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Cox proportional hazard Models for PFS: role of MRD-negativity after 12 month of follow-up.

Purpose: The role of the minimal residual disease (MRD) in follicular lymphoma is still debated. In this study, we assessed whether the BCL2/IGH rearrangement could have a prognostic role in patients receiving R-CHOP, R-FM, or R-CVP.Experimental Design: DNAs from 415 patients among the 504 cases enrolled in the FOLL05 trial (NCT00774826) were centralized and assessed for the BCL2/IGH at diagnosis, at the end of treatment, and after 12 and 24 months.Results: At diagnosis, the molecular marker was detected in 53% of cases. Patients without molecular marker or with a low molecular tumor burden (−4 copies) showed higher complete remission (CR) rate and longer progression-free survival (PFS; 3-year PFS 80% vs. 59%; P = 0.015). PFS was significantly conditioned by the PCR status at 12 and 24 months, with 3-year PFS of 66% for MRD− cases versus 41% for those MRD+ at 12 months (P = 0.015), and 84% versus 50% at 24 months (P = 0.014). The MRD negativity at 12 and 24 months resulted in an improved PFS both in CR and in partial remission (PR) patients (3-year PFS = 72% for cases CR/PCR− vs. 32% for those CR/PCR+ vs. 62% for those PR/PCR− and 25% for patients in PR/PCR+; P = 0.001). The prognostic value of MRD at 12 and 24 months of follow-up was confirmed also in multivariate analysis.Conclusions: In this study, standardized molecular techniques have been adopted and applied on bone marrow samples from a large cohort. Data reported show that the MRD detection is a powerful independent predictor of PFS in patients with follicular lymphoma receiving conventional chemoimmunotherapy. Clin Cancer Res; 20(24); 6398–405. ©2014 AACR.

Supplemental Figure Legend from Minimal Residual Disease after Conventional Treatment Significantly Impacts on Progression-Free Survival of Patients with Follicular Lymphoma: The FIL FOLL05 Trial

CONSORT diagram of the study.

Background In addition to morphological and cytogenetic features, acute myeloid leukemias are characterized by mutations that can be used for target-therapy; also the minimal/measurable residual disease (MRD) could be an important prognostic factor. The purpose of this retrospective study was to investigate if somatic mutations could represent an additional prognostic value in respect of MRD alone. Method At baseline, 98 patients were tested for NPM1, FLT3, and for WT1 expression; 31 for ASXL1, TET2, IDH1, IDH2, N-RAS, WT1, c-KIT, RUNX1, and DNMT3A. The same genes have been also tested after induction and consolidation. Results Overall, 60.2% of our patients resulted mutated: 24.5% carried mutations of FLT3-ITD, 38.7% of NPM1, 48.4% of c-KIT, 25.8% of N-RAS and 19.3% of IDH2. The probability of achieving a complete response (CR) was higher for younger patients, with low ELN risk score, NPM1-mutated, with low WT1 levels, and without FLT3. The presence of additional mutations represented a poor predictive factor: only 19% of these cases achieved CR in comparison to 43% of subjects without any of it. Concerning survival, it was conditioned by a lower ELN risk score, younger age, reduction > 1 log of the NPM1 mutational burden, disappearance of FLT3 mutations and lower WT1 expression. Regarding the role of the additional mutations, they impaired the outcome of 20% of the already MRD-negative patients. Concerning the possibility of predicting relapse, we observed an increase of the NPM1 mutational burden at the time-point immediately preceding the relapse (about 2 months earlier) in 50% of subjects. Similarly concerning WT1, an increase of its expression anticipated disease recurrence in 64% of cases. Conclusions We demonstrated that additional somatic mutations are able to impair outcome of the already MRD-negative subjects. About MRD, we suggest a prognostic role also for the WT1 expression. Finally, we considered as relevant the assessment of NPM1 quantity clearance instead of the presence/absence of mutations alone. Still remains in doubt the utility in terms of long-term prognosis of a baseline more complex mutational screening; we could hypothesize that it would be useful for those patients where other markers are not available or who reached the MRD negativity. Electronic supplementary material The online version of this article (10.1186/s12935-019-0807-0) contains supplementary material, which is available to authorized users.

SARS-CoV-2 is the viral agent responsible for the pandemic that in the first months of 2020 caused about 400,000 deaths. Among compounds proposed to fight the SARS-CoV-2-related disease (COVID-19), tyrosine kinase inhibitors (TKIs), already effective in Philadelphia-positive acute lymphoblastic leukemia (Ph+ ALL) and chronic myeloid leukemia (CML), have been proposed on the basis of their antiviral action already demonstrated against SARS-CoV-1. Very few cases of COVID-19 have been reported in Ph+ ALL and in CML Italian cohorts; authors suggested that this low rate of infections might depend on the use of TKIs, but the biological causes of this phenomenon remain unknown. In this study, the CML model was used to test if TKIs would sustain or not the viral replication and if they could damage patient immunity. Firstly, the infection and replication rate of torquetenovirus (TTV), whose load is inversely proportional to the host immunological control, have been measured in CML patients receiving nilotinib. A very low percentage of subjects were infected at baseline, and TTV did not replicate or at least showed a low replication rate during the follow-up, with a mean load comparable to the measured one in healthy subjects. Then, after gene expression profiling experiments, we found that several “antiviral” genes, such as CD28 and IFN gamma, were upregulated, while genes with “proviral” action, such as ARG-1, CEACAM1, and FUT4, were less expressed during treatment with imatinib, thus demonstrating that TKIs are not detrimental from the immunological point of view. To sum up, our data could offer some biological explanations to the low COVID-19 occurrence in Ph+ ALL and CML patients and sustain the use of TKIs in COVID-19, as already proposed by several international ongoing studies.

Isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) interfere with cellular metabolism contributing to oncogenesis. Mutations of IDH2 at R140 and R172 residues are observed in 20% of acute myeloid leukemias (AML), and the availability of the IDH2 inhibitor Enasidenib made IDH2 mutational screening a clinical need. The aim of this study was to set a new quantitative polymerase chain reaction (PCR) technique, the drop-off digital droplet PCR (drop-off ddPCR), as a sensitive and accurate tool for detecting IDH2 mutations. With this technique we tested 60 AML patients. Sanger sequencing identified 8/60 (13.5%) mutated cases, while ddPCR and the amplification refractory mutation system (ARMS) PCR, used as a reference technique, identified mutations in 13/60 (21.6%) cases. When the outcome of IDH2-mutated was compared to that of wild-type patients, no significant difference in terms of quality of response, overall survival, or progression-free survival was observed. Finally, we monitored IDH2 mutations during follow-up in nine cases, finding that IDH2 can be considered a valid marker of minimal residual disease (MRD) in 2/3 of our patients. In conclusion, a rapid screening of IDH2 mutations is now a clinical need well satisfied by ddPCR, but the role of IDH2 as a marker for MRD still remains a matter of debate.

We here describe a novel method for MYD88L265P mutation detection and minimal residual disease monitoring in Waldenstrom macroglobulinemia, by droplet digital polymerase chain reaction, in bone marrow and peripheral blood cells, as well as in circulating cell-free DNA. Our method shows a sensitivity of 5.00×10−5, which is far superior to the widely used allele-specific polymerase chain reaction (1.00×10−3). Overall, 291 unsorted samples from 148 patients (133 with Waldenstrom macroglobulinemia, 11 with IgG lymphoplasmacytic lymphoma and 4 with IgM monoclonal gammopathy of undetermined significance) were analyzed: 194 were baseline samples and 97 were followup samples. One hundred and twenty-two of 128 (95.3%) bone marrow and 47/66 (71.2%) baseline peripheral blood samples scored positive for MYD88L265P. To investigate whether MYD88L265P detection by droplet digital polymerase chain reaction could be used for minimal residual disease monitoring, mutation levels were compared with IGH-based minimal residual disease analysis in 10 patients, and was found to be as informative as the classical, standardized, but not yet validated in Waldenstrom macroglobulinemia, IGH-based minimal residual disease assay (r2=0.64). Finally, MYD88L265P detection by droplet digital polymerase chain reaction on plasma circulating tumor DNA from 60 patients showed a good correlation with bone marrow findings (bone marrow median mutational value 1.92×10−2, plasma circulating tumor DNA value: 1.4×10−2, peripheral blood value: 1.03×10−3). This study indicates that droplet digital polymerase chain reaction assay of MYD88L265P is a feasible and sensitive tool for mutation screening and minimal residual disease monitoring in Waldenstrom macroglobulinemia. Both unsorted bone marrow and peripheral blood samples can be reliably tested, as can circulating tumor DNA, which represents an attractive, less invasive alternative to bone marrow for MYD88L265P detection.

Background. Immunochemotherapy is effective in follicular lymphoma (FL), but most patients (pts) eventually relapse. MRD analysis, based on the detection of Bcl-2/IGH rearrangement by highly sensitive PCR-based tools, is effective in identifying pts at risk of relapse [Ladetto Blood 2012; Pott EHA23]. However, several issues are still unresolved, including: i) which is the best tissue source and the most reliable technique; ii) which are the most predictive time points; iii) which is the role of disease kinetics during the long natural history of FL. The FIL FOLL12 prospective, phase III randomized clinical trial (EudraCT: 2012-003170-60) included a systematic MRD analysis on both peripheral blood (PB) and bone marrow (BM) taken at eight different pre-planned time points, by both nested and real time quantitative (RQ)-PCR. Therefore, it allows addressing these unresolved issues. Methods. The FOLL12 compared conventional rituximab maintenance [Salles et al, Lancet 2010] vs a combined PET/MRD response-based post-induction approach in pts with advanced FL after first line chemo-immunotherapy. Clinical results have been already reported [Luminari et al, ICML16]. PB and BM samples were centralized at four Italian Euro-MRD certified laboratories. MRD was assessed with consensus primers on Bcl-2/IGH rearrangements (MBR, mcr and minor rearrangements) by both nested and RQ-PCR at eight time points: baseline, end of induction (EoI) and every six months thereafter till month 36. MRD data were treated as a time-varying covariate and analyzed by means of flexible parametric survival model (Parmar-Royston) with the log cumulative baseline hazard function. MRD data were modeled with restricted cubic spline as function of time. Effect of fixed covariates and landmark analysis were performed with the Cox PH regression. Any estimation was reported with its 95%CI. Results. Overall, 10,702 analytical results were generated, (3,000 for marker screening and 7,702 for MRD). 780 of 786 eligible pts (99%) were screened at baseline for the presence of a molecular marker. 443/780 (57%) had a detectable Bcl-2/IGH rearrangement, as expected. High rates of MRD negativity were observed at EoI, with similar results by both techniques (87% in BM and 95% in PB by nested-PCR, 90% in BM and 95% in PB with RQ-PCR). Overall, the presence of one MRD positive result was associated during the entire follow-up period with an increased risk of relapse in the subsequent six months interval (HR for PFS 2.82, 95% CI 1.84-4.34, p Conclusions. This comprehensive MRD study in FL clearly indicates that: i) punctual MRD analysis is predictive of poor outcome at multiple pre-planned time points taken over a 36 months period; ii) both nested and RQ-PCR performed adequately, the latter being preferable as broadly used and internationally standardized; iii) BM allows better prediction at the early time points but, starting from month 12 after EoI PB is superimposable to BM, allowing effective and reliable long-term non-invasive MRD monitoring; iv) the high predictive value of punctual time point analysis is further improved by a kinetic approach to the interpretation of MRD results. Figure 1 Figure 1. Disclosures Ladetto: AbbVie, Jazz, Gentili, Incyte, ADC Therapeutics, Acerta, Pfizer: Honoraria; Roche, J&J, Celgene, Novartis, Amgen, Gilead, Beigene, GSK: Honoraria. Ferrero: Servier: Speakers Bureau; EUSA Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Morphosys: Research Funding; Incyte: Membership on an entity's Board of Directors or advisory committees; Gilead: Research Funding, Speakers Bureau; Clinigen: Membership on an entity's Board of Directors or advisory committees. Del Giudice: Tolero: Membership on an entity's Board of Directors or advisory committees; Astrazeneca: Membership on an entity's Board of Directors or advisory committees. Galimberti: Incyte: Speakers Bureau; AbbVie, Janssen: Honoraria, Other: Travel grants. Gattei: abbVie: Research Funding; Janssen: Research Funding; Menarini: Research Funding. Mannina: Janssen,Takeda: Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees. Falini: Rasna Therapeutics: Honoraria. Luminari: Roche, Celgene, Teva Pharmaceuticals, Gilead Sciences, and Takeda Pharmaceuticals: Honoraria.

