Clonal Abnormalities Research Articles

FISH Analysis of CDKN2A/B Deletion in Chronic Lymphocytic Leukemia

Introduction: The course of chronic lymphocytic leukemia (CLL) ranges from indolent to rapid disease progression, including transformation to an aggressive lymphoma (Richter transformation, RT). A subset of CLL pts have been shown to acquire deletions of the tumor suppressor CDKN2A/B. RT has been associated with deletion of CDKN2A/B, however, prior studies’ sample populations were limited or not primarily focused on those with this deletion. Due to the rarity of this genetic abnormality in CLL and severity of RT, additional studies concerning this subgroup should be investigated. Here, we examine a larger cohort of CLL pts with CDKN2A/B deletion by fluorescence in situ hybridization (FISH) to provide further clarity on this genetic lesion. Methods: We conducted a retrospective case study for all pts for whom cytogenetic clinical samples were submitted for extended CLL FISH panel analysis over a 3.5 year (yrs) period, between 2015 and 2018 at The Ohio State University. Pts were screened for CDKN2A/B deletion with a new or follow up diagnosis of CLL or small lymphocytic lymphoma (SLL). Cytogenetic testing was performed on cells stimulated with pokeweed mitogen, phorbol myristate acetate (PMA), and CpG oligonucleotides. FISH for D13S319, D12Z3, ATM, TP53, BCL6, MYC, REL, SEC63, MYB, and CDKN2A/B was performed via manufacturer's recommendations. IGHV mutational status was determined using PCR. A 50-gene hematologic sequencing panel was performed via Ion Torrent. Overall survival (OS) from first detection of abnormal CDKN2A/B results were examined. Cox regression model was used to evaluate OS post CDKN2A/B deletion. The Fine and Gray Model was used to examine correlation of variables with regard to RT, treating death without RT as a competing risk. Results: 636 pts had sample(s) submitted for extended CLL FISH panel analysis. Of these, 43 (6.8%) pts with confirmed CLL or SLL were found to have loss of CDKN2A/B by FISH, including 6 pts within one year of diagnosis. Median age at diagnosis was 54.9 yrs (range 41.9-77.7). Loss of CDKN2A/B was detected at a median of 7.8 yrs (range diagnosis-26.1) from diagnosis. Of those with known IGHV status (n=33), 91% were unmutated. A complex karyotype (>3 clonal chromosomal abnormalities) was found in 90% percent of pts. In regard to CDKN2A/B deletion, 77%, 16%, and 7% of pts had heterozygous, homozygous, or subclonal populations of heterozygous as well as homozygous loss, respectively. Additional FISH testing showed 23% had gain of REL, 16% had BCL6 gain/rearrangement, 16% had deletion 6q, 37% had gain/rearrangement of MYC, 16% had loss of ATM, 14% had trisomy 12, and 58% had deletion 13q. Deletion of TP53 was seen in 63%. Sequencing data was available for 15 pts (35%), of note, 10 pts had mutations in TP53, with 4 also having mutations in SF3B1. In terms of OS, there were 30 deaths in the cohort, with median OS of 10.8 yrs from diagnosis (95% CI 8.0-14.4). With a median follow-up of 4.9 yrs among survivors, the median OS from time of detection of CDKN2A/B deletion was 1.7 yrs (95% CI 0.3-4.7). Multivariable analysis of OS post CDKN2A/B detection was significant for increasing beta-2-microglobulin measured at first visit at our institution (p=0.0044, hazard ratio (HR) 1.32 (95% CI 1.09-1.60)) and CDKN2A/B subclonal heterozygous and homozygous loss (p=0.0024, HR 9.81 (95% CI 2.25-42.73)). Of the 43 pts with loss of CDKN2A/B, 21 (49%) progressed via RT (n=20) or prolymphocytic leukemia (PLL) (n=1). The Cumulative Incidence Rate of RT development 12-months post CDKN2A/B deletion was 23.5% (95% CI 10.9-38.9), at 24-months, it was 29.4% (15.1-45.3). Deletion of TP53 was seen in 85.7%. Albeit there was limited pts with detection of CDKN2A/B deletion prior to RT, the multivariable model for RT found loss of TP53 (p=0.02, HR 4.53 (95% CI 1.33-15.43)) and homozygous and heterozygous loss of CDKN2A/B (p=0.0008, HR 6.08 (95% CI 2.13-17.39)) were independently significant. Conclusion: Here we show that deletion of CDKN2A/B in CLL is a very rare occurrence and was seen predominantly in pts with highly aggressive disease characteristics. Close to half of our cohort progressed with RT or PLL. Additional investigation of serial samples is needed to establish when CDKN2A/B deletion occurs relative to RT. Our results indicate that prospective FISH analysis for CDKN2A/B deletion, in conjunction with other high-risk genetic alterations, to establish RT prognostication in high-risk CLL is warranted.

Hematological Response to Avatrombopag Switch in Thrombopoietin Receptor Agonist-Refractory Aplastic Anemia

