Supplementary Tables 1 and 2. Supplementary Table 1 describes Summary of FDG PET/CT positivity criteria Supplementary Table 2 contains Univariate analysis of baseline and post-treatment variables adversely affecting progression-free survival and overall survival

ABSTRACT Background No studies have investigated changes of substance or alcohol consumption for non-problematic drug users within a period without restrictions in comparison with a period of lockdown. Objectives Identification of changes in habits and in substance consumption styles during the lockdown due to COVID-19 among socially integrated people who use drugs. Methods Semi-structured questionnaire administered via video calls. Results During the lockdown we highlight a reduction in consumption and expenditure on illegal substances (cocaine, MDMA, ketamine) and alcohol (spirits) and a drastic drop in alcohol misuse. Many interviewees have stopped or have passed from acontextual consumption of substances, connected to sociality, to asolitary use motivated by boredom (alcohol and cannabis), habit (alcohol), search for particular sensations (cannabis) and escape from reality (cocaine). Substance consumption alone increased. Conclusion Particular attention should be paid to the study of the dissemination and socialization of individual harm reduction practices among socially integrated people who use drug.

The current study presents data on the antimicrobial resistance (AMR) patterns of 102 S. enterica subsp. enterica (72 Salmonella ser. Typhimurium and 30 monophasic S. Typhimurium serovar) and 79 Escherichia coli (and their phylogenetic group determination) isolates from different species of bivalve molluscs and from the water samples collected from the sub-areas of a mollusc production area near Ferrara (Italy). These areas were classified as Long-line, Lupini, B-Out, B-in, and Sacca. A retrospective evaluation was performed to assess the spatial trends of the resistance patterns of Salmonella and E. coli and the temporal trend for Salmonella; the role of molluscs as AMR indicators and the potential use of E. coli as a microorganism indicator of AMR occurrence in a seawater environment were also investigated. Overall, 81% of Salmonella spp. and 75% of E. coli isolates were resistant to, at least, one antimicrobial agent (AA) and 44% and 38% of the isolates were multidrug resistant (MDR), respectively. The resistance levels of Salmonella were influenced by the investigated serovars. Monophasic S. Typhimurium serovar showed the highest resistance value with 70% of MDR isolates, in contrast with only 33% in S. Typhimurium. In monophasic S. Typhimurium versus S. Typhimurium, twofold resistance levels were observed to streptomycin (97 versus 43%), ampicillin (80 versus 40%) and tetracyclines (67 versus 36%). Regarding the temporal trend for Salmonella, strains were resistant to, at least, one AA, but this resistance was significantly lower during the first years of this 17-year sampling; however, in parallel MDR isolates, the resistance increased from 23% to a maximum level of 57% during the 2008–2012 period. On assessing the spatial trends, the Sacca area was found to show the lowest number of Salmonella spp. and E. coli strains resistant to, at least, one AA and MDR. For E. coli, the most commonly observed resistance was towards ampicillin (56%), streptomycin (52%), sulphonamides (30%) and ceftiofur (24%). The great majority (65%) of E. coli isolates belonged to the commensal phylogroups A and B1, with B1 as the dominant one, whereas most MDR isolates belonged to phylogroup C. Molluscs may be an efficient tool for antimicrobial resistance monitoring, and E. coli could be used as a microorganism indicator of the occurrence of antimicrobial resistance in seawater environment.

Objective: Mutation carriers (Mut+) in DNA mismatch repair genes are predisposed to cancer of various organs and to adenomatous polyps; however, they may remain asymptomatic and cancer or polyp-free for several years. We purposed to analyse the clinical follow-up of individuals carrying constitutional mutations in the MLH1, MSH2 or MSH6 genes who were unaffected by benign polyps or malignant tumours at diagnosis.Material and Methods: Mut+subjects (n.81) were members of Lynch syndromes in whom mutations were detected between 1993 and 2015; all were asymptomatic at diagnosis. They were informed of the cancer risk and surveillance was suggested. As controls, 113 nongene carriers (Mut-) in the same Lynch families were identified.Results: About one-fourth of the mutation carriers developed polyps, mostly adenomas; polyps were less (12%, p<.05) in Mut-subjects, and hyperplastic lesions were the prevalent histology. More polyps were detected in MLH1 vs. MSH2 mutation carriers. In Mut+, 21 malignant tumours developed in 14 carriers vs. 4 tumours in 3 patients among Mut- (p<.001). Tumours were mostly of the Lynch spectrum; however, three glioblastomas were developed, together with neoplasms of various organs (duodenum, thyroid, skin, lung and cervix). Mean age of tumour occurrence was 43.0 years in Mut+vs. 53.0 among Mut-.Conclusions: Cancer developed more often in Mut+, with no consistent difference between MLH1 and MSH2 carriers. More polyps (mostly adenomas) were detected in MLH1 carriers. The majority (13 of 21) of malignant tumours occurred in organs for which there is no recommended surveillance, and were lethal in three patients.

