Additional Karyotypic Abnormalities Research Articles

Clinical significance of cytogenetic and molecular genetic abnormalities in 634 Chinese patients with myelodysplastic syndromes.

PurposeTo explore the relevance of cytogenetic or molecular genetic abnormalities to clinical variables, including clinical and laboratory characteristics and prognosis in Chinese patients with myelodysplastic syndromes (MDS).MethodsA total of 634 consecutive patients diagnosed with MDS at The First Affiliated Hospital, Zhejiang University School of Medicine from June 2008 to May 2018 were retrospectively included in this study. All patients had evaluable cytogenetic analysis, and 425 patients had MDS‐related mutations sequencing.Results38.6% of patients displayed abnormal karyotypes. The most common cytogenetic abnormality was +8 (31%). Sole +8 was related to female (p = 0.002), hemoglobin >10 g/dL (p = 0.03), and <60 years old (p = 0.046). TP53 mutations were associated with complex karyotype (CK) (p < 0.001). DNMT3A mutations correlated with ‐Y (p = 0.01) whereas NRAS mutations correlated with 20q‐ (p = 0.04). The overall survival (OS) was significantly inferior in patients with +8 compared with those with normal karyotype (NK) (p = 0.003). However, the OS of sole +8 and +8 with one additional karyotypic abnormality was not different from NK (p = 0.16), but +8 with two or more abnormalities had a significantly shorter OS than +8 and +8 with one additional karyotypic abnormality (p = 0.02). In multivariable analysis, ≥60 years old, marrow blasts ≥5% and TP53 mutations were independent predictors for poor OS (p < 0.05), whereas SF3B1 mutations indicated better prognosis. Male IDH1 and IDH2 mutations and marrow blasts ≥5% were independent risk factors for worse leukemia free survival (LFS) (p < 0.05).ConclusionIn this population of Chinese patients, trisomy 8 is the most common karyotypic abnormality. Patients with +8 showed a poorer OS compared with patients with NK. Sole +8 and +8 with one additional karyotypic abnormality had similar OS with NK, whereas +8 with two or more abnormalities had a significantly shorter OS. DNMT3A mutations correlated with ‐Y and NRAS mutations correlated with 20q‐. TP53 mutations were associated with CK and had a poor OS. SF3B1 mutations indicated a favorable OS. IDH1 and IDH2 mutations independently indicated inferior LFS.

Several different cytogenetic clones arising during treatment of Philadelphia positive chronic myeloid leukemia with tyrosine kinase inhibitors lead to the progression into Philadelphia negative acute myeloid leukemia

Introduction. Additional karyotype abnormalities in the Philadelphia-positive (Ph+) clone can emerge during the progression of chronic myeloid leukemia (CML) and are of-ten associated with the resistance to treatment with tyrosine kinase inhibitors (TKI). Sometimes, during the TKI treatment, karyotype abnormalities can appear in the Philadelphia-negative (Ph-) cells as well but do not seem to adversely affect the outcome except for chromosome 7 abnormalities. Case report. The patient presented was in the chronic phase of Ph+ CML with highly diverse karyotype abnormalities. The abnormalities appeared in three unrelated clones during the TKIs treatment, followed by the evolution of the disease into acute myeloid leukemia (AML). The primary Ph+ clone was revealed during the chronic phase of CML, and therapy with imatinib mesylate was commenced. After a three-year hematologic and cytogenetic remission period, the evolution of the primary clone was noticed. Nilotinib was introduced, leading to a good molecular response and the disappearance/loss of the Ph+ clone with additional abnormalities but with the appearance of the Ph-clone with trisomy 8. Finally, after 5.5 years of nilotinib therapy, the Ph-clone with monosomy 7 occurred during the deep molecular response for BCR-ABL. At that time, the FISH analysis for trisomy 8 was negative, but the rise in blast count was noticed in the bone marrow, and the diagnosis of the secondary AML was established soon after. Conclusion. The achievement of the deep molecular response in CML patients does not rule out regular cytogenetic testing of their bone marrow. This is of crucial importance for detecting adverse karyotype abnormalities leading to the development of the myelodysplastic syndrome and AML.

Additional cytogenetic abnormalities in chronic myeloid leukaemia; an experience from Pakistan.

To evaluate the presence and characteristics of additional karyotype abnormalities in chronic myeloid leukaemia cases. The cross-sectional study was conducted at the Department of Cytogenetics and Molecular Pathology, National Institute of Blood Diseases and Bone Marrow Transplant, Karachi, from May 2010 to September 2016 and comprised diagnosed chronic myeloid leukaemia patients regardless of age and gender. Baseline cytogenetic evaluation was done on overnight, 24-hrs un-stimulated and 72-hrs stimulated bone marrow cultures, and karyotypes were defined according to the International System for Human Cytogenetic Nomenclature2013. Data was analysed using SPSS 23. There were 222 cases with a median age of 38 years (range: 12-84 years). The male-to-female ratio was 1.8:1. Chronic myeloid leukaemia was detected in 18(8.1%) patients having additional cytogenetic abnormalities. Among the patients found positive, cytogenetic type was minor in 10(55.55%), major 3(16.66%), complex 3(16.66%), and variant 2(11.11%). Additional cytogenetic abnormalities were found in 8% of the sample.

