BackgroundAtypical BCR-ABL1 transcripts are detected in less than 5% of patients diagnosed with chronic myeloid leukaemia (CML), of which e19a2 is the most frequently observed, with breakpoints in the micro breakpoint cluster region (μ-BCR) and coding for the p230 BCR-ABL1 protein. p230 CML is associated with various clinical presentations and courses with variable responses to first-line imatinib.Case presentationHere we report a case of imatinib resistance due to an E255V mutation, followed by early post-transplant relapse with a T315I mutation that achieved a persistent negative deep molecular response (MR5.0) after treatment with single-agent ponatinib. Using CastPCR, we could trace back the presence of the T315I mutation to all the RNA samples up to the detection of T315 mutation by Sanger sequencing shortly after allogeneic hematopoietic stem cell transplantation (HSCT).ConclusionThis case illustrates the major interest of ponatinib as a valid treatment option for e19a2 CML patients who present a T315I mutation following relapse after HSCT.Electronic supplementary materialThe online version of this article (10.1186/s12885-018-5100-4) contains supplementary material, which is available to authorized users.

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Chronic Graft versus Host disease (cGVHD) is a major limiting factor for the success of allo-HSCT. In this prospective study we aimed to evaluate the association between the kinetics of Regulatory T cells (TREG) and Conventional CD4+T cells (TCON) reconstitution and the emergence of cGVHD. We performed a detailed phenotypic analysis by multiparametric flow cytometry using fresh blood from 39 patients undergoing unrelated donor HSCT after a reduced intensity conditioning regimen containing ATG over a 2 year period, representing a total of 213 samples analyzed. GVHD prophylaxis consisted of cyclosporine plus mycophenolate mofetil. 11 patients were excluded due to disease relapse and/or death due to infection or acute GVHD in the first 9 months post-HSCT. We observed indiscriminately low numbers of TREG (CD4+CD127lowFoxp3+CD25bright) until mo 6 after HSCT and reduced TREG numbers in patients developing cGVHD (GVHD+) versus those who did not (GVHD-) (p=0.02 at mo 9). We further studied the dynamics of TREG subset reconstitution. CM TREG (CD45RA-CD62L+) was the predominant population in both patient groups. EM (CD45RA-CD62L-) was the second most abundant TREG subset. CM and EM TREG numbers were similar between patient groups. EMRA (CD45RA+CD62L-) TREG remained very low throughout the follow-up but were significantly increased at mo 9 in GVHD+ (p= 0.03). Interestingly, Naïve TREG (CD45RA+CD62L+CD95-) started to emerge at mo 9 and were significantly reduced in GVHD+ patients at mo 12 (0.71 vs 0.14 cells/µl; p=0.02) The Stem Cell Memory subset (SCM), identified as CD45RA+CD62L+CD95+, is thought to be self renewing and multipotent, being able to differentiate into CM, EM and TEMRA memory subsets. While in GVHD- SCM TREG started to emerge at mo 9, this subset remained low in GVHD+. Statistically significant differences were observed at mo 18 (0.91 vs 0.15 cells/µl; p=0.02). To ascertain the role of thymic output in TREG reconstitution we quantified CD31+ naïve TREG. Recent Thymic Emigrant cells (RTE) TREG were significantly reduced in GVHD+ at mo 12 (0.8 vs 0.16cells/µl; p=0.02) and at mo 18 (1.93 vs 0.28 cells/µl; p=0.02). In order to clarify whether both thymic output and peripheral expansion were contributing factors to decreased Naïve TREG, we quantified proliferation using Ki-67. TREG from GVHD+ patients proliferated less from months 2 to 18 reaching statistical significance at mo 9 and 12 (p= 0.003 and 0.02, respectively), suggesting that decreased TREG numbers are at least partly due to reduced peripheral expansion. Taken together, these observations suggest a compromised peripheral expansion and de novo generation of TREG through thymic output in cGVHD patients. Of note, susceptibility to apoptosis was not increased in TREG from GVHD+ patients, as Bcl-2 levels tended to be higher in relation to GVHD- patients. In the CD4+Foxp3- TCON population, we observed a clear predominance of EM in all patients. These cells, together with CM, are