Cladribine Efficacy in a Patient with Hairy Cell Leukemia and Severe Renal Insufficiency.

BACKGROUND: Hairy cell leukemia commonly presents with pancytopenia, indolent course, and predisposition as infectious complications. Current first-line therapeutic options are purine analogues, particularly cladribine, with a high percentage of complete responses and durable remissions. However, their use is poorly investigated in patients affected by severe chronic renal insufficiency. CASE PRESENTATION: Here, we describe a case of HCL in a 68-year-old man affected by multiple comorbidities, including severe chronic renal failure. After a course of interferon-α, the patient received therapy with Cladribine every other week, obtaining a complete hematological remission and improvement of renal function. DISCUSSION: With a different soft schedule of cladribine, the patient was treated adequately, obtaining a complete remission. CONCLUSION: Cladribine can be administered with caution, even in patients with renal failure, with good results.

Minimal Residual Disease Detection at RNA and Leukemic Stem Cell (LSC) Levels: Comparison of RT-qPCR, d-PCR and CD26+ Stem Cell Measurements in Chronic Myeloid Leukemia (CML) Patients in Deep Molecular Response (DMR).

A Deep Molecular Response (DMR), defined as a BCR::ABL1 transcript at levels ≤ 0.01% by RT-qPCR, is the prerequisite for the successful interruption of treatment among patients with Chronic Myeloid Leukemia (CML). However, approximately 50% of patients in Treatment-Free Remission (TFR) studies had to resume therapy after their BCR::ABL1 transcript levels rose above the 0.1% threshold. To improve transcript detection sensitivity and accuracy, transcript levels can be analyzed using digital PCR (dPCR). dPCR increases BCR::ABL1 transcript detection sensitivity 10-100 fold; however, its ability to better select successful TFR patients remains unclear. Beyond the role of the immune system, relapses may be due to the presence of residual leukemic stem cells (LSCs) that are transcriptionally silent. Flow cytometry can be used to identify and quantify circulating bone marrow Ph+ LSCs CD34+/CD38- co-expressing CD26 (dipeptidylpeptidase-IV). To date, the significance of circulating Ph+ LSCs in TFR is unclear. The aim of this work is to compare and examine the values obtained using the three different methods of detecting minimal residual disease (MRD) in CML at RNA (RT-qPCR and dPCR) and LSC (flowcytometry) levels among patients in TFR or exhibiting a DMR. The twenty-seven patients enrolled received treatment with either imatinib (12), dasatinib (6), nilotinib (7), bosutinib (1), or interferon (1). Twelve patients were in TFR, while the rest exhibited a DMR. The TFR patients had stopped therapy for less than 1 year (3), <3 years (2), 6 years (6), and 17 years (1). Blood samples were collected and tested using the three methods at the same time. Both d-PCR and LSCs showed higher sensitivity than RT-qPCR, exhibiting positive results in samples that were undetectable using RT-qPCR (17/27). None of the patients tested negative with d-PCR; however, 23/27 were under the threshold of 0.468 copies/µL, corresponding to a stable DMR. The results were divided into quartiles, and the lowest quartiles defined the lowest MRD. These data were strongly correlated in 15/27 patients, corresponding to almost half of the TFR patients. Indeed, the TFR patients, some lasting up to 17 years, corresponded to the lowest detectable DMR categories. To the best of our knowledge, this is the first attempt to analyze and compare DMRs in a CML population using standard (RT-qPCR) and highly sensitive (dPCR and LSCs) methods.

The prognostic role of gene polymorphisms in patients with indolent non-Hodgkin lymphomas and mantle-cell lymphoma receiving bendamustine and rituximab: results of the 5-year follow-up study.

The variability in disease outcome for indolent non-Hodgkin lymphomas (iNHL) and mantle-cell lymphoma (MCL) could be related to single nucleotide polymorphisms (SNPs) in genes that affect immune and inflammatory response. We investigated SNPs that could have a prognostic role for patients receiving bendamustine and rituximab (BR). All samples were genotyped for the IL-2 (rs2069762), IL-10 (rs1800890, rs10494879), VEGFA (rs3025039), IL-8 (rs4073), CFH (rs1065489) and MTHFR (rs1801131) SNPs by allelic discrimination assays using TaqMan SNP Genotyping Assays. We report a long-term follow-up analysis of 79 iNHL and MCL patients that received BR. Overall response rate was 97.5% (CR rate 70.9%). After a median follow-up of 63 months, median PFS and OS were not reached. We report a significant association between SNP in IL-2 (rs2069762) and reduced PFS and OS (p<.0001). We suggest a role for cytokine SNPs in disease outcome, while SNPs seem not related to long-term toxicity or secondary malignancies.

