ALBERT

Description: The pseudo tyrosine kinase receptor 7 (PTK7) is an orphan tyrosine kinase receptor assigned to the planar cell polarity pathway. It plays a major role during embryogenesis and epithelial tissue organization. Here we found that PTK7 is also expressed in normal myeloid progenitors and CD34+ CD38− bone marrow cells in humans. We performed an immunophenotyping screen on more than 300 patients treated for hematologic malignancies. We demonstrated that PTK7 is expressed in acute myeloid leukemia (AML) and is mostly assigned to granulocytic lineage differentiation. Patients with PTK7-positive AML are more resistant to anthracycline-based frontline therapy with a significantly reduced leukemia-free survival in a multivariate analysis model. In vitro, expression of PTK7 in cultured leukemia cells promotes cell migration, cell survival, and resistance to anthracycline-induced apoptosis. The intracellular region of PTK7 is required for these effects. Furthermore, we efficiently sensitized primary AML blasts to anthracycline-mediated cell death using a recombinant soluble PTK7-Fc protein. We conclude that PTK7 is a planar cell polarity component expressed in the myeloid progenitor compartment that conveys promigratory and antiapoptotic signals into the cell and that represents an independent prognosis factor of survival in patients treated with induction chemotherapy.

Description: Invasive Pulmonary Aspergillosis (IPA) is a major cause of morbidity in patients with hematological malignancies. The aim of this single centre retrospective study was to determine the impact of prior IPA on outcome after RIC Allo-SCT. All cases of proven or probable IPA diagnosed prior to performance of RIC Allo-SCT at the Paoli-Calmettes Institute Cancer Centre from 1 January 2000 through 31 December 2007 were included. 28 patients were identified among 434 patients undergoing Allo-SCT. Patients’ data were collected from a maintained database and by retrospective clinical chart review. Twenty eight cases were identified. Gender: M/F (16/12); median age at diagnosis was 53 years (18–65). 23 patients (82%) had acute myeloid leukemia. In all patients, IPA was diagnosed according to standard procedures. IPA therapy included: 20 patients (71%) were receiving Voriconazole; 4 patients Itraconazole, and 4 patients with an association of different antifungal drugs including Caspofungine, Liposomal Amphoteracine-B, or Posaconazole. The median duration of treatment prior to RIC was 8 months range (1–16). The median time between the diagnosis of IPA and transplantation was 6 months (1–27); 20 patients (71%) received a graft from a family donor, 4 patients had a MUD, and 4 patients received cord blood cells donors. 21 patients (75%) received a conditioning regimen with Fludarabine Busulfan and ATG, and 4 patient (14%) received a conditioning with Fludarabine Cyclophosphamide and TBI, 3 patients (11%) Fludarabine and TBI, Most patients (n=25; 89%) received or continued a secondary prophylaxis against aspergillosis at time of Allo-SCT: 17 (68%) had a prophylaxis with Voriconazole; 3 patients Itraconazole, 2 patients Posaconazole. After RIC transplantation only 4 patients (14%) had reactivation of their IPA. The latter 4 patients were experiencing severe acute GVHD treated with high dose corticosteroids. None of these patients died of IPA. Actually 18 patients (64%) are still alive with a median follow up of 23.5 months (12.6–48.5). Overall survival at 2 years was 59% [95%CI, 43–83]. In all, 3 patients died from other causes not directly related to IPA, 2 patients because of relapse or disease progression, and one due extensive chronic GVHD. In comparison to literature in standard myéloablative allo-SCT setting, these data suggest that adequately treated and controlled IPA prior to RIC Allo-SCT do not worsen outcome. The latter is likely due to multiple changes in transplantation practice, including the RIC nature of the conditioning regimens, the infusion of peripheral blood stem cells, rapid diagnosis of IPA, and use of modern efficient antifungal drugs.