Monoclonal (M) protein can be detected in the serum and/or urine of patients with multiple myeloma (MM) as either intact immunoglobulins (Ig) or free light chains (FLC). Current guidelines recommend the use of serum and urine electrophoresis plus immunofixation to evaluate and monitor response to therapy in MM patients with measurable M-protein. In addition, measurement of serum FLC (sFLC) ratio (sFLCR) is required to fulfill the definition of stringent complete response and to define response or progressive disease (PD) in oligosecretory or non-secretory MM. Nonetheless, sFLC assay might be an appropriate tool also in secretory MM, since an imbalance in sFLCR can be detected in approximately 90% of patients with Ig-secretory MM (Ig-MM) and in almost all patients with light chain MM (LC-MM). Moreover, it is well recognized that sFLC escape might occur before, or at the time of, relapse and increasing levels of sFLC at progression have been reported to predict a worse prognosis. However, most of these studies are biased by the lack of serial assay measurements. Furthermore, the prognostic significance of increased levels of sFLC in the absence of any additional parameter defining PD or clinical relapse (Rel) remains an area of investigation. To address this issue, we analyzed a cohort of 100 MM patients at our center who received first-line, fixed-duration, novel agent-based therapies and for whom sFLCR measurements after treatment were available every 3-4 months until relapse. sFLC assay was performed by BN II nephelometer as part of routine clinical care. International Myeloma Working Group (IMWG) criteria were used for the definition of measurable disease, PD and Rel. Criteria defining PD in oligo/non-secretory MM according to sFLC levels were used to identify patients with secretory MM who showed rising sFLC levels in the absence of any additional parameter consistent with PD or Rel. Time to second progression (2 TTP) and overall survival (OS) after relapse were calculated from the date of first progression to the date of second progression or death, respectively, or of last follow up. Time to secondline therapy was the interval between the date of first progression and the date when salvage treatment was started. At diagnosis, 80 patients were classified as having IgMM, 15 LC-MM and 5 oligo/non-secretory disease. sFLC measurements were available at baseline in 81 patients, of whom 63 had sFLC measurable disease, as defined by an abnormal sFLCR and involved sFLC levels 100 mg/L or more. First-line treatments included the proteasomeinhibitor (PI) bortezomib in 45 patients, the immunomodulators (IMiDs) thalidomide or lenalidomide in 25 patients, and both bortezomib and IMiDs in the last 30 patients. Fifty patients received a single or double autologous stem cell transplantation. Median follow up was 63 [interquartile range (IQR) 38-83] months from diagnosis and 23 (IQR 13-37) months from progression. Best response rates were: complete response, 32%; very good partial response, 35%; partial response, 27%, and stable disease or PD, 6%. Overall, in 66 patients, sFLCR after up-front therapy was in the normal range. According to IMWG consensus recommendations, 88 patients required a second-line therapy due to Rel (n=72) or paraprotein relapse (n=16). The remaining 12 patients did not receive salvage therapy due to a paraprotein relapse not fulfilling criteria for restarting treatment (n=8) or early death (n=3) or a concomitant solid tumor (n=1). Serial monitoring of serum/urine M-protein and sFLC levels throughout the follow-up phase after first-line treatment allowed 4 different patterns of relapse to be identified. These were characterized by: 1) an increase in both M-protein and sFLC (n=30); 2) an increase in M-protein only (n=42); 3) an increase in sFLC levels only (n=15), as established according to IMWG criteria for oligo/nonsecretory MM; 4) the presence of one or more criteria defining Rel, without any concurrent change in M-protein or sFLC (n=13). Patterns of relapse characterized by an increase in sFLC levels, with or without concomitant rise in M-protein, were observed more frequently in patients with abnormal versus normal sFLCR at baseline (62% vs. 17%; P=0.001). Among the 80 patients with IgMM, patterns of relapse were: 32% both M-protein and sFLC (n=26), 48% M-protein only (n=38), 10% sFLC only (n=8), and 10% Rel without change in M-protein or sFLC (n=8) compared with 27% both M-protein and sFLC (n=4), 27% M-protein only (n=4), 40% sFLC only (n=6) and 6% Rel without change in M-protein or sFLC (n=1) for the 15 patients with LC-MM. Patients with LC-MM had a higher frequency of relapse characterized by isolated sFLC increasing than those with Ig-MM (P=0.043). In the subgroup of 14 patients with secretory-MM (8 IgMM and 6 LC-MM) and an sFLC only pattern of relapse, an increase in sFLCs preceded by 2.3 months (IQR 1.76.1) the onset of any conventional parameter defining PD, including Rel with organ damage in 10 (71%) of them, and by 4.0 (IQR 2.8-9.3) months the start of subsequent salvage therapy. Median increase in the difference between involved and uninvolved sFLC levels was 224.1 mg/L (IQR 138.3-437.9) at the time of escape and 687.5 mg/L (IQR 224.1-1819.8) at the time of PD or Rel. Overall, patients who experienced a relapse with an increase in both M-protein and sFLC or sFLC only had higher creatinine levels than those with an increase in Mprotein only or Rel without change in M-protein or sFLC (P=0.025). Conversely, no differences between these subgroups were seen with respect to the other diseaseand tumor-related characteristics that could potentially influence clinical outcomes. Second-line treatments including IMiDs (47%), PI (23%) or both these agents (30%) were equally distributed among patients with different patterns of relapse. Time to second-line therapy was shorter for patients relapsing with both M-protein and sFLC, sFLC-only or Rel without increase in M-protein or sFLC levels, than for those relapsing with M-protein only (P=0.001, trend P=0.0001). Moreover, patterns of relapse characterized by an increase in sFLC, with or without a concomitant rise in M-protein levels, correlated with worse 2 TTP and OS after relapse as compared to the other patterns. In particular, patients relapsing with both M-protein and sFLC or sFLC only had a 2-fold increase in