ATG combined with CsA is the first-line treatment for patients with SAA who cannot choose HLA-matched HSCT because of various conditions. The HR rate is only approximately 60-80%, and the CR is not high. The lack of residual HSC in patients is an important reason for this unsatisfactory effect. RhTPO/TPO-RA combine with HSC membrane receptors to promote their proliferation and differentiation, expand multipotent hematopoietic progenitor cells in vivo. Studies have found that TPO-RA eltrombopag (EPAG) combined with IST can significantly increase the HR rate and improve the quality of the HR. From 40-50% hematologic improvement can also be obtained with refractory SAA. However, TPO-RA treatment is ineffective in some patients. Avatrombopag (APAG) acts on the transmembrane region of the TPO receptor, has a stronger effect than EPAG, and has no obvious liver toxicity. We analyzed the efficacy and safety of IST combined with rhTPO and/or EPAG in patients with SAA of switching to APAG. We retrospectively analyzed the data of 10 patients who were hospitalized between January and December 2021. All patients have been diagnosed with V/SAA. They received IST combined with rhTPO/TPO-RA treatment, failed to obtain a HR, and were converted to APAG. The patients were treated with IST for at least 6 months, combined with rhTPO and/or EPAG for at least 3 months, and did not get HR. None of the patients included in this study was suitable for undergo allogeneic HSCT. APAG was administered orally at a dose of 40 mg/day once a day for at least 6 months.The efficacy criteria for refractory SAA were defined as follows: one-line response included platelet response (HI-P), neutrophil response (HI-N), and erythroid response (HI-E). The 2-line HR could be a combination of any two of the aforementioned one-line responses. A trilineage response was defined as a patient with simultaneous platelet, erythroid, and neutrophil responses, and the quality of the trilineage response was the same as that of the initial treatment with SAA/TDNSAA. Ten patients were included in the study, with a median age of 54 (14-68) years, including five males and five females. All patients received IST for a median of 8.5 (6-40) months. Four patients received rhTPO for 1-2 months followed by EPAG for 2-8 months. Six patients received only EPAG for 3-4 months. All the patients received rhTPO/TP-RA for a median of 3.5 (3-10) months. Five patients with PNH clones were positive by flow cytometry, and there was no hemolytic or biochemical evidence of hemolysis. Two patients had clonal chromosomal abnormalities: one with 47,XY; +8 [2/20] and the other with 45,XY; -Y [10/20]. Ten patients were converted to APAG treatment for a median of 6 (6-10) months. After 3 months of follow-up , seven patients obtained a HR (7/10, 70%), of which two patients obtained a HI-P and HI-N, and five patients (5/10, 50%) obtained a three-line HR (1-CR, 3-GPR, 1-PR). Seven patients (7/10, 70%) obtained HR after 6 months of follow-up, of which one patient obtained HI-P/N and six patients (6/10, 60%) obtained three-line HR (3-CR, t2-GPR, 1-PR). The patient with two-line HR further improved after 8 months of APAG, reaching a three-line blood cell response to PR. At the end of the last follow-up, the HR rate of all patients with APAG conversion therapy was 70%, and all of them achieved three-line blood cell responses (4-CR, 1-GPR, 2-PR). During the follow-up period, no APAG-related adverse events of grade 2 or above were found in any patient, no thrombotic events occurred, and no fibrous tissue hyperplasia was found in the bone marrow pathology review after 6 months of treatment. Among the five patients with PNH clones before APAG conversion therapy, three patients did not change significantly. Otherwise one patient's granulocyte PNH clone decreased from 16.2% at baseline to 6.3%, and the other patient's PNH clone increased from 9.8% at baseline to 19.6%, whereas there was no symptomatic hemolysis in all patient. One patient (1/10, 10%) with normal chromosomal before therapy get a new clonal chromosomal abnormality after 3 months. It was 46XX, t(X;4 )(p11.4;q21) [4/20]; the HR was assessed as GPR. In conclusion, patients with SAA refractory to TPO-RA, drug conversion therapy can be based on the safety, pharmacological, and pharmacodynamic characteristics of TPO-RA drugs. Therefore, APAG is a better choice for switch therapy in these patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Outcomes of Hematologic Complications in Shwachman Diamond Syndrome: High Risk Features and Implications for Surveillance

Background Shwachman Diamond syndrome (SDS) is characterized by genetic predisposition to bone marrow failure and TP53-mutated myeloid malignancies. Data are scarce to guide surveillance strategies. Objectives This study aims to characterize the presentation and outcomes of hematologic complications in 218 patients with SDS with the goal of informing surveillance. Design/Method Data was obtained from medical records of 218 study subjects with biallelic mutations in SBDS/DNAJC21/ EFL1, or heterozygous mutations in SRP54 consented to the SDS Registry or the Bone Marrow Failure/MDS study which are longitudinal prospective cohort studies. Results The median age of the study cohort was 12.2 years (yr) (range 0.1-52.8 yr). Of 204 patients with SBDS mutations, 12 developed severe BMF requiring transplant (median age 2.9 yr, range 0.4-39.5), 16 developed MDS (median age 15.9 yr, range 0.5-45), 11 developed AML (median age 27.7 yr, range 9.4-47.3) and 2 developed lymphoma (ages 17.0 and 23.8 yr). Of the 48 subjects with SBDS mutations who were adults >18 years (median 25.5, range 18.5-52.8), 16 (33%) developed a myeloid malignancy. Of 20 patients with an SRP54 mutation, 1 developed AML at age 15.2. One patient with biallelic EFL1 mutations died of BMF at age 0.7 yr. Further analysis focused on patients with SBDS mutations. Five patients with MDS and 1 patient with AML were on G-CSF at the time of malignancy diagnosis. Only 2 patients who developed AML had had surveillance, but neither included somatic mutation analysis. The diagnosis of SDS was unrecognized prior to malignancy diagnosis for 5 with MDS (age range 13.8 to 21.5 yr) and 2 with AML (age 37.8 and 47.3 yr). Three patients were diagnosed with MDS on surveillance marrow exam. All patients with AML presented with symptoms which led to diagnosis. Of the 16 patients who initially presented with MDS, 5 progressed to AML. Median survival for those who presented with MDS was 1.3 yr (range 0.2-14.9). Ten were deceased, 4 were alive with median follow up of 3.4 yr (range 1.0-14.9). Two additional patients were <100 days out from HSCT. Of 9 patients with MDS who proceeded to HSCT without prior cytoreductive therapy, 4 were alive with a median follow up of 3.1 yr (range 0.2-14.4) post-HSCT, 5 were deceased from treatment related mortality (TRM) (n=2), relapsed/refractory disease (n=2) or osteosarcoma at 10 years post-HSCT (n=1). Of 6 patients who received cytoreductive therapy, 3 died prior to HSCT with active disease (n=2) or TRM (n=1). Of the 3 who proceeded to HSCT post-cytoreductive therapy, 2 are alive at 0.1 and 0.7 yr post HSCT, and 1 died of TRM. One patient who received no therapy died 7.7 years post-diagnosis. Median survival following AML diagnosis was 1 year (range 0.2-5.6). Of the 11 patients with AML, 8 died with relapsed/refractory disease, 1 died of TRM, and 2 survive in remission at 2 and 5.6 years post-AML diagnosis. Of the two patients who proceeded directly to HSCT, one is alive 5.6 years post-HSCT. Nine patients received a variety of cytoreductive regimens for AML, including AraC/Idarubicin, hypomethylating agents +/- venetoclax, of whom 6 were able to proceed to HSCT. None of the 9 adult patients >18 years with SBDS mutations and AML survived. Eight patients with SDS undergoing surveillance marrow exams developed high risk features (HRF), including progressive dysplasias, dropping blood counts with rising marrow cellularity, high risk clonal cytogenetic abnormalities, and large or rapidly growing TP53 clones. Median age of patients with HRF was 19.7 yrs (range 1.3-40.9). All 6 patients transplanted for HRF are alive and well with a median follow up of 1.8 yr (0.1-4.5) post-BMT (Figure 1); 2 patients are currently undergoing evaluation for HSCT. None of the patients transplanted for HRF developed grade 3 or 4 GVHD. One patient with a large TP53 clone prior to transplant is alive without a detectable TP53 clone 0.9 years post-BMT. Conclusions AML carries a poor prognosis for patients with SBDS mutations, so early referral for novel therapies should be considered. Marrow surveillance including morphology, cytogenetics, FISH, and somatic mutation analysis identified patients who developed HRF. Survival was excellent for patients transplanted for HRF. Although SDS is rare and patient numbers are small, these data show that regular comprehensive marrow surveillance can identify HRF to inform the need for closer monitoring or transplant prior to progression to malignancy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The Independent Adverse Prognostic Significance of 1q21 Gain/Amplification in Newly Diagnosed Multiple Myeloma Patients