// Elena Ciabatti 1,3 , Angelo Valetto 2 , Veronica Bertini 2 , Maria Immacolata Ferreri 2 , Alice Guazzelli 2 , Susanna Grassi 1,3 , Francesca Guerrini 1 , Iacopo Petrini 4 , Maria Rita Metelli 1 , Maria Adelaide Caligo 2 , Simona Rossi 2 and Sara Galimberti 1 1 Department of Clinical and Experimental Medicine, Section of Hematology, University of Pisa, Pisa, Italy 2 Laboratory of Medical Genetics, Azienda Ospedaliero-Universitaria Pisana, S. Chiara Hospital, Pisa, Italy 3 GenOMec, University of Siena, Siena, Italy 4 Department of Translational Research and New Technologies in Medicine, University of Pisa, Pisa, Italy Correspondence to: Elena Ciabatti, email: // Keywords : myelodysplastic syndromes, aCGH, cytogenetics, TP53, SF3B1 Received : August 23, 2016 Accepted : March 14, 2017 Published : March 25, 2017 Abstract In this study we present a new diagnostic workup for the myelodysplastic syndromes (MDS) including FISH, aCGH, and somatic mutation assays in addition to the conventional cytogenetics (CC). We analyzed 61 patients by CC, FISH for chromosome 5, 7, 8 and PDGFR rearrangements, aCGH, and PCR for ASXL1, EZH2, TP53, TET2, RUNX1, DNMT3A, SF3B1 somatic mutations. Moreover, we quantified WT1 and RPS14 gene expression levels, in order to find their possible adjunctive value and their possible clinical impact. CC analysis showed 32% of patients with at least one aberration. FISH analysis detected chromosomal aberrations in 24% of patients and recovered 5 cases (13.5%) at normal karyotype (two 5q- syndromes, one del(7) case, two cases with PDGFR rearrangement). The aGCH detected 10 “new” unbalanced cases in respect of the CC, including one with alteration of the ETV6 gene. After mutational analysis, 33 patients (54%) presented at least one mutation and represented the only marker of clonality in 36% of all patients. The statistical analysis confirmed the prognostic role of CC either on overall or on progression-free-survival. In addition, deletions detected by aCGH and WT1 over-expression negatively conditioned survival. In conclusion, our work showed that 1) the addition of FISH (at least for chr. 5 and 7) can improve the definition of the risk score; 2) mutational analysis, especially for the TP53 and SF3B1, could better define the type of MDS and represent a “clinical warning”; 3) the aCGH use could be probably applied to selected cases (with suboptimal response or failure).

Notwithstanding the introduction of Tyrosine Kinase Inhibitors (TKIs) revolutionized the outcome of Chronic Myeloid Leukemia (CML), one third of patients still suspends treatment for failure response. Recent research demonstrated that several BCR/ABL1-independent mechanisms can sustain resistance, but the relationship between these mechanisms and the outcome has not yet been fully understood. This study was designed to evaluate in a “real-life” setting if a change of expression of several genes involved in the WNT/BETA-CATENIN, JAK-STAT, and POLYCOMB pathways might condition the outcome of CML patients receiving TKIs. Thus, the expression of 255 genes, related to the aforementioned pathways, was measured by quantitative PCR after 6 months of therapy and compared with levels observed at diagnosis in 11 CML patients, in order to find possible correlations with quality of response to treatment and event-free-survival (EFS). These results were then re-analyzed by the principal component method (PCA) for tempting to better cluster resistant cases. After 12 months of therapy, 6 patients achieved an optimal response and 5 were “resistant;” after application of both statistical methods, it was evident that in all pathways a significant overall up-regulation occurred, and that WNT was the pathway mostly responsible for the TKIs resistance. Indeed, 100% of patients with a “low” up-regulation of this pathway achieved an optimal response vs. 33% of those who showed a “high” gene over-expression (p = 0.016). Analogously, the 24-months EFS resulted significantly influenced by the degree of up-regulation of the WNT signaling: all patients with a “low” up-regulation were event-free vs. 33% of those who presented a “high” gene expression (p = 0.05). In particular, the PCA analysis confirmed the role of WNT pathway and showed that the most significantly up-regulated genes with negative prognostic value were DKK, WNT6, WISP1, and FZD8. In conclusion, our results sustain the need of a wide and multitasking approach in order to understand the resistance mechanisms in CML.

