Normal Cytogenetics Research Articles

Acute Myeloid Leukemia (AML) Patients with an Isolated Trisomy Display Increased Frequency of Spliceosome Mutations

Abstract Introduction: The molecular landscape of patients (pts) with cytogenetically normal (CN) AML has been extensively evaluated; however, there are limited data providing comprehensive mutation analysis of AML pts with sole somatic trisomy. A few analyses with small numbers of pts found frequent mutations of spliceosome encoding genes (Eisfeld et al, Leukemia 2016, Herold et al, Blood 2014). Methods: Review of the Cancer and Leukemia Group B/Alliance for Clinical Trials in Oncology cytogenetics database found 138 de novo adult AML pts with isolated trisomy of chromosomes 4, 8, 11, 13 or 21. We compared clinical and molecular data between pts within each isolated trisomy group as well as with 131 pts who had ≥1 chromosome abnormality in addition to a trisomy (non-sole trisomy) and with 716 pts with CN-AML. Results: Spliceosome encoding genes, mainly SRSF2 and U2AF1, were often mutated in pts with isolated trisomy (Figure 1). SRSF2 was mutated in 28% of +8 pts and 35% of +21 pts. SRSF2 mutations were the most common mutation in +13 pts, seen in 48% of +13 pts. U2AF1 was mutated in 15% of +8 pts and in 43% of +11 pts. Other genes commonly mutated in each trisomy group included DNMT3A, FLT3 -ITD, IDH2, NRAS, RUNX1 and TET2. Notably, pts with +4 lacked mutations in ASXL1, BCOR, STAG2 and WT1 genes, all of which were mutated in pts in the other sole trisomy groups. NPM1 mutations, often seen in the +4, +8, +13 and +21 groups, were not seen in +11 pts. There were no differences in complete remission rates, relapse rates, disease-free or overall survival among trisomy groups. For pre-treatment clinical features of the 138 de novo isolated trisomy AML pts see Table 1. The median age was 61 years (range, 19-84 years). We saw several differences in the mutation spectrum between older (aged ≥60 years) and younger (aged We next studied molecular differences between the 138 sole trisomy pts and 131 pts with non-sole trisomy. We saw more mutations in SRSF2 (29% vs 14%, P=.003) and U2AF1 (14% vs 14%, P=.003) in sole trisomy versus non-sole trisomy pts. Several genes were often mutated (≥5%) in both sole and non-sole trisomy groups; most common were spliceosome genes SRSF2 , U2AF1, ZRSR2, and others were ASXL1, BCOR, DNMT3A, FLT3 -ITD, FLT3 -TKD, IDH1, IDH2, NPM1, NRAS, RUNX1, STAG2, TET2. TP53 mutations were often seen in non-sole trisomy pts, but were rare in sole trisomy pts (17% vs 1%, P Comparing sole trisomy pts to 716 CN-AML pts, we found spliceosome encoding genes more often mutated (48% vs 19%, P Conclusions: This is the largest series of de novo AML pts with isolated trisomy that provides comprehensive mutation analysis. These pts display a distinct molecular landscape from AML pts with non-sole trisomy and from CN-AML pts. Mutations affecting spliceosome encoding genes were seen in almost 50% of pts with isolated trisomy suggesting that aberrant RNA splicing may be implicated in the pathogenesis of these pts. Support: U10CA180821, U10CA180850, U10CA180882, U24CA196171; ClinicalTrials.gov Ids: NCT00048958, NCT00900224 Download : Download high-res image (236KB) Download : Download full-size image Disclosures Kolitz: Gilead, Magellan, Novartis, Pharmacyclics, and Seattle Genetics: Consultancy; Boehringer Ingelheim, Cantex, Erytech, and Millennium: Research Funding; Gilead, Novartis, and Seattle Genetics: Other: Travel Support; Celgene, Jazz: Equity Ownership; Gilead, Magellan, and Novartis: Honoraria. Powell: Rafael Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees. Byrd: Acerta Pharma: Research Funding; Genentech: Research Funding; The Ohio State University: Patents & Royalties: OSU-2S; Janssen: Research Funding; Pharmacyclics: Research Funding.

Inotuzumab Ozogamicin (InO) for Relapsed/Refractory Acute Lymphoblastic Leukemia (R/R ALL) in the Global Phase 3 INO-VATE Trial: Efficacy By MLL Status

Inotuzumab Ozogamicin (InO) for Relapsed/Refractory Acute Lymphoblastic Leukemia (R/R ALL) in the Global Phase 3 INO-VATE Trial: Efficacy By MLL Status

The Effect of Bortezomib on Renal Dysfunction in African American Patients with Multiple Myeloma