Prospective monitoring of chronic myeloid leukemia patients from the time of TKI discontinuation: the fate of peripheral blood CD26+ leukemia stem cells.

Introduction: In chronic myeloid leukemia (CML), about half of the patients achieving a deep and stable molecular response with tyrosine kinase inhibitors (TKIs) may discontinue TKI treatment without disease recurrence. As such, treatment-free remission (TFR) has become an ambitious goal of treatment. Given the evidence that deepness and duration of molecular response are necessary but not sufficient requisites for a successful TFR, additional biological criteria are needed to identify CML patients suitable for efficacious discontinuation. Leukemia stem cells (LSCs) are supposed to be the reservoir of the disease. Previously, we demonstrated that residual circulating CD34+/CD38-/CD26+ LSCs were still detectable in a consistent number of CML patients during TFR. Methods: CML LSCs could be easily identified by flow-cytometry as they express the CD34+/CD38-/CD26+ phenotype. In this study, we explored the role of these cells and their correlation with molecular response in a cohort of 109 consecutive chronic phase CML patients prospectively monitored from the time of TKI discontinuation. Results: After a median observation time of 33 months from TKI discontinuation, 38/109 (35%) patients failed TFR after a median time of 4 months, while 71/109 (65%) patients are still in TFR. At TKI discontinuation, peripheral blood CD26+LSCs were undetectable in 48/109 (44%) patients and detectable in 61/109 (56%). No statistically significant correlation between detectable/undetectable CD26+LSCs and the rate of TFR loss was found (p = 0.616). The incidence of TFR loss based on the type of TKI treatment was statistically significant for imatinib treatment compared to that of nilotinib (p = 0.039). Exploring the behavior of CD26+LSCs during TFR, we observed fluctuating values that were very variable between patients, and they were not predictive of TFR loss. Discussion: Up to date, our results confirm that CD26+LSCs are detectable at the time of TKI discontinuation and during TFR. Moreover, at least for the observation median time of the study, the persistence of "fluctuating" values of residual CD26+LSCs does not hamper the possibility to maintain a stable TFR. On the contrary, even patients discontinuing TKI with undetectable CD26+LSCs could undergo TFR loss. Our results suggest that factors other than residual LSCs "burden" playing an active role in controlling disease recurrence. Additional studies evaluating CD26+LSCs' ability to modulate the immune system and their interaction in CML patients with very long stable TFR are ongoing.

"Friends and foes" of multiple myeloma measurable/minimal residual disease evaluation by next generation flow.

Next Generation Flow (NGF) represents a gold standard for the evaluation of Minimal Residual Disease (MRD) in Multiple Myeloma (MM) patients at any stage of treatment. Although the assessment of MRD is still not universally employed in clinical practice, numerous studies have demonstrated the strength of MRD as a reliable predictor of long-term outcome, and its potential to supersede the prognostic value of CR. The possibility to acquire millions of events, in combination with the use of standard reagents and a good expertise in the analysis of rare populations, led to high chance of success and a sensitivity of 10-6 that is superimposable to the one of Next Generation Sequencing molecular techniques. Some minor bias, correlated to the protocols applied, to the quality of samples and to the high heterogeneity of plasma cells phenotype, may be overcome using standard protocols and having at disposition personnel expertise for MRD analysis. With the use of NGF we can today enter a new phase of the quantification of residual disease, switching from the definition of "minimal" residual disease to "measurable" residual disease. This review takes account of the principle "friends and foes" of Myeloma "Measurable" Residual Disease evaluation by NGF, to give insights into the potentiality of this technique. The optimization of the quality of BM samples and the analytic expertise that permits to discriminate properly the rare pathologic clones, are the keys for obtaining results with a high clinical value that could be of great impact and relevance in the future.

