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Background: Secondary or treatment-related acute myeloid leukemia (sAML) is associated with poor outcomes. Although allogeneic hematopoietic cell transplantation (alloHCT) is the treatment of choice, patient eligibility criteria and optimal conditioning regimen remain under study. We have previously shown an advantage of a myeloablative conditioning regimen with reduced toxicity (Fludarabine 150mg/m2, Treosulfan 42g/m2, FluTreo) compared to a reduced intensity regimen. However, the long-term effects of this regimen remain unknown, especially in comparison to patients not receiving alloHCT. Aims: We hypothesized that patients transplanted with FluTreo would have a long-term survival advantage over other treatment alternatives. Methods: We retrospectively studied consecutive patients treated for sAML in our center over the last two decades (1998-2018). Exclusion criteria were: age>70 years, ECOG performance status≥3 at presentation, induction regimens≤2, and autologous or haploidentical HCT because these were performed in individual patients. Since 2013, we have introduced FluTreo for patients with a suitable donor that would have been previously eligible only for reduced intensity conditioning/RIC. The following factors were studied in the whole cohort: age, type of disease (treatment-related or post myelodysplastic syndrome/MDS), previous intensive chemotherapy cycles, cytogenetic risk, overall (OS) and disease-free survival (DFS). In transplant recipients, we also recorded HCT-comorbidity index/CI score, cumulative incidence (CI) of graft-versus-host disease (GVHD), and treatment-related mortality (TRM). Follow-up was calculated from diagnosis in the whole cohort; and from date of transplant in the sub-group analysis of transplant recipients. Results: We studied 19 FluTreo recipients compared to 46 recipients of other conditioning regimens (38 myeloablative and 8 RIC), and 52 patients treated only with chemotherapy. Complete remission (CR) had been achieved in all FluTreo recipients before HCT, compared to 53% of other transplants, and 44% of chemotherapy only patients (p Within transplanted patients, there was no significant difference in GVHD, relapse and TRM between FluTreo and other transplants. Within the FluTreo group, acceptable rates of CI in severe acute and extensive chronic GVHD were observed (15.2% and 18.4%, respectively). Similarly, 4-year CI of TRM reached 30.4%, despite a median HCT-CI of 3 (0-7), age of 59 (51-67) years, 4 lines of treatment (3-6), and a majority of matched unrelated donors (13/19). Conclusion: Our real-world study confirms that alloHCT with FluTreo expands the transplant population with similar safety and efficacy to previous transplant modalities in sAML patients. Achievement of CR remains a major predictor of OS in these patients. The choice of alloHCT in this unique patient population of a rather older age and comorbidity index needs to be revisited. Figure 1 Disclosures Gavriilaki: Omeros Pharmaceuticals: Consultancy.

Recent evidence suggests that complex karyotype (CK) defined by the presence of =3 chromosomal aberrations (structural and/or numerical) identified by chromosome banding analysis (CBA) may be relevant for treatment decision-making in chronic lymphocytic leukemia (CLL). However, many challenges towards routine clinical application of CBA remain. In a retrospective study of 5290 patients with available CBA data, we explored both clinicobiological associations and the clinical impact of CK in CLL. We found that patients with =5 abnormalities, defined as high-CK, exhibit uniformly dismal clinical outcome, independently of clinical stage, TP53 aberrations (deletion of chromosome 17p and or TP53 mutations, TP53abs) and the expression of somatically hypermutated (M-CLL) or unmutated (U-CLL) immunoglobulin heavy variable genes (IGHV). Thus, they contrasted CK cases with 3 or 4 aberrations (low-CK and intermediate-CK, respectively) who followed aggressive disease courses only in the presence of TP53abs. At the other end of the spectrum, patients with CK and +12,+19 displayed an exceptionally indolent profile. Building upon CK, TP53abs and IGHV gene somatic hypermutation status, we propose a novel hierarchical model where patients with high-CK exhibit the worst prognosis, while M-CLL lacking CK or TP53abs as well as CK with +12,+19 show the longest overall survival. In conclusion, CK should not be axiomatically considered unfavorable in CLL, representing a heterogeneous group with variable clinical behavior. High-CK with =5 chromosomal aberrations emerges as prognostically adverse, independently of other biomarkers. Prospective clinical validation is warranted before finally incorporating high-CK in risk stratification of CLL.