Introduction -Renal dysfunction (RD) is a major negative prognostic factor in multiple myeloma (MM). Approximately 50% of patients have RD throughout the course of their disease, which is associated with a more aggressive clinical course, poorer quality of life, and shortened overall survival. Bortezomib, a potent proteasome inhibitor, has shown significant anti-myeloma activity and is known to have a protective renal effect. In addition, it is well known that African Americans (AA) are twice as likely to be diagnosed with MM compared to Caucasians and tend to have worse outcomes. At our cancer center, which is in a large urban hospital, nearly 85% of MM patients are AA. To date, there has been no study specifically looking at the reversibility of RD in this population. We evaluated the effectiveness of bortezomib on RD in AA patients with MM in a large urban hospital.Methods -We conducted a retrospective analysis to investigate the outcome of bortezomib based therapy in 51 AA patients diagnosed with MM and RD from 2004-2015, including 4 cases requiring hemodialysis. Renal dysfunction was defined as Cr ³ 2.0 mg/dL or GFR < 60 mL/min/1.73m2. Patients required at least 3-6 months of continuous treatment with a bortezomib based regimen to be eligible for analysis. Laboratory data was collected prior to initiation of treatment, and again at 3 months and 6 months. Both newly diagnosed and relapsed or refractory patients were included.Results -Approximately 75% of patients treated with bortezomib were > 65 years old, with an equal distribution of males and females. At presentation, 80% had anemia (Hb<10 g/dL), 14% hypercalcemia (Ca>11 mg/dL), 37% elevated LDH, 61% elevated B2 microglobulin. The majority of patients had normal risk cytogenetics and 33% were ISS stage III. Myeloma subtype was IgM 65%, IgA 16%, IgM 2%, light chain only 16%. Sixty percent were kappa light chain subtype. The median M spike was 2.54 (range 0.5-16), creatinine 2.41 mg/dL (range 1.1-10.48), GFR 38 mL/min/1.732 (range 6-60). Treatment was bortezomib based in 100% patients: 6% had bortezomib, 45% bortezomib/dexamethasone (VD), 22% VD/cyclophosphamide (VCD), 12% VD/lenalidomide (VRD), 12% VD/melphalan (VMP), 2% VD/thalidomide (VTD), 2% VD/doxorubicin (PAD). A partial response or better was seen in 55% of patients by month 3 and 63% of patients by month 6.Improvement of RD was observed in 85% of patients by month 3. The average creatinine improved by approximately 24.5% (p<0.001), GFR improved by 52.6% (p<0.001). A plateau effect was noticed by month 6, as creatinine and GFR improved by 19.5% and 44.7%, respectively. Newly diagnosed patients had a greater reversal of RD compared to relapsed/refractory disease (p=0.032). None of the 4 patients who began hemodialysis were able to discontinue this therapy.Conclusion -A statistically significant improvement in RD was observed in AA patients with MM based on Cr and GFR treated with a bortezomib based regimen. The greatest response was seen in the first 3 months of therapy which plateaued by month 6. These differences were magnified in de novo compared to relapsed/refractory disease. No patients were able to discontinue hemodialysis if already initiated prior to starting treatment. Bortezomib is a safe and effective drug in the subset of AA patients with impaired renal function. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Conventional Karyotyping Is Still an Important Prognostic Marker for Myeloma in the Era of R-ISS Staging

Introduction: The Revised International Staging System (R-ISS) has been widely adopted to prognosticate Multiple Myeloma (MM). As a result, the continued utility of conventional karyotyping, has been called into question.Methods: A multi-centre study for newly diagnosed MM patients who received novel agent/s at induction from year 1999 at the National University Hospital Singapore, Singapore General Hospital, Alfred Hospital, Melbourne, Australia, was conducted. Conventional karyotype information was categorized based on ploidy as follows: normal, hyperdiploid (48-74 chromosome numbers), non-hyperdiploid (other abnormal karyotype). We evaluated the impact of ploidy on Overall Survival (OS) including a multivariate analysis, taking into account the R-ISS stages, transplant status, age, and novel agent/s used at induction. We also evaluated if it is possible to substitute FISH with conventional karyotyping in identifying high-risk patients. Results were validated in an independent cohort from China.Results: There were 303 patients evaluable. Ploidy significantly affected OS of patients with R-ISS stage II (N=212; p=0.026), with non-hyperdiploid patients doing worst. For R-ISS stage I patients (N=20), the number of patients in each cytogenetic group was too small to draw any conclusion. Non-hyperdiploid karyotype had no prognostic impact on patients with R-ISS stage III (N=71; p=0.990). In the multivariate analysis, ploidy was significantly associated with OS (p=0.018).Based on our results, we divided the R-ISS stage II patients into groups with or without non-hyperdiploid karyotype. The R-ISS stage I group and the R-ISS stage II group without non-hyperdiploid karyotype had similar median OS (median OS of 92.1 and 96.0 months respectively; p=0.001). They were grouped together into a standard-risk group. The R-ISS stage II group with non-hyperdiploid karyotype and the R-ISS stage III group had similar OS and were categorized together as a high-risk group (median OS of 45.0 months and 45.1 months respectively; p=0.001). As high-risk myeloma are of most clinical significance, we proposed a new prognostic staging system that incorporates ploidy into the R-ISS. In this R-ISS+K(Karyotype) staging system, patients were divided into standard and high-risk groups.. Patients in the 2 groups had significantly different OS (median OS of 92.1 versus 45.0 months, p=0.000).We replaced FISH with conventional karyotyping in the K(Karyotype)-ISS staging system, where stage I consisted of patients with ISS stage I with normal LDH and cytogenetics, stage III consisted of patients with ISS stage III with raised LDH or abnormal cytogenetics, and stage II consisted of all others. Of the R-ISS stage III patients, 83.0% were also K-ISS stage III. R-ISS stage II patients who were K-ISS Stage III had median OS of 45.0 months, shorter than K-ISS stage II patients that were R-ISS stage III (median OS of 60.0 months). In this K-ISS staging system, K-ISS stage III patients had median OS of 38.0 months, shorter than the median OS (45.1 months) of the R-ISS stage III group.These results were validated with an independent cohort of 382 patients from the Institute of Hematology, Peking University, China. The median OS in the R-ISS+K standard risk group was 127.1 months compared to 43.0 months for the high risk group (p=0.000). Similarly, K-ISS stage III patients had median OS of 43.1 months, compared to 46.1 months for the R-ISS stage III patients (p=0.000).Conclusion: Ploidy by conventional karyotyping is an independent prognostic factor even in the setting of R-ISS. Ploidy can be incorporated into R-ISS staging system to better identify high-risk patients (R-ISS+K). In countries where FISH is not available, conventional cytogenetics can be used instead of FISH (K-ISS) to identify high-risk patients. [Display omitted] DisclosuresSpencer:Amgen: Consultancy, Honoraria, Research Funding; Janssen: Honoraria, Research Funding. Chng:Janssen China R&D: Research Funding.

