5q Deletion Research Articles

Young Adults with Multiple Myeloma Have Distinct Clinical and Molecular Characteristics Compared to Older Patients

Introduction Multiple Myeloma (MM) is an incurable plasma cell malignancy with a median age of 65 years at diagnosis. Although previous studies in "young” MM have been performed, they focus on the clinical characteristics. Within this study, we aim to understand the clinical characteristics and genomic drivers of young MM using the Weill Cornell/New York Presbyterian (WCM) and CoMMpass (MMRF) registry. Methods Young MM was defined as onset of MM at age <= 45. The WCM MM registry was queried for patient meeting the diagnosis for MM based on International Myeloma Working Group (IMWG). A control population of MM was created by randomly sampling the patients within the registry that were >45 years old. Clinical features and outcomes were recorded for each patient. The MMRF study is an observational international registry of newly diagnosed MM. A total of 1143 patients had clinical data. Baseline demographic, clinical and laboratory data, along with treatment and genomic (whole exome sequencing, RNA sequencing) was collected, and patients were followed prospectively every 6 months. Patients were divided into <= 45-year group and those above the median age in this cohort (>63 year). The primary end point of our analysis was progression free survival (PFS). Kaplan Meier and univariable Cox proportional hazard model was performed. Multivariable Cox model was created based on the results of univariable model. Land-mark analysis was used to control for time-dependent covariates. All analysis was performed using R software, version 4.1 Results The WCM cohort consisted of a total of 179 patients for this study. 61 patients were <= 45 years, and 98 patients belonging to an older control group. The younger-patients group was enriched for individuals of African American descent (40% vs 10%, p = 0.04), stage I ISS (51% vs 34%, p 0.01), more use of autologous stem cell transplant (ASCT), and less frequent prior diagnosis of smoldering myeloma (SM) (2.3% vs 18%, p = 0.017). There was a trend of higher rates of EMD and higher free light chain ratio in the young group, but this was not statistically significant. The median PFS for each group was 7.92 years (95% CI 4.50 - NA) for the younger group and 3.25 years (95% CI 1.92 - 10.67) for the >45-year group. In the univariable model the >45 year had worse PFS (HR 2.4, 95CI 1.2-4.6, p < 0.01). The association of worse PFS in the >45 year group (HR 6.3, 95%CI 2.3-17.2) persisted in the multivariable analysis, in addition to the presence of EMD (HR 3.2, 95% CI 1.4-7.3), and ISS stage (Table 2). In the MMRF registry, 65 and 548 patients <= 45 years and >63 years respectively were identified. The young group had higher proportion of African American (33% vs 15%, p 0.01), lower ISS (stage I 46% vs 31%, p = 0.04), and less frequency of deletion 13q (35% vs 50%, p 0.03) and gain 1q (22% vs 42%, p < 0.01). t(4;14) was seen more frequently in the young group, but this was not statistically significant (18% vs 9%, p = 0.056). DEA and GSEA showed a total of 1945 (3.5%) and 2164 (3.9%) over and under expressed genes, with the young group being enriched for the CD-1 transcriptional signatures (NES 1.86, adjusted p 0.028). There were no differences in the rates of mutation in the top 23 mutated genes in MM. The median PFS for the young group was 5.0 years (95% CI 2.3 - 5.9) vs 5.4 (95% CI 5.4 - 5.8) years, HR 1.56 (95% CI 1.05-2.3, p = 0.02) on the univariable analysis. Conclusions Young patients with MM have distinct clinical and demographic characteristics, including higher rates in African Americans, lower ISS stage, and lower rates of deletion 13q and gain 1q and enrichment in the CD-1 MM signature suggesting a potential role genetic and environmental factors in the early presentation at diagnosis. However, young patients had comparable to improved PFS than older patients that could be attributed to both biologic drivers, or factors related to patient age, such as better performance status, aggressive treatment, and potentially better tolerance leading to more exposure to antimyeloma therapy. The impact of EMD and ISS have been well described in MM, and the presence of EMD in this population remains an important prognostic factor. Lastly, although we used an arbitrary cut-off of 45 years old, we acknowledge this population may well represent biological spectrum of disease subtypes with potential differences in disease kinetics. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Del5q with One Additional Cytogenetic Abnormality Other Than Del20q Identifies a Higher Risk Population of MDS Patients