A multivariate model for predicting induction response and prognosis in core binding factor acute myeloid leukemia

Objective: To evaluate the efficacy and prognostic factors in core binding factor (CBF) acute myeloid leukemia (AML) under current therapy modalities, therefore optimizing the treatment strategies. Methods: Standard cytological and immune methods including next generation sequencing (NGS) were used for risk stratification. Complete remission (CR) rate, disease-free survival (DFS) and overall survival (OS) were assessed by multivariate Logistic and Cox regression models in a total of 206 adults (aged 16-65 years) with CBF-AML, including 152 AML patients with t(8;21) and 54 with inv(16). Results: The CR rate of inv(16) patients after first course was 54/54(100%), significantly higher than that of t(8;21) patients [127/147(86.4%), P=0.005]. The fusion transcript level and KIT mutation were independent factors related to CR rate in t(8;21) patients (P=0.044 and 0.027; respectively). DFS and OS in inv(16) patients tended to be more superior than that in t(8;21) patients (P=0.066 for DFS; P=0.306 for OS; respectively). Multivariate Cox identified negative expression of CD(19) and female gender the independent predictors of inferior DFS in t(8;21) patients (P=0.000 for CD(19); P=0.006 for sex; respectively). Analysis of combining CD(19) with gender indicated that females/CD(1)(9-)subpopulation had significantly poor DFS than did males/CD(19)(+) ones (Bonferroni-P<0.000 01). The number of mutations in each patient, FLT3-ITD and additional karyotype abnormalities did not affect CR rate and DFS (all P>0.05). Conclusions: Patients with inv(16) have better induction response than those with t(8;21). High level of fusion transcripts and positive KIT mutation are associated with low CR rate in t(8;21) patients. Negative CD(19) expression and female gender are independent predictors of inferior DFS in t(8;21) patients.

Genetic characteristics and clinical outcomes of pediatric acute myeloid leukemia with NUP98-NSD1 fusion gene

Objective: To investigate the genetic characteristics and clinical outcomes of pediatric acute myeloid leukemia patients with NUP98-NSD1 fusion gene. Methods: A total of 80 pediatric AML patients were enrolled in this study, and bone marrow specimens were collected at initial diagnosis and relapse. NUP98-NSD1 was screened by fluorescence in situ hybridization (FISH) and PCR. Other laboratory test results and clinical outcomes were further analyzed for the NUP98-NSD1 positive cases. Results: A total of eight patients (10.0%) were positive for NUP98-NSD1, which were all fusions of NUP98 exon12 and NSD1 exon 6. There were two M2, three M4, and three M5 cases according to the French-American-British classification. Seven patients had karyotype results at the time of initial diagnosis, and none of them had complicated karyotype abnormalities. Among these patients, two cases had normal karyotype, three cases had trisomy 8, one case had trisomy 6, and two cases had anomalies involving 9q13 or 9q21. Additional karyotypic abnormalities and clonal evolutions were observed during disease progression or relapse, five cases had 9q13 or 9q32 abnormalities. Five cases (62.5%) were positive with FLT3-ITD mutation. Patients were treated with DAE/NAE/HAE/IA chemotherapy. Three cases did not achieve remission after several courses of chemotherapy, and five cases achieved remission but relapsed in 1 to 19 months. Five cases underwent salvage allogeneic hematopoietic stem cell transplantation (allo-HSCT). Among whom, four died in 40 days to 4 months after transplantation, and one survived 8.5 months till the last follow-up. Conclusions: NUP98-NSD1 is a recurrent genetic abnormality with significant clinical prognostic significance, and this group of disease has unique clinical and genetic characteristics. NUP98-NSD1 should be screened by FISH or PCR for children with AML who are newly diagnosed or refractory and relapsed to identify the high-risk genetic marker.

Allogeneic Stem Cell Transplantation for Myelodysplastic Syndrome Patients with a 5q Deletion

The deletion (5q) karyotype (del [5q]) in patients with myelodysplastic syndrome (MDS) is the most common karyotypic abnormality in de novo MDS. An increased number of blasts and additional karyotypic abnormalities (del [5q]+) are associated with a poor outcome. We analyzed the outcome of allogeneic hematopoietic cell transplants (HCT) in patients suffering from MDS with only del (5q) or del (5q)+ . A total of 162 patients, of median age 54 years (range, 9 to 73), having MDS and del (5q) abnormalities received HCT from identical siblings (n = 87) or unrelated donors (n = 75). The cumulative incidence of nonrelapse mortality and relapse incidence at 4 years was 29% (95% CI, 22 to 36) and 46% (95% CI, 38 to 54), whereas the estimated 4 year survival, relapse-free and overall, was 25% (95% CI, 18 to 33) and 30% (95% CI, 23 to 38), respectively. In a multivariate analysis patients with del (5q) and a blast excess displayed poorer survival (hazard ratio, 2.38; 95% CI, 1.44 to 3.93; P < .001), whereas female recipient sex resulted in improved survival (hazard ratio, .61; 95% CI, .41 to .90; P = .01). We conclude that allogeneic HCT can cure a subset of patients with MDS and a del (5q) abnormality.

Prevalence and Impact on Outcomes of Additional Karyotypic Abnormalities in Patients (Pts) with Myelodysplastic Syndromes (MDS) and Del(5q) from the MDS-003 and MDS-004 Studies

Prevalence and Impact on Outcomes of Additional Karyotypic Abnormalities in Patients (Pts) with Myelodysplastic Syndromes (MDS) and Del(5q) from the MDS-003 and MDS-004 Studies

Rare but authentic Philadelphia-positive acute myeloblastic leukemia: Two case reports and a literature review of characteristics, treatment and outcome