Introduction: Endothelial dysfunction is a common denominator of graft-versus-host disease (GVHD) and transplant-associated thrombotic microangiopathy (TA-TMA). The latter is also characterized by excessive complement activation. Recent studies have introduced the Endothelial Activation and Stress Index (EASIX) as a potential predictor of survival in patients with GVHD. We hypothesized that EASIX would predict complement activation and survival in patients with GVHD and TA-TMA. Methods: We enrolled consecutive adult TA-TMA (International Working Group/IWG criteria), acute and/or chronic GVHD and control allogeneic hematopoietic cell transplantation (HCT) recipients in a 1:1:1 ratio (January 2015-December 2018). Plasma was collected and stored immediately at -80oC at the first day of confirmed TA-TMA or GVHD diagnosis and at a similar post-transplant period in control recipients. EASIX [lactate dehydrogenase (U/L) × creatinine (mg/dL)/thrombocytes (10⁹ cells per L)] was calculated at day 0, 30, 100 and last follow-up. Complement activation was detected measuring soluble C5b-9/membrane attack complex (ELISA, Quidel). Results: We studied 20 TA-TMA, 20 GVHD and 20 control patients (Table 1). TA-TMA developed at a median of 125 post-transplant day (range 9-2931); whereas the first day of confirmed GVHD diagnosis was at a median of 78 post-transplant day (range 16-145). EASIX at day 100 and last follow-up differed significantly among groups (p=0.014 and p=0.001, Table 1), although there was no significant difference between TA-TMA and GVHD patients. In contrast, soluble C5b-9 was significantly higher in TA-TMA compared to GVHD (p=0.008) and control patients (p Furthermore, EASIX at day 0 and last follow-up was significantly associated with overall survival (p=0.013 and p=0.046). Among other pre-transplant factors studied (age, disease type and phase at transplant, donor), EASIX at day 0 was an independent predictor of overall survival (beta=2.627, p=0.029). Conclusion: Our study shows for the first time that EASIX predicts complement activation and overall survival in patients with endothelial dysfunction syndromes and control HCT recipients. Our findings suggest that EASIX may be a useful dynamic marker reflecting the course of endothelial dysfunction in these patients. Disclosures No relevant conflicts of interest to declare.

Chronic myeloid leukemia (CML) is rare in childhood (less than 5% of all childhood leukemias). The main characteristic is the Philadelphia chromosome (BCR-ABL1 positive) and the tyrosine kinase inhibitor imatinib mesylate (Gleevec) is the treatment of choice, with the target oral dose being 440 mg/m2/day asdetermined by the COG-P9973 and COG-ADVL0122 trials, while allogeneic stem cell transplantation is postponed until CML becomes refractory to the drug. We administer a treatment dose of 400mg/m2/day but we have observed high toxicity levels associated with prolonged treatment. We present a girl with CML, with persistent residual disease (MRD), even two years following diagnosis, and serious side effects (dry skin, significant hair loss, gastrointestinal discomfort and diarrhoea) that affected her quality of life. The patient was tested for polymorphisms in the tyrosine kinase and was found negative. Careful interviewing of the family revealed that the persistent MRD was due to poor compliance of the patient to the therapeutic regimen. The child was unhappy due to the side effects and refused to take her pills (Gleevec), hence the poor compliance. Therefore, taking into consideration the child’s wellbeing and psychological welfare, it was decided that she would receive the drug on alternate months (one on/one off). Gleevec was discontinued when the patient completed two years of being MRD negative. The patient remains in complete molecular remission four years after the discontinuation of Gleevec. To date, there are few reports on childhood CML so most data come from studies in adults. Even though Gleevec is currently implemented as the primary treatment method in children, there are still doubts as to whether it can result in a permanent cure and of the potential complications of long-term use in the growth and development of these children. No specific guidelines have been set on the dosage and duration of treatment with Gleevec, especially for childhood CML patients facing a potentially lifelong treatment, who might also be faced with a wide range of unknown side effects. Psychological factors should also be taken into account and special attention should be given in avoiding adverse effects that interfere with the quality of life and the psychological welfare of this extremely fragile population. Overall, in our case, despite the persistent MRD, intermittent dosing of Gleevec proved to be an efficient method both in keeping toxicity levels to a minimum and in achieving complete and continuous remission. Persistent MRD levels in this case were due to the interrupted treatment regime, i.e. due to poor compliance, and not due to additional cytogenetic abnormalities that were resistant to Gleevec. Future clinical trials in children should investigate whether intermittent dosing of the drug produces fewer side effects during the course of treatment and whether it may present a more favourable option when considering the normal growth development of the children treated for CML. Disclosures: No relevant conflicts of interest to declare.

