Secondary Chromosomal Abnormalities Research Articles

Abstract B114: Population differences in the associations between chromosomal abnormalities and overall survival among Blacks and Whites with multiple myeloma

Abstract Background: Several primary and secondary cytogenetic abnormalities (e.g., translocation t(4;14) and 1q copy number changes) are associated with poor prognosis and worse survival of multiple myeloma (MM). Some of the aberrations (e.g., t(11;14), t(14;16), and t(14;20)) have been suggested, albeit inconclusively, to be enriched in Blacks. However, whether the associations between chromosomal abnormalities and overall survival (OS) of MM differ by race/ethnicity remains largely unknown. Methods: We prospectively enrolled 586 patients with newly diagnosed MM between 2010-2019 at the University of Chicago Medical Center. Of the 561 subjects identified as Black or White, 181 had chromosomal abnormalities determined by fluorescence in situ hybridization (FISH), which used CD138+ plasma cells enriched from bone marrow aspirates collected within 180 days of diagnosis. We analyzed data for 19 aberrations, including IGH/FGFR3 fusion [t(4;14)(p16,q32)]; IGH/CCND1 fusion [t(11;14)(q13,q32)]; IGH/MAF [t(14;16)(q32,q23)]; IGH/MAFB [t(14;20)(q32,q12)]; trisomies of chromosome 3, 7, 9, and 12; chromosome 13 deletion; loss of ATM [11q22.3]; gain of ATM; loss of TP53 [17p13.1]; gain of TP53; loss of CDKN2C [1p32.3]; gain of CKS1B [1q21]; IGH translocation with uncommon chromosome partners; IGH locus rearrangement; loss of IGH; and tetraploidy. Vital status was ascertained using the National Death Index. Baseline clinical and laboratory data were retrieved from electronic medical records. We used proportions and Chi-square tests to compare the distributions of cytogenetic abnormalities in Blacks and Whites. In each population, we evaluated the associations between cytogenetic abnormalities and OS using hazard ratios (HRs) and 95% confidence interval (CIs) from Cox proportional hazards models, adjusting for prognostic factors including age, international staging system, elevated lactate dehydrogenase, and estimated glomerular filtration rate. We used the Wald test of the cross-product terms to assess potential heterogeneity of effect in the HRs by race. Results: Of the 181 MM patients, 55 (30.39%) were Black and 126 (69.61%) were White. The median follow-up was 69 months. The frequencies of the 19 cytogenetic abnormalities did not differ between Blacks and Whites. However, we observed population differences in the associations between two chromosomal abnormalities and OS: the presence of t(4;14) was associated with worse OS in Blacks (HR=14.34 [2.17-94.58]; N=38) but not Whites (HR=2.04 [0.70-5.88]; N=99, p-heterogeneity = 0.08), whereas gain of 1q was associated with poorer OS in Whites (HR: 3.38, 95% CI: [1.25- 9.09], N=77), but not Blacks (HR=1.32 [0.31-5.68]; N=32; p-heterogeneity=0.02). Conclusions: Although we found similar frequencies of primary and secondary chromosomal abnormalities between Blacks and Whites, our data show population differences in the associations between two established high-risk cytogenetic abnormalities and the OS of MM. Our findings suggest that cytogenetic features may have varying impacts on the OS of MM based on race. Citation Format: Bei Wang, Benjamin A. Derman, Jason Cheung, Wei Zhang, Andrzej Jakubowiak, Brian C. Chiu. Population differences in the associations between chromosomal abnormalities and overall survival among Blacks and Whites with multiple myeloma [abstract]. In: Proceedings of the 16th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2023 Sep 29-Oct 2;Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(12 Suppl):Abstract nr B114.

IS THERE A PROLIFERATIVE ADVANTAGE FOR NORMAL CELLS ASSOCIATED WITH MARKER CHROMOSOMES OVER INTERPHASIC NUCLEI POSITIVE FOR PML-RARA FUSION?

