ALBERT

Description: INTRODUCTION: Hematopoietic stem cell transplantation (HSCT) is an effective treatment for malignant and nonmalignant diseases. Improved long-term survival after HSCT translates into coming across secondary neoplasms. Contributory factors include primary disease, male sex, young age, prior therapies, conditioning regimens. The secondary neoplasms are particularly solid tumors, as well as lymphoproliferative disorders. Chronic graft-versus-host disease (GvHD) and immunosupressive therapy have also been reported to contribute to neoplasia risk. OBJECTIVE: The purpose of this study was to evaluate the frequency and distribution of the posttransplant secondary neoplasms in our center, to determine the possible contributory factors and relative effect of GvHD. METHODS: From 457 patients who received a HSCT between 1994-2014 clinical records of 312 patients were available to review retrospectively. 21 patients diagnosed with a secondary neoplasia in the posttransplant period are included in the study. Age, sex, primary diagnosis and treatment, time of HSCT, GvHD and immunosupressive treatments, localisation of neoplasms and outcomes were reported. RESULTS: Twenty-one cases of secondary neoplasms were observed (%6,7). The median age at diagnosis and transplantation were 44 and 47, respectively. The median follow-up time was 122 months (32-304). The most common primary diagnosis was Hodgkin's disease (HD). The most commonly used pretransplant conditioning regimen was BEAM. There were no cases of acute GvHD, chronic GvHD was observed in 3 cases. The most common secondary neoplasm was skin cancer followed by urogenital system cancers. The secondary malignancies seen in cases with chronic GvHD are concordant with GvHD sites. Three patients had benign neoplasms comprising fibroadenoma, mol hydatiforme and hibernoma; 2 patients developed preinvasive lesions of vulva (VIN 3) and oral cavity (squamous papilloma). For 20 patients the median time interval between the date of HSCT and diagnosis of a secondary neoplasia is 62 months (5-118); data is missing for 1 case. Two deaths were observed, 1 due to disease progression, 1 due to secondary colorectal malignancy. %90,4 of the study group are alive and in remission. Details are listed in Table 1 and 2. CONCLUSION: Patients undergoing HSCT have an increased risk of secondary cancers later in life. Known risk factors are primary disease, age at transplantation, pretransplant therapies, pretransplant conditioning regimens like total body irradiation, chronic GvHD and immunosuppressive therapies. Our study group is small to comment on these risk factors. Coherent with the literature skin cancer was the most common secondary cancer in our cohort as well. Interestingly we observed a trend towards increased urogenital cancers in comparison to reported data. This finding can be incidental because of the small number of study population or needs to be clarified yet. The increased risk of secondary neoplasms over time after transplantation and the greater risk among younger patients indicate the need for lifelong surveillance. Table 1 Characteristics of patients with secondary neoplasms after HSCT Characteristic n (%) Sex Female 10 (%47) Male 11 (%53) Primary diagnosis Acute myeloid leukemia 1 (%4,7) Acute lymphoblastic leukemia 3 (%14,3) Chronic myelogeneous leukemia 3 (%14,3) Multiple Myeloma 5 (%23,8) Hodgkin’s disease 6 (%28,6) Non-Hodgkin’s lymphoma 3 (%14,3) Prior therapy Chemotherapy 9 (%42,7) Chemoimmunotherapy 3 (%14,3) Chemoradiotherapy 5 (%23,7) Combination treatment* 3 (%14,3) Type of HSCT Allogeneic 5 (%23,8) Related 5 Unrelated 1 Autologous 15(%71,5) Both 1(%4,7) Conditioning regimens TBI+Cy 3 (%14,3) BEAM 8 (% 38,1) MEL 6 (% 28,6) BU+Cy 2 (% 9,5) Unknown 2 (%9,5) Acute GvHD 0 Chronic GvHD 3 (%14,3) Skin 3 Oral cavity 2 Eye 2 Pulmonary 1 Hepatic 1 Acute GvHD prophylaxis 7 (%33,3)  CsA 1 MTX+CsA 6 Chronic GvHD treatment 3 (14,3) Steroid+CsA 2 CsA 1 Table 2 Characteristics and risk factors of patients who had secondary malignancy undergoing HSCT Breast Skin GIS Urogenital Lung Lymphoma Risk factors (n:2) (n:6) (n:2) (n:3) (n:2) (n:1) Prior therapy Chemotherapy 0 4 0 2 1 0 Chemoimmunotherapy 1 1 1 1 0 0 Chemoradiotherapy 1 0 0 0 1 1 Combination 0 1 1 0 0 0 Allogeneic Related 1 3 0 1 0 0 Unrelated 0 1 0 0 0 0 Autologous 1 3 2 2 2 1 TBI+Cy 0 1 0 0 0 0 BEAM 1 2 1 0 1 1 MEL 0 1 1 2 1 0 BU+Cy 0 1 0 1 0 0 Unknown 1 1 0 0 0 0 Chronic GvHD 0 2 0 0 0 0 Sex Female 2 2 2 1 0 0 Male 0 4 0 2 2 1 Disclosures No relevant conflicts of interest to declare.

