Additional Chromosomal Changes Research Articles

Leukemic presentation and progressive genomic alterations of MCD/C5 diffuse large B-cell lymphoma (DLBCL).

Diffuse large B-cell lymphoma (DLBCL) is a heterogenous group of lymphoid malignancies. Based on gene expression profiling, it has been subdivided into germinal center (GC)-derived and activated B-cell (ABC) types. Advances in molecular methodologies have further refined the subclassification of DLBCL, based on recurrent genetic abnormalities. Here, we describe a distinct case of DLBCL that presented in leukemic form. DNA sequencing targeting 275 genes revealed pathogenically relevant mutations of CD79B, MyD88, TP53, TBL1XR1, and PIM1 genes, indicating that this lymphoma would be best classified as MCD/C5 DLBCL, an ABC subtype. Despite an initial good clinical response to BTK inhibitor ibrutinib, anti-CD20 antibody rituxan, alkylating agent bendamustine, and hematopoietic stem-cell transplant, the lymphoma relapsed, accompanied by morphologic and molecular evidence of disease progression. Specifically, the recurrent tumor developed loss of TP53 heterozygosity (LOH) and additional chromosomal changes central to ABC DLBCL pathogenesis, such as PRDM1 loss. Acquired resistance to ibrutinib and rituxan was indicated by the emergence of BTK and FOXO1 mutations, respectively, as well as apparent activation of alternative cell-activation pathways, through copy-number alterations (CNAs), detected by high-resolution chromosomal microarrays. In vitro, studies of relapsed lymphoma cells confirmed resistance to standard BTK inhibitors but sensitivity to vecabrutinib, a noncovalent inhibitor active against both wild-type as well as mutated BTK. In summary, we provide in-depth molecular characterization of a de novo leukemic DLBCL and discuss mechanisms that may have contributed to the lymphoma establishment, progression, and development of drug resistance.

BAALC-Expressing Leukemia Hematopoietic Stem Cells and Their Place in the Study of CBF-Positive Acute Myeloid Leukemias in Children and Adults

Background. Due to changing views on pathogenesis, risk factors and therapy strategies in prognostically favorable CBF-positive acute myeloid leukemias[1] (AML), the expression monitoring of RUNX1/RUNX1T1 or CBFB/MYH11 fusion genes, as an additional evaluation of treatment outcomes, appears to be insufficient. This indicates the need to improve the monitoring of the CBF+ AML course by means of parallel measurements of BAALC expression levels which roughly correlate with the mass of BAALC-expressing leukemia hematopoietic stem cells (BAALC-e LHSC).
 Aim. To improve the quality of assessing treatment outcomes with due account for expression levels of RUNX1/RUNX1T1 or CBFB/MYH11 fusion genes and the mass of BAALC-e LHSC and on this basis to pave the way for personalized CBF+ AML treatment.
 Materials & Methods. This study enrolled 39 adult patients aged 20–81 years (median 32 years) and 8 children aged 2–18 years (median 12 years). Among them there were 20 females and 27 males. AML with inv(16)(p13;q22)/t(16;16) was identified in 19 patients, t(8;21)(q22;q22) was detected in 28 patients. BAALC, WT1, RUNX1/RUNX1T1, CBFB/MYH11 expression levels were measured by quantitative real-time PCR and related to the expression of the ABL1 expert gene.
 Results. In 23 patients, inv(16) and t(8;21) appeared to be isolated. Additional multidirectional chromosomal changes were observed in 24 patients with inv(16) and in 18 patients with t(8;21). All enrolled patients showed increased BAALC expression. In the course of therapy, it was decreasing to the threshold value in 16/18 (89 %) patients. The evaluation of the mean BAALC expression levels in the pooled groups of children and adults with isolated findings of either inv(16) or t(8;21) showed the decrease of the BAALC-e LHSC mass only in children (p = 0.049). The comparison of the mean WT1 expression levels in the pooled groups of children and adults with isolated and additional chromosomal abnormalities revealed their significant decrease in patients with complicated variants (p = 0.023).
 Conclusion. The case reports provided in this paper show that the molecular monitoring with serial measurements of fusion genes and BAALC gene expression levels in CBF+ AML patients can lay the basis for further improvement of personalized treatment strategies for these patients. In all likelihood, parallel measurements of the above gene expression levels will allow to establish the framework for decision-making concerning treatment extent and timely HSC transplantation.

EP056: Myelodysplastic syndrome/myeloproliferative neoplasm with highly complex intrachromosomal rearrangements resulting from multiple 5q15q32 deletions, 5’PDGFRβ deletion, pericentric inversion and TP53 inactivation

Myelodysplastic syndrome/myeloproliferative neoplasms (MDS/MPNs) are a heterogeneous group of clonal disorders defined by overlapping elements of peripheral cytopenias and/or elevated cell counts and generally show morphologic dysplasia in the bone marrow and carry an increased risk of transformation to acute myeloid leukemia. Chromosome 5q deletions are common in MDS occurring either as isolated deletions or associated with additional chromosomal changes. PDGFRβ rearrangements in MDS/MPN are rare but delineate a distinct type of myeloid neoplasm with characteristic clinicopathologic features for most defined rearrangements.