Abstract 3915 Although chromosomal translocations (CTRAs) are detected relatively frequently, CLL is conspicuous for the paucity of recurrent CTRAs. The biological and clinical significance of CTRAs in CLL remains uncertain, likely reflecting the underlying genetic heterogeneity. Here we reappraise CTRAs in CLL in a cohort of 893 patients with reliable classic cytogenetic data, selected with an intentional bias towards cases carrying CTRAs. The study group included 568 males and 325 females with a median age of 65.4 years; information about Binet stages at diagnosis was available for 761 cases: A, 650/B, 76/C, 35; 419/646 (64.8%) cases with available data carried mutated IGHV genes (M-IGHV). The median time from diagnosis to classic cytogenetic analysis was 2.3 months; FISH data was available for 556/893 cases. Overall, 311 cases were found to carry ≥1 CTRAs; 193/311 (62%) cases carried balanced CTRAs whereas the remaining 118 cases (38%) carried ≥1 unbalanced CTRAs. The most frequent chromosome breakpoints were 13q (14% of all translocation partners, TPs), followed by 14q (6%), 18q (5%), 17q (3.5%) and 17p (3.5%); notably, CTRAs involving chromosome 13q showed a wide spectrum of TPs. When compared to CTRA- cases (n=582), CTRA+ cases exhibited a significantly higher prevalence of complex karyotype (three or more structural and/or numerical aberrations; 112/311 CTRA+ cases vs. 46/582 CTRA- cases, p Disclosures: No relevant conflicts of interest to declare.

Abstract 1587 Monoclonal B-cell lymphocytosis (MBL) with an immunophenotype consistent with marginal zone origin (MBL-MZ), that can be either CD5− or CD5+ but atypical for CLL, and also lacking an IGH/CCND1 translocation, is an increasingly recognised entity with poorly understood biological background and clinical significance. In particular, it is not yet clarified whether it represents a precursor state to one of the distinct lymphoma entities recognized by WHO as deriving from MZ cells or whether it constitutes a novel entity, likely with similar ontogeny. To obtain insight into this issue we retrospectively evaluated a series of 71 patients (male/female: 35/36, median age: 73.3 years) with lymphocytosis (median lymphocyte count: 5.77 × 109/l) detected incidentally on a routine blood test. No case had lymphadenopathy, organomegaly or any clinical features to suggest a concurrent marginal zone lymphoma. Hemoglobin and platelet counts were normal in all cases; 15/57 (26%) cases had paraproteinemia. Peripheral blood immunophenotyping revealed the presence of a clonal B-cell population with Matutes score Disclosures: No relevant conflicts of interest to declare.

Abstract 4961 Background: Platelet increase under azacitidine in patients with myelodysplastic syndrome (MDS) has been acknowledged as an early predictive factor of response to treatment. However, extreme thrombocytosis under azacitidine has not been reported. Methods: We studied consecutive patients with MDS or MDS/myeloproliferative neoplasm (MDS/MPN) who had platelet counts near or over 1, 000 G/L under azacitidine. Results: Four patients, sex ratio 1:1, with median age of 65 years, had extreme thrombocytosis under azacitidine. Baseline characteristics were: WHO classification RAEB-2/CMML-1/CMML-2 in 2/1/1 patients, median platelet count 248 G/L (15% at baseline. A median number of 8 cycles of azacitidine was administered. Individual platelet counts reached 2, 960 G/L, 800 G/L, 1, 188 G/L and 2, 740 G/L. Thrombocytosis occured early after treatment onset or resumption (Figure 1). Histologic findings under treatment were: Increased cellularity (N=4), micromegakaryocytes and other signs of megakaryocytic dysplasia (N=4), reticulinic fibrosis grade I and II in 1 and 2 patients, respectively. JAK2 V617F mutation was detected in 1 patient (with maximum platelet count of 2, 900 G/L) and was undetectable in the remaining patients. None had a thrombotic or hemorrhagic event. Two patients had a concomitant increase of WBC count. Response to azacitidine was CR, PR and stable disease in 1/1/2 patients. Three patients received hydroxyurea (HU) in addition to azacitidine and one patient underwent hematopoietic stem cell transplantation (HCT). AML transformation occurred in 1 patient 25 months after azacitidine onset. Median overall survival after azacitidine onset was 25 months. Conclusion: Extreme thrombocytosis of the range of essential thrombocytosis, with megakaryocytic dysplasia and hyperplasia, was noted under azacitidine in 4 patients with MDS-MDS/MPN and normal baseline platelet count. Hypothetically, azacitidine may induce the expression of critical genes of megakaryopoiesis or platelet release in patients with rare mutations. Notably, JAK2 mutation was detected in only one patient. Alternatively, demethylation could unmask an underlying unclassified MDS/MPN similar to RARS-T. Disclosures: No relevant conflicts of interest to declare.