Background Multiple myeloma (MM) is a hematological malignancy with proliferation of monoclonal plasma cells which has complex genetics and prognostic heterogeneity. It involves a variety of primary genetic abnormalities, including t (4; 14), t (11; 14), t (14,16) and hyperdiploid karyotypes, as well as secondary genetic abnormalities, including chromosome copy number abnormalities (1q21+, 1p-, 13q-, 17p-, etc) and secondary translocations like MYC. As the most common secondary genetic event, 1q21+ occurs in about 30-40% of NDMM patients while the proportion is especially higher in Chinese population, accounting for 59.7% (96/161) by a domestic study in 2022, it is closely related to the situation that MM patients may have long disease duration and late stage when initial diagnosed in China. Despite its frequency, considerable debate remains regarding to the prognostic impact of 1q21+ in NDMM, 1q21 status hasn't been included among the high-risk markers listed by the IMWG R-ISS stage, and neither in Chinese guidelines. If 1q21+ is included as one of the poor prognostic factors, it will greatly increase the proportion of high-risk patients in NDMM patients. So, our center has begun to take 1q21 seriously as a heterogeneity problem for clinical discussion. Methods 248 NDMM patients admitted in The First Affiliated Hospital of Soochow University from September 01, 2018 to August 31, 2021 of a VRD clinical study, with VRD in combination with ASCT or VRD treatment for 8 cycles, were retrospectively analyzed. 135 cases (54.4%) had 1q21+ by CD38-sorted fluorescence in situ hybridization (FISH), among which 153 patients were compared for the involved genes by CytoScan, focusing on the clinical characteristics, treatment response and prognostic significance by combining FISH and CytoScan technology complementary. Results Among the 248 NDMM patients, 54.4% patients with 1q21+ were more likely to have anemia, hypoalbuminemia, renal insufficiency, high lactate dehydrogenase and high proportion of R-ISS-III stage, and are more likely to be combined with 13q14 deletion, t(4;14), and 1p deletion，and they are all at middle-risk or high-risk groups defined by Prognostic Index score. Multivariate analysis showed that 1q21 gain/amplification was an independent adverse prognostic factor (PFS HR=2.358, 95%CI 1.286-4.324, P=0.006; OS HR=2.598, 95%CI 1.050-6.425, P=0.039). 1q21+ subgroup had an inferior outcome (PFS P=0.0133, OS P=0.0293) (Figure 1a). According to the mSMART3.0 stages, double-hit and triple-hit group had the worst prognosis, with a median PFS of 22 months and a median OS of 32 months. Both PFS and OS were clinically significant (P<0.0001), as shown in Figure 1b. Furthermore 1q21 amplification with more than 4 copy numbers had a shorter PFS than 1q21 gain with 3 copy numbers (24 months vs not reached, P=0.0403), but the OS difference hasn't been not clinically significant (Figure 1c). The positive proportion of 1q21 had no effects on prognosis，while the status of 1q21 in the main clone rather than in the subclone defined by CytoScan when the proportion of abnormal clone populations are differed more than 30%, was an adverse factor affecting the prognosis (PFS P=0.0172, OS P=0.1260). (Figure 1d). ASCT plays an important role in the prognostic survival of 1q21 gain and amplification patients (P<0.05). Outcomes of 1q have been widely discussed in CRD/CTD scheme of Myeloma XI, KRd/KCD scheme of FORTE, VRD/PAD of HOVON65 and so on, our study focusing on the patients with VRD +/- ASCT is also unique, demonstrating that the prognosis of 1q21+ in VRD scheme is also poor, but ASCT can overcome the adverse effects significantly. Conclusions Patients with 1q21+ have clinically significant end-stage organ damage and higher tumor burden, more likely to combine 13q14-, t(4;14), 1p32- and other cytogenetic abnormalities. 1q21+ is an independent high-risk cytogenetic factor for poor prognosis in NDMM patients, of which 4 or more copy numbers and main clone position significantly associated with prognosis results. By combining FISH, CytoScan and other technologies with related abnormalities such as 1q21+，17p-, 1p32-，t (4;14), t(14;16), etc., clinical characteristics, efficacy evaluation, and prognosis can be analyzed more accurately, aiming to identify patients with poor prognosis earlier and enhancing the possibility of moving forward to the treatment strategy based on risk stratification in the future. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Cytogenetic profile of adult acute myeloid leukemia in Egypt: a single-center experience