QUANTITATIVE REAL-TIME PCR (RT-QPCR) COMPARING THE RELATIVE EXPRESSION LEVELS OF GENE TRANSCRIPTS INVOLVED IN A CRYPTIC THREE-WAY TRANSLOCATION T(9;11;19): AN ORIGINAL CASE OF AN INFANT WITH DISMAL PROGNOSIS ACUTE LYMPHOBLASTIC LEUKEMIA

KMT2A gene aberrations are more frequent in infants less than one year (yr) of age, accounting for about 70% of acute lymphoblastic leukemia (ALL) patients, and about 30% are diagnosed with acute myeloid leukemia (AML). The most common abnormality found in these patients is the translocation t(11;19)(q23;p13.3), corresponding to 22% of KMT2A rearranged ( KMT2A -r) positive cases. Related literature data shows that infants <1 yr with t(11;19)(q23;p13.3) present with up to 50% additional chromosomal changes. In this context, Meyer and coworkers (2007) showed that 25% of the patients with t(11;19)(q23;p13.3) and the KMT2A - MLLT1 fusion transcript presented three-way or more complex fusions, associated with a worse prognosis. In such cases, KMT2A -r must be assessed by more sensible techniques combining high-resolution cytogenetics and molecular approaches, providing a precise characterization for the correct choices in risk-adapted therapy. In this work, we show a cryptic three-way translocation t(9;11;19) harboring a KMT2A - MLLT1 and the novel SEC16A - KMT2A fusion, associated with SEC16A and KMT2A aberrant expression levels, in an infant with B-ALL presenting a poor prognosis. At diagnosis, a bone marrow sample from a 2-month-old boy was referred to the Laboratory of Cytogenetics – INCA. Immunophenotyping showed 94% of blast cells presenting lymphoid morphology and the diagnosis of pro-B lymphoblastic, being treated under a high-risk BFM INTERFANT 06 protocol. The patient could not experience any treatment response and died due to an early relapse. G-banding and FISH were performed on bone marrow and peripheral blood samples under standard protocols. LDI-PCR assays were used to identify the KMT2A partner genes and their corresponding breakpoints. RT-qPCR analyses were performed to verify the levels of transcript expression of genes involved in the rearrangements. G-banding showed the karyotype: 47,XY,+X,t(9;11)(q34;q23). The FISH analysis revealed a KMT2A -r. Molecular cytogenetic analyses revealed a cryptic three-way translocation. The LDI-PCR sequencing revealed a KMT2A - MLLT1 and the novel out-of-frame SEC16A - KMT2A . RT-qPCR showed standard expression levels for MLLT1 , whereas revealed a SEC16A reduced expression (75%), and KMT2A overexpression (3-fold). To relate the abovementioned data to the patient's clinical presentation, we hypothesized relation between the expression profiles of the fusion transcripts and the patient's dismal prognosis. Thus, we compared the relative expression among three different KMT2A regions, donor X patient. The first region, spanning exons 3 and 4, is present in both samples and was attributed the value 1. Spanning exons 11 and 12 ( SEC16A - KMT2A fusion region), despite its lower expression in the patient, no significant difference was observed. On the other hand, spanning exons 14 and 15 (downstream transcript of the KMT2A fusion) was significantly lower expressed in the patient (0.4-fold). These results confirm that increased KMT2A expression in the patient depends on the wild-type allele and that the KMT2A transcripts with fusions are probably expressed. Our case illustrates the importance of molecular tests in selecting cases for further investigations of posttranscriptional regulation mechanisms, which could open perspectives regarding novel therapeutic approaches for poor prognosis childhood leukemias.

38. Cytogenomic profiling of 26 cases with iAMP21 acute lymphoblastic leukemia

B-cell acute lymphoblastic leukemia (B-ALL) with intrachromosomal amplification of RUNX1 on chromosome 21, or iAMP21 B-ALL, is a rare subtype with a reported prevalence of 2% of B-ALL pediatric cases and is associated with poor outcomes. We report a study looking at the genomic profile of 26 pediatric iAMP21 B-ALL cases using conventional cytogenetics, FISH, and cytogenomic microarray approaches. FISH was used to identify these 26 cases of iAMP21 B-ALL with an amplification of 5-20 copies of RUNX1. Cytogenetic testing showed a highly variable abnormal chromosome 21 including several atypical aberrations amongst all patients. Additional chromosomal changes were observed in 35% of cases including gain of chromosome X, distal 7q deletions, and co-existence of BCR-ABL1 in one case. Eight of the cases underwent microarray analysis which showed chromosome 21 regions of amplification ranging from 18 to 37 Mb in size, with variable segments of copy number changes. A variable size loss of distal 21q was observed in six of the eight cases. This underlines highly complex intrachromosomal 21q rearrangements in these patients. Microarray showed additional gene deletions involving IKZF1, CDKN2A/B, ETV6, and RB1 and three of the cases showed partial loss of heterozygosity (LOH) of chromosome 12q. Our results substantiate and expand the cytogenomic features of pediatric iAMP21 B-ALL and sheds additional light on its genomic profile.

Cell dynamics during differentiation therapy with all-trans retinoic acid in acute promyelocytic leukemia.

The introduction of all-trans retinoic acid (ATRA) has made acute promyelocytic leukemia (APL) a curable disease; however, early death prior to the completion of treatment remains a problem. In quantitative evaluation of response to ATRA treatment, lymphocytes must be excluded as they do not originally have t(15;17). We categorized peripheral blood leukocytes by nuclear morphology into polymorphonuclear cells (PMNs) comprising segmented granulocytes, and non-polymorphonuclear cells (NPMs) which includes lymphocytes, monocytes, band cells, and immature myeloid cells. We consecutively evaluated the ratio of t(15;17)-positive cells using fluorescence in situ hybridization in eight newly diagnosed patients with APL. We confirmed the differentiation of APL cells until cytogenetic complete remission; the association of a decrease of t(15;17)-positive NPMs and an increase of t(15;17)-positive PMNs was followed by a decrease of t(15;17)-positive PMNs. The kinetic pattern of t(15;17)-positive NPMs and PMNs was consistent in most patients, irrespective of leukocyte counts at diagnosis, additional chromosomal changes, and ATRA with or without chemotherapies. Kinetic analysis enables us to evaluate treatment response and the recovery of normal hematopoiesis in individuals.