Background: According to the IPSS-R cytogenetic risk classification for myelodysplastic syndrome (MDS), patients with isolated deletion 5q (del5q), isolated deletion 20q (del20q), and del5q with an additional cytogenetic abnormality are classified as having good cytogenetics, similar to those with a normal karyotype. However the international database study, on which the IPSS-R cytogenetic risk classification is based, has its limitations in the small number of patients with del5q plus one additional cytogenetic abnormality (n=46). We sought to further define the prognostic significance of del5q with one additional cytogenetic abnormality in an evaluation of 86 patients. Methods: We performed a retrospective analysis of 857 MDS patients with a karyotype of isolated del20q (n=160) or any karyotype involving del5q seen at a single tertiary cancer center between 1987-2021. Patients with del5q (n=697) were divided into 3 cohorts: isolated del5q (n=147), del5q with concomitant del20q (del5q+del20q, n=8), and del5q with a non-del20q cytogenetic abnormality (del5q+1, n=78). The remainder of del5q patients with complex karyotypes were excluded. Patient characteristics, laboratory values, and bone marrow data, including blast percentage and cytogenetics, were reviewed. Median OS was estimated using the Kaplan-Meier method and two-tailed log-rank test. Results: The median age of patients was 70 years, with the majority being male (58%). Patients were more likely to present with lower risk disease (IPSS low to int-1, 78% of patients). The median follow-up was 2 years. Patients with del5q+1 had an inferior survival when compared to patients with isolated del5q (median OS 1.78 years vs. 3.87 years, p=0.0047). Del5q+1 patients also had a higher risk of AML transformation (26% vs. 16%, p=0.098, table I) with a shorter median time to AML transformation (1.07 years vs. 3.46 years, p<0.0001, figure 1). A multivariate analysis of the combined del5q and del5q+1 cohort was performed, which evaluated age >60, percent blasts in the bone marrow, IPSS score, and cytogenetics. The only statistically significant prognostic factors for OS with a p-value <0.01 were age>60 (HR: 3.67 (2.24-6.0), p<0.0001 ) and cytogenetics (HR for del5q+1: 1.96 (1.3-2.9), p=0.001). The only prognostic factor for time to AML transformation with a p-value <0.01 was cytogenetic classification (HR for del5q+1: 5.2 (2.0-13.2), p=0.0006). Patients with del20q had an OS between that of del5q (2.63 years vs. 3.87 years, p=0.14) and del5q+1 (2.63 years vs. 1.78 years, p=0.18). Del20q patients were less likely to transform to AML when compared to del5q+1 patients (11% vs. 26%, p=0.0035) with a trend towards longer time to AML transformation (2.1 years vs. 1.07 years, p=0.09). There were no statistically significant differences in AML transformation between the del5q and del20q cohorts (20% vs. 11%, p=0.1; median time to AML transformation: 3.46 years vs. 2.1 years, p=0.1). The evaluation of OS and risk of AML transformation in patients with del5q and a contaminant del20q was limited due to low patient numbers (n=8, 1.1%). The median follow-up for these patients was 2.67 years, and none of the patients transformed to AML during this time. Two patients (25%) expired from the non-disease related causes of presumably underlying heart failure and massive pulmonary embolism following surgery. Interestingly, both deceased patients had >10% bone marrow blasts and were IPSS intermediate risk. The median OS for these 8 patients was not reached. However, the median follow-up time of 2.67 years may suggest their OS is better than those with del5q+1 (median OS ≥2.67 years vs. 1.78 years). Conclusion: Contrary to the suggested IPSS-R cytogenetic risk stratification scheme, this analysis demonstrates a significant difference in terms of OS and risk of AML transformation when patients with isolated del5q or isolated del20q were compared to patients with del5q+1. Patients with del5q+1 had inferior median OS and a shorter time to AML transformation, suggesting more aggressive management may be warranted in this population. Though rare, patients with del5q+del20q may have better clinical outcomes than those with del5q+1, warranting further investigation into this group of patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The first comprehensive genomic characterization of rectal squamous cell carcinoma

BackgroundRectal cancers represent 35% of colorectal cancers; 90% are adenocarcinomas, while squamous cell carcinoma accounts for 0.3% of them. Given its rarity, little is known concerning its pathogenesis, molecular profile and therapeutic management. The current treatment trend is to treat rectal squamous cell carcinoma by analogy to anal squamous cell carcinoma with definitive chemo-radiotherapy, setting aside surgery in case of local recurrence.MethodsWe performed an in-depth genomic analysis (next-generation sequencing, copy number variation, and human papilloma virus characterization) on 10 rectal squamous cell carcinoma samples and compared them in silico to those of anal squamous cell carcinoma and rectal adenocarcinoma.ResultsRectal squamous cell carcinoma shows 100% HPV positivity. It has a mutational (PIK3CA, PTEN, TP53, ATM, BCL6, SOX2) and copy number variation profile (3p, 10p, 10q, 16q deletion and 1q, 3q, 5p, 8q, 20p gain) similar to anal squamous cell carcinoma. PI3K/Akt/mTOR is the most commonly affected signaling pathway similarly to anal squamous cell carcinoma. Most commonly gained or lost genes seen in rectal adenocarcinoma (FLT3, CDX2, GNAS, BCL2, SMAD4, MALT1) are not found in rectal squamous cell carcinoma.ConclusionThis study presents the first comprehensive genomic characterization of rectal squamous cell carcinoma. We confirm the existence of this rare histology and its molecular similarity with anal squamous cell carcinoma. This molecular proximity confirms the adequacy of therapeutic management based on histology and not localization, suggesting that rectal squamous cell carcinoma should be treated like anal squamous cell carcinoma and not as a rectal adenocarcinoma.