We report the case of an aggressive non-Hodgkin lymphoma transmission from a live renal donor to her recipient. Soon after donation, the donor developed a high-grade B cell lymphoma, and the disease became evident also in the recipient three months thereafter, showing the same biological characteristics as confirmed by IgH rearrangement, karyotype and chimerism analyses. In the recipient, the IgH clone appeared in circulating cell free DNA (cfDNA), but not in peripheral blood nor in bone marrow, one month earlier than the imaging evidence of disease. This case supports the fundamental predictive role of molecular analysis of the plasma compartment in the diagnosis and putatively follow up of lymphomas

Introduction Zinc plays an important role in thymic function and immune homeostasis. We performed a prospective clinical trial using a high-dose zinc oral supplementation to improve the immune reconstitution after hematopoietic stem cell transplant (HSCT). Patients and methods We enrolled 18 patients undergoing autologous HSCT for multiple myeloma. Nine patients were randomized to receive only a standard antimicrobial prophylaxis; whereas, nine patients received in addition 150 mg/day of zinc from day +5 to day +100 after transplant. Results CD4+ naive lymphocytes and TRECs showed a significant increase from day +30 until day +100 only in the zinc-treated group. Moreover, the load of Torquetenovirus, a harmless virus that replicates in course of immunedepression, increased at day +100 only in the control group. No severe adverse events were reported during the zinc consumption. Conclusion First data from the ZENITH trial suggest that high-dose zinc supplementation is safe and may enhance the thymic reconstitution after HSCT. Registered: http://Clinicaltrials.gov (NCT03159845); and EUDRACT: 2014-28 004499-47.

Aim: Imatinib is a tyrosine kinase inhibitor that has revolutionized the treatment of chronic myeloid leukemia (CML). Despite its efficacy, about a third of patients discontinue the treatment due to therapy failure or intolerance. The rational identification of patients less likely to respond to imatinib would be of paramount clinical relevance. We have shown that transmembrane transporter hOCT1 genotyping predicts imatinib activity. In parallel, Polycomb group genes (PcGs) are epigenetic repressors implicated in CML progression and in therapy resistance. Patients & methods: We measured the expression of eight PcGs in paired pre- and post-imatinib bone marrow samples from 30 CML patients. Results: BMI1, PHC3, CBX6 and CBX7 expression was significantly increased during imatinib treatment. Post-treatment levels of CBX6 and CBX7 predicted 3-month response rate. Measurement of post-treatment BMI1 levels improved the predictive power of hOCT1 genotyping. Conclusion: These results suggest that the expression levels of PcGs might be useful for a more accurate risk stratification of CML patients.

We here describe a novel method for MYD88L265P mutation detection and minimal residual disease monitoring in Waldenström macroglobulinemia, by droplet digital polymerase chain reaction, in bone marrow and peripheral blood cells, as well as in circulating cell-free DNA. Our method shows a sensitivity of 5.00×10−5, which is far superior to the widely used allele-specific polymerase chain reaction (1.00×10−3). Overall, 291 unsorted samples from 148 patients (133 with Waldenström macroglobulinemia, 11 with IgG lymphoplasmacytic lymphoma and 4 with IgM monoclonal gammopathy of undetermined significance) were analyzed: 194 were baseline samples and 97 were followup samples. One hundred and twenty-two of 128 (95.3%) bone marrow and 47/66 (71.2%) baseline peripheral blood samples scored positive for MYD88L265P. To investigate whether MYD88L265P detection by droplet digital polymerase chain reaction could be used for minimal residual disease monitoring, mutation levels were compared with IGH-based minimal residual disease analysis in 10 patients, and was found to be as informative as the classical, standardized, but not yet validated in Waldenström macroglobulinemia, IGH-based minimal residual disease assay (r2=0.64). Finally, MYD88L265P detection by droplet digital polymerase chain reaction on plasma circulating tumor DNA from 60 patients showed a good correlation with bone marrow findings (bone marrow median mutational value 1.92×10−2, plasma circulating tumor DNA value: 1.4×10−2, peripheral blood value: 1.03×10−3). This study indicates that droplet digital polymerase chain reaction assay of MYD88L265P is a feasible and sensitive tool for mutation screening and minimal residual disease monitoring in Waldenström macroglobulinemia. Both unsorted bone marrow and peripheral blood samples can be reliably tested, as can circulating tumor DNA, which represents an attractive, less invasive alternative to bone marrow for MYD88L265P detection.

R-Bendamustine is an effective treatment for follicular lymphoma (FL). Previous large trials demonstrated the prognostic role of the molecular minimal residual disease (MRD) during the most frequently adopted chemotherapeutic regimens, but there are not yet conclusive data about the effect of combination of rituximab (R) and bendamustine in terms of MRD clearance. Thus, the aim of this retrospective study was to assess if and in what extent the combination of rituximab and bendamustine would exert a significant reduction of the molecular disease in 48 previously untreated FL patients. The molecular marker at baseline was found in the 62.5% of cases; no significant differences were observed between patients with or without the molecular marker in respect of the main clinical features. Moreover, the quantization of the baseline molecular tumor burden showed a great variability: the median value was 1.4x10-2 copies, ranging from 3x10-5 to 4x104. The initial molecular tumor burden did not correlate with clinical features and did not impact on the subsequent quality of response. After treatment, 93% of cases became MRD-negative; the median reduction of the BCL2/JH load was 4 logs. The 2-years PFS was 85%; it was significantly longer for patients in complete than for those in partial response (91% vs 57%; p=0.002), and for cases with lower FLIPI-2 score (88% vs 60%; p=0.004). On the contrary, PFS did not differ between patients with or without the molecular marker at baseline; a molecular tumor burden 15 times higher was observed in the relapsed subgroup in comparison to the relapse-free one, but this difference did not change the PFS length. The 2-years OS was 93.6%; the only variable that significantly impacted on it was the FLIPI-2 score; the presence of the molecular marker at baseline or its behavior after treatment did not impact on survival. This study, even if retrospective and conducted on a small series of patients, would represent a proof of concept that R-bendamustine is able to so efficaciously eradicate MRD that it could be able to by-pass the prognostic significance of MRD already demonstrated for other chemotherapeutic regimens in follicular lymphoma.