Description: Background and Aim BCR-ABL1 mutation testing is recommended for chronic myeloid leukemia (CML) patients who have suboptimal response and/or treatment failure with tyrosine kinase inhibitor (TKI) therapy. BCR-ABL1 mutations in the kinase domain (KD) of ABL1 account for at least 40-50% of all TKI resistant cases. Thus, detection of low-level mutations after development of resistance may offer critical information to guide subsequent therapy selection. The current gold standard for BCR-ABL1 mutation detection is Sanger sequencing (SS), which has an analytical sensitivity of approximately 10-20%. In this study, our aim was to detect low level BCR-ABL1 variants in follow up samples of CML patients with TKI resistance using next-generation sequencing (NGS) approach. Methods Eight patients with CML who were resistant to imatinib had been routinely sequenced with SS for BCR-ABL1 KD mutations between December 2009 and December 2012. We then retrospectively analyzed these samples with NGS. RT and long range PCR was performed to amplify BCR-ABL1 fusion transcripts and the PCR products sequenced bidirectional after library preparation. We performed a fusion transcript based BCR-ABL1 mutation assay using Roche 454 amplicon deep-sequencing technology that is suited for detecting low level variants in pooled amplicon samples. Sequencing data was analyzed with GS Amplicon Variant Analyzer (AVA) software, and the variant frequency cut-off was adjusted to 1%. Results Clinical features, sequencing results, and the outcomes of the patients were summarized in Table 1. Four patients were male, and the median age was 37 years (range, 20-60 years). The patients were all in chronic phase at the time of the diagnosis. After imatinib resistance, 4 patients had received dasatinib (DAS), and 2 were given nilotinib (NIL) as second line TKI treatment. The remaining two patients had both received DAS and NIL (Table 1). In a set of 20 clinical samples, at different time points, NGS not only identified all the mutations detected by SS, but additionally identified low level variants present between 1 – 28.12 %. T315I and E255K/V were the most common mutations, which were detected in four patients, both by SS and NGS at the same time points (Table 1). Two patients (patient #1 and #4) had T315I, and they both progressed to blastic phase and died. E255K was detected in patients #2 and #3, and patient #2 had achieved and maintained complete cytogenetic and major molecular responses with 100 mg daily DAS, whereas patient #3 had received both NIL and DAS, but she was deceased due to myeloid blastic crisis. Among 4 patients (patients #5, #6, #7, and #8), mutation analysis was performed at eleven different time points, and these patients were wild-type with SS. We also did not detect any clinically significant mutations in these patients by NGS. Most probably mechanisms other than KD mutations were responsible for the TKI resistance among these four patients. Conclusions Polyclonal mutations in BCR-ABL1 KD are commonly identified in TKI resistant patients. Thus, detection of low-level mutations after development of resistance offers critical information to guide subsequent therapy selection. An inappropriate kinase inhibitor selection could highly increase the risk of treatment failure with clonal expansion of the resistant mutant. In our imatinib resistant cohort, we detected low level variants accompany to known mutations which may constitute background genetic variations. Although we had expected to detect mutations earlier by NGS (i.e. before these mutations can be detected by SS), we did not observe such finding in our patients. The patients' samples may not show a stable mutation spectrum between time points. Hence, it is not always possible to spot a mutation before patients show resistance to therapy. Regular NGS analysis might detect these mutations in earlier phases, which might help clinicians to choose the most suitable individual treatment modality for the patients. Acknowledgment The authors would like to thank the Interlaboratory Robustness of Next-generation sequencing (IRON) Phase II study group members, especially to Simona Soverini and Alexander Kohlmann who designed BCR-ABL primers and plates. We also would like to thank the Research Fund of the Istanbul University (Project no. 24244) and Turkish Society of Hematology for supporting the study. Disclosures: Sayitoglu: Roche Diagnostics: Research Support Other.