Mutational features of immunoglobulin heavy chain variable region gene in patients with chronic lymphocytic leukemia

Objective: To investigate the mutational features of the immunoglobulin heavy chain variable region (IgHV) gene in patients with chronic lymphocytic leukemia (CLL) using immunophenotypic and molecular genetic methods. Methods: The laboratory results of 266 CLL patients who underwent IgHV gene examination at Sino-US diagnostics laboratory from February 2020 to February 2021 were analyzed for the IgVH mutational status and presence of specific IgVH fragments. In addition, their immunophenotypic, molecular, chromosomal karyotypic, and FISH profiles were investigated and correlated with the IgVH mutational status. Results: Among 266 patients, 172 were male and 94 were female, with a media age of 67 years (20-82 years).There were more patients with mutated IgHV (m-IgHV) than unmutated IgHV (un-IgHV) (69.2%∶30.8%). There was association of VH family and the presence of gene fragments: the overall incidence of VH families including VH3 family (142/266, 53.4%), VH4 family (75/266, 28.2%), and VH1 family (34/266, 12.8%) was about 95%, among which the proportion of VH4-34 (26/266, 9.8%), VH3-23 (25/266, 9.4%), VH3-7 (24/266, 9.0%), and VH4-39 (16/266, 6.0%) was about 35%. VH3-20 and VH3-49 only occurred in un-IgHV (P<0.05). In addition, the expression rates of CD38 (26.3% vs. 3.0%), CD79b (71.1%∶45.5%) and 11q deletion (25.5%∶5.3%) were higher in un-IgHV, and single trisomy 12 (37.9%∶5.6%) were more commonly found in m-IgHV (P<0.05). MYD88 was one of the major mutation genes in m-IgHV, while ATM had the highest mutation rate in un-IgHV. Conclusion: CLL patients have differential expression in terms of IgHV gene mutations, correlating to their immunophenotype and genetics characteristics.

Outcomes after allogeneic hematopoietic stem cell transplantation in acute myeloid leukemia patients with der(1;7)(q10;p10).

The prognosis of acute myeloid leukemia (AML) patients with der(1;7)(q10;p10) who underwent allogeneic hematopoietic stem cell transplantation (allo-SCT) is unclear due to its rarity. We retrospectively analyzed 151 AML patients with der(1;7)(q10;p10) and compared the findings with those of 853 AML patients with monosomy 7 or chromosome 7q deletion (-7/del(7q)) using Japanese nationwide registry data. The der(1;7)(q10;p10) group showed significantly better transplant outcomes than the -7/del(7q) group. In the multivariate analysis of the der(1;7)(q10;p10) group, additional chromosomal abnormalities and a poor performance status significantly influenced the survival. In conclusion, allo-SCT is a feasible treatment option for AML patients with der(1;7)(q10;p10).

53. Clinical whole exome/whole transcriptome (WES/WTS) analysis detects copy number and structural rearrangements important

<h3>Background</h3> Chromosomal copy number alterations (CNAs) and structural rearrangements (SR) are a key part of risk stratification and management of patients with MM. We developed a clinical paired tumor/normal WES/WTS assay that detects CNAs at one-copy difference from focal to whole genome, and gene fusion at 20% and 10% limit of detection, respectively. <h3>Patients and Methods</h3> We performed WES/WTS analysis utilizing DNA/RNA extracted from CD138+ enriched cells and buccal swab from 92 MM patients. Plasma cell content was not available for most cases. We compared the detection of CNAs, and SR with other techniques currently in use including FISH, arrays and molecular analysis. <h3>Results</h3> Molecular alterations (MA) were present in 82/92(89.1%) cases, whereas 65/92(69.5%) cases had CNAs. In cases with CNAs (indicating sufficient tumor content), deletions of (1p32), (1p12), or both were detected in 7(10.8%), 12(18.5%), and 5(7.7%) cases, respectively. Gain of (1q22), and hyperdiploidy were detected in 19(29.2%) and 32(49.2%), respectively. Deletions of 13q, 14q, 16q, and 17p(TP53) were detected in 32(49.2%), 14(21.6%), 16(24.6%), and 13(20%) cases, respectively. In cases with MA, 10/82(10.8%), and 3(3.6%) cases had TP53 mono and bi-allelic inactivation, respectively. Translocations (4;14)(p16;q32)(IGH::NSD2/FGFR3) and t(14;16)(q32;q23)(IGH::MAF) were detected in 11(11.9%), and 3(3.7%) cases with MA, respectively. Overexpression of CCND1 (research use only) was observed in 8/82(9.7%) cases. <h3>Discussion</h3> Our WES/WTS detected abnormalities in this series similarly to what has been reported by techniques currently needed in combination such as FISH, arrays or molecular analysis. By incorporating RNA expression analysis, WES/WTS may reliably detect all clinically-relevant alterations in MM.