Abstract 3596 Immune phenotyping plays a critical role in the diagnosis and classification of acute leukemia. Several studies have reported a variable proportion of patients with acute myeloid leukemia (AML) expressing lymphoid-associated antigens (LAA). The exact frequency and true clinical significance of this phenomenon remains undefined due to inconsistencies between series, likely related to methodological aspects or potential case selection biases. We retrospectively evaluated the expression of LAA in blast cells from 278 consecutive and unselected patients with AML diagnosed in our Department between 2002 and 2010. The patient cohort included 168 males and 110 females with a median age of 61 years (range, 10–88); 146/278 cases were above the age of 60. Within this cohort, 190 cases (68%) had de novo AML, whereas the remaining 88 cases (32%) concerned secondary AML (sAML) to either MDS (n=80) or other non-hematologic malignancies (n=8). Patients were treated uniformly according to age with Aracytin/Idarubicin induction regimens (“3+7” or “2+5” for ages \q60 or ≥60, respectively). The immunophenotype was determined by flow cytometric analysis of (mainly) bone marrow aspirate and/or peripheral blood samples utilizing a primary CD45/side scatter (SSC) gating procedure with antibodies against CD7, CD13, CD19, CD33, CD4, CD10, CD34, CD117, CD64, HLA-DR, CD20, CD2, CD15, CD56, CD14, CD8, MPO, CD3, CD79a, CD22, TdT and lysozyme; a cut-off value for positivity of 20% was adopted. Overall, we identified 153/278 cases (55%) expressing at least one LAA. The most commonly expressed LAAs were CD4 (outside AML with monocytic differentiation), CD56, CD7, CD2, CD10 and CD79a (in 39%, 33%, 29%, 14%, 10% and 8% of LAA+ AML cases, respectively); interestingly, all CD79a-positive cases co-expressed at least one more LAA. A significant association was identified between LAA expression and cytogenetic profile: in particular, at least one LAA was detected in 37/50 cases (74%) with adverse cytogenetics (SWOG unfavorable and/or monosomal karyotype), compared to 24/41 (58%) cytogenetically favorable cases and 68/134 (51%) cytogenetically intermediate risk cases (p=0.01). No other statistically significant associations were found for LAA expression (positive vs. negative) in respect to age and complete remission (CR) rate. Furthermore, the frequency of LAA-positive cases was identical (55%) in both de novo AML (105/190 cases) and sAML (48/88 cases). Monoparametric statistical analysis was also performed individually for each of the six more frequent LAAs. Significant associations (p Disclosures: No relevant conflicts of interest to declare.