Tetraploidy in Acute Myeloid Leukemia Is Associated with Poor Outcomes Regardless of the Presence of Favorable Risk Features, and Requires an Allogeneic Stem Cell Transplant in First Remission

Introduction: While tetraploidy (92 chromosomes) and near-tetraploidy (>80 chromosomes) confer favorable prognosis in B-cell lineage pediatric acute lymphoblastic leukemia, their prognostic significance in acute myelogenous leukemia (AML) remains ill-defined. Moreover, the mechanisms underlying their pathobiology and impact on known prognostic factors in AML are unknown. Combining data collected at our institution along with a review of the literature, we provide the most complete analysis of tetraploidy and near-tetraploidy in AML including data on biology, treatment outcomes, impact on other prognostic markers, and survival to assist in clinical management.Patients and methods: A systematic literature search was performed and included studies published from June 1st, 1994 to June 1st, 2017 in PubMed, Embase, and Web of Science. We used the key words tetraploidy, near-tetraploidy, acute myeloid leukemia, acute myelogenous leukemia, and AML. Data was tabulated regarding the following seven variables: Fluorescence in situ hybridization (FISH) analysis, molecular analysis, karyotype, risk status, overall survival (OS), history of allogeneic stem cell transplantation, and chemotherapy. Data was reported as percent and total number of cases with available data of each variable.Results and discussion: We identified 104 AML cases with confirmed tetraploidy or near-tetraploidy (102 cases in 24 articles, and 2 cases from our institution). Incidence was nearly equal between tetraploidy (55/104) and near-tetraploidy (49/104). The median age at diagnosis was 54. A notable male predominance was observed with a male-to-female ratio of 2.2:1 (71 males and 33 females). While insufficient molecular data were available to include in risk stratification, cytogenetics including FISH and karyotype were available in all cases. We stratified our cases irrespective of tetraploidy or near-tetraploidy into favorable risk (defined by the presence of the following genetic alterations in core-binding factors: inv(16) or t(16;16) or t(8;21), or t(15;17)), unfavorable risk (defined by complex karyotype [≥3 clonal chromosomal abnormalities], monosomal karyotype, 5q-, 7q-, -5,-7, t(6;9), inv(3), t(3;3), 11q23 - non t(9;11), t(9;22)) and intermediate risk (the rest). Consistent with the literature, cases with other cytogenetic abnormalities in addition to those defined in the favorable-risk group, were included within the favorable-risk category (Byrd Blood 2002). Interestingly, t(8;21) was the only favorable risk feature represented, seen in 13 patients (12.5%), thus grouped under favorable-risk AML. 51 patients (49.0%) were grouped under unfavorable-risk AML and included 24 patients with complex karyotype, 9 patients with -7, 7 patients with 5q-, 4 patients with -5, 1 patient with 7q-, 1 patient with t(9;22), 3 patients with both -5 and -7, 1 patient with both 5q- and -7, and 1 patient with both -5 and 7q-. Intermediate risk was observed in 40 patients (38.5%) who were found to have normal or non-defined cytogenetics. We used the Kaplan-Meier method to stratify survival data, and the log-rank test to compare the survival between subgroups. The overall outcomes were poor, the median OS (mOS) of the whole cohort was 6 months (M) (Figure 1A). The prognosis was equally poor for both tetraploidy (mOS 6.2M) and near-tetraploidy (mOS 6M) (P=0.81; Figure 1B). Surprisingly, risk stratification showed no impact on overall survival (P=0.57; Figure 1C), which suggest that the unfavorable risk imposed by tetraploidy or near-tetraploidy may overcome favorable-risk features, such as t(8;21), challenging current data on AML risk stratification. Twenty-one patients (20.2%) had an allogeneic stem cell transplantation. Interestingly, we noticed a significant improvement in outcomes (mOS 17M) in patients who received an allogeneic stem cell transplantation compared to those who did not (mOS 4M) (P=0.0085; Figure 1D).Conclusion: Regardless of the risk status, the prognosis of tetraploid or near-tetraploid AML is dismal and should be incorporated within the unfavorable risk group. Prompt evaluation for allogeneic stem cell transplant should be initiated at diagnosis. Indeed, longer survival can be achieved with chemotherapy followed by allogeneic stem cell transplantation at first complete remission. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Constitutional SAMD9L Mutations Cause Familial Myelodisplastic Syndrome with Monosomy 7 and Stable Revertant Mosaicism

Familial myelodysplastic syndromes (MDS) arise from haploinsufficiency of genes involved in hematopoiesis, show variable penetrance and are primarily associated with MDS in younger patients. The same genes involved in familial MDS might also cause sporadic manifestation, e.g. in GATA2 deficiency where ~2/3 of identified germline mutations arise de novo . In this study we aimed to discover the genetic cause in 7 patients of 4 unrelated pedigrees (Fig. A). The phenotypic denominator was MDS with progressive or transient loss of chromosome 7 and remarkably, spontaneous long-lasting remission was observed in 2 cases. Dysmorphic features or neurological symptoms were absent. Median age at diagnosis was 2 (1-42) years, all patients presented with thrombocytopenia with or without additional cytopenias and a hypocellular marrow without increase of blasts. There was no spontaneous growth in an in vitro GM-CSF hypersensitivity assay performed in 2 patients.Genomic studies identified constitutional mutations p.H880Q, p.R986H, p.R986C and p.V1512M in SAMD9L gene on 7q21 cytoband, with decreased allele frequency in hematopoiesis. Interestingly, similar (p.I981T, p.C1196S) or identical (p.H880Q, p.R986C) SAMD9L mutations had been recently reported in 4 families with Ataxia-Pancytopenia syndrome. Family testing in 3 pedigrees (Fig A), revealed an incomplete penetrance in 33% (3/9) of mutation carriers. In comparison with other germline variants, SAMD9L missense mutations showed significantly lower median allelic frequencies in blood across all patients (53% vs. 16%, p<.05). The nonrandom loss of mutated SAMD9L allele was attained through monosomy 7, deletion 7q, uniparental isodisomy 7q, or acquired truncating SAMD9L variants p.R1188X and p.S1317RfsX21 (Fig. B). TA-cloning confirmed cis orientation of both SAMD9L mutations in P1. Additionally, acquired mutations were found in oncogenes in P1 at CMML disease stage (SETBP1 p .D868N, EZH2 p.V582M, KRAS p.Q61P) and in P5 (RUNX1 c.413_427+5dup20bp). Functional studies in cell lines confirmed growth-restrictive effect of SAMD9L mutations p.R986C and p.V1512M. Double-mutated SAMD9L p.V1512M-p.R1188X vector resulted in stable expression of a truncated protein lacking p.V1512M (suggesting a compensatory effect).Leukemic progression from MDS to CMML was observed after 3.6 years in P1. In contrast, spontaneous remission occurred in P2 and P7, who showed transient monosomy 7 that had been replaced by large UPD7q clones with wildtype SAMD9L allelic duplication. Both patients maintained normal marrow morphology, cytogenetics, and blood counts until over 10 and 20 years from initial diagnosis.In conclusion, our study establishes constitutional SAMD9L mutations as a novel cause of hereditary MDS without accessory phenotypes and point to the notion that MDS with chromosome 7 loss can be the sole and common manifestation of SAMD9L-related disease. The clinical outcomes range from leukemic progress to spontanous remission. This is the first observation of sustained revertant mosaicism due to UPD7q and etiologic explanation for transient monosomy 7. Further studies will be essential to define the role of SAMD9L in hematopoiesis and explore the observed variable penetrance. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Comprehensive Profiling of Somatic Mutations Which Define Primary Disease and Relapse in Various Acute Leukemia Subtypes

