Revisiting Diagnostic performances of serum erythropoïetin level and JAK2 mutation for polycythemias: analysis of a cohort of 1090 patients with red cell mass measurement.

We assessed the diagnostic performances of erythropoietin and JAK2 mutations in 1,090 patients with suspected polycythemia who were referred for red cell mass (RCM) measurement. In patients with a high haematocrit and/or haemoglobin level, a low erythropoietin level (<=3·3 mUI/ml) and JAK2 mutation showed comparable positive predictive value (PPV) for true polycythemia (RCM>=125%), 92·1% and 90% respectively. A very-low erythropoietin level (<=1·99 mUI/ml) had a PPV of 100% for polycythemia vera (PV) diagnosis. We confirmed the correlations between RCM, erythropoietin and JAK2 variant allelic frequency in PV patients. This study prompts the need to revisit the role of EPO in PV diagnostic criteria.

Human-Derived α1-Antitrypsin is Still Efficacious in Heavily Pretreated Patients with Steroid-Resistant Gastrointestinal Graft-versus-Host Disease.

Almost one-half of patients developing graft-versus-host disease (GVHD) will not respond to standard first-line steroid treatment. Alpha-1 antitrypsin (AAT) is able to induce tolerance in preclinical models of GVHD. AAT alters the cytokine milieu, promotes a tolerogenic shift of dendritic cells, and skews effector T cells toward regulatory T cells. Gastrointestinal steroid-refractory (SR)-GVHD is a protein-losing enteropathy that might represent the optimal setting in which to use AAT. Here we analyze the outcomes of 16 patients treated with human-derived AAT in advanced-stage gut SR-GVHD, with two-thirds of the patients having failed at least 1 treatment for SR-GVHD. The overall response rate (ORR) was 44%, with a complete response (CR) rate of 27%. Gastrointestinal response was observed in 61% of patients. The median time to best response was 21 days (range, 6 to 26 days). At day 56 after AAT treatment, all CRs were maintained, and the ORR was 39%. The 1-year overall survival was 48% (95% confidence interval, 26% to 74%). Ancillary studies showed that AAT serum levels were in the normal range at the beginning of treatment, whereas fecal loss was elevated. AAT levels consistently rose after exogenous administration, but no correlation was found between serum levels and response. REG3α and IL-33 levels were associated with response while, in contrast to previous reports, regulatory T cells decreased during AAT treatment. This retrospective analysis supports a previous report of AAT as a promising agent in the management of gut SR-GVHD and should prompt its evaluation at an earlier stage.

Synergistic effects of PRIMA-1Met (APR-246) and 5-azacitidine in TP53-mutated myelodysplastic syndromes and acute myeloid leukemia.

Myelodysplastic syndromes and acute myeloid leukemia with TP53 mutations are characterized by frequent relapses, poor or short responses, and poor survival with the currently available therapies including chemotherapy and 5-azacitidine (AZA). PRIMA-1Met(APR-246,APR) is a methylated derivative of PRIMA-1, which induces apoptosis in human tumor cells through restoration of the transcriptional transactivation function of mutant p53. Here we show that low doses of APR on its own or in combination with AZA reactivate the p53 pathway and induce an apoptosis program. Functionally, we demonstrate that APR exerts these activities on its own and that it synergizes with AZA in TP53-mutated myelodysplastic syndromes (MDS)/acute myeloid leukemia (AML) cell lines and in TP53-mutated primary cells from MDS/AML patients. Low doses of APR on its own or in combination with AZA also show significant efficacy in vivo Lastly, using transcriptomic analysis, we found that the APR + AZA synergy was mediated by downregulation of the FLT3 pathway in drug-treated cells. Activation of the FLT3 pathway by FLT3 ligand reversed the inhibition of cell proliferation by APR + AZA. These data suggest that TP53-mutated MDS/AML may be better targeted by the addition of APR-246 to conventional treatments.

Next-generation sequencing for JAK2 mutation testing: advantages and pitfalls.