Description: Immune thrombocytopenia (ITP) is an autoimmune disease resulting from inhibition of megakaryopoiesis and destruction of platelets by platelet reactive autoantibodies. Adult-onset ITP usually exhibits a relapsing chronic course and is often associated with other disorders of autoimmune or infectious origin, including systemic lupus erythematosus (SLE), lymphoproliferative diseases, common variable immunodeficiency (CVID) disease, and human immunodeficiency virus (HIV) infection. The exact pathophysiology of ITP has not been fully elucidated. Heterogeneity in disease presentation and in response to immunosuppressive therapy suggests different underlying mechanisms; and thus justifies the investigation of relevant genetic associations. Here we tried to unravel the genetic background of the immune dysfunction in ITP by using SNP array technology. We analyzed the blood samples of 12 adults (6 males and 6 females) with primary refractory ITP, by SNP array approach to identify candidate genomic regions. None of the patients showed clinical and laboratory findings of autoimmune diseases, malignancy or infections. All individuals were genotyped using Illumina Human HumanCytoSNP-12 BeadChip (300K). Whole genome SNP genotyping data were delineated using GenomeStudio software platform. The genotyping data was obtained by further analyzes in terms of copy number variation (CNV). In total we observed 14 CNVs in 12 patients. All CNVs were on autosomal chromosomes; 8 duplications and 6 deletions (gain 2p, 9q, 13q, 14q, 17p, 17q, 20q; loss 5q, 7p, 7q, 15q, 17q, 19q). The rearrangements sizes were between 45.42 Mb and 1.08 Mb. Moreover in five patients the duplications were in the form of mosaic structural genomic rearrangements. These mosaic duplications were found at 17q21.31 (0.2 Mb), 2p23.2 (0.34 Mb), 9q21.2 (10.89 Mb), 9q31.2 (17.77 Mb), 14q13.2 (0.37 Mb), 17p12 (1.46 Mb), 20q11.21 (14.24 Mb). None of the detected CNV regions could be demonstrated in two independent cohorts of 30 individuals with non-hematological disorders and healthy controls. Little is known on the genetic background of immune thrombocytopenia. Our aim was to investigate the impact of CNVs on the pathophysiology of ITP. We found several gains/losses in different chromosomal regions. However, the most striking result of our study was the detection of mosaic patterns that have been reported to most commonly associate with hematological cancers. These mosaic patterns may be associated with clonal expansion of T- or B-cells, leading to the immune dysfunction seen in ITP. Evaluation of the detected regions and their relation to B and T- cell clonality is ongoing. These preliminary results provide early evidence for the presence of predisposing CNVs in adult onset ITP. Disclosures No relevant conflicts of interest to declare.