Description: Elderly patients with acute myeloid leukemia (AML) have a very poor prognosis, explained by the biologic characteristics of the disease but also by host-related factors including comorbidities. The objective of this study was to determine the prognostic role of comorbidities in this population. Between 1995 and 2004, 133 patients aged 70 years or older with newly diagnosed acute non promyelocytic leukemia, were treated with intensive induction chemotherapy (“3+7” regimen) at a single institution. Comorbidities at diagnosis were retrospectively evaluated using an adapted form of the Charlson Comorbidity index (CCI) (Sorror et al., Blood2004;104:961–968). Seventy-five patients achieved a complete response (CR) (56.4%) and 23 patients died during induction chemotherapy (17.3%). Comorbidity scores at diagnosis were 0 for 83 patients (68%), 1 for 16 patients (13.1%), and 〉 1 for 23 patients (18.7%). The median overall survival (OS) was 9 months (95% CI: 6–13) and was significantly poorly affected by high leukocyte count (≥ 30 G/l) and elevated serum creatinine level (〉1.5 x upper normal limit). By multivariate analysis, we identified 4 adverse prongnostic factors for CR: unfavorable karyotype, leukocytosis ≥ 30 G/l, expression of CD34 on blast cells and a CCI 〉 1. Age was not found to be a significant prognosic factor for either CR or survival. When a score of 0 or 1 was affected to each prognostic variable, patients could be separated into a low-risk group (score= 0–1; 22% of patients), an intermediate-risk group (score= 2; 41% of patients) and a high-risk group (score≥3 ; 37% of patients), having 87%, 63% and 37% chance of achieving CR following conventional induction chemotherapy, an 8-week mortality rate of 9%, 22% and 34% (p=0.02), and a 2-year overall survival rate above 50%, less than 30% and 15%, respectively (p=0.01). Our results show that comorbidity is an important prognostic factor for elderly patients with AML which may help the physicians’ decision making. Its capture at diagnosis should be prospectively evaluated in larger study for its future use for risk-stratification among elderly patients treated for AML.

Description: GVHD remains a matter of concern after RIC allo-SCT. Our knowledge of aGVHD risk factors is still primarily based on historical analyses performed in the myeloablative allo-SCT setting. Thus, modern approaches to predict the occurrence/severity of aGVHD are needed. This study aimed to identify a plasma protein signature correlating with occurrence of early aGVHD. We performed Surface-Enhanced Laser Desorption/Ionization-time (SELDI) of flight mass spectrometry profiling of plasma from 88 patients who received a RIC allo-SCT from HLA-identical siblings. Patients characteristics were: median age was 51 (range, 18–70) y. 41 patients (47%) had a myeloid malignancy, whereas 30 patients (34%) had a lymphoid malignancy. The remaining 17 patients (19%) were treated for metastatic non-hematological malignancies. The majority of patients (n=70, 80%) had an advanced disease with high risk clinical features precluding the use of standard myeloablative allo-SCT. All patients (100%) received G-CSF-mobilized PBSCs. The RIC regimen consisted of fludarabine, busulfan and ATG in 53 patients (60%) and low dose irradiation in 35 patients (40%). With a median follow-up of 400 (range, 127–829) d, 20 patients (23%) experienced early (prior to day 35 after allo-SCT) grade 2–4 acute GVHD (12 grade 2 and 8 grade 3–4). Denatured plasma samples (collected at a median of 28 days after allo-SCT) were incubated with H50 and CM10 ProteinChip arrays and subjected to SELDI analysis. Patient population was divided into a training (n=59) and a validation set (n=29). In the training set, 36 protein peaks were differentially expressed according to early aGVHD occurrence. By combining partial least squares and logistic regression methods, we built a multiprotein model that correctly predicted outcome in 96% of patients (14/14 patients with early aGVHD; specificity, 96%). The observed correct prediction rate in the validation set was 69% with a sensitivity of 67%, and a specificity of 70%. While negative predictive value of the model was only 36%, predictive positive value was estimated to 89% in the validation set. The performances of the model remained very similar after iterative (500 times) random resampling (correct prediction rate: 74%, median sensitivity: 48%, median specificity: validation set: 83%). Univariate and multivariate analyses of known risk factors (demographic features, diagnoses and transplant procedures) for occurrence of an early grade 2–4 acute GVHD did not show any statistically significant difference between the group of 20 patients who had early grade 2–4 aGVHD as compared to the remaining patients, and suggested that the multiprotein index is likely to be the only independent prognostic parameter. Major components of this multiprotein index are currently being characterized and will be presented. Obviously, larger prospective studies are still needed, but our results already suggest that proteomic analysis of plasma will prove increasingly important in the early and clinical diagnosis of aGVHD allowing improving patient outcome.