Outcomes after Allogeneic Hematopoietic Stem Cell Transplantation in Patients with Acute Myeloid Leukemia Harboring t(7;11)(p15;p15)

Introduction:Acute myeloid leukemia (AML) with chromosomal translocation t(7;11)(p15;p15) [t(7;11)], which results in a fusion between NUP98 and HOXA9, is uncommon, and classified as intermediate risk (IR) in National Comprehensive Cancer Network (NCCN) Guidelines. Previous studies have reported that the patients with AML harboring t(7;11) exhibited a worse clinical outcome than the other AML groups. These reports included mostly patients who underwent chemotherapy rather than allogeneic hematopoietic stem cell transplantation (allo-HSCT), and the number of patients in each study was very small. Moreover, the transplant outcomes of patients with AML harboring t(7;11) compared to patients with other AML have not been reported.Therefore, we investigated the transplant outcomes among 91 patients with AML harboring t(7;11) compared to the patients with IR (n=7,308) and poor risk (PR) (n=2,406) AML, except for t(7;11). Moreover, we evaluated the risk factors for survival in patients with AML harboring t(7;11) who underwent allo-HSCT.Patients and Methods:During 1997 and 2014, 91 patients with AML harboring t(7;11) were identified from the nationwide registration data of the Japan Society for Hematopoietic Cell Transplantation.Cytogenetic risk group stratification was performed using the NCCN guidelines for AML in 2016.Univariate and multivariate analysis for survival were performed in all patients cohort and only among patients with AML harboring t(7;11). Univariate models for overall survival (OS) and disease-free survival (DFS) included age at allo-HSCT (age 55 ≥ years vs. 55 < years), sex, performance status (PS) (2-4 vs. 0-1), the hematopoietic cell transplantation-comorbidity index (0-2 vs. ≥ 3), additional chromosomal change, disease status at allo-HSCT (first complete remission [CR]: CR1 vs. second CR:CR2 or non-CR at allo-HSCT), conditioning regimen (total body irradiation [TBI] ≥ 8Gy vs. others), and stem cell source (related donor vs. cord blood or unrelated donor). Factors associated with at least borderline significance (p < 0.20) on univariate analyses were subjected to multivariate analysis.Results:Patient CharacteristicsWe evaluated the clinical characteristics of patients with t(7;11), IR, and PR.The median follow-up period for survivors was 1,124 days (range, 1-8,758). Patients with t(7;11) were younger (median age: 45 vs. 48 vs. 50 years, p<0.01), included more females (49.5 vs. 42.6 vs. 36.2%, p<0.01), and more frequently had PS less than 2 (82.5 vs. 80.2 vs. 76.3%, p<0.01), unrelated donor (40.7 vs. 38.3 vs. 33.9%, p<0.01), received myeloablative TBI (52.4 vs. 42.3 vs. 34.2%, p<0.01), and underwent allo-HSCT at CR1 (48.4 vs. 40.0 vs. 30.9%, p<0.01). Among the patients with t(7;11), 64 (70.3%) had French-American-British classification of M2, and 8 (8.7%) had additional chromosomal change.Transplantation OutcomesAt 3 years after allo-HSCT, OS and DFS in the t(7;11) group were 34.4% and 32.8%, respectively, and tended to be lower than those in the IR group after adjusting background characteristics (47.9% [hazard ratio {HR}=0.79, p=0.15] and 43.5% [HR=0.79, p=0.14]). However, OS and DFS in the t(7;11) group were similar to those in the PR group (33.9% [HR=1.18, p=0.3] and 29.2% [HR=1.18, p=0.27]) (Figure 1).Interestingly, the estimated cumulative incidence of relapse (CIR) at 3 years in the t(7;11) group was higher than that in the IR group (35.8% vs. 21.1% [HR=0.59, p<0.01]), but comparable to that in the PR group (29.8% [HR=0.8, p=0.24]) (Figure 2). In contrast, the estimated cumulative incidence of transplant-related mortality (TRM) in the t(7;11) group was similar to that in the IR/PR group (26.4% vs. 34.2% [HR=1.18, p=0.52] and 43.8% [HR=1.55, p=0.09]).In multivariate analysis among the t(7;11) group, the only factor influencing DFS was disease status (CR2: HR=2.78 [p=0.03], non-CR: HR=2.23 [p<0.01]) (Figure 3). In addition, patients in the CR1 group showed a lower CIR (21.0%) compared with patients in the CR2 or non-CR group (45.0% [HR=2.25, p=0.16], 44.9% [HR=2.31, p=0.03]) at 2 years.Conclusion:OS and DFS for the AML with t(7;11) following allo-HSCT were lower than those in the IR group due to higher CIR, but similar to those in the PR group. Among the patients with AML harboring t(7;11), disease status was the only independent prognostic factor for survival in the setting of allo-HSCT. Allo-HSCT at CR1 may overcome the poor prognosis of AML with t(7;11). [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Role of additional chromosomal changes in the prognostic value of t(4;14) and del(17p) in multiple myeloma: the IFM experience

AbstractIn multiple myeloma, cytogenetic changes are important predictors of patient outcome. In this setting, the most important changes are deletion 17p, del(17p), and translocation of chromosomes 4 and 14, t(4;14), conferring a poor outcome. However, a certain degree of heterogeneity is observed in the survival of these high-risk patients. We hypothesized that other chromosomal changes may impact the outcome. We retrospectively analyzed a large series of 242 patients displaying either t(4;14) (157 patients) or del(17p) (110 patients), 25 patients presenting both abnormalities, using single nucleotide polymorphism array. In patients with t(4;14), del(1p32), del22q, and >30 chromosomal structural changes negatively impacted progression-free survival (PFS). For overall survival (OS), del(13q14), del(1p32), and the number of chromosomal structural changes worsened the prognosis of patients. For patients with del(17p), del6q worsened the prognosis of patients, whereas trisomy 15 and monosomy 14 were found to have a protective effect on PFS. For OS, del(1p32) worsened the prognosis of patients, whereas having >8 numerical changes was found to have a protective effect on survival. This study, which is the largest series of high-risk patients analyzed with the most modern genomic technique, identified 1 main factor negatively impacting survival: del(1p32).