Acute promyelocytic leukemia (APL) is usually related to a good prognosis, however, about 20% of patients whose outcome is not favorable, early complications cause mortality in 10%, and disease relapse or drug resistance affects the other 10%. Prompt diagnosis and early initiation of treatment are critical to lowering the risk of early mortality. Although the hallmark of APL is the PML-RARA fusion, generated from t(15;17)(q24;q21), it is common to observe additional chromosomal abnormalities, with a clinical impact not yet established, associated with this neoplasm, which arouses the interest of different study groups in the literature about additional (cryptic) anomalies related to the disease. In this work, we present a FISH positive PML-RARA case, but negative in chromosome banding analysis, associated with a very poor prognosis in an adolescent with APL. A 14-year-old girl was admitted to the pediatric hematology service of the Lagoa Federal Hospital, with a history of 30 days of edema, pain, and intense gingival bleeding, associated with fever and pancytopenia. A bone marrow (BM) sample was referred to the Laboratory of Cytogenetics of CEMO – INCA. FISH and molecular biology tests confirmed the PML-RARA fusion, helping to define the diagnosis of AML-M3, and the patient was treated under the BFM-AIOPE-2012 protocol. Treatment with ATRA was initiated, but on the fourth day, she evolved with pulmonary condensation and desaturation and chemotherapy was suspended. Computed tomography revealed multiple hemorrhage sites. Hydreia was administrated to reduce white blood cell count and treatment with ATRA and ATO was resumed. However, on the fourth day of treatment, the adolescent evolved with low saturation being then intubated, in addition to worsening of bleeding even with intense transfusional support. The patient died 20 days after the initial diagnosis. The patient's BM sample was studied by G-banding and FISH assays. The karyotype was 46,XX, with a marker chromosome. FISH analysis revealed the PML-RARA gene fusion in 90% of the interphase nuclei analyzed, besides an extra RARA gene signal, but this abnormality was not observed in metaphase spreads. Complementary FISH with the RARA break apart probe showed a RARA gene rearrangement in 90% of the interphase nuclei analyzed, without the involvement of the secondary chromosomal abnormality. Molecular studies detected a breakpoint within PML intron 3 (bcr 3) producing a short PML-RARA isoform. Although secondary cytogenetic abnormalities occur in about 30% of cases of APL, their prognostic importance has not been determined, and these additional cytogenetic abnormalities are often complex. We could not eliminate the secondary alteration as a cryptic abnormality, however, through FISH (in interphase nuclei), the gold standard method, we showed the PML-RARA fusion. In the past, before the advances brought by FISH methodology for detecting PML-RARA fusion, the hallmark of APL, there was controversy about the culture time needed to diagnose the t(15;17)(q24;q21). In our patient, we observed a chromosome marker that may confer a proliferative advantage for normal cells over aberrant interphasic nuclei positive for PML-RARA. Our findings confirm that FISH, regardless of the culture time used, is the fastest and most effective method to confirm the diagnosis of this hematologic urgency. However, molecular studies in metaphases with the observed marker chromosome will be important to elucidate/eliminate the involvement of cryptic abnormalities present in the karyotype of the patient in this study. Besides, the influence of PML-RARA isoform on patient outcomes remains controversial.

Chromosome microarray characterisation of chromosome arm 12p loss associated with complex molecular karyotype and recurrent adverse cytogenetic markers in multiple myeloma

Copy number loss within chromosome 12 short arm (12p) has gained attention as an adverse cytogenetic marker in multiple myeloma. The prognostic significance and characterisation of the common minimal deleted region remains controversial between various studies with loss of CD27 proposed as the putative critical gene. We aimed to determine the frequency of 12p loss, its correlation with adverse cytogenetic markers further to define and characterise 12p deletions. Our study included a prospective cohort of 574 multiple myeloma patients referred for cytogenetic testing, including interphase fluorescence in situ hybridisation for IGH (14q32.33) translocations and chromosome microarray. Loss of 12p was detected in 54/574 (9.4%) patients and when compared with the non-12p loss group [520/574 (90.6%)], 12p loss patients demonstrated a statistically significant association with specific recurrent cytogenetic markers: complex molecular karyotypes (98.1% vs 45.2%), 1p loss (50.0% vs 20.2%), t(4;14) (20.4% vs 7.7%), 8p loss (37.0% vs 15.0%), 13/13q loss (70.4% vs 41.7%), and 17p loss (33.3% vs 6.5%). The size and location of 12p losses were heterogeneous with a common 0.88 Mb minimally deleted region that included ~9 genes from ETV6 to CDKN1B in 52/54 (~96.3%) patients but did not include CD27. Our findings support 12p loss being a secondary chromosome abnormality frequently co-occurring with adverse cytogenetic markers and complex molecular karyotypes indicative of chromosome instability.

Structure and signifi cance of cytogenetic abnormalities in patients with multiple myeloma

Introduction. Cytogenetic and genomic traits of tumour cells are considered the key mediating factors in multiple myeloma (MM). Selected chromosomal abnormalities are prognostic of therapeutic response and patient survival in MM.Aim — to assess of the diversity and rate of chromosomal abnormalities in MM patients and their association with the disease course.Materials and methods. The study enrolled 134 MM patients with pre-treatment bone marrow FISH assay screening for chromosomal abnormalities: t(11;14), t(4;14), t(14;16), t(14;20), t(6;14), hyperdiploidy, del13q14/-13, del17p13/TP53, amp1q21, t(8q24)/cMYC. The studied criteria at the MM onset were: hemogram, lactate dehydrogenase (LDH) activity, calcium, β2-microglobulin and creatinine concentrations, punctate cytology, bone marrow trephine biopsy and/or soft tissue biopsy histology, bone X-ray, immunochemical variant of MM, disease staging. A median follow-up was 20 months (3.2–77.4).Results. The primary chromosomal abnormality rate was 82.9 %, among them t(14q32)/IGH — 29.1 %, multiple trisomies — 46.3 % and their combination — 7.5 %. The rates of particular t(14q32)/IGH): t(11;14) — 16.4 %, t(4;14) — 12.7 %, t(14;16) and t(14;20) — 3.7 and 2.2 %, respectively. The secondary chromosomal abnormality rate was 69.4 %, among them del13q14/-13 — 40.3 %, amp1q21 — 39.6 %, t(8q24)/cMYC — 17.2 %, del17p13/TP53 — 12.7 %, del1p32 — 2.2 %. Analyses of the primary–secondary abnormality combinations showed that del13q14/-13 is more frequently combined with t(4;14) and less frequently with trisomies (p < 0.05). Amp1q21 occurs more frequently with t(4;14) and less — with t(11;14) (p<0.05). Patients with t(4;14) more frequently (p < 0.05) had anemia at a hemoglobin level<100 g/L, and the presence of amp1q21 and del17p13/TP53-enhanced serum LDH activity (p < 0.05). Abnormality t(8q24)/cMYC more often co-occurred with higher serum β2-microglobulin concentrations (p < 0.05). A three-year overall survival (OS) in del17p13/TP53-positive patients was 35.5 vs. 71.3 % in the negative (p = 0.002) and 50.8 vs. 67 % — in t(8q24)/cMYC-positive and negative patients, respectively (p = 0.001). Patients without amp1q21, with one, with two or more additional 1q21 copies had a five-year OS 79.4, 67.3 and 20.9 %, respectively (p = 0.0016), and a two-year progression-free survival (PFS) 83, 50 and 0 %, respectively (p = 0.005).Conclusion. We establish a negative impact of del17p13/TP53 and t(8q24)/cMYC on patients’ OS in MM, as well as unfavourable effect of amp1q21 on OS and PFS in the presence of two or more additional copies of 1q21 loci.