Abstract 5025 Background: Data on outcomes of patients (pts) with myelodysplastic syndromes/myeloproliferative neoplasms (MDS/MPN), especially MDS/MPN-unclassified (MDS/MPN-U), are scarce. Patients/methods: We retrospectively studied pts followed in our center, with MDS/MPN according to WHO 2008 criteria. Because of overlap characteristics of MPN and MDS, pts with systemic mastocytosis associated with MDS (SM/MDS) were also included. Pts with previous MDS or MPN were excluded. Response and disease progression were defined according to IWG 2006 criteria. Results: Twenty-five pts with MDS/MPN were included. Median age was 70 y (range 19–79). Male/female ratio was 1.77/1. Diagnosis was CMML-1 N=7, CMML-2 N=7, JMML N=1, MDS/MPN-U N=8, systemic mastocytosis (SM)/MDS N=2, with one additional pt with CMML subsequently developing SM. At diagnosis, median WBC count was 18.8 G/L (range 3–120), ANC 15.5 G/L (0.6–70), monocytes 1.9 G/L (0.1–16), left shift 16% (0–28), Hb 11.2 g/dL (6–17), platelets 99 G/L (10–680), peripheral and bone marrow (BM) blasts 5% (0–17) and 7% (2–19), respectively (resp.). 25% of pts had platelets count ≥400 G/L. Splenomegaly, B-symptoms and BM fibrosis were present in 23%, 57% and 27% of pts, resp. Karyotype was fav, int and unfav in 55%, 36% and 9% of pts, with −7, +8, del(12)(p11), del(12)(q14;q21), +10, +21, and previously unreported t(9;12)(q13;q13) in 3, 6, and 1 pt each, resp., while +21 and i(17)(q10) appeared during disease progression other than AML transformation. IPSS was low/int-1 and int-2/high in 50% and 50% of pts, resp. JAK2 V617F and CKIT D816V mutations were detected in 2/6 pts and 2/2 SM/MDS pts, resp. 70% and 29% of pts were transfused at diagnosis with PRBC and platelets, resp. Treatment included erythropoiesis stimulating agents (ESAs), low dose chemotherapy, intensive chemotherapy (IC) and azacitidine (AZA) in 40%, 36%, 16% and 48% of pts resp. Response rate to ESAs, IC and AZA was 60%, 14% and 14% resp. Response rate to AZA in CMML-1 pts was 33%. Dasatinib yielded no response in 1 SM/MDS pt with CKIT D816V. 3-year cumulative incidence of AML and median overall survival (OS) in pts with CMML-1, CMML-2 and MDS/MPN-U were 20%, 40% and 0 (P=0.059) and 39, 8, and 20 mo (P=0.50), resp. The pt with JMML died from AML transformation 3 months after diagnosis. 2/3 pts with SM/MDS died from disease progression w/o AML at a median of 10 mo after diagnosis. Median survival after disease progression other than AML transformation was 35, 15 and 14 mo in pts with CMML-1, CMML-2 and MDS/MPN-U, resp. (P=0.88). Cause of death was disease progression other than AML, AML transformation and unrelated to disease in 50%, 50%, and 0 and 80%, 0 and 20% of cases in CMML and MDS/MPN-U, resp. (P=0.10). Percentage of circulating blasts ≥5% was the only independent factor affecting risk of AML transformation in the overall population (P=0.0004). Diagnosis other than CMML-1, WBC ≥30 G/L, % of circulating blasts ≥5% and IPSS high/int-2 were associated with worse survival in univariate analysis (P=0.06, 0.03, 0.04 and 0.08, resp.). No predictive factor of OS was found in multivariate analysis. Conclusion: MDS/MPN are heterogeneous disorders with respect to disease progression and AML transformation. MDS/MPN-U tended to differ from CMML-1 by shorter survival after disease progression other than AML, and from CMML-2 by lower risk of AML transformation. Mortality of pts with MDS/MPN-U was mainly attributed to disease progression without AML transformation. Alternatively to hypomethylating agents, therapeutic options in pts with MDS/MPN-U could include JAK2 inhibitors. Disclosures: No relevant conflicts of interest to declare.