Population Based Study of Cytogenetic Abnormalities in Addition to Philadelphia (Ph) Chromosome in Patients with Chronic Myeloid Leukaemia (CML) and Impact on Survival

Ph chromosome is the hallmark of CML. However there are few reports of additional chromosomal abnormalities at the time of diagnosis and the impact this has on overall survival (OS). [NT1] The Cytogenetic laboratory at this hospital provides a regional service as a single facility. The data from this laboratory was combined with survival information to evaluate the impact of additional chromosomal changes on outcomes in patients with CML.Methods: This is a retrospective population based study, it was not possible to obtain consent from individual patients and details about haematological parameters or treatments delivered were not available. The aim was to evaluate if cytogenetic changes should be considered in addition to established risk scoring systems. Patients were classified as complex-Philadelphia (Ph) if they had t(9;22)(q31;q34) with additional chromosomal abnormalities. Impact of individual additional abnormalities was analysed and then the effect was stratified according to presence of chromosomal gains, deletions or translocations. Few cases who had normal cytogenetics on their first sample as they were initially treated elsewhere.Results: 1129 patients were diagnosed with CML between 1985 and 2013, with 4760 samples analysed. The median age of the patient was 52.4 years (4.3-103, 602 male; 511 female; 16 unknown). Median follow up was 6.4 years [0-26.8 years, 725/1129 (64.2%) had follow-up more than 10 years]. End point for analysis was probability of survival at 10yr. 194/1129 (17.2%) had complex-Ph at diagnosis, 759/1129 (67.2%) had standard Ph, 77/1129 (6.8%) had negative cytogenetics and in 34/1129 (3%) cytogenetic analysis failed at diagnosis. Patients with standard Ph translocation had significantly better chance of achieving cytogenetic CR than those with complex-Ph (23.4% vs. 13.4%, p<0.001). OS was significantly better in patients below the age of 45 (65% vs 25% p <0.0001). OS was also better in patients diagnosed after 2000 (67 % vs 40 %, p<0.0001). In univariate analysis OS was significantly lower with trisomy 8 (10% vs 50%, p<0.0001), del(5q) [NT2] (20% vs 50%, p=0.001), other deletions (12% vs 48%, p=0.0005), del 17( 48% vs. 0%, p<0.0001), add 21 (50% vs. 0%, p<0.0001), any translocations (50% vs. 22%, p<0.0001), der 22 or iso 17 (48% vs. 10%, p<0.0001), any deletions (50% vs. 20%, p<0.0001) and variant Ph translocations (50% vs 22%, p=0.004). In multivariate analysis, excluding year of diagnosis, age group (HR 1.93, 95% CI:1.6-2.4 P=<0.0001), complex t(9;22;v) (HR 1.8, 95% CI:1.0-3.1, P=0.035), del(17q) (HR 3.8, 95% CI:1.1-12.6, P=0.033), translocations (HR:1.6, 95% CI: 1.1-2.25, p=0.013), trisomy 8 (HR: 1.76, 95% CI: 1.19-2.65, p=0.005), add 21 (HR: 3.21, 95% CI: 1.65-6.25, p=0.001) and der 22 or iso17 (HR: 1.57, 95% CI: 1.1-2.25, p=0.013) were independently associated with inferior OS. Number of risk factors in individual patients was used to design a scoring system. Patients with 0 risk factor (70% OS at 10 yr.), 1 risk factor (40% OS at 10 years), 2 risk factors (22% OS at 10 years) or more than 3 risk factors (12% OS at 10 years) had incrementally reduced OS. This was true of patients diagnosed before and after 2000.This analysis suggests that incorporating nature of karyotype at diagnosis can refine established scoring systems. However this data needs to be analysed with larger patient population to include all established risk factors and the effect of therapeutic measures. DisclosuresCavet:Novartis: Research Funding; BMS: Research Funding.

Prognostic Impact of 3q21 and 3q26 Cytogenetic Abnormalities in Acute Myeloid Leukemia.