Comprehensive Characterization of Cytogenetic and Mutational Analysis of Acute Promyelocytic Leukemia: Is PML-Rara Everything?

Acute promyelocytic leukemia (APL) is characterized by PML-RARA fusion caused by the t(15;17)(q24;q21) translocation. Although PML-RARA fusion explains the dedifferentiation in most of APL patients, it still does not entirely represent the unique cause of all the clinical manifestations of the disease failing to determine the full leukemic phenotype. Up to 40 % of APL patients have an additional chromosomal abnormality other than PML-RARA. Murine studies have reported that additional cytogenetic abnormalities and secondary somatic mutations (for instance FLT3-ITD) might contribute to leukemia progression. Indeed, mice expressing mutant PML-RARA develop definitive leukemia after one year, suggesting that additional hits are required for transformation. Moreover, no distinct genetic signature has been characterized by next generation sequencing (NGS). This confirms that no gene has been reproducibly identified. APL respond to all-trans retinoic acid (ATRA) in the great majority of patients. However, one quarter of APL develop ATRA resistance suggesting that additional secondary chromosomal abnormalities might be evolving resistance. Combination of low dose arsenic, modify certain epigenetics, with ATRA decreased resistance potential and improved response. In the line with other possible factors involved in ATRA resistance, is the broad nature of the targets of ATRA. A molecular core network of ATRA's targets has been clustered in differentiation, growth factors and nuclear receptors possibly cooperating with PML-RARA and additional chromosomal abnormalities. Herein, we aimed to characterize the gene mutations and chromosomal abnormalities playing key roles in cellular differentiation and epigenetic regulation and to correlate the occurrence of these alterations with treatment response and survival outcomes in APL. We took advantage of a large cohort of APL patients (n=145). Median age of the cohort was 50 yrs (19-85); equal gender distribution; median blood counts were: [WBC 6.2 x 109/L (0.4-155); 37% had leukopenia], hemoglobin [9.8 g/dL (2.7-16.2); 32% had anemia] and platelets [29 x 109/L (range of 0-228); 93% had thrombocytopenia]. In terms of karyotype, 15% of the patients carried +8, 7% had complex karyotyping (≥3 cytogenetic abnormalities), 2% had -7/del (7q) or del (12p), 1% had -17/del(17p), and 1 patient had -5. Mutational analysis of 30 genes panel, identified 141 mutations carried by 65% (94/145) of APL patients. The most frequent mutations were observed in FLT3-ITD (61/143; 43%), WT1 (26/139; 23%), and ASXL1 (7/136; 5%) genes. Less frequent mutations were found in 3.7% of CEBPA, KRAS, and NRAS genes as well as in CBL, EZH2, TET2 (3% each) genes. Additionally, we noted that all mutations were recurrent in specific functional pathways and patients carried mutations in more than 1 gene of the same pathway. Of note, cell signaling and proliferation genes (CBL, NRAS, KRAS, KIT, FLT3) were the most frequently mutated (77/141, 55%) and impacted OS (HR: 1.7, P=0.02). Moreover, transcriptional factors which are often mutated in AML (e.g. CEBPA, TP53, NPM1, RUNX1, WT1) as well as major determinants of cell's fate were markedly mutated (38/141, 27%) suggesting that genetic impairment of signaling and transcription might contribute to the lack of differentiation observed in APL phenotypes. Mutations in epigenetic genes and histone methyltransferases (ASXL1, BCORs, DNMT3A, EZH2, IDH1/2, TET2) were also found in 18/141 (13%) while genes regulating cell proliferation and RAS family (CBL, NRAS, KRAS, NPM1) were enriched in 16/132 (12%) of APL cohort. We then analyzed the genetic picture of remission (APLRm, n=131, 90%) and relapsed (APLR, 1sr relapsed to ATRA, n=14, 10%) patients. Acknowledging the low number of APLR, we observed that molecular mutations did not make a key difference in APLRvs. APLRm [except for a complete lack of mutations in epigenetic pathways (0% vs. 13%)]. Contrarily, specific cytogenetic abnormalities were more common in APLR compared to APLRm as the case of +8 (36% vs. 11%; P= .02) and -17/del(17p) (2/14 vs. 0/131; P= .008). In sum, our study demonstrates that PML-RARA might be accompanied by additional acquired chromosomal change with a variety of genetic mutations in key pathways driving cellular differentiation. These molecular/ cytogenetic associations could determine resistance to ATRA and overall APL patients' survival. Disclosures No relevant conflicts of interest to declare.