BackgroundAcute myeloid leukemia (AML) is a diverse disease characterized by the expansion of blasts of myeloid lineage. Cytogenetic testing is the cornerstone for risk stratification of AML patients. Geographical and environmental factors may play a very important role in the development of leukemia and several differences in genetic profile may be seen among different ethnicities. In our study, we evaluated cytogenetic findings of adult AML patients in South Egypt.MethodsCytogenetic testing (karyotyping and M-FISH) was performed for 120 adult patients with AML. Twenty metaphases were analyzed for each patient.ResultsIn our study, the median age of AML patients was 36.5 years, with an age range between 18 and 86 years. 56.7% of patients had normal karyotypes and 43.3% of patients had clonal cytogenetic abnormalities. t (15;17) was the most detected structural abnormality, and + 8 was the most detected numerical abnormality. Regarding cytogenetic risk stratification, 65% of patients were in the intermediate-risk category.ConclusionThe cytogenetic profile of AML patients in our locality showed some differences and some similarities with cytogenetic profiles in different Arab, Asian and Western countries. Further studies are needed using advanced techniques such as next-generation sequencing and optical genome mapping to elucidate more ethnic and geographic genetic heterogeneity among different countries.

33714 Palisaded neutrophilic and granulomatous dermatitis in the setting of SRSF2-mutated chronic myelomonocytic leukemia: Case report and review of the literature

Palisaded neutrophilic and granulomatous dermatitis (PNGD) is a rare cutaneous histopathologic reaction pattern associated with several underlying disorders. Few cases of PNGD have been associated with hematologic malignancies, in particular with chronic myelomonocytic leukemia (CMML), a malignant hematopoietic disorder with features of myeloproliferative neoplasm and myelodysplastic syndrome. CMML is characterized by peripheral blood monocytosis and bone marrow dysplasia, and can be supported by an acquired clonal cytogenetic abnormality most commonly in TET2, SRSF2, ASXL1, RUNX1, NRAS, and CBL. We present a patient with a papulosquamous rash on the neck, chest, and shoulders with histomorphological features on the spectrum of PNGD. Subsequent lab workup demonstrated a persistent mild monocytosis, raising concern for CMML. The patient was referred to hematology-oncology for a bone marrow biopsy, which ultimately led to her diagnosis. Cytogenic studies of the bone marrow biopsy demonstrated mutations in SRSF2, IDH2, and ASXL1, which were strongly supportive of this diagnosis. After discussion at a multidisciplinary tumor board, treatment directed at the skin eruption alone was recommended. She was started on prednisone taper and demonstrated marked clinical improvement. PNGD in the context of CMML has been scarcely reported, with only 4 prior reports in the literature. Our patient is the fifth reported case, and the fourth case with confirmed underlying SRSF2 mutation. This is likely a novel and reproducible clinical-histopathologic-molecular subtype of reactive granulomatous disease. The findings in this case strengthen the previously made association between PNGD and SRSF2-mutated CMML, and may help better define a unique recognizable subtype for dermatopathologists.

Spectrum of stable and unstable rearrangements in lymphocytic chromosomes investigated in Bhopal population 30 years post MIC disaster amid co-exposure to lifestyle, living, and occupational hazards.

Immediate assessment of genetic damage in methyl isocyanate (MIC) gas-exposed population in small and heterogeneous samples using diversified study designs and solid-stained metaphases could not depict the actual genetic impact of MIC on accidentally exposed individuals. The outcome of the then large multi-center genetic screening program was not available to the public and scientific community. Also, the routine and regular epidemiological health survey does not capture the genetic and long-term effect of MIC. Therefore, genetic screening was carried out 30 years post disaster during 2015-2017 with a view to screen the present status of chromosomal consequences in lymphocytic cells. Participants were recruited from moderate (34) and severely (78) exposed and unexposed (35) cohorts with their informed consent. Analysis of ~100 mitotic cells and karyotyping of at least 10-15 and all abnormal metaphases detected structural and numerical alterations, including stable and replicable ones. Clonal abnormalities were detected with monosomal and complex karyotypes, trisomy 8, del5q/20q, loss of Y, etc. Among all, X-chromosome was frequently involved in numerical alterations. Structural aberrations appeared higher in the then exposed populations, though abnormalities cannot be linked directly to MIC exposure 30 years post disaster. Collectively, all rearrangements were markedly higher in the severely exposed population. Altogether, the detected abnormalities appeared random and indicated genomic instability, suggesting follow-up at shorter intervals for the individuals detected with clonal aberrations. G-banding has facilitated recognition of chromosomal involvement and their breakpoints and classification of structural rearrangements. The present data has been derived from the 30-year post-disaster genetic screening.

Labial Mucosa Stem Cells: Isolation, Characterization, and Their Potential for Corneal Epithelial Reconstruction.