105. Clinical whole-genome sequencing identifies NSD3 as the correct fusion partner of NUP98 in a patient with acute myeloid

<h3>Background</h3> Only rare cases of acute myeloid leukemia (AML) have been shown to harbor a t(8;11)(p11.2;p15.4). This translocation is believed to involve the fusion of NSD3 or FGFR1 with NUP98; however, apart from targeted mRNA quantitative PCR analysis, no molecular approaches have been utilized to define the chimeric fusions present in these rare cases. <h3>Case Presentation</h3> Here we present the case of a 51-year-old female with AML with myelodysplastic-related morphologic changes, 13q deletion and t(8;11), where initial fluorescence in situ hybridization (FISH) assays were consistent with the presence of NUP98 and FGFR1 rearrangements, and suggestive of NUP98/FGFR1 fusion. Using a streamlined clinical whole-genome sequencing approach, we resolved the breakpoints of this translocation to intron 4 of NSD3 and intron 12 of NUP98, indicating NUP98/NSD3 rearrangement as the underlying aberration. Furthermore, our approach identified small variants in WT1 and STAG2, as well as an interstitial deletion on the short arm of chromosome 12, which were cryptic in G-banded chromosomes. <h3>Conclusions</h3> NUP98 fusions in acute leukemia are predictive of poor prognosis. The associated fusion partner and the presence of co-occurring mutations, such as WT1, further refine this prognosis with potential clinical implications. Using a clinical whole-genome sequencing analysis, we resolved t(8;11) breakpoints to NSD3 and NUP98, ruling out the involvement of FGFR1 suggested by FISH while also identifying multiple chromosomal and sequence level aberrations.

123. Chromosomal microarray assists interpretation of cytogenetic abnormalities in hematopoietic malignancies

Chromosome analysis is a first-line ancillary test performed on bone marrows to evaluate for hematopoietic malignancy. While characteristic cytogenetic findings correlate with pathology in most cases, the chromosomal abnormalities are sometimes too complex for complete characterization, limiting interpretation of the results. Additionally, chromosomal abnormalities may lack correlation with the hematopathology findings. Here, we present two cases where chromosomal microarray was performed, which aided interpretation of chromosome analysis. Case 1: A patient with a history of acute myeloid leukemia (AML) with NPM1 mutation had a bone marrow evaluated for residual disease. Although chromosome analysis identified a mosaic balanced translocation between chromosomes 7 and 17, concurrent ancillary studies were unrevealing. The significance of this finding was unclear given the unremarkable morphology and flow cytometry. Chromosomal microarray identified deletions of NF1 and SUZ12 on chromosome 17 as well as deletion of ASXL1 on chromosome 20, which may be a driver for leukemogenesis in this case. Case 2: A patient presented with circulating blasts and was diagnosed with AML. Chromosome analysis demonstrated deletion of chromosome 5q and loss of chromosome 7. However, additional complex chromosomal abnormalities were also identified, including a jumping translocation. Chromosomal microarray showed complex abnormalities of chromosome 1p, gain of chromosome 5p including TERT and IL7R, deletion of IKZF1 on chromosome 7, and deletion of TP53 on chromosome 17. These chromosomal microarray findings provide greater insight toward AML pathogenesis. These case reports demonstrate the utility of chromosomal microarray in further defining chromosomal findings with unclear significance to further understand disease pathogenesis.

Rare Germline ATM Variants Influence the Development of Chronic Lymphocytic Leukemia.

Germline missense variants of unknown significance in cancer-related genes are increasingly being identified with the expanding use of next-generation sequencing. The ataxia telangiectasia-mutated (ATM) gene on chromosome 11 has more than 1,000 germline missense variants of unknown significance and is a tumor suppressor. We aimed to determine if rare germline ATM variants are more frequent in chronic lymphocytic leukemia (CLL) compared with other hematologic malignancies and if they influence the clinical characteristics of CLL. We identified 3,128 patients (including 825 patients with CLL) in our hematologic malignancy clinic who had received clinical-grade sequencing of the entire coding region of ATM. We ascertained the comparative frequencies of germline ATM variants in categories of hematologic neoplasms, and, in patients with CLL, we determined whether these variants affected CLL-associated characteristics such as somatic 11q deletion. Rare germline ATM variants are present in 24% of patients with CLL, significantly greater than that in patients with other lymphoid malignancies (16% prevalence), myeloid disease (15%), or no hematologic neoplasm (14%). Patients with CLL with germline ATM variants are younger at diagnosis and twice as likely to have 11q deletion. The ATM variant p.L2307F is present in 3% of patients with CLL, is associated with a three-fold increase in rates of somatic 11q deletion, and is a hypomorph in cell-based assays. Germline ATM variants cluster within CLL and affect the phenotype of CLL that develops, implying that some of these variants (such as ATM p.L2307F) have functional significance and should not be ignored. Further studies are needed to determine whether these variants affect the response to therapy or account for some of the inherited risk of CLL.