The Philadelphia chromosome (Ph+), corresponding to translocation t(9;22), is found in chronic myeloid leukemia (CML) and acute lymphoblastic leukemia. Several cases of Ph+ acute myeloid leukemia (AML) have been reported in the literature.A retrospective study of Ph+ AML between 2001 and 2012 was conducted through a review of the literature. Among 400 AML patients, two cases of Ph+ AML (0.5%) were identified and treated with conventional chemotherapy with or without tyrosine kinase inhibitors (TKIs), followed by allogeneic hematopoietic stem cell transplantation (allo-HSCT). One patient had a complex karyotype including 7 monosomy (−7) and p190 BCR-ABL fusion transcript. Both patients remain in complete molecular remission.To date, 21 Ph+ AML cases treated with TKIs have been described in the literature with a median overall survival of 18months. One-third of the patients had additional karyotypic abnormalities, and 14% had −7. Molecular analysis showed 59% p210 and 41% p190 fusion protein. Relapse rate was observed in 38% of patients with p190 compared to 10% in patients with p210. Allo-HSCT was performed in eight patients; two relapsed (25%).Cytogenetic (−7) and molecular features help to distinguish Ph+ AML from CML. Survival improved with TKIs, particularly in association with conventional chemotherapy and allo-HSCT. Further studies of Ph+ AML patients are needed to better define this entity, its prognostic value, and therapeutic strategy.

Arsenic trioxide in front-line therapy of acute promyelocytic leukemia (C9710): prognostic significance of FLT3 mutations and complex karyotype

The addition of arsenic trioxide (ATO) to frontline therapy of acute promyelocytic leukemia (APL) has been shown to result in significant improvements in disease-free survival (DFS). FLT3 mutations are frequently observed in APL, but its prognostic significance remains unclear. We analyzed 245 newly diagnosed adult patients with APL treated on intergroup trial C9710 and evaluated previously defined biological and prognostic factors and their relationship to FLT3 mutations and to additional karyotypic abnormalities. FLT3 mutations were found in 48% of patients, including 31% with an internal tandem duplication (FLT3-ITD), 14% with a point mutation (FLT3-D835) and 2% with both mutations. The FLT3-ITD mutant level was uniformly low, < 0.5. Neither FLT3 mutation had an impact on remission rate, induction death rate, DFS or overall survival (OS). The addition of ATO consolidation improved outcomes regardless of FLT3 mutation type or level, initial white blood cell count, PML–RARA isoform type or transcript level. The presence of a complex karyotype was strongly associated with an inferior OS independently of post-remission treatment. In conclusion, the addition of ATO to frontline therapy overcomes the impact of previously described adverse prognostic factors including FLT3 mutations. However, complex karyotype is strongly associated with an inferior OS despite ATO therapy.

Pattern of chromosome involvement in childhood hyperdiploid pre-B-cell acute lymhoblastic leukemia cases from India.

BACKGROUND:Hyperdiploid pre-B-cell acute lymhoblastic leukemia (pre-B-ALL) is a common form of childhood leukemia with very good prognosis with present day chemotherapy. However, the chromosomal composition of the hyperdiploidy has not been extensively studied and possible mechanism for this pathology remains so far conjectural.OBJECTIVE:To analyze the pattern of chromosome involvement in a cohort of childhood hyperdiploid pre-B-ALL from India and from this pattern to develop an understanding on the causation of such pathology. Whether such patients also carry translocations and FLT3 mutations in addition to their hyperdiploid karyotype.MATERIALS AND METHODS:One hundred and twenty-six childhood pre-B-ALL patients were studied. Bone marrow aspirate of these patients were evacuated for morphology, FAB classification, immunophenotyping and both conventional and molecular cytogenetics.RESULTS:Of 126 patients with pre-B-ALL (age 2-15 years), 90 patients with abnormal karyotype showed 50 with hyperdiploid karyotype (50/90 i.e. 55.5%). Chromosomes 9, 10, 14, 17, 18, 20 and 21 were more often involved in hyperdiploidy. Chromosome 21 duplication was present in 92% of the cases. Chromosomes 5, 15, 16, 17 and Y were less often involved (18-20%) in hyperdiploidy. About 44% of patients with hyperdiploidy had additional karyotypic abnormality of which TEL-AML1 was predominant (24%). Chromosome loss was rare and accounted for 20% of the cases only. We did not find any FLT3 mutation in our patients.CONCLUSION:In this study, the pattern of chromosome involvement in hyperdiploid karyotype of ALL patients is same as other studies except some chromosomes like 1, 6, 11, 12, 19 and 22 have some more frequent involvement than other studies. This study also showed the occurrence of TEL/AML1 fusion is more (19.8%) than other reports from India.

How We Treat Chronic Myeloid Leukemia Patients During Daily Practice? Case Studies From The Real Life Experience Of Two Genoa Centers Of Hematology