Recent studies have shown that high ATP levels exhibit direct cytotoxic effects on several cancer cells types. Among the receptors engaged by ATP, P2X7R is the most consistently expressed by tumors. P2X7R is an ATP-gated ion channel that could drive the opening of a non-selective pore, triggering cell-death signal. We previously demonstrated that acute myeloid leukemia (AML) cells express high level of P2X7R. Here, we show that P2X7R activation with high dose ATP induces AML blast cells apoptosis. Moreover, P2X7R is also expressed on leukemic stem/progenitor cells (LSCs) which are sensitive to ATP-mediated cytotoxicity. Conversely, this cytotoxic effect was not observed on normal hematopoietic stem/progenitor cells (HSCs). Notably, the antileukemic activity of ATP was also observed in presence of bone marrow stromal cells and its addition to the culture medium enhanced cytosine arabinoside cytotoxicity despite stroma-induced chemoresistance. Xenotransplant experiments confirmed ATP antineoplastic activity in vivo.Overall, our results demonstrate that P2X7R stimulation by ATP induced a therapeutic response in AML at the LSC level while the normal stem cell compartment was not affected. These results provide evidence that ATP would be promising for developing innovative therapy for AML.

Biosimilar G-CSFs are widely employed today for different clinical indications including hematopoietic stem cell mobilization, despite lack of robust evidence in this setting. Different single-institution retrospective studies compared mobilization kinetics of patients given biosimilar G-CSFs or originator compound, overall reporting equivalent activity.1-3 However, extremely little evidence is available about comparative efficacy of biosimilar and originator compounds when G-CSF is combined with plerixafor to rescue patients at high risk of mobilization failure.4 We herein report the results of a retrospective analysis of 296 patients affected by multiple myeloma (MM), non-Hodgkin (NHL), or Hodgkin lymphoma (HL) who underwent PBSC mobilization with G-CSF and plerixafor as part of the mobilizing strategy at 22 italian centers from January 2008 to December 2016 (patient characteristics are presented in Supporting Information Table 1). G-CSF compound employed was either originator (Neupogen®, Amgen) or biosimilar filgrastim (Zarzio®, Sandoz Industrial Products; Tevagrastim®, Teva Pharmaceutical Industries). Originator or biosimilar filgrastim were administered subcutaneously at a dosage of 5 μg/kg/day (following chemotherapy) or 10 μg/kg/day (in case of steady-state mobilization). Plerixafor was given at a dosage of 240 mcg/kg body weight. Peripheral blood CD34+ (PB-CD34+) were measured at hematopoietic recovery (in case of chemo-mobilization) or at day 4 of G-CSF administration (in case of steady-state mobilization), and then daily until stem cell collection completion or failure. The threshold of PB-CD34+ cells for starting apheresis procedure was established at 20 × 106/L. Given the retrospective nature of the study, a propensity score weighted analysis was conducted in order to soften inherent limitations as imbalances between the two study groups in terms of patient and mobilization characteristics.5 A total of 296 patients were included in the analysis. Forty-two percent of patients (n = 123) were affected by MM, 49% (n = 143) by NHL and 9% (n = 29) by HL. Forty percent of patients (n = 118) underwent chemo-mobilization, while 60% (n = 178) steady-state mobilization. One hundred and ninety-seven patients (67%) received originator filgrastim combined with plerixafor (OR + PLX), while 99 patients (33%) were given biosimilar filgrastim and plerixafor (BIO + PLX). The median PB-CD34+ count before and after plerixafor administration were 8/mcl (IQR 3–12), and 33/mcl (IQR 15–58), respectively, with an average 6-fold increase. Patients