PurposeThe purpose of this study was to characterize labial mucosa stem cells (LMSCs) and to investigate their potential for corneal epithelial reconstruction in a rabbit model of total limbal stem cell deficiency (LSCD).MethodsRabbit LMSCs (rLMSCs) and human (hLMSCs) LMSCs were derived from labial mucosa and characterized in terms of their proliferation activity by the evaluation of proliferation index (PI) and colony forming efficiency (CFE), cell senescence, and differentiation abilities. The expression of various limbus-specific, stem cell-specific, and epithelial markers was assessed via immunocytochemistry. Flow cytometry was used to evaluate mesenchymal and hematopoietic cell surface markers expression. Chromosomal stability of the derived cells was examined using the conventional GTG-banding technique. To assess the impact of LMSCs on corneal epithelial reconstruction, rLMSCs were seeded onto a decellularized human amniotic membrane (dHAM), thereafter their regeneration potential was examined in the rabbit model of total LSCD.ResultsBoth rLMSCs and hLMSCs showed high proliferation and differentiation abilities, entered senescence at later passages, and expressed different stem cell-specific (ABCB5, ALDH3A1, ABCG2, and p63α), mesenchymal (vimentin), and epithelial (CK3/12, CK15) markers. Cell surface antigen expression was similar to other described mesenchymal stem cells. No clonal structural chromosome abnormalities (CSCAs) and the low percentage of non-clonal structural chromosome abnormalities (NSCAs) were observed. Transplantation of rLMSCs promoted corneal epithelial reconstruction and enhanced corneal transparency.ConclusionsLMSCs have significant proliferation and differentiation abilities, display no detrimental chromosome aberrations, and demonstrate considerable potential for corneal repair.

POLQ suppresses genome instability and alterations inDNA repeat tract lengths.

DNA polymerase theta (POLQ) is a principal component of the alternative non-homologous end-joining (ANHEJ) DNA repair pathway that ligates DNA double-strand breaks. Utilizing independent models of POLQ insufficiency during telomere-driven crisis, we found that POLQ–/– cells are resistant to crisis-induced growth deceleration despite sustaining inter-chromosomal telomere fusion frequencies equivalent to wild-type (WT) cells. We recorded longer telomeres in POLQ–/– than WT cells pre- and post-crisis, notwithstanding elevated total telomere erosion and fusion rates. POLQ–/– cells emerging from crisis exhibited reduced incidence of clonal gross chromosomal abnormalities in accordance with increased genetic heterogeneity. High-throughput sequencing of telomere fusion amplicons from POLQ-deficient cells revealed significantly raised frequencies of inter-chromosomal fusions with correspondingly depreciated intra-chromosomal recombinations. Long-range interactions culminating in telomere fusions with centromere alpha-satellite repeats, as well as expansions in HSAT2 and HSAT3 satellite and contractions in ribosomal DNA repeats, were detected in POLQ–/– cells. In conjunction with the expanded telomere lengths of POLQ–/– cells, these results indicate a hitherto unrealized capacity of POLQ for regulation of repeat arrays within the genome. Our findings uncover novel considerations for the efficacy of POLQ inhibitors in clinical cancer interventions, where potential genome destabilizing consequences could drive clonal evolution and resistant disease.

Evaluation of miR-145 and miR-146a as potential biomarkers for diagnosis of Myelodysplastic Syndrome

Objective: Myelodysplastic syndromes (MDS) are a group of heterogeneous hematopoietic stem cell disorders characterized by ineffective hematopoiesis, bone marrow dysplasia, and peripheral cytopenias. microRNAs (miRNAs) are small non-coding RNAs that play key roles in post-transcriptional regulation of gene expression and have been determined potential in disease diagnostics and therapeutics owing to their stability. Recent evidence suggests that haploinsufficiency of the miR-145 and miR-146a, encoded from 5q Common Deleted Region (CDR) may contribute to the phenotype in MDS. Although, interstitial del(5q) is the most common chromosomal abnormality in MDS, these findings are inconsistent in Turkish patients. Therefore, we aimed to investigate assess the diagnostic value of miR-145/miR-146a and their relation with del(5q) or monosomy 5 in MDS. Methods: In order to determine the association between del(5q) and expression miR-145/miR-146a, conventional cytogenetics (CC), FISH, and qRT-PCR methods were performed for 24 patients with MDS and 20 healthy individuals. Additionally, ROC curves were generated to evaluate putative diagnostic value of miRNAs. Results: Cytogenetic examination revealed clonal cytogenetic abnormalities in 43.4% of cases. miR-146a decreased in 23 of 24 patients regardless of chromosome 5 abnormalities (p