Introductionthe natural history of Chronic Myeloid Leukemia (CML) has changed after the introduction of first generation tyrosine kinase inhibitors (TKIs). Data from clinical trials suggest that about 30% of patients fails imatinib treatment and 18% never achieves complete cytogenetic remission (CCyR). However, CCyR and major molecular response (MMR) can be achieved with second generation TKIs in many patients. AimWe have analized 119 CML patients that have received at least one course of TKI and their outcomes. We have calculated how many patient are alive and continuing to receive first TKI therapy or patients who are alive but had to switch to alternative TKI therapy. Patients and methodsFrom May 1987 and May 2013 among chronic phase CML patients diagnosed, we have analized 119 patients who received at least one course of first, second or third generation TKIs. At diagnosis patients characteristic were: mean age 52 years (range 17-80); comorbidities unrelated to CML were found in 55 patients (i.e. arterial hypertension, ischemic heart disease, atrial fibrillation and chronic obstructive pulmonary disease (grading 1-2). Nine patients had a past history of epythelial cancer. Additional karyotype abnormalities were present in 7 patients (5%). Subjects were stratified according to Sokal score: 32 high risk, 39 intermediate risk and 48 low risk. Thirty five patients were diagnosed in pre-TKI era. Thus, they received second line imatinib, after conventional chemotherapy. Sixty-nine patients were treated with frontline imatinib, 14 subjects received frontline nilotinib and 1 frontline ponatinib in. The median follow up is 102,5 months (range 3-312). ResultsResponse to treatment was defined in according to 2009 European LeukemiaNet guidelines In our series 20 patient died (17%) because of: disease progression in imatinib (= 2; 1.7%), acute myocardial ischemia during imatinib therapy (= 2), respiratory failure in 1 case and second tumor in 9 subjects. Of nine additional patients, three were lost at follow up and 6 subjects died.Overall 96/119 patients are alive (80,7%). Patients receiving frontline TKI are 55 ( 42in imatinib; 12 in nilotinib and 1 in ponatinib). Patients treated with second-line TKI are 30 (19in imatinib post chemoterapy, 6 in Nilotinib and 5 in dasatinib). Subjects receiving third-line TKI are 10 (9 in dasatinib and 1 in imatinib post allograft transplant). Only 1 patient receives ponatinib as fourth-line TKI. Responses are: CCyR in 96% of alive patients of whom 81% have a molecular response (MMR or better).Switch to TKI other than imatinib (24 patients) was due to failure to achieve optimal response or loss of response according to LeukemiaNet guidelines. Moreover, three patients did not tolerate first line TKI. Nilotinib was generally well tolerated: we did not observe any cardiovascular adverse event. However we found that previous failure to imatinib predicted subsequent failure to nilotinib (=11). As previously reported, dasatinib induced three pleural effusions, resolved with medical therapy without requiring switch of therapy, except for one patient. ConclusionWe noticed that Sokal score does not correlate with response to TKI therapy. Imatinib is well tolerated, only few patients discontinued because of side effects.However 24% of patients switched to second-line TKI due to loss of response especially among those patients previously treated with chemotherapy. Frontline nilotinib induced a sustained optimal response in 85,7%(=12) without adverse event. Our results about percentage of patient who switched therapy are slightly lower than those of clinical trials. This result support the notion that CML patients enrolled in clinical trials are not always fully representative of a general population referred to outpatient clinic. Disclosures:No relevant conflicts of interest to declare.

Bone Marrow Research in USA

Histology of CML The hypercellular marrow of CML shows panmyelosis and a virtual absence of adipocytes. There is granulocytic and/or megakaryocytic proliferation, eosinophilia and basophilia, occasionally with pseudoGaucher/sea-blue histiocytes and fibrosis. Past studies divided CML into two histological subtypes: a commoner granulocytic variant (~66%) showing unilinear granulopoietic hyperplasia, and a bilinear megakaryocytic type (~33%) additionally showing megakaryocytic hyperplasia, altered topology and pleomorphism with ineffective diminished erythropoiesis and grade 2-4 reticulin [1]. The clinical significance of this distinction (apart from the higher grade of fibrosis encountered in the megakaryocytic-type) remains controversial. Fibrosis in CML Manifest myelofibrosis is an adverse morphological factor associated with larger spleens, increased circulating blast percentages, lower hemoglobin levels and additional karyotypic abnormalities. Grading of collagen density, whether semiquantitative or by computerized morphometry, reveals that even slight increase in reticulin (compatible with doubled normal values) is associated with significantly worsened prognosis [2]. Thiele and Kvasnicka recommended that myelofibrosis should be included in any staging system in CML relating to survival Histological Changes in Therapy Imatinib mesylate therapy significantly decreases cellularity, neutrophil granulopoiesis, abnormal micromegakaryocytes, microvessel density and cell proliferation indices with regression of myelofibrosis. There is an increase in erythroid precursors and reactive lymphoid nodules with enhanced apoptosis. Myeloblasts, CD34+ cells and immature myelomonocytic cells also decrease in patients who go into complete or partial remission Imatinib-associated marrow aplasia has been described. Interferon-alpha induces apoptosis, resulting in reduced cellularity, expansion of normal erythropoiesis with increased iron-laden histiocytes and reticulum cells. Busalfan promotes myelofibrosis while hydroxyurea prevents it in a significant number of patients. In a study on 363 BMBs taken sequentially before and after allogeneic bone marrow transplantation, there was a significant correlation of marrow fibrosis, CD61+ cells and peripheral blood platelet counts with delayed haemopoietic reconstitution and leukemic relapse Ancillary Studies on the BMB Apart from assessing cellularity and fiber content, the BMB is also suitable for immunohistochemisty (angiogenesis, apoptosis, and proliferation indices) and morphometric studies as well as Fluorescent In-Situ Hybridization (FISH). Application of FISH to paraffin sections for demonstrating bcr/abl in intact cells, though not recommended for diagnosis, has been used as an adjunct to cytogenetic studies and to demonstrate the common stem cell origin of the CML clone. FISH for bcr/abl on BMBs was also found to be useful to monitor therapeutic efficacy of Imatinib Conclusion In conclusion, the BMB remains a valuable investigation in CML, both for the wealth of prognostic information conveyed as well as its usefulness in investigating scientific queries.