Description: Background: Drug-induced pulmonary arterial hypertension (PAH) can be observed as an adverse event (AE) during the administration of dasatinib (DAS), which is a second generation tyrosine kinase inhibitor (TKI), used in the treatment of chronic myeloid leukemia (CML) and Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL). The occurence of DAS-induced PAH at a late onset in most of the cases suggests a chronic pathological mechanism rather than an acute inflammatory or cardiac etiology. The treatment strategies of DAS-induced PAH include the cessation of the drug and PAH-specific therapies. Aim: The aim of the study was to evaluate the frequency, clinical features, management strategies and outcomes of patients with DAS-induced PAH among a cohort consisted of CML and Ph+ ALL patients who had received DAS as a salvage treatment after imatinib (IM) failure or intolerance. Patients and Methods: Forty patients with Ph+ leukemias who received second-line DAS were enrolled. Patients' demographics, Sokal risk scores, molecular and cytogenetic responses, comorbidities [including preexisting cardiac disease, renal insufficiency, hypertension and chronic obstructive pulmonary disease (COPD)], DAS dose, dosing intervals and treatment durations, durations of IM therapy prior to DAS, and if any, treatments prior to IM (interferon (IFN), cytarabine (Ara-C), and hydroxyurea (HU)) and follow-up periods were noted retrospectively. TKI response criteria were based on the recommendations of European LeukemiaNet, and the definitions of the CML phases and responses were as described elsewhere. Results: Twenty-four patients were male, and the median age was 45 years (range, 18-81 years). There were 39 patients with CML and one with Ph+ ALL. Among the thirty-nine CML patients, 3 were in accelerated phase (CML-AP), two with blast crisis (CML-BC), and the rest were in chronic phase (CML-CP). The percentanges of low, intermediate, and high Sokal risk scores were 46%, 33%, and 21%, respectively. Thirteen patients received only IM prior to DAS, whereas the others had used HU, IFN and Ara-C prior to IM. After a median duration of 41.5 months (range, 1-93 months) of IM, the reason for switching to DAS were IM failure and intolerance in 37 and 3 patients, respectively. DAS was administered with a median of 50 months (range, 2-78 months). During DAS treatment hematological AEs were observed in 6 patients, whereas in twenty-one pulmonary complications including exacerbation of COPD and pneumonia (n=1), pleuro/pericaridal effusions (n=19), PAH (n=5) and gastrointestinal bleeding (n=1) were detected. DAS therapy was ceased in 13 patients, of which ten were switched to nilotinib (NIL) due to AEs (n=7) and failure (n=3). Also, two patients received cytotoxic treatment due to BC and one had allogeneic hematopoietic stem cell transplantation (allo-HSCT). Five patients (12.5%) had DAS-induced PAH (Table 1). Four of them were in CML-CP at diagnosis, and one was in CML-AP. All cases received DAS due to IM failure. At the time of DAS initiation, 4 cases were in CML-CP and one in CML-BC. PAH was diagnosed by transthoracic echocardiography (TTE) in 3 patients, and by right heart catheterization (RHC) in 2, and it was observed after a median of 8 months (range, 2-25 months) of DAS. Three patients had accompanying pleuro/pericardial effusions. All patients with DAS-induced PAH were alive at the time of the analysis, and the management of PAH included dose reduction in two, and DAS was switched to NIL in 2 cases and allo-HSCT was performed in one. Conclusion: DAS-induced PAH seems to be reversible with the cessation and/or modification of DAS ± PAH-specific treatments. As pulmonary vascular toxicity related to DAS is thought to be molecule-related rather than class-related, it seems reasonable to switch to another TKI. The patients in our cohort had good responses to dose modification and drug cessation and none received PAH-specific therapy. Although DAS-induced PAH is mainly defined as a late complication, we detected that PAH can be observed even after 2 months of drug exposure. PAH can be observed during DAS treatment and physicians should be aware of this AE. Routine cardiopulmonary evaluation prior to and/or during DAS may be beneficial. Mechanisms under this pathological condition, preceding and prognostic factors, and treatment strategies are needed to be evaluated with prospective trials. Disclosures No relevant conflicts of interest to declare.