1p36 deletion is associated with TNFRSF14 mutations in primary cutaneous follicle center lymphoma

Background: Primary cutaneous follicle center lymphoma (PCFCL) is an indolent variant of follicular lymphoma (FL) with limited information available on the genetic background of the disease. The genetic hallmark of nodal FL, the t(14;18) translocation, affecting the BCL2 gene, is rare in PCFCL. Loss of 1p36, the most common secondary chromosomal abnormality in nodal FL, has been recently reported in 16.7% of PCFCL cases. The mutation analysis of TNFRSF14, the candidate gene in the 1p36 region has not yet been performed.

Concomitant 1p36 deletion and TNFRSF14 mutations in primary cutaneous follicle center lymphoma frequently expressing high levels of EZH2 protein.

Primary cutaneous follicle center lymphoma (PCFCL) is an indolent variant of follicular lymphoma (FL) with limited information available on the genetic background of the disease. The genetic hallmark of nodal FL, the t(14;18) translocation, affecting the BCL2 gene, is rare in PCFCL. Loss of 1p36, the most common secondary chromosomal abnormality in nodal FL, has been recently reported in 16.7% of PCFCL cases. In order to further characterize PCFCL, 21 cases were analyzed using interphase fluorescence in situ hybridization with BCL2 break apart and 1p36/1q25 dual color probes. Sanger sequencing was used to investigate TNFRSF14 and EZH2 mutations and immunohistochemistry to assess BCL2, EZH2 protein expressions.1p36 deletion occurred in 22% (5/21), BCL2 gene break in 10% (2/20) of the PCFCL cases. Mutations of the candidate tumor suppressor gene of the 1p36 region, TNFRSF14 mutations were detected in 4/17 (23.5%) cases with 2 cases presenting with concurrent 1p36 deletion. EZH2 hotspot mutations at Y641, A682, and A692 were not found. High EZH2 protein expression associated with a BCL2 negative phenotype was observed in 43% (9/21) of the cases. BCL2 gene break or 1p36 deletion did not impact the prognosis; however, they showed association with advanced stages at diagnosis (p = 0.016) and a tendency with shorter event free survival (p = 0.052).In conclusion, 1p36 deletion co-occurs with acquired TNFRSF14 mutations, suggesting a role of this tumor suppressor gene in the development of a subgroup of PCFCL. High EZH2 protein expression associated with BCL2 negative phenotype is common and might represent an ideal therapeutic target.

Cytogenetic analysis of acute myeloid leukemia with t(8;21): Its clinical correlation with loss of X Chromosome and Del (9q)

BACKGROUND: Translocation (8;21), t(8;21), is one of the most common cytogenetic abnormalities in adult de novo acute myeloid leukemia (AML) patients. It is usually associated with secondary chromosomal abnormalities; however, it's unclear whether these abnormalities affect the clinical characteristics of t(8;21) patients. OBJECTIVES: To investigate the effect of additional aberrations; loss of X chromosome and deletion of the long arm of chromosome 9 (del 9q)on the clinicopathological and immunophenotypic characteristics and prognostic behavioral of t(8;21) de novo AML. METHODS: Fifty six adults with de novo AML-M2 were enrolled. Detection of loss of X-chromosome and del 9q were performed using fluorescent in situ hybridization (FISH). RESULTS: More than half of the patients (53.6%) harbored a secondary chromosomal abnormality in addition to t(8;21). Del 9q was found in 17.9% of the patients. A significant association was found between this chromosomal aberration and age, hepatomegally, high total leucocytic count and low platelets count (P< 0.05). Most patients had poor clinical outcome, high tendency for resistance to therapy and significantly shorter survival. On the other hand, loss of X chromosome was found in 25% of the studied patients and was not related to clinicopathological features or prognostic markers except for high platelets count and low expression of aberrant CD19 (P< 0.05). CONCLUSIONS: 9q deletion associated t(8;21) could be considered as an adverse prognostic predictor being associated with poor disease outcome and shorter survival of the patients. Thereafter the use of these cytogenetic aberrations would be recommended to guide therapeutic regimens.

Abstract IA06: Intratumor heterogeneity and its role in therapeutic escape in multiple myeloma