AbstractAbstract 2594 Background:Abnormalities affecting long arm of chromosome 3 are rare but recurrent in Acute Myeloid Leukemia (AML) and are detected in a variable percentage of AML patients according to different series. The 2008 World Health Organization classification recognizes AML with inv(3)(q21q26.2) or t(3;3)(q21;q26.2) as a distinct subtype characterized by a poor prognosis. Allogeneic stem cell transplantation seems to improve outcome in eligible patients with these aberrations. Inappropriate expression of the ecotropic viral integration site 1 (EVI1) was demonstrated in virtually all patients with t(3;3)(q21;q26.2) and inv(3)(q21q26.2); as well as in a majority of patients with other 3q26 rearrangements. Other chromosome 3 abnormalities are rarely recognized in AML patients; clinical and prognostic relevance of these alterations is not yet defined. The aim of this study is to assess the prognostic impact of chromosome 3 abnormalities on disease characteristics and treatment outcome in AML. Patients and methods:A total of 580 consecutive adult patients were diagnosed with AML at our institution between February 2002 and July 2012. Conventional cytogenetic analysis performed on diagnostic bone marrow samples detected the presence of 3q abnormalities in 16 patients (2.7%). Two patients were lost to follow-up and were not evaluated for survival analysis. Molecular status of FLT3 and NPM1 was also performed and results are available for 10 patients. Median follow-up time for patients in this series was 47 months ( range 6–125). Results:There were 10 male and 6 female patients, the median age being 64.5 years (range 33–81), 10 patients had de novo AML while 6 evolved from a previously diagnosed myelodysplastic syndrome (MDS). Karyotype from MDS phase was available in 2 patients; both acquired 3q rearrangement at time of progression to AML. At time of diagnosis median haemoglobin value was 9.0 g/dL (range 4–11); median leucocyte count was 10.5 × 103′/L (range 2.3 – 431). Median platelet count was 116 × 109′/L (range 28 – 529), consistently with previous studies, which have shown that these patients present with higher platelet count at diagnosis when compared with no 3q rearranged ones. Regarding cytogenetic features 3 patients had t(3;3)(q21;q26), 3 patients had inv(3) (q21; q26), 3 patients showed a balanced rearrangement involving 3q26, while 6 patients harbored a del3q. One patient showed monosomy 3. Additional chromosomal changes were demonstrated in 5 patients, two of them had a complex karyotype (see Table 1), 3 had a monosomy 7. Thirteen patients out of 14 received intensive induction chemotherapy; complete remission (CR) was achieved in 5 patients (CR rate: 35.7%), the remaining 7 patients were resistant to induction as well as to salvage chemotherapy. Four patients underwent autologous stem cell transplantation. Median overall survival in this series is 5.5 months (range 0 – 20). At present only one patient is still alive and in CR, 20 months after diagnosis. Median disease free survival (DFS) for patients achieving a CR was 9 months (range 6–20). Median overall survival for patients resistant to first-line therapy was 3 months (range 0–6). Clinical features and treatment outcome of the patients are summarized in Table 1. Conclusions:The incidence of 3q abnormalities in our single institution series is 2.4%, in keeping with previous studies. Our findings confirm the association between these alterations and thrombocytosis at diagnosis, preceding MDS or multilineage dysplasia, presence in all FAB subtypes (except M3), association with additional chromosomal abnormalities as well as the poor response to conventional chemotherapy (CR rate 35.7%), and very short DFS in spite of obtaining CR. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

CML Shows Characteristic Pattern by Flow Cytometry Analysis of Peripheral Blood

Abstract 4430A total of 75 chronic myeloid leukemia (CML) cases from peripheral blood (40 cases) and bone marrow (BM, 35 cases) with adequate flow cytometry (FC) data, smear, blood cell count, and the presence of t(9;22)/BCR-ABL by fluorescence in-situ hybridization (FISH) studies were analyzed for immunophenotypic pattern. The FC pattern in CML was compared with benign (healthy) controls (blood, 20 cases; BM, 20 cases), blood with reactive neutrophilia (15 cases), myelodysplastic syndrome (BM, 15 cases), and blood with eosinophilia (15 cases). CML showed a characteristic pattern by FC in blood, which can be easily differentiated from reactive neutrophilia or eosinophilia, regardless of WBC count. The identification of distinct population of blasts, basophilia, lack of CD10, CD11b, CD13 and/or CD16 on subset of granulocytes, decreased granularity, and/or aberrant expression of CD56 on granulocytes and monocytes, can be easily identified by routine FC analysis. We suggest using FC analysis of blood as a screening tool for patients with leukocytosis (neutrophilia) with follow-up FISH studies in cases with the phenotypic features suggestive of CML. Patients with confirmed CML diagnosis by FISH will undergo marrow biopsy for differential count including blast and basophil enumeration (to exclude accelerated phase or blast crisis), degree of reticulin fibrosis and cytogenetic studies (for additional chromosomal changes present at diagnosis). This approach, in our opinion, allows to diagnose early (unsuspected) CML and eliminates the need for unnecessary cytogenetic/FISH testing, and especially bone marrow biopsy in patients with reactive leukocytosis or eosinophilia. Disclosures:No relevant conflicts of interest to declare.

A Four-Way Translocation t (1;17; 9; 22) (p?35; q24; q34;q11) in a Newly Diagnosed Chronic Phase CML Patient: A Case Report