Lenalidomide Therapy of Myelodysplastic Syndromes

Partial or complete deletion of the long arm of chromosome 5 [del(5q)], with or without additional karyotypic abnormalities, is present in 10-15% of myelodysplastic syndromes (MDS). Anemia in these MDS responds less often to erythroblastic stimulating agents. However, immunomodulatory, anti-cytokine, and anti-angiogenic agent Lenalidomide (CC5013, Revlimid®) leads to red blood cells transfusion independence of low risk MDS with del(5q). The low risk del(5q) MDS is now recognized as a distinct pathologic subtype of MDS with markedly better clinical responses with lenalidomide treatment compared to non del(5q) MDS patients. Several mechanisms of action are believed to contribute to the therapeutic effect of lenalidomide. They include the effect on the immune system, with cytokine production, T- and natural killer cells co-stimulation, stimulation of erythropoiesis, substantional improvement in the hematopoiesis-supporting potential of bone marrow stroma and significant decrease in the adhesion of bone marrow CD34+ cells, and anti-inflammatory effects and angiogenesis inhibition. The exact mechanism of action of lenalidomide on del(5q) clones is not known, but there appears to be several candidate (tumor suppressor) genes whose expression may be modulated by lenalidomide treatment. The addition of lenalidomide inhibited the in vitro proliferation of erythroblasts harboring del(5q) while the proliferation of cells from normal controls and cells without 5q deletion was not affected. Patients with mutated TP53 were shown to have poorer erythroid and cytogenetic responses to lenalidomide and a higher potential for acute myeloid leukemia (AML) evolution. The mechanism of lenalidomide action is different in non-del(5q) MDS, where lenalidomide restores and promotes effective erythropoiesis without direct cytotoxic effect. Recent trials have focused on combining lenalidomide with other agents active in MDS.

Clinical, Cellular and Molecular Differences Between Newly Diagnosed and Relapsed Patients with Acute Promyelocytic Leukemia: Insights Into Mechanisms of Resistance

Abstract 1390We undertook a comprehensive prospective study to compare the clinical, cellular and molecular differences between newly diagnosed (ND) and relapsed (Rel) acute promyelocytic leukemia (APL) patients. 98 ND cases and 28 Rel cases (5 second relapses) were enrolled in this study. 85 (86.7%) of the ND cases were treated with a single agent arsenic trioxide (ATO) regimen. Five (17.8%) Rel cases were discharged against medical advice, the rest were treated with a combination of ATO, ATRA and chemotherapy and advised a SCT in molecular remission.Table 1 summarizes the major clinical differences between ND and Rel cases. Rel patients had a significantly lower WBC counts and higher platelet counts at diagnosis. Coagulopathy was less prominent in Rel cases as evident by the significantly lower number of cases with IC bleeds in this group and the significantly lower platelet rich concentrate requirement in induction (Table 1). Immunophenotyping of the malignant promyelocytes and blasts revealed a significantly increased CD34 expression and significantly reduced CD13 and CD38 expression in Rel cases (Figure 1A). Cytogenetic (CTG) analysis revealed that relapsed patients had a trend to greater proportion of cases with additional karyotypic abnormalities (Table 1).Evaluation of the malignant promyelocytes and blasts’ capacity to concentrate ATO intracellularly as shown in Figure 1B (level of ATO in 107 cells after 24 hour culture with 0.5μM ATO was measured using Atomic Absorption Spectrometry) was not significantly different. Similarly an in-vitro assay of cytotoxicity of ATO (IC-50 measured using standard MTT cell viability assay) was not significantly different between ND and Rel cases (median IC-50= 5.64 ± 3.8 vs. 4.59±4.5 μM; P= 0.28). We had previously noted a protective effect of stromal cell co-culture on the apoptotic action of ATO on malignant promyelocytes. We compared this effect in newly diagnosed and relapsed cases and noted that this protective effect was seen in both groups and was not significantly different (Figure 1C).A microarray expression profile on 8 randomly selected ND and 8 Rel cases was done. Total RNA extracted from BMMNCs with 90% promyelocytes + blasts and above (enriched when required) was used and microarray was performed with a standard one color Agilent–44K array platform. Differentially regulated genes were clustered using hierarchical clustering based on Pearson coefficient correlation, and 1744 genes were 2 fold and above differentially expressed between at relapse and at diagnosis (Figure 1D). 26 differentially expressed genes were validated by RQ-PCR using ABI Taqman assay system (data not shown). Differential gene list were classified based on functional category and pathways using Biointerpreter Software. The most prominent differentially regulated pathways were cell adhesion, cell survival, cell cycle, immune regulation, stem cell regulation, ubiquitinproteasome degradation system and ion transporters. The heat map of the one such prominent pathway (adhesion) is shown in the figure 1E.In conclusion, the Rel cases have significant clinical, immunophenotypic and molecular differences when compared to the newly diagnosed cases. The potential reason for relapse and poor prognosis in the Rel group appears to be multi-factorial. Exploring the pathways and genes differentially regulated between these two groups may provide insights in identifying potentially novel therapeutic targets. Preliminary in-vitro data from our centre, consistent with these observations, has shown that stromal cell adhesion mediates resistance to ATO and that inhibition of this by drugs such as bortezomib can overcome it. [Display omitted] Table 1Comparison of the baseline demographic data of newly diagnosed and relapsed acute promyelocytic leukemia patientsDemographic ParametersNewly Diagnosed N=98 N (%)/Median (Range)Relapsed N=28 N (%)/Median (Range)P-valueAge (years)28 (2-60)31 (8-54)0.997Sex: Male48 (49)25 (89.3)0.000WBC x 109/Lt14.5 (0.5-290)3.5 (0.5-24.90)0.000Platelet x 109/Lt17 (3-85)31.5 (6-248)0.006RT-PCR: bcr159 (60.2)21 (75)bcr22 (2)00.311bcr337 (37.8)7 (25)Lactate dehydrogenase (IU/Lt))748 (291-2700)554 (284-1155)0.006Fibrinogen mg%161 (25-689)163 (70-1122)0.975Additional Cytogenetic finding34 (36.6)11 (61.1)0.068IC Bleed in induction: Yes16 (16.3)0 (0%)0.022Platelet rich concentrates in induction (units)24 (0-85)16 (0-48)0.027 Disclosures:No relevant conflicts of interest to declare.

