Search

A role of endothelial cells (ECs) in Primary Myelofibrosis (PMF) was supposed since JAK2 mutation was found in endothelial precursor cells (EPCs) and in ECs captured by laser microdissection. By Cell Search method, the circulating endothelial cells (CECs) from 14 PMF patients and 5 healthy controls have been isolated and compared by NGS with CD34+Hematopoietic stem and progenitors cells (HSPCs) for panel of 54 myeloid-associated mutations. PMF patients had higher levels of CECs. No mutation was found in HSPCs and CECs from controls, while CECs from PMF patients presented several somatic mutations. 72% of evaluable patients shared at least one mutation between HSPCs and CECs. 2 patients shared the JAK2 mutation, together with ABL1, IDH1, TET2 and ASXL1, KMT2A, respectively. 6 out of 8 shared only NON MPN-driver mutations: TET2 and NOTCH1 in one case, individual paired mutations in TP53, KIT, SRSF2, NOTCH1 and WT1, in the other cases. In conclusion, 70% of PMF patients shared at least one mutation between HSPCs and CECs. These latter harbored several myeloid-associated mutations, besides JAK2V617F mutation. Our results support a primary involvement of EC in PMF and provide a new methodological approach for further studies exploring the role of the “neoplastic” vascular niche.

Melanoma is an immunogenic neoplasm infiltrated by T cells, although these adaptive T cells usually fail to eradicate the tumor. Plasmacytoid dendritic cells (PDCs) are potent regulators of the adaptive immune response and can eliminate melanoma cells via TLR-mediated effector functions. The PDC compartment is maintained by progressively restricted bone marrow progenitors. Terminally differentiated PDCs exit the bone marrow into the circulation, then home to lymph nodes and inflamed peripheral tissues. Infiltration by PDCs is documented in various cancers. However, their role within the melanoma immune contexture is not completely known. We found that in locoregional primary cutaneous melanoma (PCM), PDC infiltration was heterogeneous, occurred early, and was recurrently localized at the invasive margin, the site where PDCs interact with CD8+ T cells. A reduced PDC density was coupled with an increased Breslow thickness and somatic mutations at the NRAS p.Q61 codon. Compared with what was seen in PCM, high numbers of PDCs were found in regional lymph nodes, as also identified by in silico analysis. In contrast, in metastatic melanoma patients, PDCs were mostly absent in the tumor tissues and were significantly reduced in the circulation, particularly in the advanced M1c group. Exposure of circulating PDCs to melanoma cell supernatant (SN-mel) depleted of extracellular vesicles resulted in significant PDC death. SN-mel exposure also resulted in a defect of PDC differentiation from CD34+ progenitors. These findings indicate that soluble components released by melanoma cells support the collapse of the PDC compartment, with clinical implications for refining TLR agonist–based trials.

Background: The heterogeneity of metastatic breast cancer (MBC) necessitates novel biomarkers allowing stratification of patients for treatment selection and drug development. We propose to use the prognostic utility of circulating tumor cells (CTCs) for stratification of patients with stage IV disease. Methods: In a retrospective, pooled analysis of individual patient data from 18 cohorts, including 2436 MBC patients, a CTC threshold of 5 cells per 7.5 ml was used for stratification based on molecular subtypes, disease location, and prior treatments. Patients with >= 5 CTCs were classified as Stage IVaggresive, those with < 5 CTCs as Stage IVindolent. Survival was analyzed using Kaplan-Meier curves and the log rank test. Results: For all patients, Stage IVindolent patients had longer median overall survival than those with Stage IVaggresive (36.3 months vs. 16.0 months, P < 0.0001) and similarly for de novo MBC patients (41.4 months Stage IVindolent vs. 18.7 months Stage IVaggresive p < 0.0001). Moreover, patients with Stage IVindolent disease had significantly longer overall survival across all disease subtypes compared to the aggressive cohort: hormone receptor-positive (44 months vs. 17.3 months, P < 0.0001), HER2-positive (36.7 months vs. 20.4 months, P < 0.0001), and triple negative (23.8 months vs. 9.0 months, P < 0.0001). Similar results were obtained regardless of prior treatment or disease location. Conclusions: We confirm the identification of two subgroups of MBC, Stage IVindolent and Stage IVaggresive, independent of clinical and molecular variables. Thus, CTC count should be considered an important tool for staging of advanced disease and for disease stratification in prospective clinical trials.

Background: The European Pooled Analysis of CTC (EPAC) in metastatic breast cancer, based on 1,944 individual data from patients with various tumor types and clinical settings (Bidard et al, Lancet Oncol 2014), has established CTC count (CellSearch) at baseline and during therapy as a level of evidence 1 independent prognostic biomarker and demonstrated its superiority over serum blood markers. As part of the study pre-planned objectives, we sought to establish nomograms allowing accurate individual survival predictions. Methods: Using individual data from 17 centers, we built simplified multivariate prognostic models taking into account the independent prognostic clinico-pathological (CP) characteristics including CTC count, dichotomized using the 5CTC/7.5ml threshold, at baseline and at 3-5 weeks after the start of a new treatment regimen, and derived nomograms for progression-free survival (PFS) and overall survival (OS) prediction at baseline and after 3-5 weeks of treatment. We report here the internal validation of these nomograms. Discrimination of the models was assessed using the c-index estimated by a jackknife procedure and the calibration was visually assessed through 10-fold crossvalidated calibration plots at 1,2,3 years for OS and 1,2 years for PFS. Results: Multivariate models at baseline for PFS and OS were fitted on 1501 and 568 individual patient data with CTC count at baseline and CTC count at baseline and after 3-5 weeks, respectively. Models include tumor subtype, the number of previous chemotherapy lines (0/1/≥2), PS, age (50-65/>65 years), metastasis-free intervals (0/>0-3/>3 years), metastatic sites (liver and CNS) and CTC count at baseline and eventually at 3-5 weeks of treatment. The C-index increased from 0.722 to 0.755 (increase in C-index:0.033, 95% CI [0.019;0.045]) when adding baseline CTC to the CP only model for OS (n=1501). For those patients with CTC values at 3-5 weeks (n=568), there was an additional increase in the C-index when adding CTC at 3-5 weeks to a model with already CP and baseline CTC from 0.731 to 0.743 (increase in C-index 0.013, 95% CI [-0.004;0.025]). The model with CP and baseline CTC counts showed a good calibration for OS at 1,2,3 years and the model with CP, baseline CTC and CTC count at 3-5 weeks a moderately good calibration. Similar results were obtained for PFS. Conclusion: From the largest database with individual CTC data, we were able to build PFS and OS survival nomograms, with satisfactory discrimination and calibration. Our planned next step is to validate the nomogram in an additional cohort. Citation Format: Bidard F-C, Peeters D, Fehm T, Nole F, Gisbert-Criado R, Mavroudis D, Grisanti S, Generali D, Garcia-Saenz JA, Stebbing J, Caldas C, Gazzaniga P, Manso L, Zamarchi R, Fernandez de Lascoiti A, de Mattos-Arruda L, Ignatiadis M, van Laere SJ, Meier-Stiegen F, Sandri M-T, Vidal-Martinez J, Politaki E, Consoli F, Bottini A, Diaz-Rubio E, Krell J, Dawson S-J, Raimondi C, Rutten A, Janni W, Munzone E, Carañana V, Agelaki S, Almici C, Dirix L, Solomayer E, Zorzino L, Reis-Filho JS, Squifflet P, Pantel K, Beije N, Sleijfers S, Pierga J-Y, Michiels S. Circulating tumor cells count-based nomograms to predict survival of metastatic breast cancer patients: Results from the European pooled analysis. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-08-08.