included in the BIO + PLX cohort were more likely to exceed the PB-CD34+ threshold of 5/mcl before plerixafor administration, as compared to the OR + PLX group, as evidenced by propensity score weighted analysis (weighted OR = 3.6; robust 95% CI 1.5–8.4). Further, patients receiving BIO + PLX showed higher probability of reaching the PB-CD34+ threshold of 20/mcl after plerixafor administration, as compared to the OR-PLX group (weighted OR = 6.8; robust 95% CI 2.6–17.6). Finally, patient mobilized with the BIO + PLX combination were more likely to collecting a stem cell dose of 2 × 106 CD34+/kg or higher (weighted OR = 6.1; robust 95% CI 1.9–18.9). The combination of BIO + PLX appeared to be more efficient in term of primary endpoints both in MM and lymphoma subgroups, although confidence intervals resulted quite wide, probably due to reduced sample size (Table 1). Patients who received BIO + PLX were more likely to reach the apheresis procedure as compared to OR + PLX group (weighted OR = 5.3; robust 95% CI 1.1–26.4) without any difference in the number of apheresis procedures (one or more) required to complete stem cell collection (weighted OR = 0.7; robust 95% CI 0.3–1.5). One hundred and seventy-eight patients (62%) received autologous transplant as planned, 79% of the patients who collected at least 2 × 106 CD34+/kg. The mobilization strategy (BIO + PLX or OR + PLX) did not influence the probability of performing the planned auto-SCT procedure (weighted OR = 1.5; robust 95% CI 0.7–3.4). Engraftment was successful in 97% of patients with no significant difference between the two study cohorts. PB-CD34+ after PLX Median (IQR) CD34+/Kg collected Median (IQR) CD34+/Kg collected >2 × 106 % of patients In the largest study comparing two different biosimilar G-CSFs and the originator drug in association with plerixafor, we observed a powerful mobilizing efficacy of the combination of biosimilar filgrastim and plerixafor, which was not inferior and, surprisingly, even more powerful as compared to the combination with originator G-CSF. Interestingly, in our series, biosimilar filgrastim resulted in higher probability of reaching the pre-plerixafor PB-CD34+ threshold of 5/mcl as compared to originator G-CSF. Further, BIO + PLX was associated with an increased likelihood to get to the threshold of 20/mcl and, as a direct consequence, to collect at least 2 × 106 CD34+/kg, as compared to OR + PLX. Our analysis confirms previous evidence of noninferiority of biosimilar to originator filgrastim,6 and suggests an even higher mobilizing power of BIO + PLX as compared to OR + PLX. Considering the constant improvement of pharmaceutical manufacturing techniques it might come as no surprise that a newer drug which is required to be “at least not inferior” actually turns out to be more efficient than the older one. It should be noted, however, that patients given the BIO + PLX combination underwent stem cell mobilization significantly more recently as compared to OR + PLX group; this might have somehow influenced data analysis, as it could be speculated that advancements in recent years brought a significant improvement in apheresis techniques, mobilization strategies and maybe even plerixafor use. In conclusion, the combination of biosimilar filgrastim and plerixafor appears to be at least equally and might be more effective as compared to originator filgrastim and plerixafor for stem cell mobilization in patients at high risk of mobilization failure. This data strongly support standard inclusion of biosimilar filgrastim in mobilizing protocols even in the challenging setting of patients who mobilize poorly, as significant cost saving seems to be accompanied by strong efficacy. Additional Supporting Information may be found in the online version of this article. Supporting Information Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

