/IGH translocations. In 10 patients bone marrow (BM) aspiration and/or lymph node (LN) biopsy were studied by FISH as well. The patients were treated at disease progression as defined by NCI criteria. Refractory disease was defined by stable disease or progressive disease during treatment or disease progression within 6 months of from antileukemic treatment using fludarabine alone or in combination with other agents. Time to chemorefractoriness was measured from date of first line treatment to date of refractoriness to fludarabine containing regimen or date of last follow-up. Overall survival was measured from diagnosis to date of last follow-up or death and from initiation of first line treatment to the date of death or last follow-up. At diagnosis 39% of the cases had 13q-, 14% had +12, 7% had 11q- and 3% had 17p-. A late-appearing 14q32/IGH rearrangement was not detected among 47 patients in group 1, whereas 7/58 cases (12,1%) in group 2 showed a 14q32/IGH break in 16–25% of the cells. These 7 patients had the following aberrations at diagnosis: 13q- and 11q- in 1 case, 13q- in 2 cases; 11q- in 1 case, +12 in 2 cases, no aberrations 1 case. The 14q32 translocation appeared after a median time of 64 months (range 51–91). It was associated with the appearance of 17p- in 3/7 cases with one of these presenting also biallelic del13q. In two cases paired BM or LN sample and PB samples were available for FISH studies and the appearance of IgH translocation in the BM or in the LN sample preceded its appearance in PB by 13–58 months. All 7 cases with late appearing 14q32/IGH translocation developed chemorefractoriness to fludarabine regimen with a median TTC of 27 months (range 12–40 months), as compared with a TTC of 67 months (range 1–143 months) in 51 treated patients who did not develop the 14q32 translocation (p=0.0002). Overall survival did not differ significantly either when measured from diagnosis or from 1st line treatment in 7 patients with 14q32 translocation as compared with the appropriate control. We arrived at the following conclusions: i) a late-appearing 14q32/IGH translocation occurred at a relatively high incidence (12,1%) in patients with relapsing disease and not in patients with stable disease, ii) this aberration involved a minority of cells and, in approximately half of the cases, it was associated with other aberrations, reflecting complex clonal evolution, iii) in 2 assessable cases it first appeared first in the BM or LN; iv) the appearance of 14q32/IGH translocation was associated with shorter TTC. Disclosures: No relevant conflicts of interest to declare.

Association of High Levels of Free Light Chain and the Presence of Igh Translocations in Multiple Myeloma and Prognostic Implications.