Newly Acquired Molecular Mutations and SNP-A Lesions Alters the Natural History of Trisomy 8 Myeloid Neoplasms

AbstractAbstract 312Prognosis in myelodysplastic syndromes (MDS) is heavily influenced by cytogenetics. Trisomy 8 (+8) is reported in 15–20% of MDS patients (pts) and is one of the most commonly identified karyotypes in this disease. Sole +8 is an intermediate-risk karyotype in MDS by the International Prognostic Scoring System (IPSS). However, pts with +8 myeloid malignancies exhibit wide clinical heterogeneity. In chronic myelomonocytic leukemia (CMML), +8 is considered high-risk, while in MDS functional data suggests an “immunopathologic” cause that is responsive to immunosuppressive agents and confers a good prognosis. Single nucleotide polymorphism array (SNP-A) and molecular technologies (next generation sequencing) have led to further refinement of prognosis in MDS, and subsequent discovery of molecular mutations with distinct effects on outcomes. We hypothesized that the differential outcomes noted in +8 MDS are primarily influenced by both clinicomorphologic features and the acquisition of new cytogenetic (isolated, +1, ≥2) and molecular defects. To further characterize the clinical and molecular behavior of +8 myeloid neoplasms, we analyzed 68 pts seen at the Cleveland Clinic with a +8 clone. Hematologic, bone marrow (BM), cytogenetic (metaphase cytogenetic [MC]/SNP-A) and survival data were collected. Survival comparisons were made by Kaplan-Meier analyses. Cox-proportional hazard ratio was used to determine factors predictive of outcomes. Response was evaluated per International Working Group 2006 criteria. Most (69%) pts were male; median age 69 years (38–89), and median follow-up 15 months. 69% (47/68) had MDS, 10% (7/68) MDS/MPN, 3% (2/68) MPN and 18% (12/68) AML. 41% of pts had an isolated +8 abnormality (+8 (i)), 16% had one additional abnormality (+8plus1), and 43% had ≥2 additional abnormalities defined as +8complex. By IPSS, Int-1=34%, Int-2=25%, high=42%. SNP-A data were available in 51% of the cases and detected new lesions in 60% of them (gains[46%], losses[43%], UPD[23%]). Clinically, pts with +8complex had higher median peripheral blood blasts (PB) compared to +8plus1 and +8(i) (3.5 vs 0 vs 0%,p=.04). By treatment, pts received high intensity (BMT±high dose chemo=35%), low intensity (hypomethylating agents [HMA]± lenalidomide=37%) or supportive therapies=28%). Overall response rate was 47%, with 14% CR. By cytogenetic grouping, +8complex had lower response rates compared to +8 with additional karyotype (30 vs 60 vs 52%; p=.06). Median overall survival (OS) was 15 months, and median event free survival (EFS) was 8 months, with worse OS noted in +8 complex compared to +8 plus1 and +8(i) (OS=11 vs 22 vs 29 months, p=.02; EFS=5 vs 10 vs 12 months; p=.04). To investigate the biologic rationale of these observations, we performing direct sequencing for poor prognostic genes in myeloid malignancies, such as ASXL1, IDH1/2, EZH2, K/NRAS, CBL and TP53 and for predictors of good outcomes/response like SF3B1/TET2. Molecular mutations were identified in 9/24 (38%) pts, with mutational frequencies within +8(i) (12 pts) of TET2 (42%), ASXL1 (42%), K-/NRAS (17%), SF3B1 (17%), and TP53 (0%). None of these mutations were detected in the +8plus1 or +8complex cohorts except for TP53 in one +8complex pt. No IDH1/2/EZH2/CBL mutations were found in the entire +8 cohort. Interestingly, mutations in TET2 conferred a better OS (88 vs 12 mos; p=.01). In +8 myeloid malignancies, ASXL1 mutations did not impact OS. We are currently performing whole exome sequencing and other immunogenetic studies in +8 pts to help identify factors that contribute to these group outcomes. To further define factors predictive of worse outcomes in +8 pts, univariate and multivariate analyses were performed and a predictive model of OS was designed. Using a simple scoring system, one point each was assigned to LDH ≥240, platelets <75, complex +8, PB blasts >5%, and 2 points to an ANC <4.5. Three prognostic groups were identified (favorable [score:0 or 1], median OS=33 months; intermediate [score:2], median OS=16 months; and unfavorable [score:>2], median OS=4.6 months; p<.0001). After incorporating SNP-A into the model, new SNP-A lesions were found to have a significant prognostic impact (p<.04). In summary, the clinical outcomes of +8 karyotype are influenced by additional karyotypic abnormalities detected by SNP-A/MC and by good/poor prognostic/predictive molecular mutations. Disclosures:No relevant conflicts of interest to declare.

Prognostic implications of additional chromosome abnormalities among patients with de novo acute promyelocytic leukemia with t(15;17)

This retrospective study performed by the Eastern Cooperative Oncology Group and the Southwest Oncology Group enrolled 140 acute promyelocytic leukemia (APL) patients with t(15;17) to determine the influence of additional karyotypic abnormalities on treatment outcome. Karyotypes were centrally reviewed by both study groups. The complete response rate after induction for patients with t(15;17) treated with chemotherapy, or all-trans retinoic acid (ATRA) as induction therapy was not affected by additional cytogenetic aberrations. Disease-free (DFS) and overall survival (OS) were unaffected by additional cytogenetic abnormalities if treatment was chemotherapy without ATRA. Patients with t(15;17) only, treated with ATRA with or without chemotherapy, had an improved DFS (P = 0.06) and a better OS (P = 0.01) compared with ATRA-treated patients with additional cytogenetic abnormalities. Patients with APL and t(15;17) alone are significantly more sensitive to treatment with ATRA than are patients with t(15;17) and additional cytogenetic abnormalities.