The JAK2V617F mutation is part of the major criteria for diagnosis of myeloproliferative neoplasms (MPN). Allele-specific quantitative PCR (qPCR) is the most prevalent method used in laboratories but with the advent of next-generation sequencing (NGS) techniques, we felt necessary to evaluate this approach for JAK2 mutations testing. Among DNA samples from 427 patients analyzed by qPCR and NGS, we found an excellent concordance between both methods when allelic burden was superior to 2% (the detection limit of our NGS assay). Only one sample among 298 was found negative by NGS while allelic burden by qPCR was 3%. Because NGS detection limit is higher, sensitivity was lower as exemplified by 21 samples found negative whereas qPCR measured allelic burdens between 0.1 and 1%. Importantly, quantitative data of samples found positive by both techniques were highly correlated (R2 = 0.9477). We also evaluated 40 samples tested for JAK2 exon 12 mutations by HRM. The concordance with NGS was of 100%. Using NGS, the full coding region of JAK2 was analyzed leading to identification of several variants outside of exon 12 and 14 which were previously described or not. Interestingly, we found one somatic mutation (c.1034A>T p.H345L) which induced constitutive activation of the JAK/STAT pathway leading to an increased proliferation of BaF/3 cells with low-dose EPO. This study showed that NGS is a robust method highly correlated to qPCR, although less sensitive, but providing the opportunity to identify other JAK2 variants with potential impact on disease initiation or evolution.

Clinical and molecular response to interferon-α therapy in essential thrombocythemia patients with CALR mutations.

Myeloproliferative neoplasms are clonal disorders characterized by the presence of several gene mutations associated with particular hematologic parameters, clinical evolution, and prognosis. Few therapeutic options are available, among which interferon α (IFNα) presents interesting properties like the ability to induce hematologic responses (HRs) and molecular responses (MRs) in patients with JAK2 mutation. We report on the response to IFNα therapy in a cohort of 31 essential thrombocythemia (ET) patients with CALR mutations (mean follow-up of 11.8 years). HR was achieved in all patients. Median CALR mutant allelic burden (%CALR) significantly decreased from 41% at baseline to 26% after treatment, and 2 patients even achieved complete MR. In contrast, %CALR was not significantly modified in ET patients treated with hydroxyurea or aspirin only. Next-generation sequencing identified additional mutations in 6 patients (affecting TET2, ASXL1, IDH2, and TP53 genes). The presence of additional mutations was associated with poorer MR on CALR mutant clones, with only minor or no MRs in this subset of patients. Analysis of the evolution of the different variant allele frequencies showed that the mutated clones had a differential sensitivity to IFNα in a given patient, but no new mutation emerged during treatment. In all, this study shows that IFNα induces high rates of HRs and MRs in CALR-mutated ET, and that the presence of additional nondriver mutations may influence the MR to therapy.

The spectrum of neutrophilic dermatoses associated with monoclonal gammopathy: Association with IgA isotype and inflammatory profile.

BACKGROUND: Neutrophilic dermatoses refer to a group of cutaneous inflammatory disorders characterized by neutrophilic infiltration of the skin. Neutrophilic dermatoses have been reported in association with various conditions including autoimmune diseases, inflammatory bowel diseases, and neoplasia. In the later condition, myeloproliferative disorders and monoclonal gammopathy (monoclonal immunoglobulin [MIg]) are the most frequent. Only few data are available in case of neutrophilic dermatoses associated with MIg regarding the pathophysiology and the clinical outcome. OBJECTIVE: We sought to gain further insight into clinical and biological aspects of neutrophilic dermatoses associated with MIg. METHODS: We report a retrospective series of 26 patients with neutrophilic dermatoses associated with MIg focusing on clinical and biological aspects, with a study of a large panel of cytokines, chemokines, and adhesion molecules. RESULTS: This study reveals an association between MIg IgA isotype and neutrophilic dermatoses, and a specific inflammatory pattern including elevated interleukin 6, vascular endothelial growth factor, monocyte chemotactic protein-1, epidermal growth factor, and intercellular adhesion molecule-1. LIMITATIONS: This is a retrospective study from a single institution with a limited number of participants. CONCLUSION: Our data highlight a strong association between IgA isotype and neutrophilic dermatoses, and the existence of a specific inflammatory profile involving several molecules.

Ovarian reserve in breast cancer: assessment with anti-Müllerian hormone.