Drug resistance and minimal residual disease in multiple myeloma.

Great progress has been made in improving survival in multiple myeloma (MM) patients over the last 30 years. New drugs have been introduced and complete responses are frequently seen. However, the majority of MM patients do experience a relapse at a variable time after treatment, and ultimately the disease becomes drug-resistant following therapies. Recently, minimal residual disease (MRD) detection has been introduced in clinical trials utilizing novel therapeutic agents to measure the depth of response. MRD can be considered as a surrogate for both progression-free and overall survival. In this perspective, the persistence of a residual therapy-resistant myeloma plasma cell clone can be associated with inferior survivals. The present review gives an overview of drug resistance in MM, i.e., mutation of ß5 subunit of the proteasome; upregulation of pumps of efflux; heat shock protein induction for proteasome inhibitors; downregulation of CRBN expression; deregulation of IRF4 expression; mutation of CRBN, IKZF1, and IKZF3 for immunomodulatory drugs and decreased target expression; complement protein increase; sBCMA increase; and BCMA down expression for monoclonal antibodies. Multicolor flow cytometry, or next-generation flow, and next-generation sequencing are currently the techniques available to measure MRD with sensitivity at 10-5. Sustained MRD negativity is related to prolonged survival, and it is evaluated in all recent clinical trials as a surrogate of drug efficacy.

Long-term CR Multiple Myeloma Patients Show Cured or MGUS-like Minimal Residual Disease Pattern by Next Generation Flow.

BACKGROUND: In the era of novel agents, many multiple myeloma patients can achieve a complete remission, but most of them relapse, and minimal residual disease detection can play a crucial role. Next-generation flow (NGF) can detect monoclonal plasma cells with a sensitivity of 10-6. Little is known about long-term remission patients (> 2 years) and in particular, if more sensitive techniques such as NGF can still detect minimal disease in those patients. OBJECTIVE: Aim of the study was to analyze patients with MM in response to NGF at > 2 years of sustained remission after several treatments. METHODS: MRD was studied by NGF in bone marrow aspirates according to Euroflow Consortium indications. RESULTS: 62 patients with sustained CR at >2 years were studied, MRD+ status was detected at a threshold cut-off of 10-6 in 32/62 (52%); 4/15 (27%) patients were MRD positive at >5 years of remission and they displayed a prevalence of normal vs abnormal monoclonal plasma cell immune-phenotype (MGUS-like). CONCLUSION: NGF is a powerful technique to detect MRD. Myeloma patients in prolonged sustained complete remission can show in high percentage an MRD negative status or MGUS like.

Anti CD38 monoclonal antibodies for multiple myeloma treatment.

CD38 is a transmembrane glycoprotein with ectoenzymatic activity and is highly and uniformly expressed on multiple myeloma (MM) cells. CD38 is expressed also at relatively low levels on normal lymphoid and myeloid cells, and in some tissues of non-hematopoietic origin. The specificity of this target has increased interest in new drugs and triggered the development of the CD38 monoclonal antibodies Daratumumab (fully human) and Isatuximab (chimeric). CD38 antibodies have pleiotropic mechanisms of action including Fc-dependent immune effector mechanisms, direct apoptotic activity, and immunomodulatory effects by the elimination of CD38+ immune-suppressor cells. Monoclonal antibody-based therapy has revolutionized MM therapy in the latest years increasing depth of response. This product review will focus on anti-CD38 monoclonal antibodies Daratumumab and Isatuximab efficacy, safety, pharmacokinetic and pharmacodynamic data from clinical trials.

CD26/DPP-4 in Chronic Myeloid Leukemia.

CD26 expression is altered in many solid tumors and hematological malignancies. Recently, it has been demonstrated that it is a specific marker expressed on LSCs of CML, both in BM and PB samples, and absent on CD34+/CD38- stem cells in normal subjects or on LSCs of other myeloid neoplasms. CD26+ LSCs have been detected by flow-cytometry assays in all PB samples of Chronic-Phase CML patients evaluated at diagnosis. Additionally, it has been demonstrated that most CML patients undergoing Tyrosine Kinase Inhibitors (TKIs) treatment still harbored circulating measurable residual CD26+ LSCs, even when displaying a consistent deep molecular response without any significant association among the amounts of BCR-ABL transcript and CD26+ LSCs. Preliminary data of our Italian prospective multicenter study showed that CML patients with a poorer response presented with a higher number of CD26+ LSCs at diagnosis. These data confirmed that CD26 is a specific marker of CML and suggest that it could be considered for the monitoring of therapeutic responses.

