The WNT Pathway Is Relevant for the BCR-ABL1-Independent Resistance in Chronic Myeloid Leukemia.

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.

The Combination of Rituximab and Bendamustine as First-Line Treatment Is Highly Effective in the Eradicating Minimal Residual Disease in Follicular Lymphoma: An Italian Retrospective Study.

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 1.4 × 10-2 copies, ranging from 3 × 10-5 to 4 × 104. 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 FL.

The Droplet Digital PCR: A New Valid Molecular Approach for the Assessment of B-RAF V600E Mutation in Hairy Cell Leukemia.

Hairy cell leukemia (HCL) is a chronic lymphoproliferative B-cell disorder where the B-RAF V600E mutation has been recently detected, as reported for solid neoplasias but not for other B-cell lymphomas. The digital droplet PCR (dd-PCR) is a molecular technique that, without standard references, is able to accurately quantitate DNA mutations. ddPCR could be an useful instrument for the detection of the B-RAF V600E mutation in HCL, where the minimal residual disease monitoring is fundamental for planning a patients-targeted treatment in the era of new anti-CD20 and anti-RAF compounds. This retrospective study enrolled 47 patients observed at the Hematology Unit of the University of Pisa, Italy, from January 2005 to January 2014: 27 patients were affected by "classic" HCL, two by the variant HCL (vHCL), and 18 by splenic marginal zone lymphoma (SMZL). The aim of the study was to compare dd-PCR to "classic" quantitative PCR (QT-PCR) in terms of sensitivity and specificity and to demonstrate its possible use in HCL. Results showed that: (1) the sensitivity of dd-PCR is about half a logarithm superior to QT-PCR (5 × 10-5 vs. 2.5 × 10-4), (2) the specificity of the dd-PCR is comparable to QT-PCR (no patient with marginal splenic lymphoma or HCL variant resulted mutated), (3) its high sensitivity would allow to use dd-PCR in the monitoring of MRD. At the end of treatment, among patients in complete remission, 33% were still MRD-positive by dd-PCR versus 28% by QT-PCR versus 11% by the evaluation of the B-cell clonality, after 12 months, dd-PCR was comparable to QT-PCR and both detected the B-RAF mutation in 15% of cases defined as MRD-negative by IgH rearrangement. Moreover, (4) the feasibility and the costs of dd-PCR are comparable to those of QT-PCR. In conclusion, our study supports the introduction of dd-PCR in the scenario of HCL, also during the follow-up.