AbstractAbstract 783Acute leukemia comprises several distinct subtypes. Apart from certain specific cytogenetic abnormalities associated with each of these subtypes, acquisition of additional somatic mutations is crucial to disease development. The present study is a comprehensive exploration of somatic mutations associated with several leukemia subtypes to catalog pattern of genetic changes associated with each category of leukemia. Moreover, the focus of the study is on identifying relapse specific mutations, thereby enabling us to understand the evolutionary relationship between clones present in primary leukemia and relapse.Whole exome sequencing was performed on 5–10 matched diagnosis, relapse and remission samples from each AML subclass characterized by either t(8;21), t(15;17), FLT3-ITD, MLL-PTD or normal cytogenetics (without FLT3-ITD and MLL-PTD) AML. Identification of somatic mutations and small indels was carried out using an automatic analysis pipeline which included 1) genome mapping using BWA, 2) calling for variant sites using SAMTOOLS, 3) annotation using ANNOVAR, and 4) filtering using Fisher exact test and sequence quality information. Candidate mutations were validated using Sanger sequencing and 5–10 nonsynonymous mutations per sample were found in each subtype, except the normal cytogenetics samples where 25–30 mutations were found, signifying a higher mutational load. In all subtypes, excluding APL, a large proportion of the mutations present at diagnosis were retained in relapse, suggesting a similar pattern of disease progression. In t(8;21) AML, we sequenced 10 paired diagnosis and relapse cases and identified 47 mutations shared between the two stages, while 10 mutations were diagnosis-specific and 35 were relapse-specific. Recurrent mutations were observed in KIT, TET2, TTN, and MGA genes in this subtype. In FLT3-ITD AML, 60 mutations in 50 different genes were found in 10 relapse and diagnosis cases. Amongst 50 mutations present at diagnosis, 38 were shared with the relapse samples. We observe recurrent mutations in several known genes including DNMT3A, WT1, RUNX1 and IDH1. In 5 cases of MLL-PTD AML, 42 genes were mutated, 24 of which were shared at both diagnosis and relapse. Similarly, a high prevalence of primary disease-associated mutations was also detected at relapse in normal cytogenetics AML, indicating persistence of disease clones during chemotherapy. In 8 cases of APL, 63 mutations in 61 genes occurred, with mutations in WT1 and PML appearing twice. Interestingly, while 4 cases shared at least one mutation between diagnosis and relapse stages, in the remaining 4 cases, the relapse resulted from independent clones.In acute lymphocytic leukemia exomes from 7 matched diagnosis and relapse pediatric cases, which should have done well but did poorly (normal cytogenetic or hyperdiploid cases), a similar rate of somatic mutations occurred as observed in myeloid malignancies. Interestingly, no non-synonymous mutations were detected in primary disease samples in 4 out of 7 cases, but each accumulated multiple mutations at relapse.The frequency of mutations in the newly identified mutant genes is currently being examined in a large cohort of diagnosis/relapse samples using custom target capture probes. We are integrating the exome sequencing data with gene expression and SNP array data for further selection of potential driver mutations. Pathway analysis is also being performed using the validated gene list from each leukemia subtype to explore affected biological processes. Finally, clonal evolution analysis using density-based clustering algorithm (DBSCAN) is being carried out to study the emergence of disease clones and its progression from primary disease to relapse stage. To our knowledge, this is the first large scale analysis of relapse leukemia in a variety of subtypes. Disclosures:Alpermann:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.

Interphase FISH Analysis Reveals High Frequence of Partial 6q Deletions in Adult Acute Lymphoblastic Leukemia (ALL) with Normal or Failed Karyotype.