Autoimmune cytopenias in chronic lymphocytic leukemia: a growing challenge in targeted therapies?

As the world’s population ages, the incidence of chronic lymphocytic leukemia (CLL) will continue to increase. CLL-related autoimmune cytopenias (AICs) are becoming a growing challenge in daily clinical practice. AICs occur in c.10% of CLL patients, and include autoimmune hemolytic anemia, immune thrombocytopenia, pure red cell aplasia, and autoimmune granulocytopenia. The complication can appear at any disease stage, both in treated and previously untreated patients. In some cases, AICs precede the diagnosis of the underlying disease. The diagnostic process of autoimmune complications is often difficult. First of all, however, it requires differentiation from bone marrow infiltration resulting from underlying disease progression. A properly established diagnosis has prognostic and therapeutic implications. Autoimmune cytopenias are more often associated with high-risk CLL (with 17p deletion, 11q deletion, wild-type variant of gene encoding immunoglobulin heavy chain variable region) and show a complex pathogenesis. CLL cells and the surrounding microenvironment are involved in autoimmune mechanisms. Treatment of AICs depends on CLL stage. In the case of isolated symptoms of AICs, without accompanying features of CLL progression, glucocorticosteroids or rituximab are recommended in the first line treatment. No response or a suboptimal response to treatment implies further therapeutic decisions. According to significant advances in the treatment of chronic lymphocytic leukemia, therapeutic strategies for autoimmune cytopenias also need to be optimized. The widespread introduction of ibrutinib, idelalisib, and venetoclax has highlighted the need for understanding the interplay between targeted therapies and AICs.

Overlapping Interstitial Deletions of the Region 9q22.33 to 9q33.3 of Three Patients Allow Pinpointing Candidate Genes for Epilepsy and Cleft Lip and Palate

Introduction: Patients carrying interstitial deletions of the long arm of chromosome 9 show similar features. These phenotypes are often characterized by developmental delay, intellectual disability, short stature, and dysmorphism. Previously reported deletions differ in size and location spanning from 9q21 to 9q34 and were mostly detected by conventional cytogenetic techniques. Methods: Based on clinical features suggesting primarily chromosomal diseases, aCGH analysis was indicated. We report on de novo overlapping interstitial 9q deletions in 3 unrelated individuals presenting neurodevelopmental disorder and multiple congenital anomalies. Results: An 8.03-Mb (90 genes), a 15.71-Mb (193 genes), and a 15.81-Mb (203 genes) deletion were identified in 9q affecting 9q22.33q33.3. The overlapping region was 1.50 Mb, including 2 dosage-sensitive genes, namely EPB41L4B (OMIM #610340) and SVEP1 (OMIM #611691). These genes are thought to be involved in cellular adhesion, migration, and motility. The non-overlapping regions contain 24 dosage-sensitive genes. Conclusion: Besides the frequently described symptoms (developmental delay, intellectual disability, skeletal abnormalities, short stature, and dysmorphic facial features) shared by the patients with interstitial deletions of chromosome 9q reported thus far, two of our patients showed distinct forms of epilepsy, which were successfully treated, and one had a bilateral cleft lip and palate. Possible candidate genes for epilepsy and cleft lip and palate are discussed.

Methylation microarray-based detection of clinical copy-number aberrations in CLL benchmarked to standard FISH analysis

Copy-number aberrations (CNAs) are assessed using FISH analysis in diagnostics of chronic lymphocytic leukemia (CLL), but CNAs can also be extrapolated from Illumina BeadChips developed for genome-wide methylation microarray screening. Increasing numbers of microarray data-sets are available from diagnostic samples, making it useful to assess the potential in CNA diagnostics.We benchmarked the limitations of CNA testing from two Illumina BeadChips (EPIC and 450k) and using two common packages for analysis (conumee and ChAMP) to FISH-based assessment of 11q, 13q, and 17p deletions in 202 CLL samples.Overall, the two packages predicted CNAs with similar accuracy regardless of the microarray type, but lower than FISH-based assessment. We showed that the bioinformatics analysis needs to be adjusted to the specific CNA, as no general settings were identified. Altogether, we were able to predict CNAs using methylation microarray data, however, with limited accuracy, making FISH-based assessment of deletions the superior diagnostic choice.