Description: Abstract 443 Background: AZA is the current standard of care for IPSS int-2 and high (“higher”) risk MDS. However, most pts will experience primary or secondary treatment failure. To date, there is no published data on the outcome of those pts. Design: 565 patients from 4 independent cohorts, who had not responded to or relapsed after response to AZA, were included. The datasets included 3 prospective trials (AZA001 (n=138; Lancet Onc, 2009), J9950 (n=26; Cancer Res 2006), J0443 (n=32; NCT00101179) trials) and data from the French ATU compassionate use program (n=369). Two cohorts administered AZA as single agent (AZA001 and French ATU) while two combined AZA with a histone deacetylase inhibitor (J9950, sodium phenylbutyrate; J0443, entinostat). 339 patients had no clinical response to AZA while 226 relapsed after initial response. The influence of pre treatment variables and salvage treatment options on the outcome after AZA failure was analyzed. Survival was measured from the date of failure of AZA. Results: The cohort included 475 MDS (including 117 RAEB-T) and 90 sAML (AML secondary to MDS). Median age was 69 years and the median number of cycles of AZA before failure was 6 (range [1-41]). Patients were randomly assigned to one learning (n=377) and one validation (n=188) set stratified on the number of deaths. There was no difference in the baseline characteristics of the 2 sets. With a median follow-up of 15 months after AZA failure, the median overall survival (OS) was 6 months and the 2-year probability of OS was 15%. The multivariate model constructed with the learning set showed that age (HR 1.02/y, 95%CI [1.01-1.03], p=0.002), sex (female 7 months vs male 5.6 months HR 1.3 [1.01-1.67] p=0.04), cytogenetics (favorable 8 months vs intermediate 5.9 months HR 1.49 [1.06-2.08] p=0.02, vs high risk 4.6 months HR 2.19 [1.63-2.91] p

Description: The outcome of classical Hodgkin lymphoma (cHL) patients may be related to the tumor microenvironment, which in turn may be influenced by Epstein-Barr virus (EBV) infection. To characterize the cHL microenvironment, a set of 63 cHL tissue samples was profiled using DNA microarrays. Their gene expression profile differed from that of histiocyte T cell–rich B-cell lymphoma (H/TCRBCL) samples that were used as controls, mainly due to high expression of PDCD1/PD-1 in H/TCRBCL. EBV+ cHL tissues could be distinguished from EBV− samples by a gene signature characteristic of Th1 and antiviral responses. Samples from cHL patients with favorable outcome overexpressed genes specific for B cells and genes involved in apoptotic pathways. An independent set of 146 cHL samples was analyzed using immunohistochemistry. It showed a significant adverse value in case of high percentage of either TIA-1+-reactive cells or topoisomerase-2+ tumor cells, whereas high numbers of BCL11A+, FOXP3+, or CD20+ reactive cells had a favorable influence. Our results suggest an antitumoral role for B cells in the cHL microenvironment and a stronger stromal influence of the PD1 pathway in H/TCRBCL than cHL. The observation of Th1/ antiviral response in EBV+ cHL tissues provides a basis for novel treatment strategies.