Abstract Multiple myeloma (MM) is a mature B cell neoplasm characterized by the accumulation of immunoglobulin-secreting plasma cells (PC) within the bone marrow. Despite treatment advances, MM remains incurable in the majority of patients and is over-represented in current cancer death rates. The lack of reliable cure in MM, despite the achievement of deep clinical responses with proteasome inhibitors (PIs), immunomodulatory drugs (IMiDs), and myeloablative melphalan, implicates the presence of intratumoral heterogeneity and the existence of drug-resistant cells with tumorigenic capacity. However, the molecular and cellular basis of this intratumoral heterogeneity remains only partially understood. Using sequential FACS and single-cell immunofluorescence-FISH or genomics strategies, we have identified clonal tumor progenitor subpopulations within the bone marrow of MM patients that recapitulate the physiologic maturation stages between post-germinal center B cells and plasma cells. These clonal MM subpopulations include tumor cells that resemble CD20+ CD27+ CD38- CD138- Irf4- Xbp1s- memory B cells, CD38+/- CD138- Irf4- Xbp1s- pre-plasmablasts, CD38+ CD138- Irf4+ XBP1s+ plasmablasts, and tumor-bulk CD38+ CD138+ IRF4+ Xbp1s+ plasma cells. Importantly, compared to tumor bulk plasma cells, early Xbp1s- tumor progenitor cells appear innately resistant to mainstay treatment with proteasome inhibitors (PI). Mechanistically, absence of Ire1-Xbp1 signaling in these primitive MM cells is associated with secretory immaturity; as Xbp1s- MM cells produce less secretory immunoglobulin, an important determinant of endoplasmic reticulum (ER) load in PCs, they experience less ER stress than Xbp1s+ cells and are thus less susceptible to induction of cytotoxic ER stress when ER-associated degradation (ERAD) is inhibited by PIs. Significantly, small numbers of Xbp1s- tumor cells can be detected in most MMs and as these progenitor cells are innately insensitive to PI-induced cytotoxic ER stress, these observations can conceivably account for a universal low level resistance that is inherent in the pathogenesis and residual progenitor structure of MM and that prevents PI-induced cure. While these findings may account for some clinical features of MM, at present it remains unknown whether or not Xbp1s- MM tumor progenitors are fully malignant or if they possess the capacity required to reinitiate MM disease. Plausibly, Xbp1s- pre-plasma cells in MM may instead represent a counterpart to the premalignant preleukemic stem cells identified in AML, CLL and HCL, or may alternatively represent dedifferentiated PCs. Although this heterogeneity in tumor cell maturation may contribute to therapeutic evasion, MM tumors also commonly contain multiple genetic subclones that show tidal dominance under therapeutic pressure that may also facilitate treatment escape. Such genetic heterogeneity is not mutually exclusive with diversity in tumor cell maturation. When MM genetic subclones with common recurrent secondary chromosomal structural abnormalities (such as del 13, del 17p, +1q or del 1p) are identified among tumor bulk PCs, similar subclones may also be identified amongst non-PC Irf4- Xbp1s- MM subpopulations; this suggests that tumor progenitor cells may play a role in the genetic evolution of tumors and may represent a reservoir for subclonal relapse. In support of a model in which MM PCs can be replenished from MM progenitor cells, primary Xbp1s- MM progenitors possess the ability to differentiate in vitro into mature Xbp1s+ MM plasma cells. Notably, however, in contrast to a model in which MM PCs are continuously replenished by MM progenitors, we have also identified some genetic events that appear to show restricted representation among tumor PCs, consistent with MM PCs comprising a self-sustaining tumorigenic population in its own right. Overall, these data support a model in which Xbp1s- progenitor subpopulations in MM represent a reservoir of innately PI-resistant cells that, following eradication of tumorigenic PCs, may give rise to clonally related clinical relapse via MM progenitor cell differentiation. Overall these findings imply that treatment strategies in MM need to better address Xbp1s- IRF4- tumor stem/progenitor cells, while also eradicating MM-bulk PCs, to achieve deeper treatment responses and cure for patients. Citation Format: Rodger E. Tiedemann. Intratumor heterogeneity and its role in therapeutic escape in multiple myeloma [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr IA06.

The role of PI3K in modulating NKT cell responses to Mantle Cell Lymphoma

Abstract Natural Killer T (NKT) cells are important in the initiation and regulation of immune responses to cancers. The observation that mantle cell lymphoma (MCL) patients have a significant reduction in both NKT cell number and function suggests that the antitumor effects mediated by these cells may be compromised. MCL is characterized by the chromosomal translocation and nuclear overexpression of Cyclin D1. Secondary chromosomal abnormalities and dysregulation of cell cycle machinery also contribute to MCL pathogenesis, such as aberrant or constitutive activation of phosphatidylinositol 3 kinase (PI3K). We hypothesize that constitutive activation of PI3K in MCL alters the repertoire of lipids in these cells, ultimately resulting in decreased CD1d-mediated NKT responses to MCL. In this study, we used small molecule pharmacologic inhibitors of PI3K (PI3Ki) to investigate the role of PI3K signaling on MCL survival and immunogenicity both in vitro and in vivo. Treatment with PI3Ki induced apoptosis in human MCL cell lines and enhanced their immunogenicity. In addition, we have found that treatment of peripheral blood mononuclear cells with isoform-specific PI3Ki does not suppress T cell activation; whereas, treatment with a pan PI3Ki, BEZ-235, completely abrogates NKT/T cell responses. These studies suggest inhibitors targeting individual isoforms may have higher therapeutic efficacy. Collectively, this work will advance our understanding of differences in the modes of PI3K signaling in MCL survival and in antigen processing and presentation.

