Circulating cell-free DNA for target quantification in hematologic malignancies: Validation of a protocol to overcome pre-analytical biases.

Circulating tumor DNA (ctDNA) has become the most investigated analyte in blood. It is shed from the tumor into the circulation and represents a subset of the total cell-free DNA (cfDNA) pool released into the peripheral blood. In order to define if ctDNA could represent a useful tool to monitor hematologic malignancies, we analyzed 81 plasma samples from patients affected by different diseases. The results showed that: (i) the comparison between two different extraction methods Qiagen (Hilden, Germany) and Promega (Madison, WI) showed no significant differences in cfDNA yield, though the first recovered higher amounts of larger DNA fragments; (ii) cfDNA concentrations showed a notable inter-patient variability and differed among diseases: acute lymphoblastic leukemia and chronic myeloid leukemia released higher amounts of cfDNA than chronic lymphocytic leukemia, and diffuse large B-cell lymphoma released higher cfDNA quantities than localized and advanced follicular lymphoma; (iii) focusing on the tumor fraction of cfDNA, the quantity of ctDNA released was insufficient for an adequate target quantification for minimal residual disease monitoring; (iv) an amplification system proved to be free of analytical biases and efficient in increasing ctDNA amounts at diagnosis and in follow-up samples as shown by droplet digital PCR target quantification. The protocol has been validated by quality control rounds involving external laboratories. To conclusively document the feasibility of a ctDNA-based monitoring of patients with hematologic malignancies, more post-treatment samples need to be evaluated. This will open new possibilities for ctDNA use in the clinical practice.

Baseline cluster of differentiation 22 fluorescent intensity correlates with patient outcome after Inotuzumab Ozogamicin treatment.

Antigen-directed target therapy for B-cell acute lymphoblastic leukemia (B-ALL) is now the standard of care for relapsed/refractory (R/R) disease. A comprehensive determination of the target itself is mandatory to aid physician's choice. We determined baseline Cluster of differentiation 22 (CD22) expression percentage and fluorescent intensity on lymphoblasts of 30 patients with R/R B-ALL treated with anti-CD22 immunoconjugate drug Inotuzumab Ozogamicin (INO) and analyzed the impact of both parameters on patient outcome. Most patients (24/30, 80%) had a high leukemic blast CD22-positivity defined as ≥90%. We did not observe a benefit in terms of complete remission, overall survival (OS) and duration of response (DoR) for patients with CD22 ≥ 90% versus CD22 < 90%. Concerning CD22-FI quartile analysis we appreciated a trend for superior response rates in higher quartiles (Q2 -Q4 ) compared to Q1 and a significant benefit in terms of OS and DoR for patients with higher CD22-FI. INO demonstrates to be effective also in patients with lower CD22 expression, but therapeutical benefits are more evident in patients with higher CD22-FI. The evaluation of both CD22 percentage and CD22-FI of the leukemic blast may help physicians in therapeutic choices for R/R B-ALL patients when multiple treatment options are available, although no CD22 expression threshold can currently be identified below which INO should be considered not effective.

Droplet digital polymerase chain reaction for the assessment of disease burden in hairy cell leukemia.

BRAFV600E mutation is the pathogenic driver of hairy cell leukemia (HCL) found in the vast majority of cases both at onset and during recurrences. The identification of the mutated allele in blood and marrow correlates with the presence of neoplastic cells and can be considered a marker of active disease. Likewise, the absence of the mutation after treatment may indicate a state of deep response. The BRAFV600E burden was measured by droplet digital polymerase chain reaction (ddPCR) and expressed as fractional abundance in 35 HCL patients at different stages of disease (onset, relapse, complete response [CR] after treatment, long-term remission) in peripheral blood and/or bone marrow (when available). Mean values of fractional abundance for patients at diagnosis, relapse and response, respectively, were 12.26%, 16.52% and 0.02% in peripheral blood and 23.51%, 13.96% and 0.26% in bone marrow. Four patients out of 6 evaluated at response were molecularly negative for BRAFV600E in peripheral blood. Mean fractional abundance in peripheral blood tested in 14 patients with long lasting CR was 0.05%, and 10 patients were BRAFV600E negative. These preliminary results suggest that ddPCR permits to assess the active tumor burden in HCL at different disease phases and support the hypothesis that some patients in CR qualify for a molecular CR.