the first to appear at similar levels in both patient groups. EMRA emerged at higher numbers in GVHD+, although the results did not reach statistical significance. Naïve and RTE TCON started to emerge after mo 6 in GVHD-. Interestingly, these cells remained much lower in GVHD+ vs GVHD- patients throughout the follow-up, reaching statistical significance at mo 12 (p=0.03 for naïve; p=0.03 for RTE TCON). SCM TCON remained very low in all patients showing a tendency to be reduced in GVHD+ when compared to GVHD-. This difference reached statistical significance at mo 18 (6.85 vs 2.13 cells/µl; p=0.02). The small number of TREG and TCON subsets in GVHD+ patients were unlikely due to increased susceptibility to apoptosis, as assessed by Bcl-2 expression. The low numbers of cells within naïve and SCM TREG gates precluded the analysis of Bcl-2 expression in these subsets. Bcl-2 tended to be increased in total TREG CM and EM in GVHD+. In TCONs, Bcl-2 levels were similar in both patient groups. In summary, our data in patients developing cGVHD suggest that decreased thymic output and increased differentiation into terminally differentiated effector cells may have a negative impact on the number of naïve and SCM TCON and TREG. We speculate that the inability to generate and maintain the more immature TREG subsets may lead to decreased TREG numbers after 6 months post transplant, potentially resulting in decreased control of effector T cells contributing to the development of cGVHD. Disclosures Ritz: Kiadis: Membership on an entity's Board of Directors or advisory committees.

Reduced-intensity conditioning (RIC) regimens is being increasingly used for allogeneic haematopoietic stem cell transplantation (HSCT) in recent years for follicular lymphoma (FL) and chronic lymphocytic leukaemia (CLL). The lower risk of transplant related toxicity associated with RIC regimens makes allogeneic transplantation applicable to patients (pt) with haematological malignancies resistant to conventional therapy and/or in poor medical condition, while preserving the putative graft-versus-tumour effect (GVT). From May 99 to January 07, 31 recipients were studied. Twenty two pt (71%) with FL: 14pt CR2; 4pt PR; 2pt resistant relapse; 1pt graft failure; 1pt advanced disease and 9pt with CLL in PR, underwent to allogeneic HSCT from HLA-matched (31) sibling donors, after treatment with fludarabine 30 mg/m2/d iv × 5 + busulfan 4 mg/Kg/d × 2 ± anti-T lymphocyte globuline (10 mgKg/d) × 4 or fludarabine 30 mg/m2/d iv × 5 + cyclophosphamide (1g/m2/d) × 3 + alentuzumab 20mg/d × 5 (pt with graft failure). Stem cell source was unmanipulated peripheral blood progenitor cells. Median number of CD34+ cells infused: 6,0 × 106/Kg (3,3–12,0). CsA 3 mg/kg/d was given from day-1 until day +90 then tapered progressively, associated with Micophenolate mofetil d0 until d+84. Results: In 31 evaluable pt hematological recovery have a median time to neutrophils ≥0.5×109/L of 13d. (3–37+) and platelets ≥20 × 109/L of 11 d. (9–105+). Transfusion requirements: median: 4 RBC units and 2 platelet transfusions per pt. Median day of discharge: d.15. Hepatic veno-occlusive disease and severe mucositis did not occur. Chimerism analysis (day +28) showed donor engraftment in all pts–mixed in 5 and full in 26 (full in graft failure pt after second transplant). 36±9,2% pts developed clinical grade 2–4 aGvHD and 68,9±10,8% developed cGvHD. Four pt had relapsed/progressive disease. Non relapse mortality occurred in 3 pts with low performance status and chemotherapy resistant disease. At 3,6 years median overall survival (OS) is 85.2±6.9%. Twenty eight pts are alive with a median follow-up of 3.6 years. Progression free survival (PFS) at 3 years is 84,1±7,1 %. Aimed to induce GvT effect, one pt with progressive disease, without GvHD was assigned to receive donor lymphocyte infusions (DLI) at regular intervals achieved stable disease. These results show that RIC regimen are well tolerated, have a low risk of transplant related mortality and are able to ensure a sustained engraftment. RIC is becoming the standard approach in allogeneic HSCT for FL/CLL and the increased risk of late disease progression might be manipulated with GvT effect associated with posterior DLI.