AbstractAbstract 4459Chronic myeloid leukemia (CML) is a myeloproliferative stem cell disorder characterized by the translocation t(9,22)(q34;q11), resulting in the Philadelphia chromosome (Ph) and the bcr-abl fusion gene. A small proportion of Ph+ CML patients (5–10%) show variant Ph translocations involving one or more chromosomes in addition to chromosomes 9 and 22. Its generation mechanism and clinical significance remains unclear. We report a novel four-way translocation detected in a newly diagnosed chronic phase CML patient. Case presentationA 51 year-old white women with no significant past medical history presented with a two-week upper left abdominal pain. She denied weight loss, fever, night sweats, fatigue or thrombosis. She has 11 siblings. On physical examination an enlarged spleen was detected, 7 cm below left costal margin. No hepatomegaly was found.Peripheral blood counts on admission showed WBC 286 × 109/l, with immature myeloid forms (21%bands, 31%neutrophils, 3%lymphocytes, 5%basophiles, 1%eosinophils, 16%metamyelocites, 14%myelocytes, 6% promyelocytes, 1% blast cells), hemoglobin 11,8 g/dl and platelet count of 986 ×109/l. LDH= 1094 IU/L. Uric acid, renal and hepatic functions were normal. Abdominal ultrasonography showed homogeneous splenomegaly (185 mm) with no focal lesions. Bone marrow aspirate demonstrated increased cellularity with myeloid hyperplasia without remarkable basophilia. Trephine demonstrated hypercellularity without fibrosis and 1% blast cells. Conventional cytogenetic analysis by GTG-banding revealed a variant Ph chromosome positive in 100 % of metaphases analysed involving chromosomes 1 and 17 inaddition to chromosomes 9 and 22: 46 XX, t (1;17; 9; 22) (p?35;q24;q34;q11)[20]. No normal cells nor additional numerical and structural chromosomal changes were detected at diagnosis. A fragment of 1605 bp was amplified with primers BCR-P1F and ABL-P1R. The sequence analysis of the amplified PCR product showed a complex BCR-ABL rearrangement formed by exon 8 of the BCR (NM_004327) and exon 2 of the ABL genes (NM_005157), with a 148 bp insertion corresponding to the whole exon 2 of the MAST2 (NM_015112, Homo sapiens microtubule associated serine/threonine kinase 2) gene.Bone marrow cytogenetical evaluation at four months do not show Philadelphia chromosome on 20 cells analysed. Interphase FISH with DUAL Colour Dual Fusion probe has a normal pattern on 300 nuclei: 2R2G. It corresponds to a complete cytogenetic response. Chronic phase CML was diagnosed and cytoreductive treatment with hydroxyurea 1,5 g/day, fluids, thromboprophilaxis and allopurinol was initiated. A good control of blood counts was obtained, as well as a complete reduction of the enlarged spleen and regression of left abdominal pain. Once cytogenetic/molecular confirmed diagnosis, treatment with Imatinib 400 mg/day was started, and hidroxiurea was discontinued. She achieved complete hematologic response at 1 month. Splenomegaly was no longer detected. At 2 months she developed neutropenia (1100/mm3) and thrombocytopenia (55000/mm3) so Imatinib was held. Two weeks later, after hematologic recovery, Imatinib was re initiated but in the following control neutropenia and thrombocytopenia were again detected. DiscussionAbout 90–95% of CML have the characteristic t(9;22)(q34;q11.2) reciprocal translocation that results in the Ph chromosome [der(22q)], the cytogenetical hallmark of this entity. The remaining cases can have variant translocations that involve 3, 4 or more chromosomes and its occurrence is not associated with disease evolution. This patient shows a four way translocation involving chromosomes 1 and 17 in addition to chromosome 9 an d 22. It is controversial whether variant translocations result in a different disease phenotype. There is no impact on the cytogenetic or molecular response or outcome, so a special approach is not recommended. ConclusionThis is a novel four way translocation described in a chronic phase CML patient, with a good response to standard therapy. Complete haematological and cytogenetic response at 4 months of treatment with Imatinib was achieved. Up to now, a differential approach does not appear to be mandatory in CML with variant translocations. However, in this patient alternative tyrosine kinase inhibitor may be of benefit due to hematologic toxicity attributable to Imatinib. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Genetic alterations in a primary medullary thyroid carcinoma and its lymph node metastasis in a patient with 15 years follow-up

BackgroundAssociation between DNA alterations and clinical parameters as recurrence, survival or prognosis has been found in a variety of tumors. A clear association between Medullary Thyroid Carcinoma (MTC) and RET oncogene mutation has been accepted. Specifically M918T RET mutation represents the main genetic event in most cases of sporadic MTC (SMTC) and limited chromosomal alterations analyses have been performed.MethodsIn the present work, a comparative genomic hybridization (CGH) study was performed using DNA from a primary tumor in a M918T RET mutation-positive SMTC patient and from its lymph node metastasis to investigate additional genetic alterations. We studied a patient with 15 years of follow-up and persistence of disease, confirmed by periodical elevated serum calcitonin (CT) levels.ResultsOnly 3 chromosomal imbalances were identified in the primary tumor, gain of 18p, and loss of 6p and 16p region, whereas 25 chromosomal imbalances were identified in the metastasis (9 gains and 16 losses).ConclusionThe chromosomal changes 6p-, 16p-, 18p + could determine in part the oncogenic phenotype in the primary M918T RET positive tumor and probably related to persistence of high serum CT levels in this patient. The additional chromosomal changes observed could be related to the metastasis phenotype. We suggest that some genes mapped at 6p, 16p and 18p chromosomal regions, could act as genes associated to cancer and could be related to persistent SMTC and good prognosis.Virtual slidesThe virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1720753793691097

Immortalization and malignant transformation of Eukaryotic cells

The process of cellular transformation has been amply studied in vitro using immortalized cell lines. Immortalized cells never have the normal diploid karyotype, nevertheless, they cannot grow over one another in cell culture (contact inhibition), do not form colonies in soft agar (anchorage-dependent growth) and do not form tumors when injected into immunodeficient rodents. All these characteristics can be obtained with additional chromosome changes. Multiple genetic rearrangements, including whole chromosome and gene copy number gains and losses, chromosome translocations, gene mutations are necessary for establishing the malignant cell phenotype. Most of the experiments detecting transforming ability of genes overexpressed and/or mutated in tumors (oncogenes) were performed using mouse embryonic fibroblasts (MEFs), NIH3T3 mouse fibroblast cell line, human embryonic kidney 293 cell line (HEK293), and human mammary epithelial cell lines (mainly HMECs and MC-F10A). These cell lines have abnormal karyotypes and are prone to progress to malignantly transformed cells. This review is aimed at understanding the mechanisms of cell immortalization by different "immortalizing agents", oncogene-induced cell transformation of immortalized cells and moderate response of the advanced tumors to anticancer therapy in the light of tumor "oncogene and chromosome addiction", intra-/intertumor heterogeneity, and chromosome instability.

Double Minute Chromosomes Associated with Philadelphia Chromosome in Meyloid Leukemia. A Case Study

The Philadelphia chromosome was the first consistent cytogenetic abnormality observed in a human cancer. Several characteristic chromosome rearrangements like double minute chromosomes are known to be associated with different subtypes of myeloid leukemia. Double Minute Chromosomes are the cytogenetic hallmarks of genomic rearrangements in cancers. Two cases, one with AML-M1 and the other with CML- blast crisis, associated with double minute chromosomes and Philadelphia chromosome is presented. The additional chromosome changes to primary chromosome abnormality may be influenced by other factor like chemotherapy. The identification of two new cases with co-expression of double minute chromosomes and Ph chromosome presented here for the first time in Iran together with large Mitelman database and other pertinent website reports is discussed.