Role of serum-free light chain assay for defining response and progression in immunoglobulin secretory multiple myeloma.

The International Myeloma Working Group (IMWG) guidelines recommend using electrophoresis and immunofixation to define response and progressive disease (PD) in immunoglobulin (Ig) secretory multiple myeloma (Ig-MM), whereas the role of serum-free light chain (sFLC) is controversial. We retrospectively analyzed the value of adding sFLC assays in the definition of response and PD according to IMWG criteria in 339 Ig-MM patients treated with a first-line novel agent-based therapy (median follow-up 54 months). sFLC PD was defined according to conventional criteria plus increased sFLC levels, or sFLC escape (sFLCe); progression/sFLCe-free survival (ePFS) was the time from the start of treatment to the date of first PD or sFLCe, or death; overall survival after PD/sFLCe (OS after Pe) was the time from first PD or sFLCe to the date of death. 148 (44%) patients achieved a complete response and 198 (60%) a normal sFLC ratio (sFLCR). sFLCR normalization was an independent prognostic factor for extended PFS (HR = 0.46, p = 0.001) and OS (HR = 0.47, p = 0.006) by multivariable analysis. 175 (52%) patients experienced PD according to the IMWG criteria, whereas 180 (53%) experienced PD or sFLCe. Overall, a sFLCe was observed in 31 (9%) patients. Median PFS and ePFS were both equal to 36 (95% CI = 32-42, and 32-40, respectively) months. sFLC PD adversely affected the OS after Pe compared to PD with increasing monoclonal Ig only (HR = 0.52, p = 0.012). Our results support the inclusion of the sFLC assay for defining response and PD in Ig-MM.

Identification of a Maturation Plasma Cell Index through a Highly Sensitive Droplet Digital PCR Assay Gene Expression Signature Validation in Newly Diagnosed Multiple Myeloma Patients.

DNA microarrays and RNA-based sequencing approaches are considered important discovery tools in clinical medicine. However, cross-platform reproducibility studies undertaken so far have highlighted that microarrays are not able to accurately measure gene expression, particularly when they are expressed at low levels. Here, we consider the employment of a digital PCR assay (ddPCR) to validate a gene signature previously identified by gene expression profile. This signature included ten Hedgehog (HH) pathways' genes able to stratify multiple myeloma (MM) patients according to their self-renewal status. Results show that the designed assay is able to validate gene expression data, both in a retrospective as well as in a prospective cohort. In addition, the plasma cells' differentiation status determined by ddPCR was further confirmed by other techniques, such as flow cytometry, allowing the identification of patients with immature plasma cells' phenotype (i.e., expressing CD19+/CD81+ markers) upregulating HH genes, as compared to others, whose plasma cells lose the expression of these markers and were more differentiated. To our knowledge, this is the first technical report of gene expression data validation by ddPCR instead of classical qPCR. This approach permitted the identification of a Maturation Index through the integration of molecular and phenotypic data, able to possibly define upfront the differentiation status of MM patients that would be clinically relevant in the future.

Halting the vicious cycle within the multiple myeloma ecosystem: blocking JAM-A on bone marrow endothelial cells restores angiogenic homeostasis and suppresses tumor progression.

Interactions of malignant multiple myeloma (MM) plasma cells (MM-cells) with the microenvironment control MM-cell growth, survival, drug-resistance and dissemination. As in MM microvascular density increases in the bone marrow (BM), we investigated whether BM MM endothelial cells (MMECs) control disease progression via the junctional adhesion molecule A (JAM-A). Membrane and cytoplasmic JAM-A levels were upregulated in MMECs in 111 newly diagnosed (NDMM) and 201 relapsed-refractory (RRMM) patients compared to monoclonal gammopathy of undetermined significance (MGUS) and healthy controls. Elevated membrane expression of JAM-A on MMECs predicted poor clinical outcome. Mechanistically, addition of recombinant JAM-A to MMECs increased angiogenesis whereas its inhibition impaired angiogenesis and MM growth in 2D and 3D in vitro cell culture and chorioallantoic membrane-assays. To corroborate these findings, we treated MM bearing mice with JAM-A blocking mAb and demonstrated impaired MM progression corresponding to decreased MM-related vascularity. These findings support JAM-A as an important mediator of MM progression through facilitating MM-associated angiogenesis. Collectively, elevated JAM-A expression on bone marrow endothelial cells is an independent prognostic factor for patient survival in both NDMM and RRMM. Blocking JAM-A restricts angiogenesis in vitro, in embrio and in vivo and represents a suitable druggable molecule to halt neoangiogenesis and MM progression.