Detection of 13q deletion in patients with chronic lymphocytic leukemia and its correlation to hematopathological parameters

Background: Chronic lymphocytic leukemia(CLL) is a monoclonal malignancy characterized by an accumulation of small and mature looking B lymphocytes in the blood, bone marrow and other tissues, and is typically characterized by expression CD5+, CD23+, CD22 -, CD79b-, with weak expression of surface immunoglobulin (sIg). Two major clinical staging systems (Rai and Binet staging), that developed to estimate prognosis in CLL. However both these systems are unable to prospectively distinguish the rapidly developing patients from those appreciated to remain with a stable disease for decades. Several publications reported the prognostic important of 13q deletion in patients with chronic lymphocytic leukemia. 13q deletion is the most important cytogenetic abnormalities detected by Fluorescence In situ Hybridization in CLL patients and represent a good prognostic marker with 60% of patients alive after 5 years as compared with 27% for patients with a normal FISH analysis. Aims of the study: To investigate 13q deletion in patients with chronic lymphocytic leukemia by using FISH technique. To correlate the presence of 13q deletion with hematological and clinical prognostic markers including complete blood picture, absolute lymphocyte count, and modified Rai staging.

CLONAL EVOLUTION TO ACUTE MYELOID LEUKEMIA FOUR YEARS AFTER ALEMTUZUMAB AND CYCLOSPORINE IMMUNOSUPPRESSION IN A PATIENT WITH VERY SEVERE APLASTIC ANEMIA

Describe a rapid clonal evolution to AML with monossomy 7 in an aplastic anemia (AA) patient. A 17-year-old patient was diagnosed with severe aplastic anemia after a history of fatigue, mucocutaneous bleeding and pancytopenia. Bone marrow (BM) aspirate was hypocellular and had no dysplastic changes. Trephine biopsy confirmed 5% cellularity and absence of increased immature progenitors; reticulin fibrosis was not observed. Conventional cytogenetics showed a 46, XY karyotype; a small paroxysmal nocturnal hemoglobinuria (PNH) clone was present in red cells, neutrophils and monocytes. Chromosome breakage analysis for Fanconi anemia was unremarkable, and telomere length was within the normal range. The patient had no HLA-matched donor, and once hATG was discontinued in Brazil and eltrombopag was unavailable, he was included in a group of patients treated with upfront immunosuppressive therapy (IST) with subcutaneous alemtuzumab and cyclosporine; The patient achieved a partial response after 3 months of treatment. Cyclosporine was gradually tapered and was completely stopped after a year of treatment. This patient was lost to follow-up visits for six months and returned after four years of IST, complaining about worsening of fatigue and gingival bleeding. Complete blood count was notable for a loss of the achieved PR, and the presence of a small population of dysplastic monocytes. Revaluation of BM showed a hypercellular smear, and the presence of 60% of immature myeloid cells composed by myeloblasts and monocytes precursors. BM immunophenotyping was compatible with acute myelomonocytic leukemia. Conventional cytogenetics detected a monosomy of chromosome 7 and a trisomy of chromosome 21. Molecular studies for FLT3 and NPM1 mutations, and RUNX1-RUNX1T1 and CBFB-MYH11 fusion genes were negative. The PNH clone size remained stable. The patient was started on standard AML chemotherapy with cytarabine and daunorubicin. An hematologic complete remission with a negative minimal residual disease was achieved. Due to an acute appendicitis during AML induction, the patient awaits consolidation therapy with an haploidentical hematopoietic stem-cell transplant (HSCT) from his brother. Frontline therapy for severe aplastic anemia patients younger than 40 years who have an HLA-matched sibling donor is HSCT. Triple IST with eltrombopag, hATG and cyclosporine is now considered the standard frontline therapy for patients older than 40 years and those who lack a suitable HLA-matched donor. Once hATG was discontinued in many countries, previous studies have reported outcomes of replacing hATG with rabbit ATG, cyclophosphamide, or alemtuzumab. Clonal evolution to myeloid neoplasms is a recognized complication of IST and patients with AA may develop secondary MDS/AML, with the main associated factors being longer disease duration, presence of mutations with an adverse prognosis, a short telomeric length and the development of cytogenetic abnormalities (CAs). The most recurrent CAs include total or partial loss of chromosome 7, trisomy 8 and 13q deletion, and, less frequently, trisomy 6, trisomy 15 and trisomy 21. T-cell-mediated autoimmune diseases, such as AA, have a higher risk of progression to AML/MDS dur to development of somatic mutations and CAs. Monosomy 7 independently represents a poor prognostic factor due to the high rate of progression to acute leukemia, such as occurred with the patient reported.

Molecular Aspects in Chronic Lymphocytic Leukemia patients with Autoimmune Cytopenias: single center experience