High-dose chemotherapy followed by autologous stem cell transplantation (ASCT) is a standard frontline therapy for multiple myeloma (MM). Therapeutic options for patients with relapsed MM after ASCT include novel agents in different combos, salvage ASCT (sASCT), and allogeneic transplant, with no unique standard of care. We retrospectively analyzed 66 MM patients who relapsed after up-front single or double ASCT(s) and received novel agent-based sASCT at five Italian centers. Median event-free survival from up-front ASCT(s) to first relapse (EFS1) was 44 months. Seventy-three percent of patients received sASCT at first disease progression. Re-induction regimens were bortezomib based in 87% of patients. Response to re-induction therapy included complete response (CR) 18%, ≥ very good partial response (VGPR) 48%, and overall response rate (ORR) 83%. Response to sASCT included CR 44%, ≥ VGPR 77%, and ORR 94%. With a median follow-up of 24 months after sASCT, 39 patients experienced disease progression. Median EFS from sASCT (EFS2) was 17 months. Median overall survival from ASCT (OS1) and sASCT (OS2) was 166 and 43 months, respectively. EFS2 and OS2 were significantly shorter in patients with EFS1 ≤ 24 months, in patients who did not receive sASCT at first disease progression and in patients with extramedullary disease (EMD). In multivariate analysis, EFS1 ≤ 24 months was associated with shorter EFS2 and OS2, EMD was associated with shorter EFS2, and < CR after sASCT was associated with shorter OS2. Novel agent-based sASCT is a safe and effective procedure for relapsed MM.

Attenuated polyposis could be defined as a variant of familial adenomatous polyposis (FAP) in which synchronous polyps of the large bowel range between 10 and 99. We analysed all cases of attenuated polyposis observed over the last 30 years with the objectives: (A) to classify the disease according to different type and proportion of polyps; (B) To ascertain the contribution of APC and MutYH genes; (C) to discover features which could arise the suspicion of mutations; (D) To obtain indications for management and follow-up. 84 individuals in 82 families were studied. Polyps were classified into four groups as adenoma, hyperplastic, other serrated lesions or others; APC and MutYH mutations were assessed. Mean age at diagnosis was 54 ± 14 years in men and 48 ± 13 in women (P = 0.005). Polyps were more numerous in women (37 ± 26 vs 29 ± 22). Sixty % of patients underwent bowel resection, mainly for cancer; the remaining were managed through endoscopy. A total of 2586 polyps were detected at diagnostic endoscopy: 2026 (80 %) were removed and analysed. Adenomas were diagnosed in 1445 (70 %), hyperplastic polyps in 541 (26 %), other serrated lesions in 61 (2.9 %). Adenomas and hyperplastic lesions were detected in the majority of patients. In 68 patients (81 %) in whom studies were executed, APC mutations were found in 8 and MutYH mutations in 10. Genetic variants were more frequent in women (12 vs 6, P = 0.039). Taking into consideration the prevalent (>50 %) histology and presence of mutations, patients could be subdivided into four groups: (1) APC mutated polyposis (AFAP), when adenomas were >50 % and APC mutations detected (no. 8, 10 %); (2) MutYH mutated polyposis (MAP), adenomas >50 % and biallelic MutYH mutations (no. 10, 12 %); (1) attenuated polyposis without detectable mutations, prevalence of adenomas, 48 cases (57 %); (1) hyperplastic-serrated polyposis, with prevalence (>50 %) of hyperplastic/other serrated lesions and no constitutional mutation (no. 18, 21 %). Aggregation of tumors, cancer in probands, distribution of polyps and other clinical characteristics showed no difference among the four groups. In conclusions, AFAP and MAP, the polyposis labeled by constitutional mutations, represented about 25 % of all attenuated polyposis. Mutation-associated cases showed an earlier age of onset of polyps and were more frequent in the female sex.