The treatment of hairy cell leukemia with a focus on long lasting responses to cladribine: A 30-year experience.

The treatment of hairy cell leukemia (HCL) has considerably changed over years. Purine analogues, namely cladribine, now represent the treatment of choice. One hundred and eighty-four patients were followed between 1986 and 2018 and treated according to era-specific guidelines. Responses were classified by combining Consensus Resolution criteria and marrow immunohistochemistry. Patients were grouped according to the number of treatment lines they received. Patients treated first line responded in 86% of cases, with complete response (CR) in 44% of cases. Response rates remained high throughout the first four lines (84%, 81%, 79% for the second line onward, with CR in 38%, 37%, 15% of cases respectively). One hundred and twenty-two patients received cladribine as first line treatment, with a response rate of 86% and a CR rate of 54%. Among the 66 CR patients, 45 (68%) have never received further therapy: 11 patients are in continuous CR between 5 and 10 years after treatment, 14 between 10 and 20 years and three patients at more than 20 years. Median time-to-next treatment (TTNT) for frontline cladribine-treated patients was 8.2 years: partial responders had a significantly shorter median TTNT than CR patients (5.3 years vs median not reached at 25.8 years, p < 0.001). Patients with HCL require subsequent lines of therapy in more than 50% of cases. Purine analogues allow significant response rates when applied first line and upon retreatment. Some patients may enjoy long lasting treatment-free intervals after one course of cladribine.

Disease-Specific Derangement of Circulating Endocannabinoids and N-Acylethanolamines in Myeloproliferative Neoplasms.

Growing evidence highlights the endocannabinoid (EC) system involvement in cancer progression. Lipid mediators of this system are secreted by hematopoietic cells, including the ECs 2-arachidonoyl-glycerol (2AG) and arachidonoyl-ethanolamide (AEA), the 2AG metabolite 1AG, and members of N-acylethanolamine (NAE) family-palmitoyl-ethanolamide (PEA) and oleoyl-ethanolamide (OEA). However, the relevance of the EC system in myeloproliferative neoplasms (MPN) was never investigated. We explored the EC plasma profile in 55 MPN patients, including myelofibrosis (MF; n = 41), polycythemia vera (PV; n = 9), and essential thrombocythemia (ET; n = 5) subclasses and in 10 healthy controls (HC). AEA, PEA, OEA, 2AG, and 1AG plasma levels were measured by LC-MS/MS. Overall considered, MPN patients displayed similar EC and NAE levels compared to HC. Nonetheless, AEA levels in MPN were directly associated with the platelet count. MF patients showed higher levels of the sum of 2AG and 1AG compared to ET and PV patients, higher OEA/AEA ratios compared to HC and ET patients, and higher OEA/PEA ratios compared to HC. Furthermore, the sum of 2AG and 1AG positively correlated with JAK2V617F variant allele frequency and splenomegaly in MF and was elevated in high-risk PV patients compared to in low-risk PV patients. In conclusion, our work revealed specific alterations of ECs and NAE plasma profile in MPN subclasses and potentially relevant associations with disease severity.

Identification of a novel large EPCAM-MSH2 duplication, concurrently with LOHs in chromosome 20 and X, in a family with Lynch syndrome.

BACKGROUND: Lynch syndrome (LS) is associated with germline mutations in one of the mismatch repair genes or EPCAM. The majority of the causative alterations are point mutations. Large genomic rearrangements represent only 5-20%. Hypothetically, the allelic imbalance, like the loss of heterozygosity, may be another high penetrance risk factor. CASE PRESENTATION: We describe the case of a patient who developed 5 tumors during her lifetime and with a family history characterized by a high frequency of tumors associated with LS. The proband was tested for mutations and copy number alterations with a panel of hereditary cancer genes and by SNP array. She showed a 187 Kb duplication including EPCAM and the first 7 exons of MSH2, plus two loss of heterozygosity (LOHs) in chromosome 20 and one in chromosome X which include many tumor suppressor genes. CONCLUSION: We found a novel large EPCAM-MSH2 duplication associated with LS and the presence of LOHs in regions containing numerous tumor suppressors, raising the hypothesis that these alterations could contribute to cancer susceptibility. Our results underline the importance to deepen the knowledge of molecular mechanisms in order to determine the role in cancer predisposition of novel genetic alterations.