Karyotypic analysis in adult ALL has provided relevant insights in the prognostic significance of chromosome abnormalities, which in turn become of potential importance for the design of risk-adapted therapeutic strategies for newly diagnosed cases. Among structural karyotypic changes, partial deletions of the long arm of chromosome 6 (6q) are reported to be between 4 and 6% of cases of adult ALL by conventional cytogenetics and depict a subset of patients with strict correlation with T-lineage ALL and an unfavorable prognostic likelihood. Recent studies at the molecular level in childhood ALL have shown 6q deletions in up to 30% of the investigated cases. With the aim of more precisely defining the exact incidence of 6q deletions in adult ALL, we started to investigate by interphase FISH patients with normal or failed karyotype included in the GIMEMA 0496 and 2000 protocols. These trials contemplated a central handling of bone marrow samples of the enrolled patients at presentation. We identified the following bacteria-derived artificial chromosome (BAC) clones with a known map location within the most common deleted region of 6q: 90G9 (6q13), 12B13 (6q15), 465M14 (6q21), 81H16 (6q26), 91016 (6q27). BAC clones were obtained from the Children's Hospital Oakland Research Institute (CHORI).Twenty-seven consecutive ALL patients have so far been analyzed by interphase FISH, 16 with T- and 11 with B-cell phenotype. Eleven cases had a normal karyotype and 16 a failed cytogenetic analysis. Among the cases with normal karyotype, 2 cases with T-cell phenotype showed a deletion in the 6q21 and 6q15 regions, whereas in the group with failed cytogenetics 1 case with B- and 3 cases with T-cell phenotype harbored deletions spanning respectively the 6q13-15 (2), 6q13-q21 and 6q15-21 regions. The median percentage of nuclei with 6q deletion was 30% (range 7–75%). T-ALL deleted cases showed the absence of the most frequent fusion genes detectable with molecular analysis, whereas a BCR-ABL rearrangement was identified in the only B-lineage ALL deleted case. Overall, partial 6q deletions were observed in 22% of the cases analyzed and in 31% of cases with T-cell phenotype. The present study shows that the probe set we have utilized is a useful tool for providing accurate cytogenetic diagnosis of partial 6q deletions and indicates that this abnormality is a frequent karyotypic change in adult ALL, particularly in T-lineage cases where it appears to represent the most relevant genetic anomaly. Since this abnormality is underestimated by conventional cytogenetic analysis, interphase FISH for 6q deletions should be included in the routine genetic screening of the ALL patients with failed or normal cytogenetics

MO802DOES ETELCALCETIDE REVERSE MYELOFIBROTIC BONE CHANGES DUE TO HYPERPARATHYROIDISM? A CASE REPORT

Abstract Background and Aims A 21 year old boy with a diagnosis of Autosomal Recessive Polycystic Kidney Disease and Caroli disease reached a final stage of chronic kidney disease (CKD) and started haemodialysis. Method After 3 years in haemodialysis he underwent a kidney transplant from a cadaveric donor. His transplanted kidney worked fairly well until the patients was 31 year old when he developed graft dysfunction (serum creatinine from 2.7 to 5 mg/dL in a very short period). There was a concomitant increase in serum phosphate levels (8.3 mg/dl) and iPTH that progressively increased to 1032 pg/ml despite a traditional therapy. Results At age 32, the patient returned to haemodialysis treatment thrice weekly with a concomitant a progressive worsening of hyperparathyroidism with bone pain . Cinacalcet at a dosage of 30 mg daily treatment was initiated, then it was increased to 120 mg daily without any benefit. Two years later, being the clinical situation without any improvement and being the patient scarcely compliant to the therapy and because a further enlargement of parathyroid glands was observed, a Parathyroidectomy (PTX) was advised. Nevertheless, PTX was not performed because of patient’s refusal. Furthermore, despite Erythropoietic Stimulating Agent (ESA) therapy, he developed severe anemia that required regular and frequent blood transfusions. iPTH increased to 4500 pg/ml [Fig.1] with a parallel rise in alkaline phosphatase &amp;gt;600 UI/L [Fig.2]. A Computed Tomography scan showed multiple bone-thickening lesions. He thus initiated Etelcalcetide 5 mg e.v. 3 times a week, after the HD session but without any benefit. The dosage was then increased to 7.5 mg but the patient gradually became frail and developed pancytopenia and low-grade fever. Hematological evaluation with bone marrow biopsy was performed in December 2019. Bone marrow histology showed severe fibrosis [Myelofibrosis (MF) grade 3] with normal bone marrow cytogenetics. Blood samples for mutations in JAK-2, CALR, and MPL and BCR-ABL rearrangement were negative. There was no evidence for a myeloproliferative neoplasm (MPN) or metastatic lesions. During the following months, while on a 7,5 mg dose of etalcalcetide, there was a gradual reduction in iPTH [Fig.1] and serum alkaline phosphatase [Fig.2], up to 500 pg/dl and 200 IU/L respectively. The patient developed asymptomatic, often severe, hypocalcemia which was managed with therapy. The patient’s clinical conditions gradually improved, anemia responded to lower doses of ESAs. A bone marrow biopsy was repeated after one year (December 2020) and it showed a reduction in fibrosis (MF grade varying from 1-2). Then etelcalcetide dosage was reduced while serum calcium and phosphate levels were in the normal limit. Conclusion Myelofibrosis secondary to renal osteodystrophy is an uncommon complication. It has been rarely reported and usually is associated with primary hyperparathyroidism. Marrow fibrosis and pancytopenia is related to the excessive iPTH that upregulates production of cytokines and paracrine factors in the bone marrow (IL-1a, IL-6, FNF-a, TGF–b, and platelet-derived growth factor) and it has an important stimulatory effect on fibroblast proliferation. It is known that surgical parathyroidectomy is associated with a reduction of bone marrow fibrosis in primary hyperparathyroidism. To our knowledge, this is probably the first case of tertiary hyperparathyroidism in which the effect of etelcalcetide is comparable to parathyroidectomy as far as on calcium-phosphate balance, and a significant improvement in bone marrow fibrosis and hemoglobin. In conclusion, etelcalcetide at least in this patient seems as effective as PTX on bone balance, bone marrow and anemia.

Wilms Tumor 1 Mutations Are Independent Poor Prognostic Factors in Pediatric Acute Myeloid Leukemia.