Abstract Introduction In most human cancer p53 impairment is a driver event, which confers a survival advantage to affected cells. In Multiple Myeloma (MM), the role of p53 clonal aberrations (mainly 17p del) is well recognised, whereas the prognostic relevance of TP53 mutations is less clear, due to the very limited frequency of clonal lesions. Here we aim at characterizing by ultra-deep sequencing (UDS) the TP53 mutational state in both newly diagnosed and relapsed MM pts, to assess the prognostic role and evolution over time of small TP53 mutated sub-clones. Pts and methods A cohort of 99 newly diagnosed MM pts treated up-front with bortezomib-based regimens and ASCT was included in this study. In 29 cases, samples were collected also at relapse(s). DNA was obtained from CD138+ highly purified plasma cells and SNPs array profiled (Affymetrix). TP53 gene was analysed by amplicon-targeted UDS approach (GSJ, 454 Roche). A specific bioinformatics pipeline was set up to discriminate between low frequency TP53 variants and sequencing errors. Results With a median coverage of 1386X, 129 correctly called TP53 variants were detected. Most newly diagnosed MM pts (55%) carried at least one TP53 sub-clonal variant (on average 1.08 variants per pts).. According to TP53 sub-clonal mutational load, pts were stratified in two sub-groups, including 28 pts with ≥2 (high load) and 71 with <2 variants (low load), respectively. The clinical impact of the TP53 sub-clonal mutational load was evaluated in 90/99 MM (median follow up = 70 months). Pts carrying high TP53 sub-clonal mutational load had significantly shorter OS and OS after relapse (% at 5 years: 38 vs 74 and 30 vs 73, respectively), as compared to the others, while no difference between these two groups was seen regarding PFS and TTP. Multivariate analysis showed that high TP53 mutational load, resulted independent factors adversely affecting OS and OS after relapse (HR = 2.38, CI: 1.31-5.98; HR = 2.6, CI: 1.01-6.42). None of the detected genomic aberrations significantly influenced the response to front-line induction therapy. The distribution of both TP53 sub-clonal variants and genomic CNAs was overall modified in longitudinally collected samples: overall 90% of relapsed pts carried at least one sub-clonal variant (on average 1.63 variants per pts). Moreover, 5 different sub-clonal lesions proved a linear increment of both TP53 VAFs (from 29.4% to 54.6%; from 7.8% to 12.4%; from 0.5% to 4.3%) and TP53 CN loss smooth signal (from 7% to 89% and from 50% to 100%). Conclusion The TP53 UDS analysis in newly diagnosed MM highlighted for the first time a high rate of variants, recurring with a wide range of frequencies among samples. The increased number of TP53 sub-clonal variants per pts in samples collected at relapse(s), compared to that seen at the onset of the disease, suggests a sub-clonal dynamics over time. Acknowledgements: Roche Diagnostics, FP7 NGS-PTL project, Fondazione Berlucchi. Citation Format: Marina Martello, Daniel Remondini, Enrica Borsi, Barbara Santacroce, Mauro Procacci, Angela Flores Dico, Annalisa Pezzi, Elena Zamagni, Paola Tacchetti, Lucia Pantani, Giulia Marzocchi, Beatrice Anna Zannetti, Katia Mancuso, Serena Rocchi, Giovanni Martinelli, Michele Cavo, Carolina Terragna. The selection of TP53 sub-clonal variants over time identifies MM patients with adverse clinical outcome. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5022.

Randomization procedure in randomized controlled trials (RCTs) permits an unbiased estimation of causal effects. However, in clinical practice, differential compliance between arms may cause a strong violation of randomization balance and biased treatment effect among those who comply. We evaluated the effect of the consolidation phase on disease-free survival of patients with multiple myeloma in an RCT designed for another purpose, adjusting for potential selection bias due to different compliance to previous treatment phases.

Abstract We retrospectively investigated the role of serial serum-free light chain (sFLC) evaluations in 150 multiple myeloma (MM) patients treated with first-line bortezomib-based regimens. Baseline sFLC ratio (sFLCR) identified three groups of patients – normal, lightly abnormal (<100), and highly abnormal (≥100) – with different progression-free survival (PFS: 3-year estimate 72% versus 61% versus 44%, respectively, p = 0.03). Moreover, the achievement of a normal sFLCR correlated with extended PFS (49 versus 17 months, p < 0.0001) and overall survival (75 versus 43 months, p < 0.0001) as compared with abnormal sFLCR, a gain maintained in a multivariate analysis for PFS. At relapse, a high sFLCR was associated with earlier start of salvage therapy compared with sFLCR <100 (3-month probability: 89% versus 64%, p = 0.0426). In 20% of patients, sFLC escape preceded the conventional relapse by a median of 3.8 months. Our results highlight the role of sFLC assay in the prognosis and follow-up of MM.