Cytogenetic study in CML

Chronic myeloid leukaemia (CML) is a clonal stem cell disorder characterized by increased proliferation of myeloid lineage. CML is the commonest adult leukaemia in India and the annual incidence ranges from 0.8–2.2/100,000 population in males and 0.6–1.6/100,000 population in females in India1. The median age of diagnosis is 38-40 years. This is a decade earlier than the median incidence in the western world. Though CML is predominantly a disease affecting adults, a minority of patients are children and young adults. CML is the first cancer in which a consistent chromosomal abnormality the Philadelphia chromosome was described by Nowell in 1962. This abnormality was later shown to be due to translocation involving t(9;22) (q34;q11.2) and involved the fusion of genes breakpoint cluster region (BCR) and the Tyrosine Kinase human homologue of the Abelson Murine leukaemia Virus (ABL). During the reciprocal translocation a segment of ABL gene (9q34) is moved into one of at least 3 well characterized breakpoints of the BCR gene in 22q112,3. This results in two fusion genes BCR-ABL and ABL-BCR. Of this, the ABL –BCR has no identified role in pathogenesis of CML. The BCR-ABL fusion gene is in frame and is translated leading to formation of an oncoprotein which is a constitutively active tyrosine kinase. Primarily three different fusion transcripts have been characterized resulting in fusion of BCR exon1 9 (e1), exons13/14 (b2/b3) and exons1-19 (e19) to ABL4. Very rarely exon6 and exon 8 are involved in BCR-ABL translocations. By contrast, the breakpoint in ABL occurs almost invariably upstream in ABL exon2 (a2) though occasionally it can occur downstream of exon2 (a3). These results in tyrosine kinase proteins of 185/190, 210 and 230 kilo Dalton sizes respectively. The 210 kilodalton protein (p210) is called the Major transcript or ‘M’ and the 185/190 Kilodalton is called the minor transcript or ‘m’. The knowledge about the fusion transcript is important when doing molecular monitoring of minimal residual disease after treatment with tyrosine kinase inhibitors. The major modality of treatment of CML inhibitors of the tyrosine kinase, the commonest used is Imatinib. These are chemical competitive inhibitors of ATP which is required for phosphorylation of tyrosine residues of downstream proteins in the signaling pathways of ABL tyrosine kinase. Newer 2nd generation tyrosine kinase inhibitors like Nilotinib and Dasatinib are used in patients resistant to Imatinib. These newer drugs have brought clinical and haematological remissions almost all patients and molecular remissions in about a third of patients. However, it has not resulted in a drug free cure of the disease. The patients have to be on long term monitoring while on therapy5–9. In this issue Anand et al10 have reported on the cytogenetic and molecular analyses of CML patients from north India. There are very few studies from India comparing cytogenetics and molecular data in CML patients in India. Cytogenetic facilities are scarce in India and restricted to predominantly academic institutes. Having a baseline karyotyping in CML is essential and additional karyotypic abnormalities predict an advanced stage of the disease and a poorer response to Imatinib. The progress in CML have brought in a paradigm shift in management of many cancers and newer approach to targeting cancers.

Acute Promyelocytic Leukemia with T(15;17): Frequency of Additional Clonal Chromosome Abnormalities and FLT3

Acute promyelocytic leukemia (APL) is a distinct subtype of acute myeloid leukemia (AML) characterized by t(15;17)(q22;q21), a balanced reciprocal translocation involving PML and RARA genes. Studies conducted in animal models with PML/RARA suggest that this gene rearrangement is not sufficient for the development of APL, emphasizing the importance of additional mutations. Another gene that could be involved in APL leukemogenesis is FLT3, a Fms-like tyrosine kinase receptor which mutations have been observed in different AML subtypes as the internal tandem duplications (ITD), in around 18–37% of APL cases or less frequently, the D835Y mutation. The association of FLT3/ITD in APL gave rise to the proposition that it could provide a common class of cooperative mutation in the development of the disease. Indeed, APL represents 21% of all cases of AML with karyotype confirmation in our institution, located in São Paulo, Southeast region of Brazil. Bone marrow samples from 92 consecutive APL patients at diagnosis were enrolled in the study. Mean age was 29.5 years old, varying from 4 months to 67 years old, with the following age distribution: 35.7% of the patients were younger than 20 years old; 55.2% were between 20 and 49 years old and only 17.2% were older than 50 years. There were 50 males and 42 females. The WBC count demonstrated a wide variation, from severe leukopenia (<500 leukocytes/μL) until marked leukocytosis (174.600/μL), most of them presenting circulating blasts. Hemoglobin level varied from 3.7g/dL to 14g/dL and platelets counts were frequently low. Cases included five (5.4%) microgranular variant patients. We observed that 23 (25%) out of 92 cases presented chromosomal abnormalities in addition to t(15;17), among which trisomy 8 was the most frequent (21.7%, 5/23). Other abnormalities included tetraploidy, trisomy 6, trisomy 21 (non constitutional), nulissomy Y, ider(17q), trisomy 11, del(9)(q22q32), add(3)(p26), del(6)(p21), abnormalities of chromosome 7 and 1, t(1;3)(q21;q29); t(7;9)(q36;q31); monosomy of chromosomes 6, 7, 8 and 9; dup(1)(q23q42), del(10)(q24); add(7)(q36) and marker chromosomes. Seventy nine cases had enough DNA for complimentary molecular studies; in the remaining 13 cases we could not retrieve any DNA amount from fixed cells. Among those, FLT3/ITD was detected in 29 (36.7%), 5 of them showing additional karyotype abnormalities and D835Y mutation was present in only 2 (2.8%) of the cases. None of the two patients with D835Y showed additional chromosomal abnormality, but one of them presented ITD and D835Y mutation simultaneously at diagnosis. No correlation was found between age, sex, WBC count, hemoglobin level, platelet count or clinical symptoms and the presence or absence of additional karyotype abnormalities, FLT3-ITD and D835Y. The frequencies of additional karyotype abnormalities as well as FLT3/ITD in Brazilian APL patients were similar to those reported in several worldwide published papers. On the other hand, the incidence of D835Y was lower than that described elsewhere (2.8% versus 7% in AML or 5.7% in APL). So although Latin American population has an increased incidence of APL, it seems not to bear a higher frequency of clonal additional chromosome abnormalities or mutations in FLT3 gene. Moreover, FLT3/ITD was not over-represented in cases with additional chromosomal abnormalities (17.2% versus 36.7%, p=0,2), thus suggesting an independent role for each FLT3/ITD and extra clonal chromosomal abnormalities in t(15;17) APL. In the present study, a significant proportion of APL patients (56%) showed additional karyotype alterations and/or FLT3/ITD or D835Y mutation. In fact, one fourth of APL cases presented additional chromosomal abnormalities at diagnosis while 1/3 of the cases showed the FLT3/ITD and only two patients presented D835Y mutation. So, roughly more than one half of the cases presented at least one of the alterations investigated, raising the possibility that these abnormalities may be complimentary events to PML/RARA in the process of leukemogenesis. The influence of additional chromosomal abnormalities predisposing to genomic instability and cooperating with genetic mutations in acute leukemia still deserves investigation, since about 45% of APL cases have not been shown to bear any detectable molecular abnormality to date.