PB1914: CYTOGENETIC FINDINGS IN 18 CHILDREN WITH MYELODYSPLASTIC SYNDROMES BY KARYOTYPIC AND FISH ANALYSIS

Background: Pediatric myelodysplastic syndromes (MDS) are rare disorders characterized by hematopoietic cell dysfunction, an increased risk of AML and a poor prognosis. Although it is known that cytogenetic abnormalities are seen in approximately half of children diagnosed with MDS, their detailed cytogenetic profile has not been elucidated yet due to the rarity of the disease. Aims: We tried to detect chromosomal abnormalities in pediatric patients with MDS, including submicroscopic aberrations such as gene alterations associated with the disease and possible malignant transformation by karyotypic and molecular cytogenetic analysis (fluorescence in situ hybridization, FISH) to contribute to the elucidation of the cytogenetic profile of pediatric MDS and the investigation of the pathogenesis of the disease. Methods: In this study 18 children with MDS are included. Karyotypic analysis was performed on 24h, 48h and 72h bone marrow (BM) cultures using GTG-banding. Karyotypic analysis was carried out on 25 at least metaphases. FISH analysis was performed on cultured BM cells using IVD probes for centromere (CEP) of chromosomes 7 and 8 and the chromosomal regions 7q22, 7q31, 8q24(MYC), 11q22.3(ATM), 11q23(MLL), and 17p13(TP53). For each sample at least 200 interphase cells (iFISH) and 5 metaphases (mFISH) were analyzed to increase sensitivity. Normal cut-off was estimated at 3%. Results: The sex ratio was 1,25 (10 M/8 F) and the mean age was 9,42 years (range 0,4y - 19y). Eleven patients had RCC, 5 MDS-EB and 2 t-MDS. Normal karyotypes were detected in 10 patients (55,6%), 6 patients with RCC, 3 with MDS-EB and 1 with t-MDS. Abnormal karyotypes were found in 8 patients (44,4%), 4 with RCC, 2 with MDS-EB and 1 with t-MDS. Monosomy 7 was observed in 2/18 patients (>70% of metaphases), +8 in 2/18 patients (5-6.6% of metaphases), inv(17)(p11.2q21) in 1/18 patients (18.75% of metaphases), complex karyotypes in 2/18 patients (31.3-73.2% of metaphases including abnormalities of 1q, 2q, 4q, 5q, 16q, 18 and Xp), in one patient after SCT in the transformation of MDS into s-AML and in the other at diagnosis with t-MDS) and chromatid and chromosome breaks in 1/18 patients, (8,6-9,6% of metaphases) in 2 different BM samples taken with an interval of 3 months, which were also found in his brother’s BM karyotype. FISH revealed -7 in 2/18 patients confirming their karyotypes, loss of one p53 allele in 3/18 patients at low frequencies (6,5%, 4,5%, 3,5%) all with hypocellular RCC and transfusion-dependency that underwent SCT, +8 in 2/18 (in 8% and 2,7% of cells) confirming trisomy 8 in their karyotypes and loss of one MLL allele and MLL rearrangement in a patient that was transformed to s-AML in 4,4% of cells. A normal pattern of hybridization was observed for the above probes in the rest cases and for MYC and ATM genes in all the examined cases. Summary/Conclusion: Karyotypic aberrations were detected in 44,4% of children including -7, +8, inv(17), complex karyotypes and chromatid and chromosome breaks, revealing that other chromosome aberrations except -7/del(7q) and trisomy 8 are characterized this disease. Clonal chromosome abnormalities can be detected at low frequencies in the karyotypes of pediatric MDS patients so except from iFISH, mFISH is also needed especially in cases with small clones or submicroscopic translocations. The low frequency of submicroscopic aberrations such as p53 deletions observed in 3 cases of hypocellular RCC must be confirmed in larger studies by molecular analysis as they may play a potential role in MDS pathogenic evolution.

Utilization and financial coverage of blood based next generation sequencing for solid tumors: A study from Augusta-Georgia.

e18592 Background: There is an increasing trend of integrating molecular profiling to devise treatment plans for solid malignancies. Tumor testing for specific clonal abnormalities is being supplanted by broad multigene panel testing from blood specimen, aptly labelled liquid biopsy. Due to varying utility across malignancies in differing contexts and issues of access and affordability, their adoption is not uniform. We attempt to characterize the pattern of utilization across tumor types and financial coverage schemes in an academic cancer institute in southeastern United States. Methods: All blood based next generation testing samples ordered by providers affiliated to Georgia Cancer center between January 1, 2017, and December 31, 2021, via Guardant platform for all solid tumors was included in this analysis. Guardant360 and Guardant360CDx assays tested for upto 83 cancer related genes in circulating cell-free DNA in patients of solid tumors. Primary cancer diagnosis, insurance information and turnaround time was obtained from Guardant. Results: 296 tests were sent from January 1, 2017, through December 31, 2021. The total number of tests performed in year 2017, 2018, 2019, 2020 and 2021 were 1, 3, 28, 99 and 165 respectively. Billing information was available for all cases, clinical information was available for 262 cases. Average turnaround time was 8.5 days overall. 221 Guardant360 was sent with average turnaround time of 10 days. 41 Guardant360CDx was sent with average turnaround time of 7 days. The frequency of malignancies in the order of dimensioning frequency were lung 50.7%, prostate 12.1%, breast 6.9%, kidney 3.8% and colorectal 3.4%. The remaining 22.1% comprised of pancreatic, melanoma, glioblastoma, and bladder malignancies. The test was covered by private insurance/employee health plan in 53.4% (158/296), Medicare in 31.1% (92/296) and Medicaid in 10.5% (31/296) (table). 4.7% (14/296) qualified for free of charge testing (financial indigent) and 0.3% (1/296) were self-pay (table). Conclusions: There is an increase in the utilization of liquid biopsy to perform comprehensive genomic analysis. The distribution of tumor types is not entirely explained by the incidence and availability of targeted therapies for each type. Private insurers accounted for most of the tests being done. The disparities in utilization of this platform across different tumor types and payment models deserve further exploration. T[Table: see text]

Cytogenetic Risk Stratification of B-Acute Lymphoblastic Leukemia and Its Correlation with Other Prognostic Factors.

Purpose of current study was to categorize WHO defined B-Acute Lymphoblastic Leukemia (B-ALL) cases into 3 cytogenetic risk groups (good, intermediate and poor) and to see their correlation with age, NCI risk criteria and treatment response. Clinical and diagnostic details were collected for 78 newly diagnosed B-ALL patients which included bone marrow morphology, flow cytometry immunophenotyping, karyotyping, FISH and RT-PCR. Study cohort comprised 44/78 (56.4%) children including 3 infants and 34/78 (43.6%) adults. Median age for paediatric group was 6 years (3 months-17 years) and for adults was 40.5 years (18 to 75 years). According to NCI risk criteria, excluding infants, 54 (72%) were high risk and 21 (28%) were standard risk. Clonal cytogenetic abnormality was detected in 59/78 cases (75.6%), while 19/78 (24.4%) cases showed normal karyotype. There was significant association of cytogenetic risk groups to age distribution (p value < 0.001) and NCI risk groups (p value < 0.001). There was no significant correlation of CNS involvement with cytogenetic risk groups (p = 0.064). Association of Day 8 steroid response and Day 15 bone marrow status with cytogenetic risk groups was significant (p = 0.006 and p = 0.003 respectively). Post treatment bone marrow status on Day 33 and Day 79 was available for 52 and 42 cases respectively. 9 adults died during induction phase. Day 33 post induction morphological remission was achieved in 51/52 cases (98%) and 1/52 (2.0%) were not in remission. Day 79 post induction morphological remission was achieved in 41/42 cases (98%) and 1/42 (2.0%) were not in remission. Day 33 or End of induction flow MRD (measurable residual disease) was negative in 39/52 (75.0%) patients and positive in 13/52 (25.0%) patients. Day 79 flow MRD was negative in 37/42 (88.1%) and positive in 5/42 (11.9%). Cytogenetic risk groups showed statistically significant Day 33 and Day 79 treatment response (morphologic remission: p = 0.009 and 0.003, flow MRD: p = 0.004 and p = 0.012 respectively). We concluded that cytogenetic risk groups showed statistically significant association with age, NCI risk criteria and treatment response.