Bortezomib-based therapy combined with high cut-off hemodialysis is highly effective in newly diagnosed multiple myeloma patients with severe renal impairment.

Multiple myeloma (MM) is often associated with renal insufficiency (RI) which adversely influences the prognosis. Several studies demonstrated that bortezomib can improve both renal function and outcome. We prospectively evaluated 21 newly diagnosed MM patients with severe renal impairment secondary to tubular-interstitial damage, most of them due to myeloma kidney, who were primarily treated with bortezomib-based therapy combined with high cut-off hemodialysis (HCOD). The median serum creatinine level at baseline was 6.44 mg dL(-1) and calculated median estimated glomerular filtration rate (eGFR), according to Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) creatinine equation, was 8 mL/min/1.73 m(2) . Serum free light chain (sFLC) median concentration was 6,040 mg L(-1) . Post induction and best stringent complete response rates were 19 and 38%, respectively. Responses were fast, occurring within a median of 1.4 months. The combination of bortezomib and HCOD led to a prompt and remarkable (>90%) decrease in sFLC levels. Sixteen patients (76%) became dialysis independent within a median of 32 days. With a median follow up of 17.2 months, the 3-year PFS and OS were 76 and 67%, respectively. No early deaths were observed. This study demonstrates that incorporation of bortezomib into induction therapy combined with HCOD is a highly effective strategy in rescuing renal function and improving outcomes in patients with MM and RI.

HIF-1α inhibition blocks the cross talk between multiple myeloma plasma cells and tumor microenvironment.

Multiple myeloma (MM) is a malignant disorder of post-germinal center B cells, characterized by the clonal proliferation of malignant plasma cells (PCs) within the bone marrow (BM). The reciprocal and complex interactions that take place between the different compartments of BM and the MM cells result in tumor growth, angiogenesis, bone disease, and drug resistance. Given the importance of the BM microenvironment in MM pathogenesis, we investigated the possible involvement of Hypoxia-Inducible transcription Factor-1 alpha (HIF-1α) in the PCs-bone marrow stromal cells interplay. To test this hypothesis, we used EZN-2968, a 3rd generation antisense oligonucleotide against HIF-1α, to inhibit HIF-1α functions. Herein, we provide evidence that the interaction between MM cells and BM stromal cells is drastically reduced upon HIF-1α down-modulation. Notably, we showed that upon exposure to HIF-1α inhibitor, neither the incubation with IL-6 nor the co-culture with BM stromal cells were able to revert the anti-proliferative effect induced by EZN-2968. Moreover, we observed a down-modulation of cytokine-induced signaling cascades and a reduction of MM cells adhesion capability to the extracellular matrix proteins in EZN-2968-treated samples. Taken together, these results strongly support the concept that HIF-1α plays a critical role in the interactions between bone BM cells and PCs in Multiple Myeloma.

Bortezomib- and thalidomide-induced peripheral neuropathy in multiple myeloma: clinical and molecular analyses of a phase 3 study.

A subanalysis of the GIMEMA-MMY-3006 trial was performed to characterize treatment-emergent peripheral neuropathy (PN) in patients randomized to thalidomide-dexamethasone (TD) or bortezomib-TD (VTD) before and after double autologous transplantation (ASCT) for multiple myeloma (MM). A total of 236 patients randomized to VTD and 238 to TD were stratified according to the emergence of grade ≥2 PN. Gene expression profiles (GEP) of CD138+ plasma cells were analyzed in 120 VTD-treated patients. The incidence of grade ≥2 PN was 35% in the VTD arm and 10% in the TD arm (P < 0.001). PN resolved in 88 and 95% of patients in VTD and TD groups, respectively. Rates of complete/near complete response, progression-free and overall survival were not adversely affected by emergence of grade ≥2 PN. Baseline characteristics were not risk factors for PN, while GEP analysis revealed the deregulated expression of genes implicated in cytoskeleton rearrangement, neurogenesis, and axonal guidance. In conclusion, in comparison with TD, incorporation of VTD into ASCT was associated with a higher incidence of PN which, however, was reversible in most of the patients and did not adversely affect their outcomes nor their ability to subsequently receive ASCT. GEP analysis suggests an interaction between myeloma genetic profiles and development of VTD-induced PN.