Abstract 4567 Background: Allogeneic hematopoietic cell transplantation (alloHCT) is a potentially curative treatment for patients (pts) suffering of AML with high risk features at diagnosis and beyond 1st complete remission (CR1) it remains the sole rescue treatment. In this retrospective study we analysed the data of all pts (149) allografted concecutively in our BMT unit for AML from 1991 to 2009. The aim of the study was to estimate the outcome in terms of overall survival (OS), disease free survival (DFS), relapse rate (RR) and non relapse mortality (NRM). Sixty-three pts of a median age 35 (8-63) years suffered from de novo (58), secondary (4) and biphenotypic AML (1) were transplanted in CR1. Cytogenetic analysis was available in 40/63 pts (intermediate 32, poor risk 8). Donors were siblings in 55, relative (1 antigen mismatch) in 3, unrelated (4 mismatched with 1–2 alleles) in 8 pts. Graft source was bone marrow (BM) in 12 and peripheral blood (PB) in 51 pts. Fifty-six received a myeloablative (MA) and 7 non- myeloablative (NMA) conditioning regimen. Eighty-six pts were allografted beyond CR1. Disease status was primary refractory (Prim. Ref) in 42/86, CR2 in 15, 1st refractory after re-induction relapse (Rel1) in 23 and advanced (CR3; Rel2+) in 6 pts. Three pts were retransplanted from the original donor for relapsed disease after alloHCT. In the cohort of pts with disease beyond CR1, cytogenetic analysis was available in 71 (favourable 4, intermediate 53, poor risk 14). Donors were siblings in 58, syngeneic (1 antigen mismatch) in 8, unrelated (3 with mismatch) in 17, unrelated double cord blood (CB) in 1, haploidentical in 5 pts. The majority of the pts received mobilized PB (72) as graft source and myeloablative conditioning regimen (82). Results: For pts transplanted in CR1 OS was 63%, NRM 23%, DFS 60% and RR 21% at 13 years. Seventeen pts transplanted before 2000 had an estimated OS and DFS 59%, RR 9% and NRM 35% whereas for forty-six pts transplanted after 1999 the OS was 64%, DFS 61%, NRM 17% and RR 25% at 9 years. DFS for pts in CR1 with an unrelated donor was 47% and 62% for siblings. Myeloablative regimen resulted in 65% DFS while NMA in lower DFS (21%) due to higher RR. According to cytogenetics OS and DFS were 62% and 64% for the intermediate risk group (n=32), 44% and 45% for the poor risk (n=8) respectively. For the cohort of pts transplanted for Prim. Ref. disease (n=42) OS was 20% (plateau at 3 years), DFS 17% (plateau at 2 years), RR 78% and NRM 34% at 12 years. Despite the small number of pts with poor risk karyotype (n=7) the prognosis seemed to be dismal (DFS and OS 0%) versus 25% and 31% respectively for the intermediate risk group (n=28). For pts transplanted in CR2 (n=15) OS was 51% and DFS 46% (plateau at 1year), RR 43% and NRM 16%. For pts in REL1 (n=23) OS was 15%, NRM 56%, DFS 4% and RR 86%. For the 6 pts transplanted for advanced disease (CR3; REL2+) OS was 17%, DFS 17%, RR 67% and NRM 50%. The 5 pts undergone haploidentical alloHCT (2 Prim. Ref., 2 CR2, 1 CR3) after TBI 8/thiotepa/fludara/ATG had OS and DFS 40% at 8 years. One of 3 pts retransplanted is alive in CR and the rest succumbed to their disease. Discussion: In 2010 when the use of alternative transplantation has been expanted the selection of pts upon the best stratification and the timing of the transplantation still remain open questions. The majority of patients are classified in the intermediate risk group with normal karyotype. According to our experience during the last two decades alloHCT for AML in early disease phase (CR1) can offer the best results and possibly cure in a significant number of patients (60%). Transplantation procedures have been continuously improved over time leading to improvement of the outcome mostly in the era of alternative donor alloHCT. For poor risk pts in CR1 and for all pts with AML beyond CR1 (apart from acute promyelocytic leukemia in molecular remission) alloHCT remains the only treatment option. In this cohort of pts results from our data indicate that pts in CR2 may attain long term survival after alloHCT (OS 51%, NRM 16%). Among pts refractory to induction and salvage treatment therapies (> CR2, Prim Ref., REL) a small proportion of pts (15-20%) may be rescued by alloHCT. Disclosures: No relevant conflicts of interest to declare.