Anti-Müllerian hormone (AMH) levels fall during chemotherapy. Treatment-induced amenorrhoea is a reversible phenomenon, but few data are available on long-term AMH changes in breast cancer. The aim of the study was to describe serum AMH levels before, during and in the long term after chemotherapy, and to show a potential AMH recovery. Between May 2010 and June 2011, we selected 134 women aged 18-43 years at the time of breast cancer diagnosis who received chemotherapy between 2005 and 2011, and had not undergone an oophorectomy or had previous cytotoxic treatment. The AMH levels were assessed before, during and 4 months to 5.5 years after the end of chemotherapy. During chemotherapy, AMH was undetectable in 69% of women. After chemotherapy, a significant increase in AMH was found, with an average magnitude of +1.2% per month (95% credibility interval: 0.7 to 1.6). Older age and 12 months of amenorrhoea were found to be associated with a lower AMH recovery rate, whereas baseline AMH and number of chemotherapy cycles were not. The process of AMH changes during and after chemotherapy is dynamic, and shows recovery after ovarian injury. Caution should be exercised in interpreting individual AMH assessment in this context.

Comprehensive analysis of mesenchymal cells reveals a dysregulated TGF-ß/Wnt/HOXB7 axis in patients with myelofibrosis.

Despite the advances in the understanding and treatment of myeloproliferative neoplasm (MPN), the disease remains incurable with the risk of evolution to AML or myelofibrosis (MF). Unfortunately, the evolution of the disease to MF remains still poorly understood impeding preventive and therapeutic options. Recent studies in solid tumor microenvironment and organ fibrosis have shed instrumental insights on their respective pathogenesis and drug resistance, yet such precise data are lacking in MPN. In this study, through a patient-sample driven transcriptomic and epigenetic description of the MF microenvironment landscape and cell-based analyses, we identify HOXB7 overexpression and more precisely a novel TGFß-Wnt-HOXB7 pathway as associated to a pro-fibrotic and pro-osteoblastic biased differentiation of mesenchymal stromal cells (MSCs). Using gene-based and chemical inhibition of this pathway we reverse the abnormal phenotype of MSCs from myelofibrosis patients, providing the MPN field with a potential novel target to prevent and manage evolution to MF.

Prognostic significance of anti-p53 and anti-KRas circulating antibodies in esophageal cancer patients treated with chemoradiotherapy.

BACKGROUND: P53 mutations are an adverse prognostic factor in esophageal cancer. P53 and KRas mutations are involved in chemo-radioresistance. Circulating anti-p53 or anti-KRas antibodies are associated with gene mutations. We studied whether anti-p53 or anti-KRas auto-antibodies were prognostic factors for response to chemoradiotherapy (CRT) or survival in esophageal carcinoma. METHODS: Serum p53 and KRas antibodies (abs) were measured using an ELISA method in 97 consecutive patients treated at Saint Louis University Hospital between 1999 and 2002 with CRT for esophageal carcinoma (squamous cell carcinoma (SCCE) 57 patients, adenocarcinoma (ACE) 27 patients). Patient and tumor characteristics, response to treatment and the follow-up status of 84 patients were retrospectively collected. The association between antibodies and patient characteristics was studied. Univariate and multivariate survival analyses were conducted. RESULTS: Twenty-four patients (28%) had anti-p53 abs. Abs were found predominantly in SCCE (p = 0.003). Anti-p53 abs were associated with a shorter overall survival in the univariate analysis (HR 1.8 [1.03-2.9], p = 0.04). In the multivariate analysis, independent prognostic factors for overall and progression-free survival were an objective response to CRT, the CRT strategy (alone or combined with surgery [preoperative]) and anti-p53 abs. None of the long-term survivors had p53 abs. KRas abs were found in 19 patients (23%, no difference according to the histological type). There was no significant association between anti-KRas abs and survival neither in the univariate nor in the multivariate analysis. Neither anti-p53 nor anti-KRas abs were associated with response to CRT. CONCLUSIONS: Anti-p53 abs are an independent prognostic factor for esophageal cancer patients treated with CRT. Individualized therapeutic approaches should be evaluated in this population.

Prospective external validation of biomarkers to predict acute graft-versus-host disease severity.