Human positive regulatory domain I binding factor 1 (PRDI-BF1 or BLIMP-1) is a transcription factor that acts as a master regulator and has crucial roles in the control of differentiation and in maintaining survival of plasma cells (PC). The PRDM1 gene, which codifies for PRDI-BF1, contains an alternative promoter capable of generating a PRDI-BF1 deleted protein (called PRDI-BF1β), which lacks 101 amino acids comprising most of the regulatory domain. PRDI-BF1β has been detected in relevant quantities especially in multiple myeloma cell lines (U266 and NCI- H929). The first aim of the study was to compare, using real time polymerase chain reaction (RT-PCR), the levels of PRDI-BF1 and PRDI-BF1β in myeloma patients and in normal human bone marrow. The second step was the examination of the expression of PRDI-BF1 and PRDI-BF1β isoform depending on disease status and treatment response. We demonstrate the correlation of PRDI-BF1 and the shorter PRDI-BF1β isoform protein levels with the clinical evolution and the management of myeloma patients.

We present on a new case of myelodysplastic syndrome characterized by array Comparative Genomic Hybridization. This technique confirmed the monosomy 7, detected by conventional cytogenetics, and revealed also a deletion on the short arm of chromosome 12. This deletion extends for about 14.8 Mb and breaks ETV6 gene. 12p deletion extents in hematological malignancies may vary, but the minimally deleted region almost invariably contains ETV6, that is considered the main candidate tumor suppressor genes within the region for tumor progression. It has been shown that levels of ETV6 were significantly decreased in cases with 12p13 deletions, whereas expression of other genes in the deleted region, like BCL2L14, LRP6, DUSP16 and GPRC5D, did not show any variation, independently of their copy number status. This observation strengthens the fact that ETV6 may play a potential role in the tumorigenesis process. The role of ETV6 in our patient myelodysplastic syndrome is showed by his clinical history and his poor prognosis.

First-line nilotinib in chronic myeloid leukemia is more effective than imatinib to achieve early and deep molecular responses, despite poor tolerability or failure observed in one-third of patients. The toxicity and efficacy of tyrosine kinase inhibitors might depend on the activity of transmembrane transporters. However, the impact of transporters genes polymorphisms in nilotinib setting is still debated. We investigated the possible correlation between single nucleotide polymorphisms of hOCT1 (rs683369 [c.480C>G]) and ABCB1 (rs1128503 [c.1236C>T], rs2032582 [c.2677G>T/A], rs1045642 [c.3435C>T]) and nilotinib efficacy and toxicity in a cohort of 78 patients affected by chronic myeloid leukemia in the context of current clinical practice. The early molecular response was achieved by 81% of patients while 64% of them attained deep molecular response (median time, 26 months). The 36-month event-free survival was 86%, whereas 58% of patients experienced toxicities. Interestingly, hOCT1 and ABCB1 polymorphisms alone or in combination did not influence event-free survival or the adverse events rate. Therefore, in contrast to data obtained in patients treated with imatinib, hOCT1 and ABCB1 polymorphisms do not impact on nilotinib efficacy or toxicity. This could be relevant in the choice of the first-line therapy: patients with polymorphisms that negatively condition imatinib efficacy might thus receive nilotinib as first-line therapy.

The welfare assessment of owned pet cats has been neglected in research. The aim of this study was to assess owners' perception and recognition of impaired welfare in their own cats. One hundred ninety-four cat owners were interviewed face-to-face by a veterinary behaviorist, completing a 42-item questionnaire. Most owners (71.1%/N = 138) correctly included both physical and psychological features within their definition of stress, but 9.8% (19) thought that stress had no consequences for the cat. When asked to rate the overall stress level of their own cats in a nonnumerical scale based on frequency, 56.7% (N = 110) chose low, 38.1% (N = 74) chose medium, and 5.2% (N = 10) chose high. Owners whose cats played little or not at all were more likely to rate the level of stress of their cats as high (90.0%) than to rate it as low or medium (33.2%/64; χ2 = 13.290; P < 0.001). Similarly, owners whose cats showed overgrooming were more likely to rate the level of stress of their cats as high (30.0%/58 vs. 7.6%/15; χ2 = 4.948; P = 0.015). The display of aggression or house soiling was not associated with the owner's rating of stress level. The number of signs recognized by respondents as potential indicators of stress in cats and the rating of the level of stress in their own cats were weakly correlated (ρ = 0.217; P = 0.002). This may be due to possible biases in the interviews as well as to an overall good welfare in the cat sample associated to a moderately good understanding of feline signs of stress in owners. A principal components analysis applied to the listed signs of stress identified 4 components which were termed: body posture, social avoidance, house soiling, and self-directed behavior. However, some of the signs that behaviorists regard as crucial in their anamnesis, such as scratching the furniture, freezing, mydriasis, and recurrent cystitis, were the least recognized signs of stress by cat owners. Only very prominent, common, or potentially disturbing behaviors such as excessive vocalization, posture with the ears back, and urinating out of the litter tray were regarded as potential signs of stress by more than two-thirds of owners. These findings suggest that owners tend to overlook certain signs and that owners' perception of stress partially depends on their false preconceptions about cat normal ethology (e.g., playfulness, social relationships, aggression, etc.). This ill-informed perception is likely to prevent owners from correctly indentifying, and intervening in, situations of poor welfare.

The present study was aimed at investigating whether imatinib pharmacogenetics is related to its pharmacodynamics in patients affected by chronic myeloid leukemia. Through a procedure based on a sequence of classical statistics methods, we investigated the possible relationships between treatment efficacy/tolerability and combinations of time-independent variables as gender and genetic covariates in the form of single nucleotide polymorphisms (SNPs) or combinations thereof. Moreover, since the drug tolerability has a strong incidence on the discontinuation of the therapy, we investigated whether the time of manifestation of the most frequent toxic effects can be related to time-independent patients’ characteristics or not. We found that a combination of two polymorphisms, namely hOCT1 c.480C>G (rs683369) and ABCB1 c.3435C>T (rs1045642), seems to play the role of predictor for imatinib in both efficacy and toxicity. Furthermore, the time of manifestation of edema toxicity is found to be associated to a combination of gender and ABCB1 c.3435C>T, whereas the time of manifestation of cramp toxicity appears related to gender. The novelty of this study is dual: the achievement of results that potentially have a significant clinical interest and the demonstration that the adoption of composed covariates may represent a unique tool to study different aspects of the treatment with imatinib.