Conventional and molecular cytogenetic characterization of Burkitt lymphoma with bone marrow involvement in Brazilian children and adolescents

Burkitt lymphoma/leukemia (BL/L) is cytogenetically characterized by the t(8;14)(q24;q32) or its variants, t(2;8)(p11;q21), and t(8;22)(q24;q11.2), which juxtapose the MYC oncogene to one of the three immunoglobulin loci. The overall cure rate of BL/L in children is 70-90%, but patients diagnosed with advanced-stage disease have a less favorable prognosis. It is possible that secondary chromosomal abnormalities contribute to this unfavorable prognosis via chemotherapy resistance, but the results of genetic studies have been inconsistent. This study aimed to identify and characterize secondary chromosomal abnormalities associated with the t(8;14) and its variants in children with French-American-British-L3 leukemia or Burkitt lymphoma with bone marrow involvement at the time of diagnosis. Chromosome analysis was based on G-banding. Fluorescence in situ hybridization technique was applied using IGH/MYC/CEP8 dual-fusion and MYC break-apart probes. Multicolor chromosome banding was performed according to standard protocol. We describe a group of BL/L with extreme adverse clinical outcome, in which secondary chromosomal abnormalities, particularly those involving the long arms of chromosomes 1 and 13, were found in 71% of cases. The IGH/MYC fusion showed molecular heterogeneity in 14% of cases and two cases exhibited three IGH/MYC fusion signals. Secondary chromosomal abnormalities were found in a high proportion of patients. We observed an extent of IGH/MYC heterogeneity not previously reported in Burkitt lymphoma, including the novel finding of three fusion signals in two cases.

Previously Unreported Chromosomal Aberrations of t(3;3)(q29;q23), t(4;11)(q21;q23), and t(11;18)(q10;q10) in a Patient with Accelerated Phase Ph+ CML

Chronic myelogenous leukemia (CML) is a clonal hematological disorder, which is characterized by the presence of the classical or variant Philadelphia translocations. During the progression to blastic phase of the disease secondary chromosomal abnormalities may emerge. Such secondary chromosomal abnormalities are nonrandom, the more frequent ones being trisomy 8 and 19, supernumerary i(17q), and extra Philadelphia chromosomes. Furthermore, a minor percentage of the patients may acquire different secondary chromosomal abnormalities including translocations between other chromosomes. We report here a patient with Ph+ CML presenting secondary chromosomal abnormalities including t(4;11)(q21;q23), t(3;3)(q29;q23) and t(11;18)(q10;q10) during the course of CML progression.

Clinical and laboratory investigation of pericentric inv(9)(p22q34) with the der(9)t(9;22)(q34;q11) in Ph-positive leukemia

To investigate clinical and molecule genetics features of four Ph-positive leukemia patients characterized by pericentric inv(9)(p22q34) with the der(9)t(9;22)(q34;q11). Cytogenetic analysis was carried out on bone marrow directly or after short-period culture. R banding was used for karyotype analysis. BCR/ABL fusion gene was detected with interphase fluorescence in situ hybridization (FISH), and chromosome painting was carried out using specific probes. RT-PCR was used to detect BCR/ABL chimeric transcripts. One patient with acute myeloid leukemia (AML) presented three clones, which included one with a normal karyotype, one with t(9;22)(q34;q11), and one with inv(9)(p22q34) involving the der(9)t(9;22) and additional t(8;12)(q12;p11). The inv(9)(p22q34) has always co-occurred with der(9)t(9;22)(q34;q11) accompanied by der(22)t(9;22)(q34;q11) in all metaphases from the three patients with chronic myeloid leukemia (CML). B3a2 transcript was detected in all patients by RT-PCR. Inv(9)(p22q34) was found in both CML and AML, and was associated with poor prognosis. Inv(9)(p22q34) is a novel, rare, but recurrent secondary chromosomal abnormality for Ph-positive leukemia. Leukemia with der(9)t(9;22) and inv(9)(p22q34) has unique clinical and laboratory characteristics.

Co-expression of t(15;17) and t(8;21) in a Case of Acute Promyelocytic Leukemia: Review of the Literature

Additional chromosomal abnormalities in acute myelogenous leukemia have been identified as one of the most important prognostic factors. Favorable chromosomal changes such as t(8;21), inv(16), and t(15;17) are associated with higher rates of complete remission and event-free survival. Translocation (15;17) characterizes acute promyelocytic leukemia (APL) (French-American-British class M3) in almost all patients. Secondary chromosomal abnormalities are also present in approximately 23%-29% of patients with newly diagnosed APL. The prognostic implications of t(8;21) and other secondary cytogenetic aberrations in APL are reviewed here. We present a 47-year-old woman diagnosed with APL whose initial cytogenetic analysis included both t(8;21) and t(15;17). The initial induction chemotherapy included 3 days of idarubicin (12 mg/m2/day) and daily all-trans retinoic acid (ATRA; 45 mg/m2/day). At the sixth week of treatment, a control bone marrow biopsy was found to be normocellular, t(15;17) bcr3 and t(8;21) were negative, and t(15;17) bcr1 fusion transcripts were reduced from 5007 (1.78525699%) copies per 1 µg RNA to 40 (0.00062020%) with real-time quantitative polymerase chain reaction. Consolidation with 4 days of idarubicin (5 mg/m2/day), ATRA (45 mg/m2/day for 15 days), and cytarabine (1 g/m2/day for 4 days) was then started. However, the patient became pancytopenic and had neutropenic fever after consolidation treatment. Unfortunately, she died 3 months after the time of APL diagnosis, due to acute respiratory distress syndrome-like respiratory problems and multiorgan dysfunction requiring respiratory support and hemodialysis. Conflict of interest:None declared.