Early consolidation with high-dose therapy and autologous stem cell transplantation is a feasible and effective treatment option in HIV-associated non-Hodgkin lymphoma at high risk

Autologous stem cell transplantation (ASCT) is a potentially curative treatment of lymphoma, but peripheral blood stem cell (PBSC) mobilization fails in some patients. PBSC mobilizing agents have recently been proved to improve the PBSC yield after a prior mobilization failure. Predictive parameters of mobilization failure allowing for a preemptive, more cost-effective use of such agents during the first mobilization attempt are still poorly defined, particularly during mobilization with chemotherapy + granulocyte colony-stimulating factor (G-CSF). We performed a retrospective analysis of a series of lymphoma patients who were candidates for ASCT, to identify factors influencing PBSC mobilization outcome. Premobilization parameters-age, histology, disease status, mobilizing protocol, and previous treatments-as well as white blood cell (WBC) and PBSC kinetics, markers potentially able to predict failure during the ongoing mobilization attempt, were analyzed in 415 consecutive mobilization procedures in 388 patients. We used chemotherapy + G-CSF in 411 (99%) of mobilization attempts and PBSC collection failed (

Background. We recently reported that Circulating Endothelial Cell (CEC) count changes represent a promising marker to monitor endothelial damage in patients undergoing allogeneic hematopoietic stem cell transplant (allo-HSCT), potentially becoming a valuable tool in the diagnostic definition of GVHD. Besides confirming an increase of CEC counts at GVHD onset, we repeatedly documented at time of engraftment statistically significant higher numbers of CEC in patients who will not manifest GVHD in comparison to patients in which GVHD will be diagnosed (Transplantation 2014,98:706-12; Bone Marrow Transplantation 2017,52:1637-42; Scientific Reports 2019,9:1-12). Recent knowledges in organ transplant pointed out that endothelial cells from the grafted organ, besides being a continuous source of alloantigens, can downregulate alloreactivity exerting tolerogenic responses. By inference to the allo-HSCT field, it could be envisaged that presence of donor CEC could induce protective effects on alloreactivity. Methods. We planned a study to test the hypothesis that at time of engraftment, CEC present in peripheral blood (PB), besides coming from cells shedding from patient vasculature, could partly belong to donor, originating from the cellular graft. Therefore, in an exploratory set, we performed FISH analysis on flowcytometry-sorted CEC (CD45neg/CD34bright/CD146pos, Lyotube #623920, BD Biosciences) (n=3) and on whole PB derived culture-expanded CEC (n=3) (EGM-2 BulletKit, Lonza), obtained at engraftment in sex-mismatched allo-HSCT. In the confirmatory set (n=15), single CEC were recovered from PB, at engraftment (T1) and at 90 days (T2) after allo-HSCT, through the DEPArrayTM technology (Menarini Silicon Biosystems), after preliminary bulk separation step carried out with the CellSearch® System. Single recovered CEC was whole genome amplified (Ampli1™ WGA Kit) and short tandem repeat (STR) profile determined (Ampli 1TM STR kit) on each single CEC. To confirm host/donor origin, single CEC STR profile was compared to that determined on patient and donor cells before allo-HSCT. Moreover, donor CEC presence was evaluated by CISH analysis on formaline fixed and paraffin-embedded biopsy sections obtained at least three months after sex mismatched allo-HSCT. Results. By positive findings of the exploratory set, we proved, at the single cell level in the confirmatory set, the presence of donor CEC at engraftment (T1) in 4 out of 15 patients (Table 1). Of them, 2 did not manifested GVHD, despite a GVHD risk score of 2, and the other 2 presented GVHD grade I. On the contrary, among the 10 patients in whom no donor CEC were detected, 6 experienced GVHD grade II-III, while 4 did not manifested GVHD, despite a 1-3 GVHD risk score. Conclusions. Our data represent the proof of principle that donor CEC may flow in host PB early on from hematopoietic recovery and seldom persist thereafter at steady-state conditions, being potentially embedded in host vascular wall. These puzzling findings suggest that neovascularization takes place in parallel with hematopoietic engraftment and could provide further clues on shedding light on tissue tolerance in the context of GVHD, opening up paradoxical scenarios on the protective role potentially played by donor CEC. Disclosures Fontana: Menarini Silicon Biosystem: Employment. Rotta:BD Biosciences Italia: Employment. Manaresi:Menarini SIlicon Biosystem: Employment, Membership on an entity's Board of Directors or advisory committees.

Introduction: Circulating Tumor Cells (CTCs) levels are dynamic indicators of prognosis and response to therapy in metastatic breast cancer (MBC) patients (pts). CTCs levels have been reported to be higher in pts with bone than visceral metastases, respectively. However, little is known about CTCs levels in pts with BM involvement. We analysed CTC patterns in patients with either visceral, bone or BM to test the hypothesis that BM involvement would facilitate tumor cells to enter the systemic circulation as CTCs. Patients and methods: Sites and extension of metastatic disease were identified with conventional imaging. Bone metastases were diagnosed with either bone scintigraphy or CT-PET and the BM involvement was documented histologically by BM biopsy and metabolically by CT-PET. A training set of 10 pts was evaluated by both BM biopsy and CT-PET to verify the diagnostic performance of CT-PET in identifying BM metastases (M+). We found a 100% concordance between BM biopsy and CT-PET and we therefore used CT-PET for further analyses. The CellSearch System (Veridex LLC, Raritan, NJ, USA) was used for the isolation and enumeration of CTCs in peripheral blood. The Student's t-test and the two-way ANOVA test were used to compare means of CTCs among patients with different metastatic patterns. Results: The CTCs mean and median scores of 129 MBC pts were analysed. Final analysis focused on 101 pts with bone M+. Thirty-three pts had bone M+ alone and 68 pts had bone and visceral M+ (including lymphnodes, brain, liver and lung/pleura). Number of metastatic sites was 1, 2, 3, 4, 5 in 30%, 37%, 25%, 6% and 2% of pts, respectively. BM involvement was documented in 27 (27%) of pts in the whole series and in 12 (33%) of pts with bone M+ alone. Mean CTCs score in the whole series was 392 (0-9993) and the median number of CTCs was 20. In the whole series, BM involvement was associated with higher mean CTCs scores (1350 vs 42, p .014). In pts with bone M+ alone, mean CTCs score was 696 (0-7000) and pts with BM involvement had higher mean CTCs scores (1846 vs 39, p 0.37). Among the different metastatic patterns, the association of bone with BM involvement and liver M+ was correlated with the highest CTCs numbers. We could not perform comparisons in the 68 pts with both bone and visceral localizations because of the variety of metastatic patterns. However, the two-way ANOVA showed a significant influence of BM involvement on mean CTCs scores in pts with different metastatic patterns (p >.001). Conclusions: This work confirms that pts with bone metastases have higher numbers of CTCs than pts with visceral metastases. Extensive bone disease with BM involvement is characterized by an increase in CTCs numbers compared to bone disease without BM involvement. Bone marrow metastases are metabolically more active by CT-PET and can be accurately detected by metabolic imaging without need of BM biopsy. Further studies are ongoing. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-07-19.