Acute graft-versus-host disease (GVHD) is still the major contributor to comorbidities and mortality after allogeneic hematopoietic stem cell transplantation. The use of plasmatic biomarkers to predict early outcomes has been advocated in the past decade. The purpose of this prospective noninterventional study was to test the ability of panels including 7 biomarkers (Elafin, HGF, IL2RA, IL8, REG3, ST2, and TNFRI), to predict day 28 (D28) complete response to steroid, D180 overall survival, and D180 nonrelapse mortality (NRM). Using previous algorithms developed by the Ann Arbor/MAGIC consortium, 204 patients with acute GVHD were prospectively included and biomarkers were measured at GVHD onset for all of them. Initial GVHD grade and bilirubin level were significantly associated with all those outcomes. After adjustment on clinical variables, biomarkers were associated with survival and NRM. In addition to clinical variables, biomarkers slightly improved the prediction of overall survival and NRM (concordance and net reclassification indexes). The potential benefit of adding biomarkers panel to clinical parameters was also investigated by decision curve analyses. The benefit of adding biomarkers to clinical parameters was however marginal for the D28 nonresponse and mortality endpoints.

PML-RARA-targeted DNA vaccine induces protective immunity in a mouse model of leukemia.

Despite improved molecular characterization of malignancies and development of targeted therapies, acute leukemia is not curable and few patients survive more than 10 years after diagnosis. Recently, combinations of different therapeutic strategies (based on mechanisms of apoptosis, differentiation and cytotoxicity) have significantly increased survival. To further improve outcome, we studied the potential efficacy of boosting the patient's immune response using specific immunotherapy. In an animal model of acute promyelocytic leukemia, we developed a DNA-based vaccine by fusing the human promyelocytic leukemia-retinoic acid receptor-alpha (PML-RARA) oncogene to tetanus fragment C (FrC) sequences. We show for the first time that a DNA vaccine specifically targeted to an oncoprotein can have a pronounced effect on survival, both alone and when combined with all-trans retinoic acid (ATRA). The survival advantage is concomitant with time-dependent antibody production and an increase in interferon-gamma (IFN-gamma). We also show that ATRA therapy on its own triggers an immune response in this model. When DNA vaccination and conventional ATRA therapy are combined, they induce protective immune responses against leukemia progression in mice and may provide a new approach to improve clinical outcome in human leukemia.

Analytical validation of eight methods of thyroglobulin measurement in fine-needle aspiration washouts.

BACKGROUND: Thyroglobulin (Tg) assay in washout fluids of fine needles, after cervical lymph nodes aspiration, is used for detecting metastases from differentiated thyroid carcinomas. Assay methods are the same as for Tg in serum. However, with non-serum samples, methods require extensive validation to notably check for the absence of matrix effect. This study fits this context. Our objectives were to assess analytic performances, in washout fluid, of eight different Tg assay methods and to compare them to validated data in serum. METHODS: Eleven medical laboratories participated in this study. The matrix tested was phosphate-buffer saline containing 1% bovine serum albumin (PBS-1% BSA). Samples used were dilutions, in this buffer, of Certified Reference Material (CRM 457). We verified, for all methods, the limit of detection, precision, linearity, trueness and accuracy. RESULTS: In PBS-1% BSA, the functional sensitivities (FS) were comparable to those expected for serum. All the methods were linear. The relative biases of trueness were between -24.5 and 10.2% around 1 µg/L. Total analytical error was ≤40% near the functional sensitivity values. CONCLUSION: No quantitatively important matrix effect was observed. All the methods showed their ability to measure Tg in PBS-1% BSA, over the concentration range of interest, with acceptable total analytical error. We validated the functional sensitivity value as a decision threshold in thyroidectomized patients after treatment and with low concentrations of serum Tg.

Ruxolitinib before allogeneic hematopoietic transplantation in patients with myelofibrosis on behalf SFGM-TC and FIM groups.