First-Line Dasatinib Plus Conventional Chemotherapy in Adults with Newly Diagnosed Philadelphia Chromosome-Positive Acute Lymphoblastic Leukemia (Ph+ ALL): Interim Analysis of the Korean Prospective Phase II Study

Abstract 1516 Background:Front-line combination of imatinib with conventional chemotherapy has demonstrated an improved complete remission (CR) rate, an increased transplantation proceeding rate with CR, and a better survival in adults with Ph+ ALL. However, in the light of disease aggressiveness and recurrence, mainly as a result of the outgrowth of leukemic subclones with imatinib-resistant mutations, an improved strategy to induce more effective leukemic cell clearance is clearly needed. Dasatinib, a potent dual BCR-ABL/SRC family kinase inhibitor, has been shown to be effective in patients with imatinib-resistant chronic myeloid leukemia and Ph+ ALL. Methods:We present the first interim results of the Korean prospective phase II study protocol designed to evaluate the clinical efficacy of first-line dasatinib plus conventional chemotherapy for adults with newly diagnosed Ph+ ALL. This study is registered at www.ClinicalTrials.gov as NCT01004497. The protocol is designed for 51 patients, and recruitment started in March, 2010. The protocol enrolls patients (15–65 years) who receive dasatinib (100 mg once daily for 4 weeks) as an alternative schedule after each conventional chemotherapy course (alternating modified hyper-CVAD and high-dose cytarabine/mitoxantrone). Patients in CR who have a suitable related/unrelated donor undergo allogeneic transplantation as early as possible (depending on the speed of coordination process). Patients without a donor continue to receive dasatinib plus conventional chemotherapy (up to 4 courses; depending on the patient’s tolerability) followed by dasatinib maintenance therapy (100 mg once daily for up to 2 years). Minimal residual disease monitoring for BCR-ABL transcript is centrally evaluated by real-time quantitative PCR (4.5 log sensitivity) through handling of bone marrow samples from all patients (Research Institute of Molecular Genetics, The Catholic University of Korea, Seoul, Korea). Results:A total of 36 patients have been enrolled to date. Of these, 3 patients are receiving the first dasatinib cycle (too early); 3 patients died before starting the first dasatinib cycle from infections. Thus, 30 patients are evaluable for assessment of response to dasatinib plus conventional chemotherapy. Median age was 47 years (range, 19 to 64 years). Karyotype analysis revealed additional chromosomal changes in 18 (60%) of the 30 patients. Twenty patients (67%) had m-BCR transcript. All patients (100%) have achieved CR with a decrease in the minimal residual disease by the first dasatinib cycle, and of these, 13 patients (43%) have achieved major molecular response [MMR; including 5 complete molecular response (CMR4.5)]. By the second dasatinib cycle, 25 patients (83%) have achieved MMR (including 11 CMR4.5). So far, no dasatinib-related serious adverse events (≥grade 3 toxicity) have been observed. Twenty-four (80%) of the 30 patients have undergone allogeneic transplantation in CR; 6 patients are receiving continuous dasatinib plus conventional chemotherapy in CR. With a median follow-up duration of 10 months (range, 5 to 17 months), 25 patients are at present alive in continuous CR, and 4 patients have died (2 infections during consolidation chemotherapy, 2 transplant-related complications). Only 1 patient who failed to achieve MMR has relapsed at 11 months from diagnosis (at 5 months after allogeneic transplantation). The estimated probabilities of disease-free survival and overall survival at 1 year were 76% and 83%, respectively. Conclusions:Our interim analysis indicates that first-line combination of dasatinib with conventional chemotherapy appears to be effective in achieving a good quality of molecular response (MMR/CMR4.5) in adults with Ph+ ALL. Whether this would translate to an improved long-term survival remains to be investigated. Disclosures:Lee:Bristol Myers Squibb: Honoraria, Research Funding. Kim:Bristol Myers Squibb: Honoraria, Research Funding.

FISH panels for pediatric acute leukemias.

Background: Conventional cytogenetics is the gold standard in the diagnostic work up of acute leukemia patients. Due to a low mitotic index, lack of sample, and poor chromosome morphology, fluorescence in situ hybridization (FISH) is widely used as a complementary method to conventional cytogenetics. Additionally, some cryptic chromosomal rearrangements, which are crucial for stratification of patients, can be detected only by FISH. Different FISH panels are proposed to obtain comprehensive cytogenetic information. Methods: Recurrent chromosomal abnormalities were detected using commercially available FISH DNA probes. Results: In 85 % of pediatric AL patients conventional cytogenetics was successfully completed, revealing chromosomal rearrangements in half of them. We introduce an algorithm for inclusion of FISH DNA probes into panel, which is based on cytomorphology and immunophenotype of leukemic blasts. Using these panels, a particular cytogenetic marker was determined in 70 % of tested samples. The difference is even more prominent in B-ALL where conventional cytogenetics is less efficient. The frequency of a particular recurrent aberration is comparable with literature data in general, although some discrepancies were observed. Besides the detection of targeted rearrangements, FISH panels frequently show additional chromosomal changes with defined diagnostic and prognostic significance. Conclusions: The proposed algorithm for inclusion of DNA-probes into FISH panels is particularly efficient in children with B-ALL. In addition, presented results confirm this approach as an appropriate in different leukemias.

Prevalence and Prognostic Impact of Allelic Imbalances Associated with Leukemic Transformation of Philadelphia Chromosome-Negative Myeloproliferative Neoplasms.