Purpose Individual variability in radiosensitivity is large in cancer patients. Single nucleotide polymorphisms (SNPs) in genes involved in DNA repair and in protection against reactive oxygen species (ROS) could be responsible for such cases of radiosensitivity. We investigated the association between the occurrence of acute reactions in 101 patients with squamous cell carcinoma of the head and neck (SCCHN) after radiotherapy (RT) and five genetic polymorphisms: XRCC1 c.1196A > G, XRCC3 c.722C > T, RAD51 (c.-3429G > C, c.-3392G > T), and GSTP1 c.313A > G. Materials and methods Genetic polymorphisms were detected by high resolution melting analysis (HRMA). The development of acute reactions (oral mucositis, skin erythema and dysphagia) associated with genetic polymorphisms was modeled using Cox proportional hazards, accounting for biologically effective dose (BED). Results Development of grade ⩾2 mucositis was increased in all patients (chemo-radiotherapy and radiotherapy alone) with XRCC1-399Gln allele (HR = 1.72). The likelihood of developing grade ⩾2 dysphagia was higher in carriers of RAD51 c.-3429 CC/GC genotypes (HR = 4.00). The presence of at least one SNP or the co-presence of both SNPs in XRCC1 p.Gln399Arg /RAD51 c.-3429 G > C status were associated to higher likelihood of occurrence of acute toxicities (HR = 2.03). Conclusions Our findings showed an association between genetic polymorphisms, XRCC1 c.1196A > G and RAD51 c.-3429 G > C, and the development of radiation-induced toxicities in SCCHN patients.

We report the case of a 67-year-old man affected by metastatic esophageal cancer. The patient developed a symptomatic heart metastasis presenting as mimicking ST-segment elevation myocardial infarction. Cardiac magnetic resonance imaging (MRI) documented the presence of a mass in the apex and septum of the left ventriculum. The dissemination of cancer was confirmed by the detection of circulating tumor cells (CTCs) in the peripheral blood, measured by the CellSearch System (Veridex, LLC, Raritan, N.J., USA). The blood sample drawn at cardiac disease progression revealed the presence of 2 CTCs per 7.5 ml of blood. This report highlights the potential role of CTCs as markers of metastatic spread.

Introduction: Autologous stem cell transplantation (ASCT) is considered safe and effective in patients with HIV-associated lymphoma (HIV-Ly). The reported studies usually analyze togheter different histological subtypes, including classical Hodgkin lymphoma (HL) and a variety of aggressive non Hodgkin lymphoma (NHL), and different indications for ASCT, and report composite outcomes. Aim: We report our single center experience on a large single institution series of patients (pts) with HIV-Ly undergoing ASCT and analyse efficacy according to histology subtypes, indications for ASCT, previous therapy and lymphoma status at transplant, to understand the settings where this treatment approach is most effective and useful. Methods: Retrospective analysis of clinical characteristics and outcome of all consecutive pts with HIV-Ly transplanted at our Institution from March 2001 to February 2018. Results: 62 pts with HIV-Ly underwent ASCT during the study period. Ninety-four % were male; median age was 46 years (range, 29-62). Lymphoma histology was HL in 11 (18%), diffuse large B-cell lymphoma in 22 (35%), Burkitt or Burkitt-like in 13 (21%) , plasmablastic in 10 (16%), T-cell aggressive NHL (3 anaplastic large cell lymphoma, ALK negative, and 1 extranodal NK/T-cell lymphoma, nasal type) in 4 (6%), and indolent B-cell lymphoma (1 follicular lymphoma, and 1 extranodal marginal zone lymphoma) in 2 (3%) pts, respectively. Median CD4+ cells count at ASCT was 208/mL (range, 55-720); 8 pts (13%) had detectable HIV viral load. Eighteen pts (29%) had concomitant hepatitis C and 2 (3%) hepatitis B infection. Indications for ASCT were primary refractory disease (26%), first or subsequent relapse (37%), and consolidation after first line therapy for partial remission (PR) or high risk complete remission (CR) (37%). At the time of ASCT, 10 pts (16%) were in first CR, 45 (73%) had chemosensitive disease (i.e. first PR, chemosensitive relapse/refractory disease), and 7 (11%) had disease refractory to the last chemotherapy (CT) received. Twenty-three patients (37%) had received only 1 line of CT before ASCT, 29 (47%) 2 lines, and 10 (16%) 3 or more lines. Conditioning regimen was BEAM in 47 patients (76%), FEAM in 13 (21%), and 1 each Mitoxantrone-Melphalan and BiCNU-Thiotepa. All pts were receiving cART throughout ASCT except 3 who suspended cART for at least 1 week, due to toxicity and/or oral mucositis. Neutrophil engraftment (neutrophils > 500/mcl) occurred in all pts at a median of 10 days (range, 8-12), and platelet engraftment (platelet count > 20.000/mcl) occurred in all pts at a median of 13 days (range, 9-46), except in one with refractory disease who died of sepsis early after ASCT (treatment-related mortality 1.6%). Twenty-three pts (70%) experienced grade 3-4 toxicity, including oral mucositis (17 patients), gastrointestinal toxicity (8), hepatic toxicity (1), and seizures (1). Twenty-five pts (41%) had an episode of fever of unknown origin. Before engraftment, 22 documented bacterial infections, 1 fungal infection, 2 herpes zoster infections and 5 CMV reactivations were recorded. After a median follow-up of 59 months (range, 1-177) from ASCT, the 5 years-progression free survival (5y-PFS) and OS (5y-OS) were 66.6% and 69.7% respectively, with no significant differences according to different histologies (figure 1). The outcome was satisfactory regardless of the indication for transplant (primary refractory disease, 5y-PFS 50% and 5y-OS 49% , relapse, 5y-PFS 62.6% and 5y-OS 70.7%, and first line consolidation, 5y-PFS 80.3% and 5y-OS 80.1%, P=NS), and number of CT lines before ASCT (1 line, 5y-PFS 80.3% and 5y-OS 80.1%, 2 lines, 5y-PFS 61.3% and 5y- 63.4%, and 3 or more lines, 5y-PFS 53.3% and 5y-OS 66.7%, P=NS). According to the status of lymphoma at the time of ASCT, pts in first CR had a 5y-PFS of 77.8% and 5y-OS of 77.8% and pts with chemosensitive disease 73.7% and 80.3%, while all chemorefractory pts died within 5 months from ASCT, with median PFS and OS of 2 and 3 months respectively (P< 0.001) (figure 2). Conclusion: This is the largest single institution series of HIV-Ly receiving ASCT. We confirm the feasibility and long-term efficacy of this treatment approach in different histological subtypes. ASCT was beneficial also in primary refractory disease and heavily pre-treated pts, provided that lymphoma proved chemosensitive to the last CT received before ASCT. Disclosures Rossi: ABBVIE: Other: ADVISORY BOARD; PFIZER: Other: ADVISORY BOARD; SANDOZ: Honoraria; GILEAD: Other: ADVISORY BOARD; AMGEN: Other: ADVISORY BOARD; SANOFI: Other: ADVISORY BOARD; JANNSEN: Other; JAZZ: Other: ADVISORY BOARD; TEVA: Other: ADVISORY BOARD; CELGENE: Other: ADVISORY BOARD; ROCHE: Other: Advisory Board; NOVARTIS: Honoraria; MUNDIPHARMA: Honoraria; BMS: Honoraria.