The aim of the study was to investigate any possible influence of polymorphisms of transmembrane transporters human organic cation transporter 1 (hOCT1), ABCB1, ABCG2 on imatinib pharmacokinetics in 33 men and 27 women (median age and range, 56 and 27-79 years, respectively) affected by chronic myeloid leukemia. A population pharmacokinetic analysis was performed to investigate imatinib disposition in every patient and the role of transporter polymorphisms. Results showed that the α1-acid glycoprotein and the c.480C>G genotype of hOCT1 had a significant effect on apparent drug clearance (CL/F) being responsible, respectively, for a 20% and 10% decrease in interindividual variability (IIV) of CL/F (from 50.1 up to 19.6%). Interestingly, 25 patients carrying at least one polymorphic c.480 G allele had a significant lower CL/F value with respect to the 35 c.480CC individuals (mean±s.d., 9.6±1.6 vs 12.1±2.3 l h-1, respectively; PG SNP may significantly influence imatinib pharmacokinetics, supporting further analyses in larger groups of patients.

Introduction: Immune reconstitution after stem cell transplantation (SCT) plays a crucial role in host defense against microbial agents. The thymus atrophy occurring with ageing represents a limit for de novo T-cell reconstitution, although the reactivation of thymic function after BMT is documented. The proper strategy to improve the thymic output is currently matter of debate. Pre-clinical and clinical evidences suggest that Zinc oral supplementation may contribute to thymic reactivation and to improve the T-mediated cellular defense against pathogens. We tested the role of Zinc oral supplementation in immune reconstitution, using as model the autologous SCT in multiple myeloma. Methods: From January 2014 to May 2016, we prospectively enrolled 18 patients (12 male, 6 female; average age: 58 years, range 43-72) undergoing single MEL 100 or 200 auto-SCT after one or two lines of therapy (VTD, lyposomal anthracycline and lenalidomide) and stem cell collection (mobilizing therapy: cyclophosphamide 3 g/sqm and G-CSF). The trial, prospective and randomized, has been approved by local ethics committee (EUDRACT: 2014-004499-47). All patients undersigned the informed consent. The clinical trial was carried out in accordance with Helsinki declaration. Randomization was effected at day 0 (day of PBSC infusion). Results: nine patients were treated from day +5 to day +100 after transplant with 600 mg/day of Zinc sulfate (uncoated tablets), whereas nine patients received only standard antimicrobial prophylaxis. Peripheral blood samples were collected in both groups at day +30 (t2) and day +100 (t3). Eight-colour flow cytometry was performed for CD3, CD4, CD8, CD45RA, CD45R0, CD27, CD28, CD25, CD127, with specific gates to identify specific lymphocyte populations (T naïve, T central memory, T effector memory, T terminal memory). The lymphocyte's population's count was statistically analysed intra- and inter-group with Wilcoxon test. Droplet-digital PCR for TRECs was performed on lymphocytes isolated by peripheral blood. A qPCR for viral load of Torquetenovirus (TTV), a harmless virus whose viral load is related to immunodepression, was performed. Zinc serum level was measured. Results: The only significant difference in the clinical features between the 2 groups was the mean age (58 years in the control group, 63 years in the sample). The recovery of naïve CD4 cells was visible in both groups from day +30 to day +100, but a significant increase was detected only in the Zinc group. Similarly, TRECs showed a significant increase in the treated group. CD8+ T cells showed a notable decrease until day 100 in the control group, specifically the CD8 naïve, memory, and effector memory populations. TTV load in the control group increased in inverse proportion to the decrease of CD8 population. In the Zinc group there were no differences between the TTV loads at the two time points. As a result, the viral load of TTV was higher in the control group than in the Zinc group at day +100. Zinc serum levels were normal in all patients. Conclusions: data show that Zinc plays a role in a faster recovery of TRECs and CD4 naïve T cells after autologous transplantation. Furthermore, Zinc seems to prevent the expected CD8 decrease of the control group, thus probably explaining the TTV reactivation. Zinc-deficiency was not observed in any of the patient; however, a Zinc supplementation may contribute to a better T-cell reconstitution. To the best of our knowledge, this is the first study describing the role of Zinc after stem cell transplantation. Disclosures No relevant conflicts of interest to declare.

This study evaluates the prognostic value of molecular monitoring of minimal residual disease (MRD) in 20 patients with multiple myeloma (MM) following autologous (peripheral blood stem cell transplantation, PBSCT) and non-myeloablative allogeneic (NMT) transplant. All patients completed their program, with a treatment-related mortality (TRM) of 20% and a 2-year progression-free survival (PFS) of 51%. After PBSCT, only 3 patients (15%) achieved PCR-negativity, versus 12 (60%) after NMT. The eradication of MRD had a favorable impact on 2-year OS. In fact, 76% of patients with no detectable MRD was still alive versus 34% of persistently IgH-positive cases (p=0.03). PCR status did not correlate with chimerism percentage: Seventy-five percent of patients achieved full donor chimerism, which was more frequently observed in cases presenting cGHVD (p=0.01). These data sustain the relevant role of molecular monitoring in MM patients undergoing NMT. MRD monitoring would assist physicians in making additional therapeutic decisions to better control this hematological malignancy.