Multi-dimensional scaling techniques unveiled gain1q&loss13q co-occurrence in Multiple Myeloma patients with specific genomic, transcriptional and adverse clinical features.

The complexity of Multiple Myeloma (MM) is driven by several genomic aberrations, interacting with disease-related and/or -unrelated factors and conditioning patients' clinical outcome. Patient's prognosis is hardly predictable, as commonly employed MM risk models do not precisely partition high- from low-risk patients, preventing the reliable recognition of early relapsing/refractory patients. By a dimensionality reduction approach, here we dissect the genomic landscape of a large cohort of newly diagnosed MM patients, modelling all the possible interactions between any MM chromosomal alterations. We highlight the presence of a distinguished cluster of patients in the low-dimensionality space, with unfavorable clinical behavior, whose biology was driven by the co-occurrence of chromosomes 1q CN gain and 13 CN loss. Presence or absence of these alterations define MM patients overexpressing either CCND2 or CCND1, fostering the implementation of biology-based patients' classification models to describe the different MM clinical behaviors.

Single-Cell DNA Sequencing Reveals an Evolutionary Pattern of CHIP in Transplant Eligible Multiple Myeloma Patients.

Clonal hematopoiesis of indeterminate potential (CHIP) refers to the phenomenon where a hematopoietic stem cell acquires fitness-increasing mutation(s), resulting in its clonal expansion. CHIP is frequently observed in multiple myeloma (MM) patients, and it is associated with a worse outcome. High-throughput amplicon-based single-cell DNA sequencing was performed on circulating CD34+ cells collected from twelve MM patients before autologous stem cell transplantation (ASCT). Moreover, in four MM patients, longitudinal samples either before or post-ASCT were collected. Single-cell sequencing and data analysis were assessed using the MissionBio Tapestri® platform, with a targeted panel of 20 leukemia-associated genes. We detected CHIP pathogenic mutations in 6/12 patients (50%) at the time of transplant. The most frequently mutated genes were TET2, EZH2, KIT, DNMT3A, and ASXL1. In two patients, we observed co-occurring mutations involving an epigenetic modifier (i.e., DNMT3A) and/or a gene involved in splicing machinery (i.e., SF3B1) and/or a tyrosine kinase receptor (i.e., KIT) in the same clone. Longitudinal analysis of paired samples revealed a positive selection of mutant high-fitness clones over time, regardless of their affinity with a major or minor sub-clone. Copy number analysis of the panel of all genes did not show any numerical alterations present in stem cell compartment. Moreover, we observed a tendency of CHIP-positive patients to achieve a suboptimal response to therapy compared to those without. A sub-clone dynamic of high-fitness mutations over time was confirmed.

Prognostic relevance of 18-F FDG PET/CT in newly diagnosed multiple myeloma patients treated with up-front autologous transplantation.

We prospectively analyzed the prognostic relevance of positron emission tomography-computed tomography (PET/CT) at diagnosis, after thalidomide-dexamethasone (TD) induction therapy and double autotransplantation (ASCT) in 192 newly diagnosed multiple myeloma (MM) patients. Presence at baseline of at least 3 focal lesions (FLs; 44% of cases), a standardized uptake value (SUV) > 4.2 (46%), and extramedullary disease (EMD; 6%) adversely affected 4-year estimates of progression-free survival (PFS; ≥ 3 FLs: 50%; SUV > 4.2: 43%; presence of EMD: 28%). SUV > 4.2 and EMD were also correlated with shorter overall survival (OS; 4-year rates: 77% and 66%, respectively). Persistence of SUV > 4.2 after TD induction was an early predictor for shorter PFS. Three months after ASCT, PET/CT was negative in 65% of patients whose 4-year rates of PFS and OS were superior to those of PET-positive patients (PFS: 66% and OS: 89%). In a multivariate analysis, both EMD and SUV > 4.2 at baseline and persistence of fluorodeoxyglucose (FDG) uptake after ASCT were independent variables adversely affecting PFS. PET/CT involvement at diagnosis, after novel agent-based induction and subsequent ASCT is a reliable predictor of prognosis in MM patients. This study is registered at www.clinicaltrials.gov as NTC01341262.