We aimed to assess the clinical validity of circulating tumour cell (CTC) quantification for prognostication of patients with metastatic breast cancer by undertaking a pooled analysis of individual patient data.We contacted 51 European centres and asked them to provide reported and unreported anonymised data for individual patients with metastatic breast cancer who participated in studies between January, 2003, and July, 2012. Eligible studies had participants starting a new line of therapy, data for progression-free survival or overall survival, or both, and CTC quantification by the CellSearch method at baseline (before start of new treatment). We used Cox regression models, stratified by study, to establish the association between CTC count and progression-free survival and overall survival. We used the landmark method to assess the prognostic value of CTC and serum marker changes during treatment. We assessed the added value of CTCs or serum markers to prognostic clinicopathological models in a resampling procedure using likelihood ratio (LR) χ(2) statistics.17 centres provided data for 1944 eligible patients from 20 studies. 911 patients (46·9%) had a CTC count of 5 per 7·5 mL or higher at baseline, which was associated with decreased progression-free survival (hazard ratio [HR] 1·92, 95% CI 1·73-2·14, p0·0001) and overall survival (HR 2·78, 95% CI 2·42-3·19, p0·0001) compared with patients with a CTC count of less than 5 per 7·5 mL at baseline. Increased CTC counts 3-5 weeks after start of treatment, adjusted for CTC count at baseline, were associated with shortened progression-free survival (HR 1·85, 95% CI 1·48-2·32, p0·0001) and overall survival (HR 2·26, 95% CI 1·68-3·03) as were increased CTC counts after 6-8 weeks (progression-free survival HR 2·20, 95% CI 1·66-2·90, p0·0001; overall survival HR 2·91, 95% CI 2·01-4·23, p0·0001). Survival prediction was significantly improved by addition of baseline CTC count to the clinicopathological models (progression-free survival LR 38·4, 95% CI 21·9-60·3, p0·0001; overall survival LR 64·9, 95% CI 41·3-93·4, p0·0001). This model was further improved by addition of CTC change at 3-5 weeks (progression-free survival LR 8·2, 95% CI 0·78-20·4, p=0·004; overall survival LR 11·5, 95% CI 2·6-25·1, p=0·0007) and at 6-8 weeks (progression-free survival LR 15·3, 95% CI 5·2-28·3; overall survival LR 14·6, 95% CI 4·0-30·6; both p0·0001). Carcinoembryonic antigen and cancer antigen 15-3 concentrations at baseline and during therapy did not add significant information to the best baseline model.These data confirm the independent prognostic effect of CTC count on progression-free survival and overall survival. CTC count also improves the prognostication of metastatic breast cancer when added to full clinicopathological predictive models, whereas serum tumour markers do not.Janssen Diagnostics, the Nuovo-Soldati foundation for cancer research.

BACKGROUND Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is burdened by life-threatening complications, with graft-versus-host disease (GvHD) being the major cause of morbility and mortality. Recently, clinical and physiopathologic evidences showed that vascular endothelium can be a target of GvHD in the early phase and circulating endothelial cells (CECs) represent surrogate markers of endothelial damage. METHODS Using the CellSearch System (Veridex LLC, Raritan, NJ), CECs were counted before (T1), after conditioning regimen (T2), at engraftment (T3), at GvHD onset (T4), and after steroid treatment (T5) in 40 patients (7 Hodgkin's Disease, 13 Acute Myeloblastic Leukemia, 5 Acute Lymphoblastic Leukemia, 8 Multiple Myeloma, 3 Chronic Lymphocytic Leukemia, 1 Non-Hodgkin Lymphoma, 1 Chronic Myeloid Leukemia, 2 Severe Aplastic Anemia) undergoing allo-HSCT. RESULTS The median CEC per milliliter at T1 was 20 (n=33, range 4-718), in comparison to a value of 2 (range, 1-14) in controls (P

High-dose chemotherapy with autologous peripheral blood stem cell rescue has been reported as feasible and effective in HIV-associated lymphoma. Although a sufficient number of stem cells seems achievable in most patients, there are cases of stem cell harvest failure. The aim of this study was to describe the mobilization policies used in HIV-associated lymphoma, evaluate the failure rate and identify factors influencing mobilization results. We analyzed 155 patients who underwent attempted stem cell mobilization at 10 European centers from 2000-2012. One hundred and twenty patients had non-Hodgkin lymphoma and 35 Hodgkin lymphoma; 31% had complete remission, 57% chemosensitive disease, 10% refractory disease, 2% untested relapse. Patients were mobilized with chemotherapy + G-CSF (86%) or G-CSF alone (14%); 73% of patients collected2 and 48%5 × 10(6) CD34(+) cells/kg. Low CD4+ count and refractory disease were associated with mobilization failure. Low CD4(+) count, low platelet count and mobilization with G-CSF correlated with lower probability to achieve5 × 10(6) CD34(+) cells/kg, whereas cyclophosphamide ≥ 3 g/m(2) + G-CSF predicted higher collections. Circulating CD34(+) cells and CD34/WBC ratio were strongly associated with collection result. HIV infection alone should not preclude an attempt to obtain stem cells in candidates for autologous transplant as the results are comparable to the HIV-negative population.

Chronic myelogenous leukemia (CML) is a clonal disorder of the hematopoietic stem cell characterized by a chimeric BCR/ABL gene giving rise to a 210-kD fusion protein with dysregulated tyrosine kinase activity. We investigated the effect of genistein, a protein tyrosine kinase inhibitor, on the in vitro growth of CML and normal marrow-derived multi-potent (colony-forming unit-mix [CFU-Mix]), erythroid (burst-forming unit-erythroid [BFU-E]), and granulocyte-macrophage (colony-forming unit-granulocyte-macrophage [CFU-GM]) hematopoietic progenitors. Continuous exposure of CML and normal marrow to genistein induced a statistically significant and dose-dependent suppression of colony formation. Genistein doses causing 50% inhibition of CML and normal progenitors were not significantly different for CFU-Mix (27 mumol/L v 23 mumol/L), BFU-E (31 mumol/L v 29 mumol/L), and CFU-GM (40 mumol/L v 32 mumol/L v 32 mumol/L). Preincubation of CML and normal marrow with genistein (200 mumol/ L for 1 to 18 hours) induced a time-dependent suppression of progenitor cell growth, while sparing a substantial proportion of long-term culture-initiating cells (LTC-IC) from CML (range, 91% +/- 9% to 32% +/- 3%) and normal marrow (range, 85% +/- 8% to 38% +/- 9%). Analysis of individual CML colonies for the presence of the hybrid BCR/ABL mRNA by reverse transcription-polymerase chain reaction (RT-PCR) showed that genistein treatment significantly reduced the mean +/- SD percentage of marrow BCR/ABL+ progenitors both by continuous exposure (76% +/- 18% v 24% +/- 12%, P ‼ or = .004) or preincubation (75% +/- 16% v 21% +/- 10%, P ‼ or = .002) experiments. Preincubation with genistein reduced the percentage of leukemic LTC-IC from 87% +/- 12% to 37% +/- 12% (P ‼ or = .003). Analysis of individual colonies by cytogenetics and RT-PCR confirmed that genistein-induced increase in the percentage of nonleukemic progenitors was not due to suppression of BCR/ABL transcription. Analysis of nuclear DNA fragmentation by DNA gel electrophoresis and terminal deoxynucleotidyl transferase assay showed that preincubation of CML mononuclear and CD34+ cells with genistein induced significant evidence of apoptosis. These observations show that genistein is capable of (1) exerting a strong antiproliferative effect on CFU-Mix, BFU-E, and CFU-GM while sparing the more primitive LTC-IC and (2) selecting benign hematopoietic progenitors from CML marrow, probably through an apoptotic mechanism.

The mechanism of dissemination of locally advanced head and neck cancer (LAHNC) is far to be resolved. Circulating tumour cells (CTC) have been identified as a prognostic factor in metastatic breast and prostate cancer. This prospective multi-centric analysis studied the possible role of CTC identification in LAHNC.CTC were searched in 73 patients with LAHNC (oropharynx, n=39; nasopharynx, n=10; larynx, n=10; paranasal sinuses, n=6, of whom 3 with sinonasal undifferentiated carcinoma, SNUC; hypopharynx, n=5; oral cavity, n=3). All of them (apart from SNUC) had squamous cell cancers. The relationship between CTC positivity and other clinical prognostic factors has been investigated. Response to treatment and survival has been related with changes in CTC number during the treatment.CTC were frequently identified in oro- and hypopharyngeal cancer and in SNUC. They were more frequent in stage IV than in stages I-III disease (18% versus 6%, p=NS (not significant)). Partial or complete response (CR) was related with the absence or disappearance of CTC during treatment (p=0.017). A decrease in the CTC number or their absence throughout the treatment seems also related with non-progressive disease, after both complete or incomplete remission and with the proportion of patients alive and NED (no evidence of disease) (p=0.009).These preliminary data suggest a possible role of CTC determination in head and neck cancer. Additional and longer follow up data need to be collected to confirm these findings.