Description: Abstract 3164 Background. Cryopreservation of Hematopoietic Stem Cells (HSC) is a standard procedure for both allogeneic cord blood and autologous HSC transplantation. PBSC are routinely used for the latter; the graft is usually thawed bedside and shortly reinfused without any manipulation. Infusion-related toxicity is usually mild, although severe reactions have been reported, mostly in patients with co-morbidities at baseline. Indeed, cell viability decreases over time after thawing. Washing the thawed graft was reported to improve both clinical tolerance and cell viability. It is however not routinely carried out as it is time-consuming and may result in clinically relevant HSC loss. The fully automated solution SmartWash™ (Biosafe, Switzerland) is designed to wash a thawed graft in a clinical setting. We report here its validation through an international, prospective, multicenter trial. Methods. A pre-clinical and a clinical study were designed and sequentially carried out in 4 European Centers (Basel, Florence, Marseille and Murcia) to assess the reproducibility and feasibility of the SmartWash™ system. During the pre-clinical phase, 10 PBSC Units/Center (total: 40 procedures) from patients who permanently lost the indication to the transplant were thawed and split in two aliquots: one was washed and the other was left unmanipulated. A shared protocol targeted to assess recovery, viability and stability of thawed PBSC at different time-points was developed; in particular, an ISHAGE-modified flow cytometry method was implemented in order to improve the evaluation of thawed CD34+ cells. Clinical grade hydroxyethyl starch (Voluven® 6%,Fresenius, Germany) was validated and used as washing solution. Following the validation of the method, a prospective, observational clinical trial was started after approval of the competent Authorities. Twenty-five consecutive patients across the 4 Centers underwent an autologous PBSC transplantation according to the local practice and indications; all the grafts were washed by Smartwash™. Cellular recovery, clinical tolerance and transplant outcome were collected according to a shared protocol. Results. All the washing procedures were completed without any laboratory problems in the participating centers. In the pre-clinical study the recovery of total CD34+ cells in the washed product was 101.2%±27.0 as compared to the pre-freezing value, with a viability of 87.2%±10.0, always showing an advantage over the unwashed samples at all considered time-points (0,1,4 and 24 hours after washing, respectively, Fig. 1). The workload of the procedure, as compared to the unwashed process, resulted in a 40 minutes prolongation for each thawed product, among which only 12 minutes were devoted to active time work, whilst 28 minutes consisted in automatic processing. In the clinical trial, patients received a median of 4.32×106 CD34+ cells/Kg (1.32–27.92), as measured before cryopreservation. Recoveries of both total and viable CD34+ cells after thawing and washing are reported in the Fig. 2, replicating the results of the pre-clinical study. Infusion-related toxicity was negligible (1 grade-1 toxicity out of 25 patients). All patients achieved both neutrophil and platelet sustained engraftment at a median of 11 (9–19) and 13 (7–19) days, respectively. Conclusions. SmartWash™ is an efficient and safe automated method to wash thawed PBSC grafts in a closed, clinical-grade system. Its feasibility and reproducibility was confirmed in an international, multicenter, clinical study. The washed product is stable and clinically feasible for several hours after thawing. Disclosures: Saccardi: Biosafe SA: Research Funding. Boieri:Biosafe SA: Research Funding. Loutan:Biosafe SA: Employment.

Description: Abstract 1180 Poster Board I-202 Benjamin Esterni, Didier Blaise Background: Allogeneic stem cell transplantation (ALLO) is the only curative therapy for many hematological malignancies. For many of these diseases, the median age at diagnosis is around the sixth decade of life, precluding myeloablative ALLO (MAC-ALLO). RIC-ALLO is less toxic and it has been performed in elderly pts, mainly affected by acute leukemia. Finally, comorbidities index seem to predict treatment related mortality (TRM) and overall survival (OS). Patients and methods: From 2001 and 2008, 67 pts older than 60 years (median age 63 y, range 60-70) received RIC-ALLO. Diseases were: acute myeloid leukemia 45%, multiple myeloma 18%, chronic lymphocytic leukemia 12%, non-Hodgkin lymphoma 10%, myelodysplasia 6%, plasmacellular leukemia 3%, others 6%. Disease status at RIC-ALLO was: complete remission 54%, partial remission 16%, and active disease 30%. RIC consisted of fludarabine-based with thymoglobulin 64%, or low-dose TBI-based 36%. Donors were: HLAid sibling 73%, matched unrelated 21%, and cord blood 