First reported case of secondary mixed phenotype acute leukemia after multiple myeloma.

In recent years the outcome of patients with multiple myeloma (MM) has significantly improved, due to new drugs. However, some agents, i.e. the alkylating drug melphalan, can be associated with an increased incidence of secondary malignancies. Myelodysplastic syndromes and acute myeloid leukemia are reported in the literature, and rarely acute lymphoblastic leukemia. Here we describe a unique case of a 56-years old female patient affected by MM since 2015 in complete remission after autologous stem cell transplant and in lenalidomide maintenance, who developed 2 years later mixed phenotype acute leukemia (MPAL). The patient, refractory to both lymphoblastic and myeloid acute leukemia regimens, achieved complete remission with bi-specific anti-CD19/anti-CD3 monoclonal antibody blinatumomab and with hypomethylating agent azacytidine plus the BCL-2 inhibitor venetoclax. She then underwent hematopoietic stem cell transplantation from HLA-identical sibling donor and she is still in complete remission after 9 months. To the best of our knowledge, there are no cases in the literature describing MPAL after autologous transplant for MM. Our patient was treated with blinatumomab and venetoclax and achieved complete remission 9 months from allogeneic transplant. The mechanism underlying the development of MPAL is not completely understood and therapies are still lacking. In this context the combination of blinatumomab, azacytidine and venetoclax successfully used in this patient may provide food for thought for further studies in this rare setting of patients.

Venetoclax in association with decitabine as effective bridge to transplant in a case of relapsed early T-cell lymphoblastic leukemia.

A case of an early-relapsed high-risk T-ALL with high BCL-2 expression on leukemic blasts was successfully treated with decitabine and venetoclax, achieving a CR. We suggest decitabine and venetoclax should be synergistic in BCL2-positive ALL.

Minimal Residual Disease in Multiple Myeloma: State of the Art and Applications in Clinical Practice.

Novel drugs have revolutionized multiple myeloma therapy in the last 20 years, with median survival that has doubled to up to 8-10 years. The introduction of therapeutic strategies, such as consolidation and maintenance after autologous stem cell transplants, has also ameliorated clinical results. The goal of modern therapies is becoming not only complete remission, but also the deepest possible remission. In this context, the evaluation of minimal residual disease by techniques such as next-generation sequencing (NGS) and next-generation flow (NGF) is becoming part of all new clinical trials that test drug efficacy. This review focuses on minimal residual disease approaches in clinical trials, with particular attention to real-world practices.

Long-Term Safety of Rapid Daratumumab Infusions in Multiple Myeloma Patients.

Multiple myeloma survival has significantly improved in recent years, due to novel agents that are available for treatment. The anti-CD38 monoclonal antibody Daratumumab is particularly efficient for patients with relapse/refractory disease, and many studies have shown its unprecedented efficacy also as a first treatment. However, to avoid the incidence of infusion reactions, long infusion schedules of 8 h at first dose and 4 h in the following doses are required, which can reduce the compliance of patients and health care professionals. A reduced infusion time of 90 min has been reported previously, but data are missing on the prolonged safety of this over time as well as the efficacy of this approach. In this work, we investigate the safety of 484 rapid Daratumumab infusions given early after the second dose over a 22 months period in 39 myeloma patients.

Flow Cytometry Assessment of CD26+ Leukemic Stem Cells in Peripheral Blood: A Simple and Rapid New Diagnostic Tool for Chronic Myeloid Leukemia.