Background: The free light chain (FLC) assay allows a nephelometric measurement of this free kappa and lambda light chains that circulate as monomers or dimers, not bound to the immunoglobulin heavy chain. Estimation of the free light chain levels and their ratio has utility in screening, prognostication and treatment response assessment in multiple myeloma, and is particularly useful in light chain myeloma. Previous studies have suggested that absence of a functional rearrangement on at least one of the IgH allele is the basis of light chain myeloma. Translocations involving the immunoglobulin heavy chain (IgH) locus on chromosome 14 can be detected using FISH in over half of the patients with myeloma. We hypothesized that presence of an IgH translocation may be associated with compromised production of heavy chains and leads to unbalanced, excess production of clonal light chain. Methods: From February 1988 to May 1992, 653 patients with previously untreated multiple myeloma were enrolled on the phase III clinical trial E9486. Serum free light chains were run on stored sera obtained at baseline in 495 patients as part of a previous study. Among these patients, 314 patients were previously studied using FISH for presence or absence of IgH translocations, p53 abnormalities and deletion 13 and were included in the current analysis. Results: The median difference between the involved and uninvolved FLC (FLCdiff) was higher among the group of patients with any IgH translocation abnormality compared to those with none (Figure 1). This was particularly evident among the patients with a t(14;16) who had the highest FLCdiff. In a multivariate analysis, we examined if the prognostic value seen with FLCdiff was independent of the presence of IgH translocations t(4:14) and t(14;16), which are associated with a worse outcome. The presence of t(4;14) or t(14;16) was associated with FLCdiff groups using a cutoff point of 185, with 13% of those with FLCd ≤185 and 27% of those with FLCd > 185 having the translocations (P= 0.008). At the cutoff value of 185, the prognostic value of the FLC-diff on progression free and overall survival appears to be independent of these high risk IgH translocations (P-value=0.03 and 0.003, respectively for PFS and OS) We then examined the prognostic value of the FLC-diff in the group of patients without these two high risk abnormalities and were able to confirm that they remained prognostic for PFS (P=0.009) and OS (p=0.005). However, among the group of patients with either of the two high risk IgH abnormalities, FLCdiff estimate added little to assessment of their outcome (P=0.97 and 0.20, respectively for PFS and OS). Conclusion: Elevated free light chain levels are associated with presence of IgH translocations and these patients are more likely to harbor the high risk genetic abnormalities. Routine FISH testing for patients with newly diagnosed myeloma will allow us to confirm these and allow better risk stratification of the patients. Additional work needs to be done to help us better understand the molecular basis for unbalanced light chain excretion by myeloma cells and the current finding of the association with IgH translocation abnormalities should form the basis for this work. Figure 1. Distribution of FLC ratio (inv vs. uninv) and FLC difference (inv − uninv) among patients. Solid squares represent patients with t(4;14) or t(14;16) or t(11;14) or other 14q32 translocation; open circles represent patients without t(4;14), t(14;16), t(11;14) and any other 14q32 translocations. The horizontal dashed lines refer to FLC ratio original values of 100 and 1000, and vertical dashed lines refer to FLC difference original values of 10 and 100. Solid line indicates significant association (P<=0.05) between the dichotomized FLC groups and translocation status. Figure 1. Distribution of FLC ratio (inv vs. uninv) and FLC difference (inv − uninv) among patients. Solid squares represent patients with t(4;14) or t(14;16) or t(11;14) or other 14q32 translocation; open circles represent patients without t(4;14), t(14;16), t(11;14) and any other 14q32 translocations. The horizontal dashed lines refer to FLC ratio original values of 100 and 1000, and vertical dashed lines refer to FLC difference original values of 10 and 100. Solid line indicates significant association (P<=0.05) between the dichotomized FLC groups and translocation status.

Translocations involving the immunoglobulin heavy-chain locus are possible early genetic events in patients with primary systemic amyloidosis

Primary systemic amyloidosis (AL) is a plasma cell (PC) dyscrasia with clinical similarities to multiple myeloma (MM) and monoclonal gammopathy of undetermined significance (MGUS), but its molecular basis is poorly understood. Translocations at the immunoglobulin heavy-chain (IgH) locus, 14q32, are likely early genetic events in both MM and MGUS and involve several nonrandom, recurrent, partner chromosomes such as 11q13, 16q23, and 4p16.3. Given the similarities between MM, MGUS, and AL, bone marrow clonal PCs were evaluated in 29 patients with AL using interphase fluorescence in situ hybridization (FISH) combined with immunofluorescence detection of the cytoplasmic light-chain (cIg-FISH) for the presence of 14q32 translocations and the t(11;14)(q13;q32). Of 29 patients studied, 21 (72.4%) showed results compatible with the presence of a 14q32 translocation, and 16 (76.2%) of those had translocation (11;14)(q13;q32) for an overall prevalence of the abnormality of 55%. IgH translocations are common in AL, especially the t(11;14)(q13;q32).

Detection of 14q32 translocations in B-cell malignancies by in situ hybridization with yeast artificial chromosome clones containing the human IgH gene locus