Chronic myelogenous leukemia (CML) is a clonal disorder of the hematopoietic stem cell characterized by the co-existence of Philadelphia-negative with Ph-positive progenitors. CML progenitor cells have been shown to be defective in adherence to marrow stroma. The present study investigated at the cytogenetic level marrow-derived CML clonogenic cells generated from the stroma-adherent cell fraction. Mononuclear marrow cells from CML patients (n = 20) were incubated with mafosfamide (100 micrograms/ml) or control medium, seeded onto marrow stromal layers, and allowed to adhere (2 h, 37 degrees C). Following a short-term (3 days) liquid culture, the cells were harvested, incorporated in methylcellulose, and individual colonies were analyzed by single colony karyotyping. On direct cytogenetic analysis, the overall mean (+/- SD) percentage of Ph-negative metaphases was 9 +/- 20%. The mean (+/- SD) percentages of Ph-negative colonies grown from the stroma-adherent and the stroma-adherent mafosfamide-treated fraction were 41 +/- 32% and 62 +/- 40% (p < or = .005), respectively. Single colony transfer experiments revealed that 50 +/- 13% stroma-adherent and 70 +/- 24% stroma-adherent mafosfamide-treated progenitors gave rise to secondary colonies. In conclusion, the present data demonstrate the possibility to select Ph-negative clones that: 1) have a maintained capability of stroma adherence; 2) are mafosfamide resistant; and 3) have high-replating potential.

Chronic myelogenous leukemia (CML) progenitor cells have been shown to be defective in their ability to adhere to marrow stroma. It was the aim of the present study to investigate at the cytogenetic level marrow-derived CML clonogenic cells fractionated on the basis of their ability to adhere to preformed, allogeneic, normal marrow-derived stromal layers. Mononuclear marrow cells from CML patients (n = 15) were incubated with mafosfamide (100 micrograms/ml) or control medium, seeded onto marrow stromal layers and allowed to adhere (3 hrs, 37 degrees C). Following a short-term liquid culture, the different cell fractions were harvested and incorporated in methylcellulose cultures. CFU-GM grown from these cultures were analyzed by single colony karyotyping. On direct cytogenetic analysis, the overall mean (+/- SD) percentage of Ph-negative metaphases was 7 +/- 20%. Following stroma adherence and shortterm suspension culture, the mean (+/- SD) percentages of Ph-negative clones were as follows: 33 +/- 25% for adherent CFU-GM, 59 +/- 40% for adherent, mafosfamide-treated CFU-GM, 12 +/- 16% for non-adherent CFU-GM, and 32 +/- 26% for non-adherent-mafosfamide-treated CFU-GM. If only the patients showing a percentage of Ph-negative clones > or = 20% were included in this analysis, the mean (+/- SD) percentages of Ph-negative clones were 47 +/- 19% for adherent CFU-GM, and 81 +/- 21% for adherent-Mafosfamide-treated CFU-GM. In contrast, the majority of pH-positive CFU-GM were detected within the stroma non-adherent cell fraction.(ABSTRACT TRUNCATED AT 250 WORDS)

The neutropenia-related morbidity and mortality occurring after autologous bone marrow transplantation (ABMT) is increased by marrow purging procedures. While phase I through III clinical trials showed the enhancing activity of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) on neutrophil recovery after ABMT with unpurged marrow, controversial results have been reported when purged marrow was used. Therefore, it was the aim of the present study to evaluate the efficacy of rhGM-CSF administration in a group of patients (n = 15) with lymphoid malignancies transplanted in complete remission with mafosfamide-purged (n = 10) or unpurged (n = 5) marrow. Mafosfamide concentrations used for marrow purging were evaluated on an individual basis by means of a recently described technique that destroys the granulocyte-macrophage (granulocyte-macrophage colony- forming units [CFU-GM]) compartment, but spares 50% of the more primitive stroma adherent colony-forming cells (CFU-Blast). rhGM-CSF (10 micrograms/kg/d) was started within 24 hours of ABMT and administered in a 4-hour infusion daily until the absolute neutrophil count (ANC) reached 500 x 10(6)/L and then for 7 more days. Patients receiving mafosfamide-purged or unpurged marrow failed to show any difference in terms of median number of days required to achieve an ANC > or = 500 x 10(6) (13 v 14.0, P > .4) cells/L. As compared with retrospective controls, granulocytic recovery was reduced by a median time of 11 (P < or = .0005) and 5 (P < or = .0005) days for patients grafted with purged and unpurged marrow, respectively. The number of CFU-GM (mean +/- SD) infused per kilogram of body weight was significantly lower in patients who received purged autografts as compared with those receiving unpurged autografts (0.85 +/- 0.79 x 10(4) v 15.7 +/- 9.2 x 10(4), P < or = .0005). The dose of CFU-GM progenitors infused per kilogram of body weight did not correlate (r = .031, P > .05) with the time required to reach an ANC > or = 500 x 10(6) cells/L. The number of CFU-Blast (mean +/- SD) infused per kilogram of body weight was not significantly different between patients who received purged or unpurged autografts (5.05 +/- 2.51 x 10(3)/kg v 6.18 +/- 2.66 x 10(3)/kg, P < or = .375). A statistically significant correlation (r = -.658, P < or = .05) was observed between the number of CFU-Blast infused and the number of days required to reach an ANC > or = 500 x 10(6) cells/L.(ABSTRACT TRUNCATED AT 400 WORDS)

Background. Outcome of HIV-associated NHL (HIV-NHL) with adverse prognostic features is not satisfactory with standard therapy. High dose therapy (HDT) and autologous stem cell transplantation (ASCT) has proven safe and active in HIV-NHL in salvage setting. Aims. To define the role of HDT and ASCT in the upfront treatment of HIV-NHL at high risk, in terms of efficacy and toxicity. We report the mature results of a multicenter prospective study including HDT and ASCT as consolidation after first-line treatment of HIV-NHL at high-risk. Patients and methods. Inclusion criteria: untreated aggressive HIV-NHL (including DLBCL, plasmablastic, anaplastic lymphoma), aa-IPI 2-3, age 18-60, CD4+ count >50/mcl and availability of effective HAART.. Patients (pts) received R-CHOP (no Rituximab for CD20 negative lymphoma) for 6 cycles and, if responsive, underwent stem cells collection after Cyclophosphamide 4gr/ms + G-CSF followed by HDT with BEAM and ASCT. Involved-field radiotherapy on previous bulky or residual disease was recommended. Patients (pts) received HAART during the entire treatment program. Results: From January 2007 to July 2014, 29 pts were registered and 25 entered the study. Median age was 48 years (range, 27-62). Nineteen pts had DLBCL, 5 plasmablastic, 1 anaplastic lymphoma. ECOG PS was >1 in 14 pts (56%); Ann-Arbor stage III/IV, 7(28%)/18(72%); B symptoms, 16 (64%); LDH >n.v., 18 (88%); aaIPI 2/3, 13 (52%)/12 (48%). Eighteen pts (72%) had a prior history of HIV-positivity, and 17 (68%) were on HAART at NHL diagnosis. In 7 pts HIV and NHL diagnosis were concomitant. Fourteen pts (56%) had detectable HIV-viremia (range 40->500.000 cp/mL). Median CD4+ count was 255/mcl (51-571). Ten pts (40%) had HCV infection. Twenty-two pts received (R)-CHOP-21 and 3 pts with plasmablastic histology received CHOP-14. One pt is on treatment and 24/25 are evaluable for (R)-CHOP response. One pt died of hepatic failure and 1 due to cerebral hemorrhage, 2 had prolonged cytopenia (plus severe hepatic toxicity in 1) and 1 infectious complications that lead to withdrawal from the trial [however 1 achieved a complete remission (CR) and 2 died of progressive disease]; 19 pts completed (R)-CHOP according to the study: 14 had CR, 4 partial remission (PR) and 1 disease progression (PD). On an intention to treat basis: ORR 79.2%, CR 62.5%, PR 16.7%. Seventeen/18 pts collected stem cells (median CD34+ cells 7.4 x 10e6/Kg, range 2.6-10.1) and 1 failed mobilization. Two pts had early disease progression after collection, 1 did not receive HDT because of cardiac ejection fraction Conclusions: This is the first prospective trial addressing the role of HDT and ASCT in first line treatment of HIV-LNH. Almost 60% of pts were able to complete the entire treatment program and the ASCT was well tolerated. The OS in this series of pts at high risk is satisfactory and no relapse occurred in pts who received ASCT, after a prolonged follow-up. Further improvement could result from an increase in the rate of patients who receive ASCT. HDT with ASCT seems an effective way to consolidate first response and improve outcome in HIV-NHL at high risk . Fig 1 Survival from study entry (25 pts) Fig 1. Survival from study entry (25 pts) Fig 2 Survival after transplant (14 pts) Fig 2. Survival after transplant (14 pts) Disclosures No relevant conflicts of interest to declare.