Hyperactivation of the Hedgehog (Hh) pathway, which controls refueling of multiple myeloma (MM) clones, might be critical to disease recurrence. Although several studies suggest the Hh pathway is activated in CD138- immature cells, differentiated CD138+ plasma cells might also be able to self-renew by producing themselves the Hh ligands. We studied the gene expression profiles of 126 newly diagnosed MM patients analyzed in both the CD138+ plasma cell fraction and CD138-CD19+ B-cell compartment. Results demonstrated that an Hh-gene signature was able to cluster patients in two subgroups characterized by the opposite Hh pathway expression in mature plasma cells and their precursors. Strikingly, patients characterized by Hh hyperactivation in plasma cells, but not in their B cells, displayed high genomic instability and an unfavorable outcome in terms of shorter progression-free survival (hazard ratio: 1.92; 95% confidence interval: 1.19-3.07) and overall survival (hazard ratio: 2.61; 95% confidence interval: 1.26-5.38). These results suggest that the mechanisms triggered by the Hh pathway ultimately led to identify a more indolent vs a more aggressive biological and clinical subtype of MM. Therefore, patient stratification according to their molecular background might help the fine-tuning of future clinical and therapeutic studies.

8000 Background: The role of upfront ASCT for newly diagnosed (ND) MM (NDMM) patients (pts) has been questioned in the novel agent era. Methods: A phase 3 study was designed to compare [random (R) 1] 4 cycles of bortezomib-melphalan-prednisone (VMP) vs high-dose melphalan (HDM) and single or double ASCT (this latter limited to centers applying a tandem ASCT policy) as intensification therapy following induction with bortezomib-cyclophosphamide-dexamethasone and subsequent collection of peripheral blood stem cells. Consolidation therapy with bortezomib-lenalidomide-dexamethasone vs no consolidation (R2) was planned after VMP and HDM, followed by lenalidomide maintenance until progression or toxicity in both treatment arms. Primary study end point was progression-free survival (PFS) from R1. A first prespecified interim analysis was performed in January 2016 when at least 33% of the required events had been observed. Results are herein reported. Results: From February 2011 through April 2014, 1503 pts aged ≤65 years with symptomatic NDMM were registered. Of these, 1308 pts were eligible for R1 and 1266 who were randomized (1:1 ratio; stratification by ISS stage) to VMP (512 pts) or HDM (1±2 ASCT) (754 pts) were analyzed. Median follow up from R1 was 24 months. PFS was significantly prolonged in pts randomized to HDM (HR=0.76; 95% CI=0.61-0.94; P=0.010), a benefit retained across predefined pt subgroups, including those with revised ISS stage III (HR=0.52; CI=0.32-0.84; P=0.008) and high-risk cytogenetics [t(4;14) ± del(17p) ± del(1p) ± 1q gain] (HR=0.72; CI=0.54-0.97; P=0.028). Superior rate of ≥ very good partial response was observed with HDM (84%) vs VMP (74%) (odds ratio=1.90; CI=1.42-2.54; P<0.0001). In a Cox regression analysis, randomization to HDM (HR=0.61, CI=0.45-0.82; P=0.001) was confirmed to be an independent predictor of prolonged PFS. Overall survival was not yet mature and no difference between the treatment groups was evident. Conclusions: Upfront ASCT still remains the preferred treatment for younger NDMM pts. Further follow-up of the study is needed. Clinical trial information: NCT01208766.

INTRODUCTION. Although remarkable advances have been reported in Multiple Myeloma (MM) therapy, mainly due to the introduction of novel agents, the disease remains incurable in most of the patients. The iperactivation of self-renewal mechanisms, like Hedgehog (Hh) pathway, which controls the refuel of the tumor clone, might be critical to disease recurrence. Whilst several studies suggestthatHh pathway is activated in the putative CD138- Myeloma Propagating Cells (MPCs), it is likely that also terminally differentiated CD138+ plasma cells might contribute to drug resistance, by reverting to an immature phenotype.