Abstract 1280Poster Board I-302Philadelphia-chromosome negative myeloproliferative neoplasms (MPNs) including polycythemia vera (PV), essential thrombocytosis (ET), and primary myelofibrosis (PMF) are defined as clonal hematopoietic stem cell disorders. These disorders show an inherent tendency for transformation into leukemia (MPN-blast phase) which is hypothesized to be accompanied by acquisition of additional genomic lesions. We, therefore, obtained a comprehensive profile of genomic alterations associated with leukemic transformation by using single-nucleotide polymorphism (SNP) array in 88 MPN patients, as well as 71 cases with MPN-blast phase, and correlated these findings with their clinical parameters.A relatively high number of genomic alterations was found in MPN after leukemic transformation with 4.6 ± 0.6 abnormalities per sample compared to only 1.4 ± 0.2 changes per patient in chronic phase (p<0.001). Compared to the cytogenetic data, SNP-chip analysis detected about 47% additional chromosomal changes in the MPN samples, and 31% more in the MPN-blast phase cases, whereas SNP-array allelokaryotyping practically captured all cytogenetic abnormalities in our study population. Several additionally altered regions were detected in patients with MPN-blast phase compared to chronic phase, including both deletion and copy-number neutral-loss of heterozygosity (CNN-LOH) on chromosome 12p (9%) and 21q (9%), involving ETV6 and RUNX1. Notably, deletion and CNN-LOH on 17p involving TP53 were diagnosed in 18% of MPN-blast phase samples, which was highly associated with preceding treatment with alkylating agents (p=0.016). Moreover, trisomy 8, as well as amplification of 8q24.21 involving the MYC gene, were detected in 13% of patients with MPN-blast phase who were almost exclusively negative for the JAK2V617F mutation. Genome-wide inspection of further critical regions with promising new candidate genes involved in the evolution to the MPN-leukemic phase included deletion and CNN-LOH on 7q22.1 (SH2B2) in 18%, duplication/amplification on 19p13.2 (PIN1, ICAM1, CDC37) in 13% and 21q22.2 (ERG) in 9% of MPN patients with blast crisis. In contrast, we detected a decreased frequency of JAK2V617F in MPN-blast phase samples (52%) compared to chronic phase (71%). Also, the percentage of patients with homozygous mutant JAK2 as a result of CNN-LOH was lower in the MPN-blast phase (43%) compared to the chronic phase (53%). Taken together, the data suggest that gain-of-function mutation of JAK2 is not a perquisite for leukemic transformation. Remarkably, CNN-LOH on either 7q or 9p was related to decreased survival after leukemic transformation (p=0.02 and p=0.012, respectively). Given the variety of allelic imbalances, our data suggest that MPN-blast phase appears to be a heterogeneous disease prone to have evolved multiple mechanisms to provide a proliferative advantage to the abnormal leukemic clone. Our analysis of MPN genomes in the chronic compared to the leukemic stage provided new prognostic insights, as well as novel causative genes which might be involved in the transformation to MPN-blast phase. DisclosuresNo relevant conflicts of interest to declare.

AML with mutated NPM1 carrying a normal or aberrant karyotype show overlapping biologic, pathologic, immunophenotypic, and prognostic features

Acute myeloid leukemia (AML) with mutated NPM1 usually carries normal karyotype (NK), but it may harbor chromosomal aberrations whose significance remains unclear. We addressed this question in 631 AML patients with mutated/cytoplasmic NPM1. An abnormal karyotype (AK) was present in 93 of 631 cases (14.7%), the most frequent abnormalities being +8, +4, -Y, del(9q), +21. Chromosome aberrations in NPM1-mutated AML were similar to, but occurred less frequently than additional chromosome changes found in other AML with recurrent cytogenetic abnormalities according to WHO classification. Four of the 31 NPM1-mutated AML patients karyotyped at different time points had NK at diagnosis but AK at relapse: del(9q) (n = 2), t(2;11) (n = 1), inv(12) (n = 1). NPM1-mutated AML with NK or AK showed overlapping morphologic, immunophenotypic (CD34 negativity), and gene expression profile (down-regulation of CD34 and up-regulation of HOX genes). No difference in survival was observed among NPM1-mutated AML patients independently of whether they carried a NK or an AK, the NPM1-mutated/FLT3-ITD negative cases showing the better prognosis. Findings in our patients point to chromosomal aberrations as secondary events, reinforce the concept that NPM1 mutation is a founder genetic lesion, and indicate that NPM1-mutated AML should be clinically handled as one entity, irrespective of the karyotype.

Imatinib mesylate efficacy in 72 previously treated Philadelphia-positive chronic myeloid leukemia patients with and without additional chromosomal changes: single-center results

Reported here are 72 previously treated Philadelphia chromosome–positive (Ph+) CML patients on imatinib (IM) therapy, with a focus on patients with additional chromosomal aberrations (CAs). At the start of IM treatment, 49 patients exhibited only the Ph chromosome (68%) and 23 patients (32%) had one or more additional CAs. The most frequent additional changes were deletions on the der(9q) (8 of 23), trisomy 8 (3 of 23), and an extra copy of the Ph chromosome (2 of 23). Five patients had a complex karyotype. At the latest follow-up, 49 of the 72 patients (68%) were alive, including 15 of the 23 patients with additional CAs (65%). Median follow-up was 6.6 years; median duration of IM treatment was 4.4 years. In all, 35 of the 49 patients with Ph only (71%) and 10 of the 23 patients with additional CAs (43%) achieved complete cytogenetic response. All patients with deletion on der(9q) achieved complete cytogenetic response. There was no statistically significant difference in the overall survival of patients with additional CAs and patients with Ph as the sole abnormality. Patients in accelerated phase had significantly worse overall survival on IM, regardless of additional CAs. The present results confirm that the majority of previously treated Ph+ CML patients benefit from starting IM therapy, including patients with defined additional changes. In contrast, patients with complex karyotypes have poor prognosis, even with IM.