Background: Clinical validity of CTCs (CellSearch®) in metastatic breast cancer (MBC) patients has previously been assessed in studies with limited statistical power. We aimed to pool all European studies to obtain high-level evidence on the prognostic value of CTCs, to investigate their effects across different clinico-pathological characteristics and therapies and to further validate the MD Anderson/Institut Curie/Fox Chase CTC-based prognostic nomogram established in first-line treated MBC patients (Giordano et al, Clin Cancer Res 2013). Material and methods: Methods were predefined in a written protocol. In December 2012, we searched for eligible studies that accrued patients in 2003-2012. We contacted all European laboratories using CellSearch®. We used likelihood ratio tests (LR) in Cox regression models stratified by study to assess the independent prognostic value of CTC when added to a clinicopathological (CP) model for progression-free (PFS) and overall survival (OS). Landmark analyses were used to assess the prognostic effect of early changes in CTC. The CTC-based nomogram (http://cancernomograms.com/CTCOnline.html) score was retrieved for every patient; we calculated C-indices, drew calibration plots and Kaplan-Meier curves according to quintiles of the nomogram score. Results: We collected individual data of 1944 MBC patients, from 20 different studies (some unpublished), from 17 centers in 7 European countries. We observed 1507 PFS events and 929 deaths. Baseline CTC count was significantly associated with several patient characteristics, such as performance status (PS, p Conclusions: This pooled analysis is the largest study ever reported on CTC in MBC, with a previously unreached statistical power. It provides a clear level-of-evidence 1 on the independent prognostic value of CTCs before and during treatment in MBC. Also, the CTC-based prognostic nomogram is independently validated. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr PD6-5.

A case of acute promyelocytic leukaemia following mitoxantrone treatment of multiple sclerosis

Abstract 3124 Background. Outcome of aggressive HIV-associated NHL (HIV-NHL) with adverse prognostic features is not satisfactory with standard therapy. High dose therapy (HDT) and autologous stem cell transplantation (ASCT) has been demonstrated safe and active in HIV-NHL in the salvage setting. Aims. To define the role of HDT and ASCT in the upfront treatment of HIV-NHL at high risk, in terms of efficacy and toxicity. We report the interim results of a multicenter study including HDT and ASCT as consolidation after first-line treatment of HIV-NHL at high-risk. Patients and methods. Inclusion criteria: untreated aggressive HIV-NHL (including DLBCL, plasmablastic, anaplastic lymphoma), aa-IPI 2–3, age 18–60, CD4+ count >50/mcl and availability of effective HAART. Burkitt and CNS lymphoma are excluded. Patients (pts) receive R-CHOP-21 (CHOP-21 or CHOP-14 for CD20 negative lymphoma) for 6 cycles and, if responsive, undergo stem cells mobilization and collection after Cyclophosphamide 4gr/ms + G-CSF followed by HDT with BEAM and ASCT. Involved-field radiotherapy on previous bulky or residual disease is recommended. Pts receive HAART during the entire treatment program. Overall survival (OS) at 2 years is the primary endpoint. Results. Since January 2007 to July 2012, 23 pts were registered and 20 entered the study. Median age was 47.5 years (range, 27–62). Fifteen pts (75%) had DLBCL, 4 (20%) plasmablastic and 1 (5%) anaplastic lymphoma. ECOG PS was >1 in 11 pts (55%); Ann-Arbor stage III/IV, 5(25%)/15(75%); B symptoms, 12 (60%); LDH > n.v., 18 (90%); aaIPI 2/3, 10 (50%)/10(50%). Fourteen pts (70%) had a prior history of HIV-positivity and 13 (65%) were on HAART at NHL diagnosis; thirteen (65%) had detectable HIV-viremia. Median CD4+ count was 283/mcl (58–571). Nine pts (45%) had HCV infection. Seventeen pts received (R)-CHOP-21 and 3 pts with plasmablastic histology received CHOP-14. One pt is still on treatment and 19/20 are evaluable for (R)-CHOP response. One had toxic death (due to hepatic failure), 2 had prolonged cytopenia (1 with severe hepatic toxicity) that lead to withdrawal of therapy and 16 completed (R)-CHOP therapy: 11 pts had complete remission (CR), 4 partial remission (PR) and 1 disease progression (PD) (ORR 79%,CR 58%, PR 21%, according to intention to treat). One pt is ongoing and 14 collected CD34+ cells after Cyclophosphamide + G-CSF (median CD34+ cells 7.35 × 10e6/Kg, range 2.65–10.0). Two pts had early disease progression after collection, 1 did not receive HDT because of cardiac ejection fraction < 50% at evaluation before BEAM and 1 is ongoing. So far, 10 pts received ASCT according to the protocol. Moreover, three pts received radiotherapy (2 on previous bulky and 1 on testicular disease). HDT-related toxicities included 2 grade II and 5 grade III GI and 1 grade III hepatic toxicity. Prior to engraftment 1 VZV infection, 1 Clostridium difficile colitis, 1 sigmoiditis and 5 episodes of FUO were registered. There were no transplant-related deaths. One case of CMV reactivation and no opportunistic infections were registered during observation. All transplanted pts are alive and relapse-free after a median of 43 ms (range, 4–58) after transplant (Figure 1). In the entire series, with a median f-up of 29.5 ms (range, 4–65), the 2-years PFS and OS of the entire series from study entry were 73% (+10.3%) and 76% (+10.6%), respectively (Figure 2). Conclusions. This is the first prospective trial addressing the role of HDT and ASCT in first line treatment of HIV-LNH. The procedure was well tolerated and the clinical results highly encouraging. Interim evaluation of OS in this very high risk series of pts is satisfactory and accrual is ongoing. Disclosures: No relevant conflicts of interest to declare.