The prognostic impact of Wilms tumor 1 (WT1) mutations remains controversial for patients with acute myeloid leukemia (AML). Here, we aimed to determine the clinical implication of WT1 mutations in a large cohort of pediatric AML. The clinical data of 870 pediatric patients with AML were downloaded from the therapeutically applicable research to generate effective treatment (TARGET) dataset. We analyzed the prevalence, clinical profile, and prognosis of AML patients with WT1 mutations in this cohort. Our results showed that 6.7% of total patients harbored WT1 mutations. These WT1 mutations were closely associated with normal cytogenetics (P<0.001), FMS-like tyrosine kinase 3/internal tandem duplication (FLT3/ITD) mutations (P<0.001), and low complete remission induction rates (P<0.01). Compared to the patients without WT1 mutations, patients with WT1 mutations had a worse 5-year event-free survival (21.7 ± 5.5% vs 48.9 ± 1.8%, P<0.001) and a worse overall survival (41.4 ± 6.6% vs 64.3 ± 1.7%, P<0.001). Moreover, patients with both WT1 and FLT3/ITD mutations had a dismal prognosis. Compared to chemotherapy alone, hematopoietic stem cell transplantation tended to improve the prognoses of WT1-mutated patients. Multivariate analysis demonstrated that WT1 mutations conferred an independent adverse impact on event-free survival (hazard ratio 1.910, P = 0.001) and overall survival (hazard ratio 1.709, P = 0.020). In conclusion, our findings have demonstrated that WT1 mutations are independent poor prognostic factors in pediatric AML.

Gene expression profiling in normal cytogenetic acute myeloid leukaemia of Malay patient

Background: Insight into recent molecular analyses of patients with acute myeloid leukaemia (AML) and a normal cytogenetic have revealed a striking heterogeneity with regard to the presence of acquired changes in gene expression. Nano String technology which is aimed to identify descriptive profiling of normal cytogenetic AML in differentially expressed genes involved in the different pathways in normal cytogenetic AML patients.&#x0D; Materials and method: Blood samples were obtained from eight at diagnosis patients with normal cytogenetics AML, two follow-up patients and two normal healthy controls prior to RNA extractions. RNA gene expression assay was performed using Nano Stringn Counter® Pan Cancer Pathway Panel (Nanostring Technologies, Seattle, Washington). Briefly, 100 ng of each total RNA sample was used as input into then Counter Pan Cancer Pathway Panel sample preparation. Data was extracted using then Counter RCC Collector and raw data output was imported into nSolver v2.6 analysis software for data analysis.&#x0D; Results: The age range was from 13-69 years, with a median age-range of 41 years. We found the most enriched up regulated genes in newly diagnosed normal cytogenetic AML enlisted MPO (7.25 log FC), FLT3 (5.02 log FC), MYCN (4.99 log FC), MYB (4.74 log FC), ITGA9 (4.48 log FC), KIT (4.41 log FC), MCM2 (3.47 log FC), RAD51 (3.40 log FC), CCNA2 (3.37 log FC) and PROM1 (3.24 log FC) which those commonly be found in AML cases. For highly expressed down regulated genes were GZMB (-5.80 log FC), IL8 (-5.79 log FC), TNFRSFSF10C (-5.03 log FC), LEF1 (-4.82 log FC), IL2RB (-4.70 log FC), IL7R (-4.44 log FC), BCL2A1 (-4.15 log FC), RASGRP1 (-4.13 log FC), IL1R2 (-4.00 log FC) and HSPA6 (-3.99 log FC).&#x0D; Discussion and conclusion: Nano String required smaller amounts of starting material, and can perform mRNA expression profiling with digital precision, therefore the results do not require further validation by another method. MPO expression was significantly associated with disease-free survival (DFS). Previous report demonstrated that the groups by the MPO expression in the intermediate cytogenetic risk group showed a significant difference in DFS (p&lt;0.001). A study by Valk et al., (2004) showed FLT3, a hematopoietic growth factor receptor, is the most common molecular abnormality in AML. The presence of such mutations in FLT3 and elevated expression of the transcription factor EVI1 confer a poor prognosis. N-MYC expression levels in AML samples from patients with favorable, intermediate, or unfavorable prognosis were compared with that in CD34+ cells from four healthy bone marrow donors. The most highly down regulated gene in newly diagnosed AML goes to Granzyme B (GZMB) which involved in cytolytic activity that showed high correlation with other transcripts expressed in activated cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells as well as lymphocyte activation-related gene validating it as a robust and specific metric of active cellular immunity. Increased expression of IL-8 and/or its receptors has been characterized in cancer cells, endothelial cells, infiltrating neutrophils, and tumor-associated macrophages, suggesting that IL-8 may function as a significant regulatory factor within the tumor microenvironment.&#x0D; Bangladesh Journal of Medical Science Vol.20(3) 2021 p.556-562

Combined chronic myeloid leukaemia and chronic lymphocytic leukaemia in five patients, including one with 17p deletion.

We report a series of five Australian cases of chronic lymphocytic leukaemia (CLL) occurring concurrently with chronic myeloid leukaemia (CML). Patient management including therapies and response together with clinical progress was obtained from medical records and laboratory information systems. Prior to CML diagnosis, all five had a preceding diagnosis of CLL. Three had received prior fludarabine. All received tyrosine kinase inhibitors (TKI). None required subsequent therapy for CLL. One patient had 17p deletion CLL and another patient had normal CLL cytogenetics. All currently have satisfactory blood counts with quantitative polymerase chain reaction for CML showing molecular response. All remain alive. Thus, such cases can be successfully managed by treating each haematological disorder in the usual manner. The control achieved in CML with the TKI enables satisfactory marrow function to recover in patients with concomitant CLL. The role for allograft in patients with dual malignancies is uncertain and needs to be individualised depending on control of each malignancy.