Expression of two MDR genes, BCRP and MDR1, was evaluated by real-time PCR technique in 51 AML patients. Fifty-six percent expressed the BCRP gene, with the 48.2% showing intermediate levels. Eighty-eight percent expressed the MDR1, with 23.8% of cases at high expression. A significant correlation between BCRP and MDR1 values was found by regression analysis. Either levels of BCRP or MDR1 did not correlate with clinical characteristics of patients at diagnosis.

A high expression of Wilms’ tumor gene (WT1) in acute myeloid leukemia (AML) seems to correlate with a poor outcome and its increased levels can be predictive of an impending relapse. WT1 has been shown in vitro to interact with the promoter of the MDR1, a gene involved in the multidrug resistance phenomenon. The aim of this study was to measure, by real-time polymerase chain reaction, levels of WT1 and MDR1 expression, in order to find a possible association between these genes, in a series of 50 newly diagnosed AML cases. Twenty-five percent of patients carried very high (>75° percentile) MDR1- and 23.3%WT1-mRNA levels. Interestingly, high levels of WT1 were significantly correlated with correspondent high levels of MDR1 gene. Nevertheless, the co-expression of these genes did not significantly influence the complete response rate to the induction therapy. Reported data confirm the existence of a co-expression of WT1 and MDR1 genes even in vivo; this may be relevant because one consequence could be the positive selection by chemotherapeutic regimens of cells with higher MDR1 levels already present before treatment. Thus, the association between these genes could suggest avoiding the use of drugs involved in the multidrug resistance (MDR) phenomenon in patients carrying high levels of WT1 at diagnosis.

Summary. To evaluate the clinical impact of minimal residual disease in multiple myeloma, apheretic products from 51 autotransplanted patients were tested by fluorescent (GeneScan) polymerase chain reaction (PCR). Sixty-nine per cent of harvests were contaminated when evaluated for IgH rearrangement. Forty-six patients responded to transplant, with 52·9% achieving complete response (CR). The clinical response of patients was significantly influenced by the number of re-infused CD34+ cells. Positive PCR results of re-infused harvests were not significantly related to patient outcome. Median overall survival (OS) was 33 months, and a significant advantage for patients transplanted by 12 months from diagnosis was observed. Moreover, OS was longer for patients receiving PCR-negative stem cells, with 72% of patients surviving to 70 months in the group receiving PCR-negative harvests vs 48% in the group transplanted with contaminated precursors (not statistically significant). Ex vivo purging caused a reduction of contamination of up to 3 logs; nevertheless, 80% of purged harvests remained PCR-positive and the purging procedure did not alter response or survival rates. Thus, the failure of a predictive role for this highly sensitive molecular method could be explained by the assumption that in vivo persisting malignant cells are the true source of relapse in MM.

Purpose: The role of the minimal residual disease (MRD) in follicular lymphoma is still debated. In this study, we assessed whether the BCL2/IGH rearrangement could have a prognostic role in patients receiving R-CHOP, R-FM, or R-CVP. Experimental Design: DNAs from 415 patients among the 504 cases enrolled in the FOLL05 trial (NCT00774826) were centralized and assessed for the BCL2/IGH at diagnosis, at the end of treatment, and after 12 and 24 months. Results: At diagnosis, the molecular marker was detected in 53% of cases. Patients without molecular marker or with a low molecular tumor burden ( Conclusions: In this study, standardized molecular techniques have been adopted and applied on bone marrow samples from a large cohort. Data reported show that the MRD detection is a powerful independent predictor of PFS in patients with follicular lymphoma receiving conventional chemoimmunotherapy. Clin Cancer Res; 20(24); 6398–405. ©2014 AACR.

Two repetitive DNA families were detected in the genomes of Palaearctic green water frogs. The first family, named RrS1, is a centromeric satellite DNA, which allows discrimination between the genomes of Rana ridibunda and Rana lessonae, thence representing a useful molecular tool to determine timing and modes of genome exclusion from the germ line cells of the hybrid R. es‐culenta. The second repetitive family, named Rana/Pol III, consists of short, tandemly arrayed sequences, scattered throughout the genomes, and resembling SINE retroposons in their structure. The Rana/Pol III family is present in the genomes of R. lessonae, R. ridibunda, and their hybrid R. esculenta, as well as in R. shqiperica, R. epeirotica, R. cretensis, and the Italian taxon. These sequences are also present in the Iberian species R. perezi, although less abundant, but appear to be lacking in the North African species R. saharica. The distribution of Rana/Pol III elements in the genomes of Palaearctic green water frogs is...