Autoimmune cytopenia's, particularly autoimmune hemolytic anemia (AIHA) and immune thrombocytopenia (ITP), complicate up to 25% of chronic lymphocytic leukemia (CLL) patients. Their occurrence correlates with a more aggressive disease. AIHA and ITP are more frequently found in patients with unfavorable biological risk factors for CLL. B lymphocytes at CLL are responsible of pathogenic mechanisms, involving aberrant antigen presentation and cytokine production. The aim of this study was evaluation of autoimmune cytopenia's in chronic lymphocytic leukemia patients from Republic of North Macedonia in correlation with genetic structure of pathologic B lymphocyte. This is a retrospective study of patients with CLL, diagnosed and followed in the period between January 2011 and January 2021. Individual data from 100 treatment naïve CLL patients were analyzed, and mutational status and configuration of IGHV-IGHD-IGHJ rearrangements and genetics were analyzed using reverse transcriptase– polymerase chain reaction (RT-PCR) and sequencing methodology at the center for bimolecular pharmaceutical analyses, faculty of pharmacy, Skopje, Republic of North Macedonia. Our evaluation have shown that 10% of CLL patients had AIHA and 4% had ITP. Most of the patients with autoimmune cytopenias had unmuteted IGHV genes. The most frequently expressed IGHV subgroup was IGHV1-69 (71%), followed by IGHV3-13 and IGHV4-4 (14%). The genetic results presented unfavorable cytogenetics with 11q deletions and NOTHCH mutation. The results of our study are consistent with published studies with specific molecular signature.

MDS-450 Impact of Number of Cells Harboring Deletion 5q Abnormality by Conventional Cytogenetics in Isolated Deletion 5q Myelodysplastic Syndromes

Isolated del 5q myelodysplastic syndrome (MDS) is defined as presence of del 5q +/- one additional chromosomal abnormality without excess myeloblasts. Lenalidomide (len) is current standard of care for those patients. The impact of percentage of cells (number of metaphases) harboring del 5q by karyotype is not known. We examined our cohort of isolated del 5q MDS patients at Moffitt Cancer Center. We assessed baseline characteristics, response to len, and outcomes based on % of cells/metaphases harboring del 5q using G-banding "conventional cytogenetics clone size." We divided patients into 2 groups based on number of cells harboring del 5q, those with <50% i.e less than 10 out 20 metaphases and 50-100% (10 or more out of 20 metaphases). We identified 142 isolated del 5q MDS patients. Baseline characteristics of the whole cohort was similar to what has been previously described in the literature. Patients with >50% metaphases harboring del 5q had more severe anemia (Hgb < 8.0 g/dl) (30% versus 13%) (p=0.05). Among patients who were treated with len (n=100), 50% achieved a response. There was a trend for higher response rate among patients with >50% del 5q metaphases with (42/80) 53% response rate compared to (8/20) 40% among those with <50%. (p=0.32). There was no difference in presence of TP53 somatic mutation based on % of metaphases harboring del 5q. There was a non-statistically significant trend for better overall survival among patients with >50% cells with detected del 5q compared to those with <50%, median OS 57 mo versus 35 mo, p=0.90. The number of cells harboring del 5q among isolated del 5q MDS patients using regular karyotyping "clone size by conventional cytogenetics" correlated with severity of anemia, trend for better response to len and trend for better overall survival. Those findings should be further confirmed in larger cohort of patients.

CLL-345 Retrospective Analysis of Real-World Outcomes of Chronic/Small Lymphocytic Leukemia (CLL/SLL) Patients Treated With First-Line Ibrutinib Versus Chemoimmunotherapy (CIT)

Ibrutinib is an oral, once-daily Bruton's tyrosine kinase inhibitor and is the only targeted treatment that has demonstrated progression-free survival and overall survival in multiple phase 3 trials across age, fitness, and high-risk markers for chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL). To describe the trends in genomic testing and compare real-world outcomes of CLL/SLL patients treated with first-line (1L) ibrutinib or chemoimmunotherapy (CIT). A nationwide, Electronic Health Record-derived de-identified database (03/04/2016-08/31/2021) was used to collect treatment and outcome data. Patient baseline characteristics before treatment were compared between the two groups. Propensity score-based inverse probability of treatment weighting was used to adjust for baseline differences between groups. Time to next treatment (TTNT) from 1L initiation was analyzed using the Kaplan-Meier (KM) method. Subgroup analyses were performed on patients with high-risk cytogenetic markers (deletion 17p, 11q, or unmutated IGHV) and those without IGHV testing. Testing for high-risk markers in CLL patients was suboptimal. Although cytogenetic testing for del17p or del11q increased from 52.3% to 74.3%, IGHV testing remained infrequent and low at 37.7%. A total of 1882 patients (61.4%) received 1L ibrutinib monotherapy (mean age 71 years, median follow-up 26 months), and 1182 (38.6%) received a CIT regimen (mean age 68 years, median follow-up 33 months). More patients treated with ibrutinib had a known high-risk cytogenetic marker than those treated with CIT (44% vs. 34%), especially those with del17p (15% vs. 4%). The median TTNT was not reached in patients treated with ibrutinib and was 39.6 months in those treated with CIT. The hazard ratios of starting second-line treatment were 0.49 (95% CI: 0.44-0.54, p<0.001) overall, 0.40 (0.34-0.47, p<0.001) among high-risk patients, and 0.51 (0.44-0.58, p<0.001) among patients without IGHV testing. Consistent with clinical trials, 1L treatment with ibrutinib for CLL/SLL significantly lowers the risk of initiating subsequent treatment compared with 1L CIT, including in patients with high-risk cytogenetic markers and patients without IGHV testing. Assessment of cytogenetic/molecular risks before initiating treatment is vital to optimizing clinical outcomes.