Correlation between eight-gene expression profiling and response to therapy of newly diagnosed multiple myeloma patients treated with thalidomide-dexamethasone incorporated into double autologous transplantation.

We performed a molecular study aimed at identifying a gene expression profile (GEP) signature predictive of attainment of at least near complete response (CR) to thalidomide-dexamethasone (TD) as induction regimen in preparation for double autologous stem cell transplantation in 112 younger patients with newly diagnosed multiple myeloma. A GEP supervised analysis was performed on a training set of 32 patients, allowing to identify 157 probe sets differentially expressed in patients with CR versus those failing CR to TD. We then generated an eight-gene GEP signature whose performance was subsequently validated in a training set of 80 patients. A correct prediction of response to TD was found in 71 % of the cases analyzed. The eight genes were downregulated in patients who achieved CR to TD. Comparisons between post-autotransplantation outcomes of the 44 non-CR-predicted patients and of the 36 CR-predicted patients showed that this latter subgroup had a statistically significant benefit in terms of higher rate of CR after autotransplant(s) and longer time to progression, event-free survival, and overall survival. These results can be an important first step to identify at diagnosis those patients who will respond more favourably to a particular treatment strategy.

Unraveling the Role of Peroxisome Proliferator-Activated Receptor ß/Δ (PPAR ß/Δ) in Angiogenesis Associated with Multiple Myeloma.

Growing evidence suggests a role for peroxisome proliferator-activated receptor ß/δ (PPAR ß/δ) in the angiogenesis, growth, and metastasis of solid tumors, but little is known about its role in multiple myeloma (MM). Angiogenesis in the bone marrow (BM) is characteristic of disease transition from monoclonal gammopathy of undetermined significance (MGUS) to MM. We examined the expression and function of PPAR ß/δ in endothelial cells (EC) from the BM of MGUS (MGEC) and MM (MMEC) patients and showed that PPAR ß/δ was expressed at higher levels in MMEC than in MGEC and that the overexpression depended on myeloma plasma cells. The interaction between myeloma plasma cells and MMEC promoted the release of the PPAR ß/δ ligand prostaglandin I2 (PGI2) by MMEC, leading to the activation of PPAR ß/δ. We also demonstrated that PPAR ß/δ was a strong stimulator of angiogenesis in vitro and that PPAR ß/δ inhibition by a specific antagonist greatly impaired the angiogenic functions of MMEC. These findings define PGI2-PPAR ß/δ signaling in EC as a potential target of anti-angiogenic therapy. They also sustain the use of PPAR ß/δ inhibitors in association with conventional drugs as a new therapeutic approach in MM.

Primary pulmonary T-cell lymphoproliferative disorders with a limited-stage, low proliferative index, and unusual clinical behavior: two cases of a rare occurrence.

Extranodal T-lymphoproliferative disorders or T-cell lymphomas (TLPD) are classified according to the WHO Classification (4th and upcoming 5th editions) (Swerdlow et al., IARC Press 1; Alaggio et al., Leukemia 36(7):1720-1748, 2) and to the International Consensus Classification Update (Campo et al., Blood 140(11):1229-1253, 3) upon several morphologic, phenotypic, and genetic features. None of those at present included has been characterized by primary pulmonary onset. We herein present two such cases which, to the best of our knowledge, have not been previously reported and that might represent another variant of T-cell proliferation at mucosal sites. The two cases share similar histological and phenotypic features, suggesting an origin from CD4 + effector memory T cells with the expression of a CD279/PD-1 antigen. They are both monoclonal, harbor few mutations, and show no disease progression outside the lung. They only differ concerning the local extension of the process and clinical setting. The two cases are examples of so far unreported primary pulmonary TLDP, with limited stage and low proliferative index. A possible relationship with a local yet unknown inflammatory trigger that might have favored the development of the T-cell clone cannot be ruled out.