Introduction. Drug transporters, such as ABCB1, hOCT1, and ABCG2, control both intracellular and systemic plasma concentrations of BCR-ABL1 inhibitors. Our group previously reported that the hOCT1 c.480C>G polymorphism significantly correlated with higher rates of adverse events and shorter event-free survival (EFS) of patients receiving imatinib. Even if nilotinib has less affinity than imatinib for the transmembrane transporters, no definitive data about the possible role of their polymorphisms are available from the "real life". Methods. Through an approach similar to the one previously used for imatinib, we investigated any possible influence of ABCB1 and hOCT1 polymorphisms on the nilotinib efficacy and toxicity in a series of 78 CML patients receiving nilotinib as first-line therapy in 8 Italian Centers from June 2012 to June 2016. Lack of CCyR at 12 months, stop of treatment for any cause, loss of MR3 or CCyR, appearance of mutations were computed as events in the EFS assessment. The following polymorphisms were tested by real-time PCR: hOCT1: rs683369[c.480C>G]; ABCB1: rs1128503 [c.1236C>T], rs2032582[c.2677G>T/A], and rs1045642[c.3435C>T]. Results. Forty-six patients were male and 32 female; the median age was 47 years (range, 18-79); Sokal score was low in 44%, intermediate in 37%, and high in 19% of cases; EUTOS was high in 30%. Efficacy: 97% of patients were in CHR and 82% in early molecular response (EMR) at 3 months; 85% achieved the CCyR at 6 months, and 88% the MR3 with a median time of 6.1 months; 78% achieved a deep molecular response (DMR), with 14% of MR5, with a median time of 26 months.With a median follow-up of 36 months, no patient lost CCyR and 9% lost MR3. All patients are still alive; 23% of patients stopped nilotinib (one third of them because of failure), and half of them reduced toT SNP, 56% of patients were heterozigous and 22% omozigous polymorphic; for the c1236 C>T SNP, 54% of patients were heterozigous and 12% omozigous polymorphic; finally, for the hOCT1 c.480 C>G, 41% of patients were heterozigous and 10% omozigous polymorphic.We found that ABCB1 and hOCT1 polymorphisms did not condition the achievement of CHR, CCyR, or MR3. Nevertheless, polymorphism of ABCB1 (c.3435C>T) was associated with a higher probability of achieving DMR (90% vs 65%; p=0.04).A longer 3-year EFS was conditioned by the achievement of EMR (90% vs 75%; p=0.012) and of MR3 (90% vs 65%: p=0.04). We found that polymorphic or wild-type status of transporters did not impact on EFS.Toxicities were observed in 41% of cases, with 40% of grade 3 or 4; ABCB1 and hOCT1 polymorphisms did not condition toxicities or nilotinib discontinuation. Conclusions. Our previous studies showed that polymorphic hOCT1 negatively conditioned CCyR and EFS during imatinib treatment, and that the combination of ABCB1 and hOCT1 polymorphisms were related to higher levels of toxicity.On the contrary, the present study demonstrated that the same polymorphisms did not condition toxicity when patients received nilotinib as first-line treatment. About efficacy, even if in a multicentric "real life" setting, it resulted superimposable to that reported in literature. We found that hOCT1 polymorphism did not have any impact on the quality of response; on the contrary, the ABCB1 c.3435C>T polymorphism (that induces a lower efflux from the leukemic cell and lower intestinal and renal excretion) was associated to a high rate of deep molecular response.On the basis of these results, we demonstrated that hOCT1 and ABCB1 polymorphisms, differently from the imatinib setting, are not relevant in patients receiving nilotinib, and that patients polymorphic for ABCB1 can even receive an advantage on DMR. Disclosures Saglio: Ariad: Consultancy; Pfizer: Consultancy; Bristol Myers Squibb: Consultancy.

Background Arsenic Trioxide (ATO) is effective in about 20% of patients with myelodysplasia (MDS); its mechanisms of action have already been evaluated in vitro, but the in vivo activity is still not fully understood. Since ATO induces apoptosis in in vitro models, we compared the expression of 93 apoptotic genes in patients’ bone marrow before and after ATO treatment. For this analysis, we selected 12 patients affected by MDS who received ATO in combination with Ascorbic Acid in the context of the Italian clinical trial NCT00803530, EudracT Number 2005-001321-28. Methods Real-time PCR quantitative assays for genes involved in apoptosis were performed using TaqMan® Assays in 384-Well Microfluidic Cards “TaqMan® Human Apoptosis Array”. Quantitative RT-PCR for expression of EVI1 and WT1 genes was also performed. Gene expression values (Ct) were normalized to the median expression of 3 housekeeping genes present in the card (18S, ACTB and GAPDH). Results ATO treatment induced up-regulation of some pro-apoptotic genes, such as HRK, BAK1, CASPASE-5, BAD, TNFRSF1A, and BCL2L14 and down-regulation of ICEBERG. In the majority of cases with stable disease, apoptotic gene expression profile did not change, whereas in cases with advanced MDS more frequently pro-apoptotic genes were up-regulated. Two patients achieved a major response: in the patient with refractory anemia the treatment down-regulated 69% of the pro-apoptotic genes, whereas 91% of the pro-apoptotic genes were up-regulated in the patient affected by refractory anemia with excess of blasts-1. Responsive patients showed a higher induction of BAD than those with stable disease. Finally, WT1 gene expression was down-regulated by the treatment in responsive cases. Conclusions These results represent the basis for a possible association of ATO with other biological compounds able to modify the apoptotic pathways, such as inhibitors of the BCL2 family.

Both arsenic trioxide (ATO) and bortezomib show separate antileukemic activity. With the purpose of evaluating whether the combination of ATO and bortezomib would be an option for patients with acute leukemia, we incubated HL60 leukemic cells with ATO alone and in combination with bortezomib. ATO and bortezomib cooperated to induce cell death and to inhibit proliferation and apoptosis in a synergistic way. The combined treatment resulted in a stronger activation of caspase 8 and 9, moderate activation of caspase 3, and increased expression of Fas and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-DR5 receptors. When bortezomib was added, some proapoptotic genes (CARD9, TRAIL) were upregulated, and some antiapoptotic genes (BCL2, BCL3, FLICE) were downregulated. When coincubated, approximately 80% of cells showed altered mitochondrial membrane permeability. Moreover, ATO alone and in combination with bortezomib abrogated DNA-binding activity of nuclear factor kappa beta (NF-kappaB). Gene expression assays showed that more deregulated genes were related to proliferation of leukocytes, tumorigenesis, control of cell cycle, hypoxia and oxidative stress, cytokines, PI3K-AKT, ERK-MAPK, EGF pathways, and ubiquitination. Finally, in three cases of acute myeloid leukemia, the addition of bortezomib to ATO significantly increased cytotoxicity. We conclude that the combination of bortezomib and ATO may be efficacious in the treatment of myeloid disorders.

The wingless-type (Wnt) signalling pathway is involved in leukemogenesis and the glycogen-synthase kinase 3 has been reported to control both -catenin and nuclear factor (NF)-B.1 Thus, if NF-B and Wnt pathways are really controlled by common genes, drugs able to modulate one or both of these signals would represent innovative therapeutic tools in treatment of myeloproliferative disorders.