CYTOGENETIC ANALYSIS OF MYELODYSPLASTIC SYNDROME PATIENTS WITH COMPLEX KARYOTYPE IN NORTHEASTERN BRAZIL

Cytogenetic abnormalities occurs in almost half of myelodysplastic syndrome (MDS) patients and are considered to be the most important prognostic factor for survival, so they have been included in the International Prognostic Scoring System (IPSS) since 2007. Complex karyotype (CK) is defined as more than or equal to 3 independent chromosomal abnormalities at the same metaphase and is an independent poor prognostic factor, usually associated with a shorter median overall survival and propensity toward malignant transformation. From January 2008 through June 2022, 1744 patients with suspected or diagnosed hematological neoplasms (including MDS/AML) who had a bone marrow evaluation and concurrent conventional karyotype were identified. Karyotyping was successfully performed on 1167 (67%) of 1744 patients. In total, 914 (54%) with an adequate karyotypic analysis carried out a diagnosis of myelodysplastic syndrome. Among the cases, 9,7% were classified as complex karyotype. Del(5q)/-5 was identified in 52% of patients with complex karyotype, followed by chromosome 11 and chromosome 7 as well as chromosome 17 abnormalities, accounting for 38%, 23% and 23%, respectively. Besides, complex karyotype patients with translocations and derivation involvements, accounted for 28% and 14%, respectively. It is likely that pathogenetic mechanisms in del(5q) may involve hemizygous mutations or haploinsufficiency and be modified by additional somatic lesions affecting genes on other chromosomes. This report shows that the presence of deletion 5q may be the first step to the development of complex karyotype and is a determinant key of the genetic and genomic complexity and clonal hierarchy in MDS with 5q abnormalities.

Molecular Cytogenetic Profiling Reveals Similarities and Differences Between Localized Nodal and Systemic Follicular Lymphomas.

Recently, we have developed novel highly promising gene expression (GE) classifiers discriminating localized nodal (LFL) from systemic follicular lymphoma (SFL) with prognostic impact. However, few data are available in LFL especially concerning hotspot genetic alterations that are associated with the pathogenesis and prognosis of SFL. A total of 144 LFL and 527 SFL, enrolled in prospective clinical trials of the German Low Grade Lymphoma Study Group, were analyzed by fluorescence in situ hybridization to detect deletions in chromosomes 1p, 6q, and 17p as well as BCL2 translocations to determine their impact on clinical outcome of LFL patients. The frequency of chromosomal deletions in 1p and 17p was comparable between LFL and SFL, while 6q deletions and BCL2 translocations more frequently occurred in SFL. A higher proportion of 1p deletions was seen in BCL2-translocation–positive LFL, compared with BCL2-translocation–negative LFL. Deletions in chromosomes 1p, 6q, and 17p predicted clinical outcome of patients with SFL in the entire cohort, while only deletions in chromosome 1p retained its negative prognostic impact in R-CHOP–treated SFL. In contrast, no deletions in one of the investigated genetic loci predicted clinical outcome in LFL. Likewise, the presence or absence of BCL2 translocations had no prognostic impact in LFL. Despite representing a genetic portfolio closely resembling SFL, LFL showed some differences in deletion frequencies. BCL2 translocation and 6q deletion frequency differs between LFL and SFL and might contribute to distinct genetic profiles in LFL and SFL.

Rare germline ATM variants of uncertain significance in chronic lymphocytic leukaemia and other cancers.

Germline pathogenic ATM (ataxia-telangiectasia mutated) variants are associated with the risk of multiple cancers; however, genetic testing reveals a large number of ATM variants of uncertain significance (VUS). Here, we studied germline ATM variants occurring in a real-world cohort of 336 patients with chronic lymphocytic leukaemia (CLL) and public cancer whole-exome/genome-sequencing datasets (445 CLL, 75 mantle cell lymphoma, 216 metastatic breast cancer, 140 lung cancer patients). We found that two-thirds of rare germline ATM variants are pathogenic (18%-50%) or VUS-predicted pathogenic (50%-82%), depending on cancer type and reaching a prevalence of up to 8%, and one-third are VUS-predicted benign. Patients with both pathogenic and VUS-predicted pathogenic variants, all heterozygous, mostly missense, are more predisposed to biallelic ATM inactivation by acquiring deletion (del)11q than patients without these variants, similar to patients with somatic ATM variants. A functional assay of ATM activity in primary CLL cells proved that VUS-predicted pathogenic ATM variants partially reduce ATM activity and concurrent del(11q) leads to complete loss of ATM activity. The rare germline variants were associated with reduced progression-free survival in CLL on novel agents, comparable to somatic ATM or TP53 disruptions. Our results highlight the need to determine the pathogenicity of VUS in clinically relevant genes such as ATM.