6%. Previous autologous transplant was performed in 59% of pts. The median number of CD34+ and CD3+ cells infused was 5 (range 1-9.4) and 296 (range 84-704), respectively. Karnofski score was 60-80% in 25% and 90-100% in 75%; HCT-CI was 0 in 33%, 1-2 in 33%, and more than 3 in 34%; PAM score was 8-16 in 9%, 17-33 in 65%, 24-30 in 22%, and more than 31 in 3%; EBMT score was 2 in 22%, 3 in 36%, 4 in 28%, more than 5 in 12%. Results: The median follow-up was 22 months. The 2-y OS and PFS were 66.8% (IC95 [55.5-80.4]) and 52.4% (IC95 [39.5-69.5]), respectively. Grade II-IV acute graft versus host disease (aGVHD) and chronic GVHD (cGVHD) incidence were 49% and 43%, respectively. Early infections were fever of unknown origin in 42% of pts, bacterial infection in 6 cases, pneumonia in 8, and viral infections in 14. The early infection-related mortality was null. Late infections were bacterial in 3 cases, pneumonia in 1, viral infections in 6, and candidemia in 1. Seven pts died from late infective complications. Overall, the cause of death was toxicities in 18 pts and disease progression in 6 pts. The 100-d and 1-y TRM were 6.35% (IC95 [0.278-12.4]) and 24.2% (IC95 [12.9-35.4]), respectively. In univariate analysis, HCT-CI, EBMT score, and PAM score did not influence TRM or OS. Furthermore, age (60-65 vs 66-70) was not related to TRM. Conclusions: The aim of this retrospective study was to verify if TRM was excessively high in elderly pts, affected from several haematological diseases and receiving ALLO from different donors and after different RIC. A secondary objective was to evaluate if several comorbidities index could predict TRM and OS. This heterogeneity should be regarded as a more realistic view of general population. TRM was acceptable and not different when compared to younger pts as reported in literature. Furthermore, neither comorbidities index nor age help segregate a group of pts with different TRM. Disclosures: No relevant conflicts of interest to declare.

Description: Background Allogeneic stem cell transplantation (SCT) remains the only curative option in poor risk chronic lymphocytic leukemia (CLL). Reduced intensity conditioning regimens (RIC) have improved outcome and enlarged number of patients eligible to SCT. However it is still controversial whether patient characteristics, CLL treatment, conditioning regimen or GVH prophylaxis influence the outcome. Here we present the analysis of our cohort of CLL patients who underwent RIC SCT. Patients and methods This is a retrospective single center study Primary endpoint was overall survival; secondary endpoints were cumulated incidence of relapse and non-relapse mortality. Results From 2000 to 2012, Thirty-nine patients with a sex ratio of 2 were transplanted. At time of transplantation, median age was 59 years (27-68), 82% patients displayed performans status (PS) of 0 and 20% had a comorbidity score ≥3. More than half patients (54%) were Fludarabine refractory and FISH 17p deletion was found in 10 out of 23 tested patients (43%). Median delay between diagnosis and transplant was 92 months (4-182). Patients received a median of 3 (1-9) prior therapies. Last treatment before transplant was fludarabine-based in 12 patients, alemtuzumab in 13, and other (RCHOP, R-Bendamustine, RDHAP) in 14. Median delay between the last treatment and transplantation was 2 months (1-33). At time of transplant, overall response rate was 82% including 33% complete response (CR). Four-color phenotypic MRD was negative (90% was obtained in all patients except one, after a median of 90d (19-180) post transplant. Acute and chronic extensive GVHD occurred in 44% and 38% patients respectively. With a median follow-up of 59 months, median overall survival (OS) was 97 months, 2- and 5-years OS were 72% and 59% respectively. Fifteen patients died, 7 from GVHD, 4 from sepsis, 1 from CLL and 3 from other causes. 2- and 5-years cumulated incidences (CI) of non-relapse mortality (NRM) were 25.6% and 38.3% respectively. Three patients relapsed after transplant, 2 are still alive 82 and 98 months post-transplant. 2- and 5-years cumulated incidences of relapse were 2.8% and 6.8% respectively. In univariate analysis, disease status at transplant and number of prior therapies were significantly associated with better OS (p=0.0009 and p=0.03 respectively), whereas a trend to correlation between PS and OS was observed (p=0.063). In multivariate analysis, PS and achievement of CR or PR before transplant correlated significantly to OS (p=0.015 and p=0.05 respectively). Clearance of MRD before transplant perhaps suggested a better survival (p=0.158). Conclusion This retrospective study highlights the ability of allogeneic transplant to improve OS in otherwise very poor prognosis CLL patients and deserve further investigations. Disclosures: Aurran-Schleinitz: Roche: Honoraria.