BACKGROUND: Recent investigations in chronic myeloid leukemia (CML) have focused on the identification and characterization of leukemic stem cells (LSCs). These cells reside within the CD34+ /CD38─ /Lin─ fraction and score positive for CD26 (dipeptidylpeptidase IV) a marker, expressed in both bone marrow (BM) and peripheral blood (PB) samples, that discriminates CML cells from normal hematopoietic stem cells (HSCs) or from LSCs of other myeloid neoplasms. CD26 evaluation could be a useful tool to improve the identification of CML LCSs by using flow-cytometry assay. METHODS: CD26+ LSCs have been isolated from EDTA PB and BM samples of patients with leucocytosis suspected for CML. Analysis of LSCs CML has been performed by using custom-made lyophilized pre-titrated antibody mixture test and control tube and a CD45+ /CD34+ /CD38- /CD26+ panel as a strict flow cytometric gating strategy. RESULTS: The expression of CD26 on CD34+ /CD38- population was detectable in 211/211 PB and 84/84 BM samples of subsequently confirmed BCR-ABL+ CP-CML patients. None of the 32 samples suspicious for CML but scoring negative for circulating CD26+ LSCs were diagnosed as CML after conventional cytogenetic and molecular testing. To validate our results, we checked for PB CD26+ LSCs in patients affected by other hematological disorders and they all scored negative for CD26 expression. CONCLUSIONS: We propose flow cytometry evaluation of CD26 expression on PB CD34+ /CD38- population as a new rapid, reproducible, and powerful diagnostic tool for the diagnosis of CML. © 2019 The Authors. Cytometry Part B: Clinical Cytometry published by Wiley Periodicals, Inc. on behalf of International Clinical Cytometry Society.

Study of gene polymorphisms as predictors of treatment efficacy and toxicity in patients with indolent non-hodgkin lymphomas and mantle cell lymphoma receiving bendamustine and rituximab.

Bendamustine is used in combination with rituximab (BR) to treat indolent non-Hodgkin lymphomas (iNHL) and mantle cell lymphoma (MCL). The variability in treatment efficacy and toxicity could be related to single nucleotide polymorphisms (SNPs) in immune response genes. We would like to show a correlation between SNPs and treatment outcome in iNHL and MCL patients receiving BR. We investigated some SNPs that had already been associated with NHL outcome. Samples were genotyped for the IL2 (rs2069762), IL10 (rs1800890, rs10494879), VEGFA (rs3025039), IL8 (rs4073), CFH (rs1065489) and MTHFR (rs1801131) SNPs by allelic discrimination assays. We enrolled 70 patients that received rituximab 375 mg/m2 and bendamustine 90 mg/m2 every 28 days, both as first-line treatment and ≥ second-line regimens. Overall response rate was 97·1% (complete response [CR] rate 73·9%). Treatment toxicity included grade 3-4 neutropenia (24/70 patients), infections (21/70 patients; 1/70 grade 3), skin rash (26/70 patients; 2/70 grade 3). After a median follow-up of 24 months we did find any correlation between the analysed SNPs, CR rate and PFS. However, we demonstrated an association between the SNP in IL2 (rs2069762) and the onset of skin rash (P = 0·0001). Our study suggests a role for cytokine SNPs in bendamustine-related toxicity, which could represent a promising research field.

Residual Peripheral Blood CD26+ Leukemic Stem Cells in Chronic Myeloid Leukemia Patients During TKI Therapy and During Treatment-Free Remission.

Chronic myeloid leukemia (CML) patients in sustained "deep molecular response" may stop TKI treatment without disease recurrence; however, half of them lose molecular response shortly after TKI withdrawing. Well-defined eligibility criteria to predict a safe discontinuation up-front are still missing. Relapse is probably due to residual quiescent TKI-resistant leukemic stem cells (LSCs) supposedly transcriptionally low/silent and not easily detectable by BCR-ABL1 qRT-PCR. Bone marrow Ph+ CML CD34+/CD38- LSCs were found to specifically co-express CD26 (dipeptidylpeptidase-IV). We explored feasibility of detecting and quantifying CD26+ LSCs by flow cytometry in peripheral blood (PB). Over 400 CML patients (at diagnosis and during/after therapy) entered this cross-sectional study in which CD26 expression was evaluated by a standardized multiparametric flow cytometry analysis on PB CD45+/CD34+/CD38- stem cell population. All 120 CP-CML patients at diagnosis showed measurable PB CD26+ LSCs (median 19.20/µL, range 0.27-698.6). PB CD26+ LSCs were also detectable in 169/236 (71.6%) CP-CML patients in first-line TKI treatment (median 0.014 cells/µL; range 0.0012-0.66) and in 74/112 (66%), additional patients studied on treatment-free remission (TFR) (median 0.015/µL; range 0.006-0.76). Notably, no correlation between BCR-ABL/ABLIS ratio and number of residual LSCs was found both in patients on or off TKIs. This is the first evidence that "circulating" CML LSCs persist in the majority of CML patients in molecular response while on TKI treatment and even after TKI discontinuation. Prospective studies evaluating the dynamics of PB CD26+ LSCs during TKI treatment and the role of a "stem cell response" threshold to achieve and maintain TFR are ongoing.