Ten adult patients with Ph-positive chronic myelogenous leukemia (CML) received autologous bone marrow transplantation (ABMT) using marrow treated ex vivo with mafosfamide. At the time of ABMT, six patients were in chronic phase and four in accelerated phase. Seven of ten patients reported herein were selected on the basis of a previous laboratory assessment of the numbers of normal and leukemic stroma-adherent progenitor cells within mafosfamide-treated marrow. Only patients showing ≥50% Ph-negative stroma-adherent progenitor cells within mafosfamide-treated marrow were considered eligible for autografting. In nine out of ten evaluable patients, the median time to achieve 500 neutrophils/μl was 32 days (range: 25–72). A platelet count of 2 × 104/μl was achieved at a median of 40 days (range: 27–97). Six out of nine analyzable patients engrafted Ph-negative. The median duration of the Ph-negative hematopoiesis, confirmed also by Southern blot analysis, was 6.5 months (range: 4–30). A good correlation was evident between the results of the in vitro preharvest screening test and the in vivo occurrence of normal hematopoiesis post-transplant. Two patients who showed 75% and 89% Ph-negative stroma-adherent progenitors engrafted Ph-positive, whereas four out of five evaluable patients who had 100% Ph-negative stroma-adherent progenitors engrafted Ph-negative. After a median follow-up of 16 months (range: 3–31), five patients evolved into blast crisis, three are alive in hematologic and cytogenetic relapse, and one died without evolving into blast crisis. In conclusion, our results demonstrate that: 1) engraftment can occur from Ph-negative stem cells selected by mafosfamide purging; 2) in selected CML patients, mafosfamide is effective in reducing the size of the malignant clone and inducing a transient period of Ph-negative hematopoiesis; and 3) modifications of the purging procedure as well as post-transplant manipulation of the immune-hematopoietic system are required to prolong cytogenetic remission or cure CML patients ineligible for allogeneic or unrelated bone marrow transplantation (BMT).

Abstract 2250 Background: Autologous stem cell (SC) transplantation (ASCT) is a potentially curative treatment for several hematologic malignancies and has been demostrated feasible and effective in HIV-related lymphoma (ARL). Peripheral blood SC collection could represent a major issue in the use of ASCT in HIV infected patients (pts). Aim: To evaluate the feasibility and efficacy of SC mobilization in HIV positive (pos) pts with lymphoma and identify factors influencing harvest results. Potential “ongoing” predictors of collection were also assessed. Patients and Methods: We retrospectively analysed 98 consecutive pts with ARL, candidates to ASCT, who underwent SC mobilization at 3 Italian and 2 Spanish centers from 2000 to 2010. A collection less than 2×106 CD34+ cells/kg was defined as “mobilization failure”, between 2–5 as “suboptimal collection” and more than 5 as “good collection”. Several parameters were evaluated for correlation with outcome: age, sex, lymphoma histopathology, disease status, WBC and Plt count at start of mobilization, type of mobilizing therapy, marrow disease, previous mobilization failure, n° of previous chemotherapy (CT) lines, months from first detection of HIV positivity, CD4 count and HIV-viremia. Moreover, circulating CD34+ and WBC count on the first day of CD34+ monitoring and their ratio (SC ratio = CD34/WBC) were assessed as “ongoing” outcome predictors. Results: A total of 127 attempts of SC harvest in 98 pts were analysed. Median age was 41.5 ys (28-65). Lymphoma diagnosis was DLBCL in 42% of cases, Burkitt 10%, plasmablastic 10%, HL 31%, anaplastic 5%, follicular lymphoma 1% and PEL 1%. Disease status was complete remission in 36%, chemosensitive disease in 53% and refractory disease in 10% of cases. In 3 cases bone marrow was involved and mobilizations failed. In 18% of cases pts received mobilizing therapy after 1 previous CT line, in 67% after 2 and in 16% after 3 or more. All pts but 2 were on antiretroviral therapy. Median CD4 count was 231/mcl (50-1146) and HIV-viremia was detectable in 22%. Median time from first HIV detection was 79.5 ms (3-295). In 24% of cases G-CSF alone (10-20 mcg/Kg) was used as mobilizing treatment, while CT + G-CSF (5-10 mcg/Kg) in 76%, including single-agent Cyclophosphamide (CTX) 1.5 gr/ms (13%), CTX >3 gr/ms (27%), platinum containing regimens (20%), ifosfamide containing regimens (11%) and others (5%). Mobilization failure occurred in 40% of procedures, a collection between 2–5 × 10^6 CD34/Kg in 24% and > 5 in 35%. Finally, of 98 pts who underwent SC mobilization, 22% failed to collect enough cell to perform ASCT, 12 pts even after repeated attempts, 33% had a suboptimal and 45% a good collection (4 and 5 pts respectively after repeated mobilizations). At univariate analysis failure was significantly associated with refractory disease, Plt < 150.000/cmm, CTX 1.5 gr/ms as mobilizing treatment, previous mobilization failure and circulating CD34+ cell < 7.4/mcl on the first day of monitoring; whereas CTX > 3 gr/ms, CD4 count and SC ratio > 0.002 were associated with a reduced risk of failure. In multivariate analysis refractory disease (p 0.002 (p 3 gr/ms, WBC count and circulating CD34+ cells >29,7/mcl at the first day of monitoring and SC ratio > 0,002, whereas G-CSF alone and previous mobilization failure were negative predictive factors. Multivariate analysis confirmed CTX > 3 (p 29,7 (p=0.0003) and SC ratio > 0,002 (0.0036) as indipendent factors for good collection. Conclusions: In this series of 98 ARL and 127 SC mobilization attempts, a substantial number of pts failed SC harvest (22%) whereas 33% had a suboptimal and 45% a good collection. Lymphoma status and mobilizing treatment seems the strongest predictors for outcome, with refractory disease and low CTX dose (1.5 gr/ms) significantly associated with failure and CTX > 3 gr/ms predictor for good collection. A high ratio between circulating CD34+ cells and WBC on the planned day of first apheresis might represent a useful “ongoing” parameter to predict the outcome. These data might help to decide the mobilizing strategy in ARL and could provide the framework to rationally explore the use of new mobilizing agents Disclosures: No relevant conflicts of interest to declare.