A Case of Aplastic Anemia and Colon Cancer With Underlying Spliceosome Mutation: Is It an Incidental Finding or a Novel Association?

Alternative splicing is an epigenetic mechanism that plays a role in the development and function of antigen-specific lymphocytes. One such is the zinc-finger-RNA-binding-motif-and-serine/arginine-rich-2 (ZRSR2), which is clinically implicated in myelodysplastic syndrome and leukemia. Here, we present a case of a young male with myriad autoimmune conditions and adenocarcinoma of the colon in the setting of ZRSR2 mutation.A 28-year-old male with common variable immunodeficiency disease, atopic dermatitis, autoimmune gastroenteropathy, inflammatory polyarthropathy, primary bone marrow failure, colon cancer, and family history of Lynch syndrome was admitted to our hospital for an acute flare of autoimmune enteropathy secondary to subtherapeutic tacrolimus levels.He initially developed pancytopenia at the age of 26 years. Workup for HIV, hepatitis, cytomegalovirus, human-herpesvirus 6, parvovirus was negative. Partial thromboplastin time (PTT), international normalized ratio (INR), d-dimer, ferritin, iron profile, antinuclear antibodies (ANA) screen was unremarkable. Direct, indirect, and super-combs antibodies were undetectable. Chromosomal study for Fanconi-related chromosomal breakage and telomerase gene panel was negative. Flow cytometry did not reveal an abnormal clone. Bone marrow biopsy showed markedly hypocellular marrow with reduced trilineage hematopoiesis and 1% blasts with normal cytogenetics, immunohistochemistry, fluorescence in situ hybridization (FISH), and negative for myelodysplastic syndrome and paroxysmal nocturnal hemoglobinuria (PNH). Cincinnati inherited children’s bone marrow transplant (BMT) panel was negative. He was diagnosed with aplastic anemia and was treated with antithymocyte globulin, cyclosporine, prednisone, and currently tacrolimus. At the age of 26 years, he was diagnosed with colon cancer. Immunohistochemistry was positive for MLH1, but the confirmatory genetic testing for Lynch syndrome was negative. He underwent total proctocolectomy and ileostomy and is currently in remission. Next-generation sequencing of bone marrow revealed a germline homozygous ZRSR2 mutation. ZRSR2 spliceosome mutations are more common in males as it’s an X-linked gene. They are seen in myelodysplastic syndrome, leukemia, increased autoimmune phenomenon, and 35 cases of colon cancer associated with this mutation are reported. In the setting of aplastic anemia and lynch negative colon cancer, we suspect our patient could have aplastic anemia due to an autoimmune phenomenon, underlying common variable immunodeficiency disease (CVID), or the new ZRSR2 mutation could be playing a role. Further studies and research is warranted to determine its true association with the disease entities. The underlying contributing factor is ZRSR2 mutation.

Radiation-free reduced-intensity hematopoietic stem cell transplantation with in vivo T-cell depletion from matched related and unrelated donors for Fanconi anemia: prognostic factor analysis

Fanconi anemia (FA) is a rare and complex genetic disorder, clinically characterized by bone marrow failure, congenital defects, and cancer predisposition. Hematopoietic stem cell transplantation (HSCT) represents the only therapeutic option to restore normal hematopoiesis after the occurrence of marrow failure or clonal hematopoietic abnormality. However, radiation exposure during transplant may increase the risk of later malignancies. In this retrospective study, we analyzed the results of HSCT with a radiation-free, busulfan-based conditioning regimen in FA patients. A total of 122 patients (median age: 8 years, range: 2-18 years) with FA who underwent HSCT between January 2008 and January 2020 were enrolled in this study and followed up to the end of 2020. The preparative regimen included busulfan (0.2 mg/kg/day, days -9 to -6), cyclophosphamide (15 mg/kg/day, days -5 to -2), and in vivo T-cell depletion with rabbit anti-thymocyte globulin. All patients received graft-versus-host disease prophylaxis with cyclosporine combined with methotrexate. We used the Kaplan-Meier method, log-rank test, and Cox proportional hazards models to analyze patient survival. Peripheral blood, bone marrow and cord blood hematopoietic stem cells were used in 84 (68.9%), 31 (25.4%) and 7 (5.7%) patients, respectively. Donors were matched siblings in 48 (39.3%), matched other relatives in 56 (45.9%), and matched unrelated persons in 18 (14.8%) patients. With a median follow-up time of 24.25 months, graft rejection occurred in only one patient. The 1- and 5-year overall survival rates were 84.14% (95% confidence interval: 76.02-89.70) and 82.16% (95% confidence interval: 73.01-88.45), respectively. Of the patient characteristics documented before transplant, the presence of cardiopulmonary, genitourinary tract, central nervous system, and limb malformations significantly affected survival rates. Our results indicate excellent outcomes in patients with FA undergoing HSCT with a radiation-free, busulfan-based conditioning regimen. It would be desirable to aim at optimizing the outcome of HSCT in FA patients in future studies.