We conducted a parallel analysis of somatic mutations in paired IG heavy (HC) and light chain (LC) chain genes in a series of 253 (165 κ- and 88 λ-LC expressing) chronic lymphocytic leukemia (CLL) cases. IGHV/IGKV/IGLV sequences were 100% homologous to germline in 32/33/27% of cases; had 97–99.7% homology in 50/27/37,5% of cases; less than 97% homology in 18/40/35% of cases. IGHV sequences were generally more mutated than the corresponding IGKV/IGKL sequences (average mutation load for IGHV/IGKV/IGLV genes: 94.4/96.4/96.8); 18/253 cases (7.1%) had more mutated LCs than HCs. The most frequent genes among 100% homologous sequences were IGHV4-39 and IGHV1-69 (10/16 and 9/15 cases, respectively) and IGKV1-39/1D-39 (9/19 cases); among IGLV genes, 3/13 IGLV3-21 sequences were unmutated and 9/13 had >98% homology. The most frequent genes in the “

Immunoglobulin kappa (IGK) locus rearrangements were analyzed in 164 κ-light chain (LC) expressing chronic lymphocytic leukemia (CLL) cases and 95 λ-LC expressing CLL cases. In κ-CLL, 151 IGKV-J transcripts were successfully amplified by RT-PCR; 147/151 were in frame; four out-of-frame IGKV-J transcripts were heavily mutated and contained one or more stop codons, presumably introduced by somatic hypermutation; in all four cases the corresponding expressed transcript was also mutated. DNA-PCR identified 24/164 κ-CLL cases (15%) with double IGKV-J rearrangements; 9/24 non-expressed IGKV-J rearrangements were in-frame; 3/9 were mutated. The most frequently used IGKV genes in expressed IGKV-J rearrangements were: 3–20, 1-39/1D-39, 1–5 and 4-1; the IGKV4-1 gene was by far the most frequent in non-expressed IGKV-J rearrangements (9/24 cases, 33%). In λ-CLL, a total of 65 IGKV-J rearrangements were amplified in 59/95 cases (62.0%); six cases had two different rearrangements. IGVK4-1 was the most frequently used gene (14/65 sequences, 21.5%), followed by IGKV1-16 (8 cases,12.3%), 1-33/1D-33 and 2-30 (7 cases each, 10.8%). IGKV-J transcripts were detected by RT-PCR in 10/59 λ-CLL cases with IGKV-J rearrangements, of which four were in-frame and six out-of-frame. Seven IGKV-J rearrangements in λ-CLL had less than 100% homology to germline; 3/7 were in-frame; 6/7 patients had mutated IGHV and 5/7 had mutated IGLV genes. In κ-CLL, biased usage of the IGKJ1/IGKJ2 genes was observed both in expressed (69%) and non-expressed rearrangements (78%); in contrast, in λ-CLL, downstream IGKJ (IGKJ3-5) usage was observed in 32/59 sequences (53%). Nonproductive rearrangements involving the kappa deleting element (KDE) that render the IGK locus inactive were also analyzed. In κ-CLL, 22/147 cases (15%) carried IGKV to KDE rearrangements, while 24/147 cases (16.5%) carried IGKJ-C- INTRON (JKI) to KDE rearrangements. In λ-CLL, IGKV-KDE rearrangements were amplified in 55/94 cases (59%); JKI-KDE rearrangements were amplified in 52/94 cases (56%). IGKV1D-43 was the most frequent gene in IGKV-KDE joints in κ-CLL (4/22 cases); in contrast, the most frequent genes in IGKV-KDE joints in λ-CLL were IGKV3-20 and IGKV2-30 (9/55 and 7/55 cases, respectively). In conclusion, the present study confirms IGK locus rearrangements in the vast majority of λ-LC expressing CLL cases. Differential usage of IGKJ genes along with significant IGKV repertoire differences in both IGKV-J and IGKV-KDE rearrangements between κ- and λ-CLL allude to prolonged IGK locus recombination before CLL clonogenic cells became λ-producers. The inactivation of productive and potentially functional IGKV-J joints by secondary rearrangements indicates a role for receptor editing in shaping the expressed IG repertoire in CLL. Finally, the identification of mutated, non-expressed IGKV-J rearrangements both in κ- and λ-CLL might be considered as evidence for secondary rearrangements occurring after the onset of somatic hypermutation, at least in a proportion of cases.

Imatinib mesylate is the new molecular targeted therapy for Chronic Myeloid Leukemia (CML). Data on fertility and pregnancy are very limited. We report the case of a 27-year old female patient having two pregnancies during therapy with imatinib. CML was diagnosed in December 2001. At diagnosis the disease was in chronic phase with 100% Philadelphia-positive metaphases in bone marrow (BM). She started therapy with IFN/Ara-C and six months later she was still with 100% Ph-positive metaphases. As she had no related transplant donor, she was started on imatinib 400mg/day. Three months later, while in complete hematologic response (CHR), she became pregnant and despite doctor’s advice, she decided not to terminate the pregnancy. At that time she had 54% Ph-positive metaphases in BM. She continued receiving imatinib during the pregnancy without any adverse effects. Ultra-sound examination revealed two normal embryos and amniocentesis yielded normal karyotypes (male and female). In 24th week of gestation she was in CHR with 20% Ph-positive metaphases in BM. Two weeks later (26th week) she delivered two newborns who both died 48 hours later due to prematurity. Laboratory tests showed no abnormalities and all parameters were normal according to their age. Seven months later and while in CHR and complete cytogenetic remission (CCR) but being BCR-ABL positive, the patient became pregnant. Once again, she continued imatinib without any adverse effects. Ultrasound parameters were normal and amniocentesis showed a normal female karyotype. After 37 weeks of gestation she delivered a healthy female newborn. Two months postpartum the child is healthy, with normal laboratory tests. The patient is in CHR, CCR and BCR-ABL positive. In conclusion we report, the case of a woman with CML in whom fertility was not affected by imatinib. In addition, Imatinib caused no abnormalities to the embryos in both pregnancies and pregnancies did not affect the therapeutic effect of imatinib. However, it is under question whether imatinib has any implication in the preterm birth of the first pregnancy.