Abstract 2149 Poster Board II-126 Background Autologous stem cell transplantation (ASCT) is a potentially curative treatment for lymphoma. Adequate stem cell (SC) collection is possible in the majority of cases but poor mobilization remains a major issue in some patients (pts). New agents were recently developed which may improve the rate of SC mobilization. Aims A retrospective analysis was conducted on a large series of lymphoma pts candidates to ASCT in order to identify factors influencing SC mobilization outcome. Potential early markers of poor mobilization were also evaluated. Patients and methods A total of 415 attempts of PBSC collection consecutively performed at 7 Italian centres in 388 pts affected by lymphoma were analyzed. Their median age was 52. A collection of less than 2×106 CD34+ cells/kg was defined as “mobilization failure” and of more than 5×106 CD34+ cells/kg as “good mobilization”. The following parameters were analysed for correlation with mobilization outcome: lymphoma diagnosis, disease status at mobilization, type of mobilizing chemotherapy, bone marrow infiltration at collection, n° of previous lines of therapy, prior use of fludarabine, alkylating agents or radiotherapy. The ratio between circulating CD34+cells/nL and total WBC/μL on the first day of CD34+ count (SCratio) was also analysed, trying to predict mobilization failure. Both univariate and multivariate statistical analyses were performed with SPSS package 13.0. Results Lymphoma diagnosis was diffuse large B cell/Burkitt in 38%, follicular in 10%, mantle in 13%, Hodgkin in 26%, T or NK/T in 6%, and other in 7% of cases. Disease status at apheresis was CR in 25%, chemosensitive in 49% and refractory in 26%. Mobilization was attempted during first-line therapy in 14%, in 37% during second-line and in 26% at third- or subsequent lines of therapy. Fludarabine, alkylating agents and extended-fields radiotherapy had been used prior to mobilization in 5%, 73% and 13% of cases respectively. Marrow infiltration by lymphoma was present in 17,7% and 7,7% of patients had failed a prior mobilization attempt. In 99% of cases (411/415) mobilizing therapy included chemotherapy and G-CSF. Two pts received AMD3100 and 2 G-CSF. Several chemotherapy programs were used for mobilization. For analysis purposes they were grouped as follows: single-agent cyclophosphamide (CTX) in 62 (15,1%); high-dose cytarabine, either single agent or in combination (HD-ARAC) in 143 (34,8%), etoposide-containing regimens in 119 (30,0%), and platinum containing regimens in 108 cases (26,3%). Mobilization failure occurred in 14% (59/415) whereas a good mobilization was obtained in 72% of cases. At univariate analysis mobilization failure was significantly associated with the use of single-agent CTX as mobilizer (p=0.000), with the n° of prior lines of treatment (p=0.006) and with failure of a previous mobilization attempt (p=0.000) whereas a diagnosis of follicular lymphoma (FL) was associated with a reduced risk of mobilization failure (p=0.007). Multivariate analysis confirmed FL as protective factor for mobilization failure (p=0.008) and single agent CTX and a higher n° of prior treatment lines as negative predictive factors (p=0.000 and 0.033 respectively). A “good mobilization” was significantly predicted at univariate analysis by HD-ARAC use as mobilizer (p=0,004) whereas single-agent CTX (p=0.000), n° of prior lines of therapy (p=0.005), prior fludarabine (p=0.035) or alkylating agents (p=0.003) were adverse predictors. Multivariate analysis confirmed HD-ARAC as independent predictive factor for good mobilization (p=0.031) and single-agent CTX and n° of prior lines of therapy as independent negative predictive factors (p=0.000 and 0.003 respectively). A SCratio of less than 10 on the first day of CD34+ count was associated with an increased risk of poor mobilization (p=0.000 risk estimate 0.638). Conclusions In this large series of pts with lymphoma the standard policy of SC mobilization was the use of chemotherapy + G-CSF. Nevertheless collection failed in a significant proportion of cases (14 %) and only 72% of cases reached an optimal SC collection. Predictive factors for mobilization outcome included disease and treatment history, type of lymphoma and type of mobilizing chemotherapy. These data provide the framework to rationally explore the use of new mobilizing agents in patients with lymphoma undergoing SC mobilization with chemotherapy and G-CSF. Disclosures: No relevant conflicts of interest to declare.

Chronic myelogenous leukemia (CML) is a clonal disorder of the hematopoietic stem cell characterized by the coexistence of Philadelphia-negative (Ph-) with Ph+ progenitors. CML progenitor cells have been shown to be defective in adherence to marrow stroma. The present study investigated at the cytogenetic level marrow-derived CML clonogenic cells generated from the stroma-adherent cell fraction. On direct cytogenetic analysis, the overall mean (+/- SEM) percentage of Ph- metaphases was 3% +/- 1%. Mononuclear marrow cells from CML patients (n = 18) were incubated with mafosfamide (100 micrograms/mL) or control medium, seeded onto marrow stromal layers and allowed to adhere (2 hours, 37 degrees C). After a short-term (3-day) liquid culture, the cells were harvested, incorporated in methyl-cellulose, and individual colonies were analyzed by single colony karyotyping. The mean (+/- SEM) percentage of Ph- colonies generated from the stroma- adherent fraction was 35% +/- 6%. As compared with marrow colony- forming unit granulocyte-macrophage plated before any manipulation, the mean (+/- SEM) percentage of Ph- clones was significantly increased by stroma adherence (35% +/- 6% v 15% +/- 4%, P < or = .005) and mafosfamide (100 micrograms/mL) incubation of marrow cells before stroma adherence (58% +/- 9% v 35% +/- 6%, P < or = .005). An additive effect was observed by combining mafosfamide treatment and stroma adherence. Single-colony transfer experiments showed that 50% +/- 4% stroma-adherent and 70% +/- 4% stroma-adherent mafosfamide-treated progenitors gave rise to secondary colonies. To further characterize the stroma-adherent fraction, experiments were performed in which CD34+ marrow cells were used. The mean (+/- SEM) output of progenitors generated by 10,000 CD34+, stroma-adherent cells was 888 +/- 188 and 570 +/- 258 for untreated and mafosfamide-treated cells, respectively. Individual colonies were analyzed by single-colony karyotyping and fluorescent in situ hybridization using a biotinylated cosmid DNA probe that hybridize to abl oncogene. The CD34+, stroma-adherent fraction contained 38% +/- 14% (untreated) and 56% +/- 18% (mafosfamide-treated) (P < or = .025) Ph- progenitors. In conclusion, the present data show the possibility to select Ph- clones that (1) have a maintained capability of stroma adherence, (2) are mafosfamide resistant, (3) are derived from the CD34+ fraction, and (4) have high-replating potential.

The introduction of highly active antiretroviral therapy (HAART) has allowed the use of intensive treatments for HIV+ patients (pts) with malignancies. In 2000 we started a multiinstitutional prospective study of high dose therapy (HDT) and autologous peripheral blood stem cell transplantation (PBSCT) as salvage treatment for HAART responding HIV+ pts with primary refractory or relapsed Hodgkin (HL) and non-Hodgkin lymphoma (NHL). Aim of the study was to test both the feasibility and efficacy in unselected pts. Pts showing chemoresistance or progressive disease (PD) during debulking treatment or failing PBSC collection were excluded and analysed according to the intention to treat. From May 2000 to Feb 2007 50 pts entered the study. 31 had NHL and 19 HL (20 refractory disease, 5 partial remission (PR), 22 first and 3 second relapse); median age was 39 (28–59); 60% stage IV disease; CD4 count was 218/mcl (17–561) and 11 pts had CD4 < 100; 22% had detectable HIV-viremia. Pts first received debulking chemotherapy (CT) at discretion of centers (2–4 courses): 2 died from toxicity and 10 had PD; 1 pt retracted and 6 failed PBSC mobilization. In 31 pts CD34+cells were collected (median 5.9x10e6/kg), after Cyclophosphamide 4g/mq + G-CSF (12 pts) or at recovery after CT (19 pts). Four pts had early PD and 27 received HDT (BEAM) according to the protocol (median time to N and PLT engraftment was 10 and 12 days). 10 pts underwent consolidation radiotherapy. Pts received HAART during the entire program (in 7 a brief discontinuance was necessary). Treatment toxicity was mainly gastrointestinal (8 grade III mucositis/enteritis and 1 grade IV mucositis) and hepatic (2 grade III). There were 9 FUO and 2 sepsis (S.epidermidis and E.coli), 1 Clostridium colitis, 1 Ps. aeruginosa pneumonia and 2 reactivation of perianal abscess. Infections in the first 2 years included 6 HZ, 2 esophagus candidosis, 1 CMV corioretinitis and 4 CMV reactivation with no disease. The CD4 count decreased after PBSCT (median 190.5/mcl pre PBSCT and 119.5 at +3), with recovery after few ms (157 at +6 and 285.5 at +12). 3 pts had PD after PBSCT (2 after a brief PR) and 3 relapsed after complete remission (CR) at +5, +8 and +12. 21 of 27 pts are alive and disease free (17 CR and 4 CRu), after a f-up of 44 mo (2–70), (OS 74% and PFS 75%). The median survival of the entire series (50 pts) from the enrolment was 33 ms with an OS of 49% and a PFS of 50% (follow-up 45 ms). Ann Arbor stage IV and CD4 100: OS 18% vs 59%;P=.001). Pts enrolled with PR or relapse had an OS of 62% compared with 32% of pts primary refractory (P=0.09). This study confirms on a large series the feasibility of HDT and PBSCT in HIV+ pts and shows its high long-term efficacy as salvage treatment in HIV-associated lymphoma. Less than 50% of pts couldn’t benefit this procedure, mainly because of advanced disease or severe immunodeficiency; earlier use of HDT in the course of their disease could be advisable.