Ectopic ILT3 controls BCR-dependent activation of Akt in B-cell chronic lymphocytic leukemia.

The high proportion of long-term nonprogressors among chronic lymphocytic leukemia (CLL) patients suggests the existence of a regulatory network that restrains the proliferation of tumor B cells. The identification of molecular determinants composing such network is hence fundamental for our understanding of CLL pathogenesis. Based on our previous finding establishing a deficiency in the signaling adaptor p66Shc in CLL cells, we undertook to identify unique phenotypic traits caused by this defect. Here we show that a lack of p66Shc shapes the transcriptional profile of CLL cells and leads to an upregulation of the surface receptor ILT3, the immunoglobulin-like transcript 3 that is normally found on myeloid cells. The ectopic expression of ILT3 in CLL was a distinctive feature of neoplastic B cells and hematopoietic stem cells, thus identifying ILT3 as a selective marker of malignancy in CLL and the first example of phenotypic continuity between mature CLL cells and their progenitors in the bone marrow. ILT3 expression in CLL was found to be driven by Deltex1, a suppressor of antigen receptor signaling in lymphocytes. Triggering of ILT3 inhibited the activation of Akt kinase upon B-cell receptor (BCR) stimulation. This effect was achieved through the dynamic coalescence of ILT3, BCRs, and phosphatidylinositol-3,4,5-trisphosphate 5-phosphatase 1 into inhibitory clusters at the cell surface. Collectively, our findings identify ILT3 as a signature molecule of p66Shc deficiency in CLL and indicate that ILT3 may functionally contribute to a regulatory network controlling tumor progression by suppressing the Akt pathway.

High CD200 expression is associated with poor prognosis in cytogenetically normal acute myeloid leukemia, even in FlT3-ITD-/NPM1+ patients.

Overexpression of CD200, a trans-membrane protein belonging to the immunoglobulin superfamily, has been associated with poor prognosis in patients with acute myeloid leukemia (AML). As few data are available in the subset of cytogenetically-normal (CN) AML, we retrospectively evaluated the correlations between CD200 expression and response to therapy in a series of 139 adults with CN-AML. CD200 was expressed in 67/139 (48%) cases; 18 of them (28%) expressed CD200 at high intensity. No differences in CD200 expression rate were observed according to age, WBC count, type of leukemia, FLT3 or NMP1 mutation, and CD56 expression. A higher incidence of CD200 expression was observed in CD34+ cases (P<0.0001) and in BCL2+ patients (P=0.04). Complete remission (CR) was evaluable achieved in 98 patients (70%): 56/71 (79%) in CD200- and 47/67 (63%) in CD200+ patients (P=0.03), with a lower CR rate in patients with high CD200 intensity (9/18, 50%). CD200 expression had a negative impact on long-term outcome. CD200 expression, per se, did not impact on disease-free survival (DFS), but cases with high CD200 expression had a lower 3-year DFS compared to CD200-negative and low-expressing ones (0% vs 65% vs 68%, P=0.019). Three-year overall survival (OS) was 51% in CD200- and 27% in CD200+ patients (P=0.01), with a significant difference among cases with low or high CD200 expression (35% vs 0%, P=0.001). CD200 high expression defined a group with very poor DFS and OS also among the 37 FLT3-/NPM1+: 3-year DFS and OS were 88% and 60% in CD200-, 50% and 32% in CD200 low and 0% and 0% in CD200 high patients, respectively (P=0.01 for DFS and P=0.05 for OS). Our data suggest a negative impact of CD200 expression in CN-AML, with a further worsening in high-expressing cases, also in the subset of FLT3-/NPM1+ patients.