Analysis of FLT3-Activating Mutations in Patients With Acute Myelogenous Leukemia in Jordan: Association With FAB Subtypes and Identification of Subgroups With Poor Prognosis

BackgroundFLT3 mutations are common in acute myeloid leukemia (AML), particularly in French–American–British M2 subtype AML and in cytogenetically normal (CN) AML; however, its incidence in Jordan is poorly studied. An FLT3 mutation implies poor prognosis in AML patients. We aimed to assess the incidence and prognostic value of FLT3 mutations in AML in Jordan. Patients and MethodsOne hundred thirty-two newly diagnosed unselected AML patients were included. Patient data were collected, including demographics as well as morphologic, cytogenetic, and molecular testing results. FLT3 mutations were detected by real-time reverse transcriptase PCR, next-generation sequencing, or both. Survival analysis and comparisons of incidence, remission rate, relapse, and survival outcomes between FLT3-mutated and wild-type groups were done and prognostic factors identified. ResultsFLT3 mutation was detected in 40% of AML patients. The highest incidence was associated with M2 subtype AML (47%) and CN-AML (50%). There was a significant negative association between FLT3 mutations and overall survival (OS), as well as a trend toward improved relapse-free survival, with 3-year OS being 19.17% vs 34.16% (P < .0001) and 33.6% vs 71.0% (P = .085), respectively. Patients with FLT3 mutation had a significantly better complete remission rate after induction (67.9% vs 63.3%, P = .001). Also, OS improved in patients with complete remission (P = .0015) and who then continued to allogeneic hematopoietic cell transplantation compared to FLT3 wild-type patients (P < .001). ConclusionFLT3 mutation is common in Jordanian AML patients, with the highest incidence occurring in patients with M2 or CN disease. It implies a poor prognosis, with poor OS and relapse-free survival, which may be abrogated by early allogeneic transplantation and/or peritransplantation provision of FLT3 inhibitors.

Inotuzumab Ozogamicin for Relapsed/Refractory Acute Lymphoblastic Leukemia in the INO-VATE Trial: CD22 Pharmacodynamics, Efficacy, and Safety by Baseline CD22.

We assessed the relationship between cluster of differentiation-22 (CD22) expression and outcomes of inotuzumab ozogamicin versus standard of care (SC) in INO-VATE (NCT01564784). Adults with relapsed/refractory B-cell precursor CD22-positive (by local or central laboratory) acute lymphoblastic leukemia were randomized to inotuzumab ozogamicin (n = 164) or SC (n = 162). Outcomes were analyzed by baseline CD22 positivity (percentage of leukemic blasts CD22 positive, ≥90% vs. <90%) and CD22 receptor density [molecules of equivalent soluble fluorochrome (MESF), quartile analysis]. Most patients had high (≥90%) CD22 positivity per central laboratory. The response rate was significantly higher with inotuzumab ozogamicin versus SC. Minimal/measurable residual disease negativity, duration of remission (DoR), progression-free survival, and overall survival (OS) were significantly better with inotuzumab ozogamicin versus SC in patients with CD22 positivity ≥90%. Fewer patients had CD22 positivity <90%; for whom, response rates were higher with inotuzumab ozogamicin versus SC, but DoR and OS appeared similar. Similar trends were evident in quartile analyses of CD22 MESF and CD22 positivity per local laboratory. Among inotuzumab ozogamicin-responding patients with subsequent relapse, decrease in CD22 positivity and receptor density was evident, but not the emergence of CD22 negativity. Rates of grade ≥3 hematologic adverse events (AEs) were similar and hepatobiliary AEs rate was higher for inotuzumab ozogamicin versus SC. No apparent relationship was observed between the rates of hematologic and hepatic AEs and CD22 expression. Inotuzumab ozogamicin demonstrated a favorable benefit-risk profile versus SC in patients with higher and lower CD22 expression. Patients with high CD22 expression and normal cytogenetics benefited the most from inotuzumab ozogamicin therapy.

How Epigenetic Therapy Beats Adverse Genetics in Monosomy Karyotype AML

The study by Greve and colleagues, in this issue of Cancer Research, provides new molecular insights into the intriguing clinical activity of DNA hypomethylating agents (HMA) in patients with acute myeloid leukemia (AML) with monosomal karyotypes. Patients with AML with adverse monosomal karyotypes are known to benefit from HMAs, but not cytarabine, a cytidine analog without HMA activity, but the specific molecular mechanisms remain poorly understood. The authors investigated the mechanistic effects of HMAs on gene reactivation in AML in the context of the most common monosomal karyotypes, genetic deletion of chromosome 7q and 5q. They identified genes with tumor-suppressive properties, an endogenous retrovirus cooperatively repressed by DNA hypermethylation, and increased genetic losses on hemizygous chromosomal regions versus normal biallelic regions in AML cell models. Treatment with HMAs preferentially induced expression of these hemizygous genes to levels similar to those of genes in a biallelic state. In addition to CpG hypomethylation, decitabine treatment resulted in histone acetylation and an open chromatin configuration specifically at hemizygous loci. By using primary blood blasts isolated from patients with AML receiving decitabine and AML patient-derived xenograft models established from patients with either monosomal karyotypes or normal cytogenetics, Greve and colleagues both validated their findings in primary patient samples and demonstrated superior antileukemic activity of decitabine compared with chemotherapy with cytarabine. These mechanistic insights into how epigenetic therapy beats adverse genetics in monosomy karyotype AML will open new therapeutic opportunities for a difficult-to-treat patient group.See related article by Greve et al., p. 834.

Study of the Age Dynamics of Morphological Subtypes of Acute Myeloid Leukemia in Adults