Description: Background With the improvement of cancer therapy, long term survivors are more exposed to the risk of secondary myeloid neoplasm including myelodysplasia and acute leukemias. The WHO 2008 classification individualize the therapy related acute myeloid leukemia (AML) as a specific entity and highlight the role of chemotherapy/radiotherapy in the pathogenesis of the disease. There is a demonstrated link between specific therapeutic agents, recurrent genetic lesions (such as t(15;17), CBF AML, monosomy 7, …), and outcome. However, most of the published series include multiple types of primary cancer and treatments. This heterogeneity may represent a problem as only limited data are available in patients with specific cancer subtypes, such as the most frequent one, breast cancer. This may be important at a public health level but also to homogenize age and types of prior treatment. Moreover, the complexity of t-AML could not be resumed to the association between treatment and karyotype, as some patients developed AML without chemotherapy, suggesting a potential predisposition to AML. In the present report, we focused on AML arising after breast cancer (BC) and describe the characteristics and outcome of this population. Patients and Methods This is a retrospective multicenter study. Patients were included if they had the diagnosis of breast cancer preceding diagnosis of acute myeloid leukemia whatever may be the interval between the 2 cancers and whatever treatment was administered for BC. All patients with AML were treated with induction chemotherapy. Clinical and biological data for both cancers were collected. Patient’s characteristics and results were compared with age, cytogenetic risk, and optionally sex matched (if possible) de novo AML with a 2/1 ratio. Results 408 patients were analyzed, including 136 AML associated with BC and 272 de novo AML. The median age at diagnosis of BC was 50 years. 47% of patients had invasive ductal carcinoma. Treatment of BC included chemotherapy in 81% of cases, radiotherapy in 91% of cases, and surgery in 99% of cases. The median time between BC and AML was 2.8 years. Median WBC was 3.4G/l and median platelets count was 49G/l. For AML-BC, Cytogenetics were abnormal in 82% of cases including 12% Complex Karyotype, 12% t(15;17), 17% CBF, and 20% MLL translocations. The recent introduction of taxanes in the treatment of BC did not seem to change the frequencies of these aberrations. With the exception of sex ratio, there was no significant difference of baseline characteristics as compared to control group. Regarding induction chemotherapy, CR rate was 81% and 8-week mortality 11.6%. In the control cohort, CR rate was 83% (p=NS) and 8 week mortality 7% (p=NS). Allogeneic transplantation was performed in 21% and 17% of patients respectively. In cytogenetic adjusted survival analysis, median overall survival and relapse free survival were similar between AML-BC and de novo AML for favorable risk (OS and RFS not reached for both groups; p=0.06 and p=0.3 respectively), and unfavorable risk (OS 12m vs. 13m p=NS, RFS 8m vs. 9m p=NS) groups. Interestingly, there was a difference for intermediate cytogenetics group, with median OS (21 months vs. 38 months p=0.01) and median RFS (14 vs. 25 months p=0.04). Difference was also confirmed for cumulative incidence of relapse (1 year probability 41% vs. 22%, p=0.04). Frequency of FLT3, NPM1, and CEBPA mutations were only available in a subset of patients. Only 1/12 pts in the intermediate AML-BC group was FLT3 mutated and 1/12 pt had isolated NPM1 mutation. In the de novo cohort, 11/28 pts had FLT3 mutations and 8/21 pts had isolated NPM1 mutation. Conclusion Our data showed that in AML arising after BC, the prior BC do not appear to impact the outcome in favorable and unfavorable cytogenetic risk groups. However, this is different for intermediate risk cytogenetics, our data suggesting a poorer outcome of AML-BC and potentially a different mutational profile. Regarding AML susceptibility, a matched pair analysis comparing the AML-BC and BC without AML will also be presented at the meeting. Disclosures: No relevant conflicts of interest to declare.