Allogeneic stem cell transplantation (allo-SCT), which provides a tumor-free graft, is an alternative approach to autologous transplantation for multiple myeloma (MM) that offers the possibility of cure through a graft vs myeloma effect (GVM). However, the inherent non relapse mortality (NRM) and the high post-transplant relapse rate are the major shortcomings of this strategy which continues to have a controversial role in MM treatment. To highlight the long-term clinical outcomes of allo-SCT, we performed a retrospective analysis on 102 patients (pts) with MM who received at our Institution either a myeloablative (MA) (74 pts) or a non-myeloablative (NMA) (28 pts) conditioning regimen between 1990 and 2014. The MA regimen consisted in low dose TBI and cyclophosphamide (cyclo) ± melphalan (mel) or busulfan-cyclo. The NMA regimen was mel-fludarabine. Graft versus host disease (GVHD) prophylaxis consisted in cyclosporine + methotrexate (83%) or mycophenolate (17%), with the addition of thymoglobulin for unrelated or related female vs male recipient donors. The median age was 42 yrs (IQR 38-47), 60% pts were male. The hematopoietic cell donors were sibling in 77 pts and unrelated in 25, while the source of stem cells was peripheral blood in 65 and bone marrow in 37 pts. Fifty-six pts received allo-SCT as first-line therapy, 31 as second-line and 13 as third or fourth-line. Median time from diagnosis to allo-SCT was 22 months. Response status at the time of transplant was at least PR in 63% of the pts, including ≥ VGPR in 36% and CR in 12%. Overall, the response rate after allo-SCT was as follows: CR 58%, VGPR 19%, PR 18%. Median CR duration was 10 years (44% at 15 years). The incidence of acute grade II-IV and III-IV GVHD was 25% and 15%, respectively. The incidence of all grades chronic cGVHD was 48%, grade ≥2: 24%, with a median onset time of 178 days. By competitive risks analysis, the cumulative incidence of cGVHD at 1 and 3 years was 20% and 32%, respectively. On univariate analysis, the gender combination of female donor-male patient resulted in significantly higher incidence of cGVHD (sub hazard ratio, SHR, 2.3, P= 0.03). The cumulative incidence of NRM was 6.6% at 100 days, 11% at 1 year and 14.4% at 3 years, with lack of statistically significant relationship with the conditioning regimen. By univariate analysis, By competitive risks analysis, the cumulative incidence of relapse was 50% at 5 years, 58% at 10 years and 59% at 15 years. On univariate analysis, sibling donor (SHR 0.54, P=0.047), all grades cGVHD (SHR 0.5, P=0.011) and ≥VGPR after allo-SCT (SHR 0.38, P=0.001) were significantly associated with extended time to progression. All grades cGVHD (SHR 0.43, P=0.005) and ≥VGPR after allo-SCT (SHR 0.36, P= 0.001) were independent predictors for a lower risk of progression on multivariate analysis. With a median follow-up of 13 years, overall survival (OS) was 43% at 5 years and 34% at 10 years. In univariate analysis, low ISS stage at diagnosis, sibling donor, absence of female donor-male patient gender combination, ≥VGPR prior to allo-SCT and first-line allo-SCT were significantly related to OS. Multivariate analysis confirmed an OS benefit for having a sibling donor (HR 0.30, P In conclusion, this retrospective analysis performed with an extended follow-up of 13 years shows that a fraction of MM pts can be long-term survivors after allo-SCT, one third of them being potentially cured. The best outcomes were obtained when allo-SCT was applied in pts in an early phase of their disease and with a small residual tumor size. The major challenge, both with MA and NMA allo-SCT, was the relatively high post-transplant relapse rate. The presence of cGVHD was protective for the risk of progression, supporting the role of GVM, but increased the risk of NRM. New approaches aimed at modulating cGVHD are warranted. In addition, incorporation of novel agents before and after allo-SCT to increase the rate and duration of high-quality responses, as well as identification of those patients mostly benefiting from this procedure, will likely contribute to improve long-term outcomes. Disclosures Zamagni:Celgene Corporation: Honoraria, Speakers Bureau; Janssen Pharmaceuticals: Honoraria, Speakers Bureau; Amgen: Honoraria, Speakers Bureau. Cavo:Sanofi: Consultancy, Honoraria; Onyx: Honoraria; Jansenn: Consultancy, Honoraria; Millenium Pharmaceuticals: Honoraria; Celgene: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; BMS: Honoraria.

Introduction. The role of TP53 tumor-suppressor gene in mediating cellular basic tumor suppressive mechanisms is crucial: its utmost importance is underscored by a 50% mutational rate among most human cancers. In MM, TP53 is rarely reported as being affected either by deletions or mutations, even if chromosome (chr) 17p13 copy number (CN) loss defines a samples subgroup with a particularly poor prognosis. Here we aim at retrospectively analyzing by Next Generation Sequencing (NGS) the TP53gene inactivation in newly diagnosed MM, assessing the mutational events’ frequency and their clinical impact.