Aim: to determine the age dynamics of morphological subtypes of acute myeloid leukemia in adult patients. Materials and methods. The study group consisted of 132 patients (including 34 aged 15 to 45 years, 46 aged 45-60 years, 52 aged over 60 years) with newly diagnosed AML. Patients with acute promyelocytic leukemia were not included in the study. The diagnosis was made in accordance with WHO recommendations and FAB classification criteria. In all cases, morphological verification was performed, including cytological, cytochemical studies and immunophenotyping. Detection of chromosomal abnormalities was performed using standard cytogenetic and real-time polymerase chain reaction methods. Point mutations were screened in 7 genes: c-KIT, DNMT3A, FLT3, NRAS, NPM1, TP53 and WT1 by direct sequencing method. Results. The distribution of morphological variants of AML according to the FAB classification was as follows: M2 — 47.7%, M4 — 26.5%, M1 — 7.6%, M0 — 4.5%, M6 — 4.5%, M5 — 3.0%, M4eo — 2.3%, blastic plasmacytoid dendritic cell neoplasm — 1.5%, acute myelofibrosis — 0.8%, acute hybrid leukemia — 0.8%. Complex genetic abnormalities were detected in 18.2%, specific chromosome abnormalities associated with a favorable prognosis — 7.6%, specific chromosome abnormalities associated with an unfavorable prognosis — 5.3%, normal cytogenetics — 28.8%. Mutations in the FLT3 gene were detected in 15.0%, NPM1 — 14.3%, DNMT3A — 12.5%, NRAS — 12.2%, TP53 — 9.6%, c-KIT — 5.7%, WT1 — 4.2%. It was revealed that the frequency of acute myeloid leukemias with monocytic differentiation (M4, M4eo and M5 according to FAB) had a statistically significant tendency to decrease with increasing age: in the subgroup of patients younger than 45 years, it was 44.1%, in the age of 45-60 years — 32.6%, in the age of 60 years and older — 23.1%. These differences correlated with the age-related dynamics of the frequency of genetic abnormalities in AML, including a decrease in the frequency of NPM1 gene mutations and specific chromosomal aberrations, as well as an increase in the frequency of TP53 abnormalities in older age groups.

Role of Molecular Mutations, Telomerase and Promoter Methylation Status of the TSGs (p15INK4b, SOCS-1, Calcitonin and FHIT) in Disease Severity, Progression and Survival in a Series of 100 MDS Patients: A Study from India

In contrast to the Western countries, Myelodysplastic syndromes (MDS) in India is being increasingly seen in young adults. The severity of the disease is more in Indian patients at time of presentation; moreover their response to treatment is poor. In the present study molecular markers which may affect the biology of MDS patients were studied.Conventional cytogenetics, molecular mutations of RAS and FLT3 genes, hTERT gene expression, telomerase activity (TA) and promoter methylation status of four tumor suppressor genes (TSGs) (p15INK4b, SOCS-1, calcitonin and FHIT) were analyzed in a series of 100 MDS patients and correlated with disease severity, progression and survival. It was found that the prevalence of MDS was higher in patients with age&lt;60 years as compared to the patients with age ≥ 60 years (75% vs. 25%). Normal cytogenetics was present in 26/51 (51%) patients and 25/51 (49%) patients had chromosomal abnormalities. The frequency of N-RAS mutation was 3% and K-RAS mutation was 9%. FLT3-ITD and FLT3-TKD mutations were absent. TA was increased in 17/100 (17%) cases. hTERT expression was present in 17/100 (17%) cases. 40 patients (40%) had p15INK4b gene methylation. p15INK4b, SOCS-1, FHIT and calcitonin gene methylation was observed in 40 (40%), 53 (53%), 43 (43%) and 58 (58%) patients respectively. After multivariate analysis, only p15INK4b gene methylation was found as an independent predictor for progression of disease in MDS patients. In conclusion, p15INK4b gene methylation may play pivotal role in the diagnosis and disease progression in younger Indian MDS patients.

Prospective Karyotype Analysis in Adult Acute Lymphoblastic Leukemia: The Cancer and Leukemia Group B Experience

AbstractThe Cancer and Leukemia Group B (CALGB) has been conducting a prospective cytogenetic companion study (CALGB 8461) to all CALGB treatment protocols for newly diagnosed adults with acute lymphoblastic leukemia (ALL). These protocols underwent a significant change in 1988 when a new intensive chemotherapy program was introduced (CALGB 8811). We asked whether karyotype continued to represent a significant prognostic factor in adult ALL patients after the change. A total of 256 patients had adequate pretreatment cytogenetic analyses: 67 before 1988 and 189 subsequently. The complete remission (CR) rate for the whole group was 80%. Patients with t(9;22), t(4;11), −7, or +8 had significantly lower probabilities of continuous CR and survival at 5 years (.11 and .12) than patients with a normal karyotype (.38 and .37) and patients with miscellaneous cytogenetic abnormalities (.52 and .49;P < .001 for each comparison). When analyzed by treatment period, the CR rate before CALGB 8811 was 63%; subsequently, it was 86% (P < .001). Patients with cytogenetic abnormalities other than t(9;22), t(4;11), −7, or +8 had better CR rates, disease-free survival (DFS), and survivals (P = .001,P = .04, and P = .004, respectively) after the change to the more intensive chemotherapy regimens. Patients with normal cytogenetics had improved CR rate but no improved DFS or survival, whereas no significant benefit for patients with t(9;22), t(4;11), −7, or +8 was seen. In a multivariate analysis, karyotype retained its prognostic significance for DFS but not for survival; it remained the most important factor for DFS. We conclude that cytogenetic analysis at diagnosis should be used to guide treatment decisions in adults with ALL.

Recurrent prenatal PIEZO1-related lymphatic dysplasia: Expanding molecular and ultrasound findings

Generalized lymphatic dysplasia (GLD), characterized by lymphedema, lymphangiectasias, chylothorax, effusions, represents a recognized cause of fetal hydrops.We describe for the first time recurrent pregnancies showing different ultrasound presentations of lymphatic dysplasia. The first fetus displayed diffuse subcutaneous cysts and septations while the second one presented fetal hydrops.Exome sequencing results at 18 gestational weeks in the second pregnancy showed compound heterozygosity for two novel PIEZO1 variants, afterwards detected also in the first fetus and in the heterozygous parents.Both ultrasound and genetic findings expand the current knowledge of PIEZO1-related GLD.We suggest exome sequencing in hydropic fetuses with normal cytogenetics and in pregnancies with recurrent hydrops/lymphatic dysplasia.

Inferior Outcomes with a High LSC17 Score Can be Improved with Flag-IDA

Inferior Outcomes with a High LSC17 Score Can be Improved with Flag-IDA