Submicroscopic genomic imbalances in burkitt lymphomas/leukemias: Association with age and further evidence that 8q24/MYC translocations are not sufficient for leukemogenesis

Chromosome banding analyses reveal secondary chromosome abnormalities in addition to the MYC translocations t(8;14)(q24;q32), t(8;22)(q24;q11), and t(2;8)(p11;q24) in 60%-80% of Burkitt lymphomas/leukemias (BL). The high incidence of such aberrations indicates that additional changes are important, perhaps necessary, for malignant transformation, i.e., the 8q24/MYC rearrangements may not be sufficient. To investigate this possibility, we performed single nucleotide polymorphism (SNP) array analysis on 20 cases of 8q24/MYC-positive BL. Nineteen (95%) harbored genomic imbalances; the only case without such aberrations displayed secondary changes by chromosome banding analysis. Thus, all BL cases had abnormalities in addition to the 8q24 translocation. The adult cases harbored more changes (median 3; range 1-21) than did the childhood cases (median 1.5; range 0-5) (P = 0.034). Several recurrent aberrations were detected by SNP array analysis, in particular losses of 6q14.1-q22.33, 9p21.3, and 13q14.2-q14.3, gains of 1q23.3-q31.3, chromosome 7, 13q31.3, and partial uniparental isodisomies for 6p12.2-pter, 9p23-pter, and 17p11.2-pter. The molecular genetic consequences of these changes include deletions of the CDKN2A and TP53 genes, and gains/losses of several genes, such as MIR17HG and E2F2K, involved in the MYC pathway. Thus, deregulation of the MYC pathway, both directly through the 8q24/MYC translocation and indirectly through secondary genomic imbalances, may be essential not only for the initiation but also for the progression of BL.

Genetic Abnormalities in Follicular Lymphoma and Transformed Follicular Lymphoma.

AbstractAbstract 2648Follicular lymphoma (FL) is an indolent lymphoma and the second most common type of non-Hodgkin lymphoma in the Western world. It is characterized by the t(14;18) chromosomal translocation, which is present in up to 90% of cases. About 40% of FL cases eventually transform into a more aggressive lymphoma (tFL), most commonly diffuse large B-cell lymphoma (DLBCL). To identify the secondary chromosomal abnormalities that contribute to the development of FL, and to its transformation, we undertook a large study using the Affymetrix 250k NspI SNP array to identify copy number abnormalities (CNAs) in 198 FL and 79 tFL samples, 75% of which have concurrent gene expression profiling studies using Affymetrix U133+2 or A+B arrays for correlative analysis. There were 22 recurrent chromosomal abnormalities that were present in over 10% of FL cases, including gains of 7, 12, 18, 21, X, 1q, 2q, 5p, 6p, 8q, 17q and loss of 6q. We also identified 20 smaller CNAs that occurred in over 5% of FL cases, the most frequent being loss of chromosome 1p36.33-p36.31 including TNFRSF14, loss of chromosome 10q23.1-q25.1 encompassing several possible cancer-related genes such as PTEN, gain of chromosome 2p16.1-p15 including REL, and gain of chromosome 8q24.13-q24.3 including MYC.Univariate Cox regression models were used to analyze the CNA regions that occurred in at least 10 FL cases as predictors of overall survival. Four recurrent CNAs were predictive of survival in univariate analysis below the p=0.05 significance level, and two were found to be borderline significant. A gain of X or the p arm of X was predictive of poor survival. Additionally, two losses on 6q (6q13–15 and 6q23.3–24.1) were associated with poor survival. The 6q23.3–24.1 loss contains TNFAIP3, which encodes a negative regulator of the NF-kB pathway, and is a frequent site of homozygous loss. Additionally, a gain of chromosome 8 that includes the MYC gene, and a loss of chromosome 9 that includes CDKN2A, were borderline predictors of poor survival. Patients with FLs that have 7 or more abnormalities had worse survival than those with fewer abnormalities.We also compared the CNAs found in tFL samples to FL samples and identified 26 abnormalities that were at least 5 times more frequent in tFL and present in at least 5% of tFLs. A gain of 3q27.3-q28 containing 5 genes including BCL6 and LPP, for example, was found in 11% of tFL case, but only 2% of FL cases. We also found differences in the deletion of Beta-2 Microglobulin (B2M) between FL and tFL. The B2M locus is deleted in 8% of FLs, but in 21% of tFLs. B2M, a subunit of the MHC class I molecule, is known to be repressed by mechanisms such as mutation and deletion in de novo DLBCL, as a way for the tumor to evade immune surveillance. HLA-A- B, and/or -C were deleted in 5% of FLs and almost 9% of tFLs. CD58, which plays a role in T- and NK-cell immune responses, was deleted in 3% of FLs and 11% of tFLs. Overall, 19% of FLs and 37% of tFLs had an abnormality in CD58, B2M, and/or HLA class I, indicating that evasion of immune surveillance is important in transformation to a more aggressive disease. We also compared CNAs from tFL cases to those found in de novo GCB-DLBCL cases and identified several that differed markedly between the 2 diseases, such as a gain of chromosome 21 which was present in 21% of tFL cases but only 3% of DLBCL cases.In conclusion, FL, tFL, and de novo GCB-DLBCL share common CNAs, but the prevalence of the individual lesions differ among the 3 entities. Functional validation of potential candidate genes will determine important pathways in the development and progression of FL, and identify possible targets for therapeutic intervention. Disclosures:No relevant conflicts of interest to declare.