We analyzed immunoglobulin light chain (IgLC) repertoire in a series of 253 typical, unselected CLL cases and compared CLL IgLC sequences to GenBank IgLC sequences from normal, autoreactive and neoplastic cells. The present series included 165 κ- and 88 λ-CLL cases. Twenty-three functional IGKV genes were used in IGKV-J rearrangements in κ-CLL; the most frequent genes were: 3-20/A27 (25 cases), 1-39-1D-39/O2-O12 (19 cases), 4-1/B3 (16 cases), 1-5/L12 (15 cases), 2-30/A17 (13 cases) and 1-8/L9 (10 cases). There were 55/165 unmutated sequences (33%), 44/165 sequences (27%) with 97-99,6% homology to germline and 66/165 sequences (40%) with less than 97% homology. KCDR3 region length ranged from 6–11 (median, 9) aminoacids (aa). N nucleotides (median 3, range 1–12) were detected in 85/165 rearrangements (51.5%). IGKJ3-5 gene usage was observed in 51/165 rearrangements (30%); interestingly, IGKJ3-5 genes were used in 7/8 IGKV3-11 and 4/5 IGKV1-9 rearrangements. Subsets with homologous and “CLL-specific” KCDR3 regions were identified: IGKV2-30, 5 mutated sequences with identical KCDR3 (MQGTYWPYT), 3/5 associated with IGHV4-34 utilizing heavy chains with a similar HCDR3 of 20 aa; IGKV1-39/1D-39, 3 unmutated sequences with identical KCDR3 (QQSYSTTPLT), all associated with IGHV4-39 utilizing heavy chains with a similar HCDR3 of 19 aa; IGKV1-5, 4 unmutated sequences with identical KCDR3 (QQYNSYPWT), 2/4 associated with unmutated IGHV4-39 utilizing heavy chains with a HCDR3 of unequal length. Twenty-six functional IGLV genes were used in IGLV-J rearrangements in λ-CLL; the most frequent genes were: IGLV2-8/1-2 (14 cases), 3-21/2-14 (13 cases), 2-14/1-4 and 1-44/1-16 (7 cases each). There were 24/88 unmutated sequences (27%), 33/88 sequences (37,5%) with 97-99,6% homology to germline and 31/88 sequences (35%) with less than 97% homology. LCDR3 region length ranged from 8-13 aa (median, 11). N nucleotides (median 3, range 1-15) were detected in 42/88 rearrangements (47.7%). The IGLJ1 gene was used in 18/88 rearrangements (20%); all other rearrangements used the IGLJ3*01/*02 genes. Subsets with homologous and “CLL-specific” LCDR3 regions were identified: IGLV1-44, 2 sequences with very similar LCDR3 (AAWDDSLNGP/QV), both associated with IGHV4-b utilizing heavy chains with a similar HCDR3 of 11 aa; IGLV3-21, 7 sequences all with identical LCDR3 (QVWDSGSDHPWV), 3/7 associated with IGHV3-21 utilizing heavy chains with a similar HCDR3 of 9 aa. These results document that IgLC repertoire in CLL is biased by both intrinsic molecular processes as well as selection after LC expression. Genes that have been reported to be overexpressed in the normal and autoimmune disorders were also found to be overrepresented in the CLL repertoire, often with “CLL-specific” molecular features. Finally, the existence of subgroups with homologous CDR3 regions associated with similar heavy chains provides further evidence for the role of antigen selection in CLL pathogenesis.