Chronic myelomonocytic leukemia: 2022 update on diagnosis, risk stratification, and management

Chronic myelomonocytic leukemia (CMML) is a clonal hematopoietic stem cell disorder with overlapping features of myelodysplastic syndromes and myeloproliferative neoplasms, with an inherent risk for leukemic transformation (~15% over 3-5 years). Diagnosis is based on the presence of sustained (>3months) peripheral blood monocytosis (≥1× 109 /L; monocytes ≥10%), usually with accompanying bone marrow dysplasia. Clonal cytogenetic abnormalities occur in ~30% of patients, while >90% have somatic gene mutations. Mutations involving TET2 (~60%), SRSF2 (~50%), ASXL1 (~40%), and the oncogenic RAS pathway (~30%) are frequent, while the presence of ASXL1 and DNMT3A mutations and the absence of TET2 mutations negatively impact overall survival. Molecularly integrated prognostic models include the Groupe Français des Myélodysplasies, Mayo Molecular Model (MMM), and the CMML specific prognostic model. Risk factors incorporated into the MMM include presence of truncating ASXL1 mutations, absolute monocyte count >10× 109 /L, hemoglobin <10g/dL, platelet count <100 × 109 /L, and the presence of circulating immature myeloid cells. The MMM stratifies CMML patients into four groups: high (≥3 risk factors), intermediate-2 (2 risk factors), intermediate-1 (1 risk factor), and low (no risk factors), with median survivals of 16, 31, 59, and 97 months, respectively. Hypomethylating agents such as 5-azacitidine and decitabine are commonly used, with overall response rates of ~40%-50% and complete remission rates of ~7%-17%; with no impact on mutational allele burdens. Allogeneic stem cell transplant is the only potentially curative option but is associated with significant morbidity and mortality.

Coexistence D’un Lymphome Lymphocytique Et D’une Leucémie Myéloïde Chronique: A Propos De Deux Observations Au Togo

This paper reported two cases of lymphocytic lymphoma associated with chronic myeloid leukemia (CML) in Togo. The first association is a simultaneous coexistence of a CML with the presence of the transcript bcr-abl, in a 32-year-old woman, at chronic myelocytic stage with polyadenopathy required and lymph node biopsy concluded for lymphocytic lymphoma. The second is the case of a 30-year-old man who developed a lymphocytic lymphoma after 6years and 5 months of following CML. These two cases are comparable to other associations of acute or chronic myeloid and lymphoid blood malignancies that was previously published. Several pathogenic hypothesis were reviewed which takes responsibility for chemotherapy. Nevertheless, the diagnosis is sometimes simultaneous with the involvement of cellular oncogenes. This is a more likely clonal abnormality of the stem cell sequentially or concomitantly expressed in the cells on both lineages. To the best of our knowledge, these are the first descriptions in South-Saharan Africa.

Autoimmune polyglandular syndrome type 1 and eye damage

Autoimmune polyendocrine syndrome type 1 (APS type 1) is a disease characterized by a variety of clinical manifestations resulting from the involvement of multiple endocrine and non-endocrine organs in the pathological process. APS type 1 is a rare genetically determined disease with autosomal recessive inheritance. Mutations in the autoimmune regulator gene (AIRE) lead to a disruption of the mechanism of normal antigen expression and the formation of abnormal clones of immune cells, and can cause autoimmune damage to organs. Within APS type 1, the most common disorders are primary adrenal insufficiency, hypoparathyroidism, and chronic candidiasis. Some understudied clinical manifestations of APS type 1 are autoimmune pathological processes in the eye: keratoconjunctivitis, dry eye syndrome, iridocyclitis, retinopathy, retinal detachment, and optic atrophy. This review presents the accumulated experimental and clinical data on the development of eye damage of autoimmune nature in APS type 1, as well as the laboratory and instrumental methods used for diagnosing the disease. Changes in the visual organs in combination with clinical manifestations of hypoparathyroidism, adrenal insufficiency and candidiasis should lead the clinical doctor to suspect the presence of APS type 1 and to examine the patient comprehensively. Timely genetic counselling will allow early identifi cation of the disease, timely prescription of appropriate treatment and prevention of severe complications.

Analysis of Clonal Cytogenetic Abnormalities in Persistent Cytopenia: Relation of Degree of Dysplasia and Prognosis

Analysis of Clonal Cytogenetic Abnormalities in Persistent Cytopenia: Relation of Degree of Dysplasia and Prognosis

The frequent and clinically benign anomalies of chromosomes 7 and 20 in Shwachman-diamond syndrome may be subject to further clonal variations

BackgroundAn isochromosome of the long arm of chromosome 7, i(7)(q10), and an interstitial deletion of the long arm of chromosome 20, del(20)(q), are the most frequent anomalies in the bone marrow of patients with Shwachman-Diamond syndrome, which is caused in most cases by mutations of the SBDS gene. These clonal changes imply milder haematological symptoms and lower risk of myelodysplastic syndromes and acute myeloid leukaemia, thanks to already postulated rescue mechanisms.ResultsBone marrow from fourteen patients exhibiting either the i(7)(q10) or the del(20)(q) and coming from two large cohorts of patients, were subjected to chromosome analyses, Fluorescent In Situ Hybridization with informative probes and array-Comparative Genomic Hybridization. One patient with the i(7)(q10) showed a subsequent clonal rearrangement of the normal chromosome 7 across years. Four patients carrying the del(20)(q) evolved further different del(20)(q) independent clones, within a single bone marrow sample, or across sequential samples. One patient with the del(20)(q), developed a parallel different clone with a duplication of chromosome 3 long arm. Eight patients bore the del(20)(q) as the sole chromosomal abnormality. An overall overview of patients with the del(20)(q), also including cases already reported, confirmed that all the deletions were interstitial. The loss of material varied from 1.7 to 26.9 Mb and resulted in the loss of the EIF6 gene in all patients.ConclusionsAlthough the i(7)(q) and the del(20)(q) clones are frequent and clinically benign in Shwachman Diamond-syndrome, in the present work we show that they may rearrange, may be lost and then reconstructed de novo, or may evolve with independent clones across years. These findings unravel a striking selective pressure exerted by SBDS deficiency driving to karyotype instability and to specific clonal abnormalities.