Lenalidomide in High-Risk Myelodysplastic Syndrome and Acute Myelogenous Leukemia Associated with Chromosome 5 Abnormalities.

Background: Treatment with lenalidomide produces remarkable responses in patients with myelodysplastic syndrome and deletion 5q 31 (5q-) cytogenetic abnormality; transfusion independence in 67% of patients, major cytogenetic responses in 44% and a median duration of response of 115.9 weeks (List et al. Abstract #251; Blood: 108). Responses have been reported in few patients with MDS and complex karyotype as well. Since chromosome 5 abnormality is considered to be an early genetic event, we designed a phase 2 study of lenalidomide in patients with high-risk MDS and relapsed/refractory acute myelogenous leukemia (AML) with any chromosome 5 abnormality.Method: The dose of lenalidomide was 25 mg/day, orally for 21 days with 7 days rest (28 day cycle). For patient with platelet count < 50,000/μl, the starting dose is 15 mg/day orally for 21 days with 7 days rest (28 day cycle). Dose interruptions/reductions are planned for ≥ grade3 neutrpenia with fever or ≥ grade 4 neutropenia (for patients starting with absolute neutrophil count [ANC] >1000/μl) and for grade 3 thrombocytopenia (for patients starting with platelet count ≥ 50,000/μl).Results: Eleven patients out of this 30 patient study have been treated so far; 6 with AML and 5 with MDS. All patients with AML and 2 patients with MDS had previous therapy. Median age is 63 years (range, 44 to 80 years). Nine patients had complex karyotypic abnormality and 2 patients had single additional chromosomal abnormality (+8 in one and 11q abnormality in another) along with 5q deletion. One patient with AML (previously treated with decitabine) and with karyotypic abnormality of trisomy 8 in addition to 5q deletion, achieved complete remission after 3 cycles of therapy. Another patient had reduction in bone marrow blasts from 21 to 5%, but could not continue lenalidomide due to profound thrombocytopenia complicated by gastrointestinal bleeding and eventually had disease progression. Four (4/6) patients with a platelet count of ≥ 30,000/μl and 5 (5/6) patients with ANC of ≥500/μl at study entry experienced worsening cytopenias. No grade 3/4 non-hematological toxicities were seen. One patient in CR currently remains on study. Reasons off study include disease progression (8 patients), stem cell transplant (1 patient) and one patient died while on study from sustained pancytopenia, infection and multiorgan failure.Conclusion: The role of lenalidomide in the treatment of high-risk MDS and AML with chromosome 5 and additional karyotypic abnormalities needs to be defined in the context of clinical trials. Future studies are needed to examine lower doses with or without growth factor support.

Multiple Granulocytic Sarcomas in Essential Thrombocythemia

A 59-year-old woman was diagnosed with essential thrombocythemia in 1988 and had been treated with hydroxyurea, mitobronitol, busulfan, and ranimustine, in that order. Hepatosplenomegaly, low-grade fever, and body weight loss manifested, and a few blasts were noted in the peripheral blood studied in March 2002. A biopsied specimen of the bone marrow showed myelofibrosis but not a leukemia in August 2004. An abnormal karyotype with der(1; 13) appeared for the first time. She was treated with low-dose prednisolone. In January 2005, she experienced left hip joint pain, and magnetic resonance scanning showed a tumoral lesion in the femoral head. Histological diagnosis of the biopsied mass revealed that it was a granulocytic sarcoma, and radiotherapy was performed. In April 2005, bone scintigraphy showed multiple lesions. She became febrile and red blood cell transfusion-dependent with hepatosplenomegaly and a small number of circulating blasts. Intravenous cytarabine (low dose) and etoposide relieved the fever and hepatosplenomegaly; however, she developed a pathologic fracture of the right humerus. An additional karyotypic abnormality (7q22 deletion) was noted. She subsequently died of infection. Granulocytic sarcoma is very rare in essential thrombocythemia, and this patient may be the first reported case of essential thrombocythemia that developed multiple lesions and a pathologic fracture without transformation to overt leukemia.