Occurrence of acute lymphoblastic leukemia and juvenile myelomonocytic leukemia in a patient with Noonan syndrome carrying the germline PTPN11 mutation p.E139D

Noonan syndrome (NS) is a common autosomal dominant condition characterized by short stature, congenital heart defects, and dysmorphic facial features caused in approximately 50% of cases by missense mutations in the PTPN11 gene. NS patients are predisposed to malignancies including myeloproliferative disorders or leukemias. We report a female NS patient carrying a PTPN11 germline mutation c.417 G > C (p.E139D), who developed in her second year of life an acute lymphoblastic leukemia (ALL) and after remission, she developed at 4 years of age a juvenile myelomonocytic leukemia (JMML). Molecular genetic analysis of lymphoblastic blasts at the time of the ALL diagnosis revealed the germline mutation in a heterozygous state, while in the myelomonocytic blasts occurring with JMML diagnosis, the mutation p.E139D was found in a homozygous state due to a uniparental disomy (UPD). These findings lead to the suggestion that the pathogenesis of ALL and JMML in our patient is due to different mechanisms including somatically acquired secondary chromosomal abnormalities.

A new dic(7;12)(p12.21;p12.2) and i(12)(q10) during the lymphoid blast crisis of patient with Ph+ chronic myeloid leukemia

Chronic myelogenous leukemia (CML) is a common myeloproliferative disease that is characterized by the clonal expansion of marrow stem cells, and is associated with the Philadelphia chromosome. As the disease progresses, additional chromosome abnormalities may arise. The prognostic impact of secondary chromosomal abnormalities in CML is complex, heterogeneous, and sometimes related to previous treatment. Here, we describe a CML patient in lymphoid blast crisis associated with a new chromosomal abnormality identified, dic(7;12)(p12.21;p12.2) and i(12)(q10) using classical cytogenetics and spectral karyotype analysis. To the best of our knowledge, this is the first report of t(7;12)(p11.1;q11.1) and i(12)(q10) in a CML patient with lymphoid evolution.

Efficacy of thalidomide- or lenalidomide-based therapy in proliferative multiple myeloma

The proliferative activity of neoplastic plasma cells (PCs) is an independent determinant of the clinical course of patients with multiple myeloma (MM). It can be measured by the plasma cell labeling index (PCLI), which denotes the percentage of bone marrow PCs in the S-phase of the cell cycle.1 Typically, MM is a slow-growing tumor with very few PCs synthesizing DNA at a given time. Proliferative myeloma, characterized by a high PCLI (1%), constitutes nearly a third of all MM cases, and has a poorer outcome.1 Although technical difficulties have limited its widespread application, the slide-based PCLI provides valuable insight into the tumor biology, with higher PCLI predictive of an aggressive course.1, 2, 3 There is a direct correlation between the cytogenetic abnormalities and the rate of PC division perhaps because these chromosomal abnormalities impart a distinct proliferative advantage to the myeloma cells.4 Conversely, it can be argued that rapid PC division leads to sufficient genetic instability to cause secondary chromosomal abnormalities, which adversely affect the prognosis of the patients.

A pericentric inv(9)(p22q34) of the der(9)t(9;22)(q34;q11.2) is a recurrent secondary anomaly in Ph-positive leukemia

A pericentric inv(9)(p22q34) of the derivative chromosome 9 that resulted from a standard t(9;22)(q34;q11.2) was identified by R-banding karyotypic analysis and fluorescence in situ hybridization (FISH) assays in 4 (0.18%) of 2,200 Philadelphia chromosome (Ph)-positive leukemia patients, including 3 with chronic myeloid leukemia (CML) in chronic phase and 1 with acute myeloid leukemia (AML) in our hospital since 2004. All four patients had two malignant clones: one with only t(9;22)(q34;q11.2) and another with der(9)t(9;22)(q34;q11.2)inv(9)(p22q34) that resulted in the separation of the ABL1/BCR fusion gene. No metaphases with only inv(9)(p22q34) were seen in any of them. FISH also found a deletion of partial sequence of BCR on der(9)t(9;22)(q34;q11.2)inv(9)(p22q34) in 67.5% of bone marrow cells in the AML patient, but did not detect the deletion of the sequence of ASS/9q34 in these four patients. Reverse transcriptase–polymerase chain reaction revealed a b3a2 type of BCR/ABL1 fusion transcript in all of them, proving their disease to be Ph-positive leukemia. On reviewing the literature, only two solitary Ph-positive leukemia patients have been noticed to have the inv(9)(p22q34) anomaly. These two patients, together with our four documented patients, indicate that inv(9)(p22q34) is a novel, rare, but recurrent secondary chromosomal abnormality for Ph-positive leukemia. Despite receiving hydroxyurea therapy ( n = 3 patients), combined chemotherapy ( n = 2), even imatinib treatment ( n = 1), three patients, including one with AML and two with CML (one of whom progressed into the lymphoblastic blast phase), died with survival times of 28 days, 13 months, and 34 months, respectively. Only one patient with CML remained alive for 5.5 months. Their negative outcome implies that inv(9)(p22q34) has an unfavorable impact on prognosis. Presently, no firm conclusions can be drawn from this study. Because the case number reported here is very small, more patients with this anomaly need to be investigated to elucidate its true prognostic significance.