Partner sites of 14q32 translocations found in B-cell malignancies were detected by fluorescence in situ hybridization (FISH) using yeast artificial chromosome (YAC) clones, Y20 and Y6, containing the human Ig heavy chain (IgH) gene locus. Y20 spans a 160-kb upstream and 40-kb downstream region of the JH segments on chromosome band 14q32.33. Y6 is 300-kb upstream of Y20, and spans a further 320-kb telomeric region. The human DNA sequences amplified by Alu polymerase chain reaction of the YAC clones were used as probes for FISH to study six patients with non-Hodgkin's lymphoma (NHL), one patient with acute lymphoblastic leukemia, and one cell line FR4 established from a plasmacytoma. Three telomeric YAC clones each specific for 3q, 8q, and 18q were also used to further characterize 14q32 translocations. The IgH YACs were successfully applied to detect cytogenetically invisible subtelomeric translocation of the IgH gene locus to each partner site in t(14;18), t(8;14), and t(14;19), and to identify t(3;14) (q27;q32.33) in three patients with 14q32 translocation of unknown origin. Furthermore, complex translocations involving more than three chromosomes were detected in an NHL patient with t(8;14), and t(3;12), and in the FR4 with der(14)t(8;14), der(8)dic(1;8), and del(1)(q21). The technique would be a useful tool in elucidating the mechanisms of a 14q32 translocation in B-cell malignancies.

14q32 translocations are associated with mixed-lineage expression in childhood acute leukemia

The frequency and characteristics of childhood acute leukemia with a 14q32 translocation [other than the t(8;14)(q24;q32)] were determined in 335 cases of newly diagnosed acute lymphoblastic leukemia (ALL) and 105 cases of acute nonlymphoblastic leukemia (ANLL). Ten children, representing 2.3% of the entire cohort, had this abnormality (1.5% of ALL patients and 4.8% of ANLL patients). By French-American-British (FAB) criteria, 4 cases were classified as L1, 1 as L2, 2 as M1, 1 as M2, and 2 as M5. Remarkably, mixed-lineage expression was found in 6 of these 10 cases, but in only 21 of the other 430 cases without a 14q32 translocation (P less than .001). Leukemic cells from 5 of these 6 cases (4 ANLL, and 1 ALL) coexpressed CD13, a myeloid-associated antigen, and CD2, a T-cell-associated antigen; blasts from the sixth case (ALL) coexpressed CD13 and CD19, a B-lineage-associated antigen. Thus, in addition to the well-described 11q23 translocations and t(9;22), 14q32 translocations also appear to be associated with mixed lineage antigen expression. Break-points of the reciprocal chromosomes from chromosome 14 were identified in five of these cases: 1q23, 6q23- q25, 7p15, 8q11, and 12q13. Of the four mixed-lineage cases that were tested, none showed rearrangement of the immunoglobulin heavy chain (IgH) gene. This suggests that the 14q32 breakpoint does not involve the IgH gene and that an unidentified important gene may reside on 14q32.

14q32 translocations are associated with mixed-lineage expression in childhood acute leukemia

The frequency and characteristics of childhood acute leukemia with a 14q32 translocation [other than the t(8;14)(q24;q32)] were determined in 335 cases of newly diagnosed acute lymphoblastic leukemia (ALL) and 105 cases of acute nonlymphoblastic leukemia (ANLL). Ten children, representing 2.3% of the entire cohort, had this abnormality (1.5% of ALL patients and 4.8% of ANLL patients). By French-American-British (FAB) criteria, 4 cases were classified as L1, 1 as L2, 2 as M1, 1 as M2, and 2 as M5. Remarkably, mixed-lineage expression was found in 6 of these 10 cases, but in only 21 of the other 430 cases without a 14q32 translocation (P less than .001). Leukemic cells from 5 of these 6 cases (4 ANLL, and 1 ALL) coexpressed CD13, a myeloid-associated antigen, and CD2, a T-cell-associated antigen; blasts from the sixth case (ALL) coexpressed CD13 and CD19, a B-lineage-associated antigen. Thus, in addition to the well-described 11q23 translocations and t(9;22), 14q32 translocations also appear to be associated with mixed lineage antigen expression. Break-points of the reciprocal chromosomes from chromosome 14 were identified in five of these cases: 1q23, 6q23- q25, 7p15, 8q11, and 12q13. Of the four mixed-lineage cases that were tested, none showed rearrangement of the immunoglobulin heavy chain (IgH) gene. This suggests that the 14q32 breakpoint does not involve the IgH gene and that an unidentified important gene may reside on 14q32.