This multicenter prospective phase 2 trial analyzed disease-free survival (DFS) in myelofibrosis patients receiving ruxolitinib for 6 months before transplantation. Seventy-six patients were recruited. Age-adjusted dynamic international prognostic scoring system was intermediate-1, intermediate-2, and high in 27 (36%), 31 (41%), and 18 (24%) patients. All patients received ruxolitinib from inclusion to conditioning regimen (fludarabine-melphalan) or to progression. A donor was found in 64 patients: 18 HLA-matched sibling donor (MSD), 32 HLA-matched unrelated (UD10/10), and 14 HLA mismatched unrelated donor (UD9/10. Among 64 patients with a donor, 20 (31%) achieved a partial response before transplantation and 59 (92%) could be transplanted after ruxolitinib therapy (18/18 MSD, 30/21 UD10/10, 11/34 UD9/10), of whom 19 (32%) were splenectomized. Overall survival from inclusion was 68% at 12 months. One-year DFS after transplantation was 55%: 83%, 40%, and 34% after MSD, UD10/10 or UD9/10, respectively. Cumulative incidence of grade 2-4 acute graft-versus-host disease (GVHD) was 66% and non-relapse-mortality was 42% at 12 months. Short course of ruxolitinib before transplantation is followed by a high rate of transplantation. With the platform used in this protocol, outcome was much better in patients transplanted with HLA-matched sibling donor as compared to unrelated donor.

Pitfalls in CALR exon 9 mutation detection: A single-center experience in 571 positive patients.

INTRODUCTION: The pathogenesis of myeloproliferative neoplasms (MPNs) is closely related to the acquisition of specific molecular alterations in JAK2, MPL, or CALR genes, the presence of which represent major diagnostic criteria in the WHO classification. The CALR exon 9 insertions and deletions are very heterogeneous, and their detection mainly relies on polymerase chain reaction (PCR) fragment length analysis. METHODS: We report on the rare nonclassical profiles that we observed among the 1382 patients analyzed by PCR fragment length analysis. In difficult cases, we tested germline DNA and performed NGS analysis. RESULTS: We faced some troubling results because of the presence of several unexpected peaks. Our investigations showed that they resulted from a mix of isolated or double somatic mutations combined with germline alterations which could be misleading for a correct diagnosis. The precise interpretation of such difficult cases is mandatory as a misinterpretation could lead to the prescription of cytoreductive drugs to nondiseased persons or to an absence of treatment in true MPN patients. CONCLUSION: Our observations showed that every mutation should be verified by direct Sanger sequencing, and we show that sometimes it may be necessary to study germline DNA and to complement with NGS analysis to precisely interpret the molecular alterations.

The Folate Cycle Enzyme MTHFR Is a Critical Regulator of Cell Response to MYC-Targeting Therapies.

Deciphering the impact of metabolic intervention on response to anticancer therapy may elucidate a path toward improved clinical responses. Here, we identify amino acid-related pathways connected to the folate cycle whose activation predicts sensitivity to MYC-targeting therapies in acute myeloid leukemia (AML). We establish that folate restriction and deficiency of the rate-limiting folate cycle enzyme MTHFR, which exhibits reduced-function polymorphisms in about 10% of Caucasians, induce resistance to MYC targeting by BET and CDK7 inhibitors in cell lines, primary patient samples, and syngeneic mouse models of AML. Furthermore, this effect is abrogated by supplementation with the MTHFR enzymatic product CH3-THF. Mechanistically, folate cycle disturbance reduces H3K27/K9 histone methylation and activates a SPI1 transcriptional program counteracting the effect of BET inhibition. Our data provide a rationale for screening MTHFR polymorphisms and folate cycle status to nominate patients most likely to benefit from MYC-targeting therapies. SIGNIFICANCE: Although MYC-targeting therapies represent a promising strategy for cancer treatment, evidence of predictors of sensitivity to these agents is limited. We pinpoint that folate cycle disturbance and frequent polymorphisms associated with reduced MTHFR activity promote resistance to BET inhibitors. CH3-THF supplementation thus represents a low-risk intervention to enhance their effects.See related commentary by Marando and Huntly, p. 1791.This article is highlighted in the In This Issue feature, p. 1775.

Therapy-resistant anaemia in congenital nephrotic syndrome of the Finnish type--implication of EPO, transferrin and transcobalamin losses.

Congenital nephrotic syndrome of the Finnish type (CNF) is due to NPHS1 mutation and is responsible for a variety of urinary protein losses. We report the case of a 4-month-old girl with a particularly severe form (proteinuria approximately 150 g/l) of CNF. She developed severe non-regenerative anaemia requiring bi-monthly blood transfusions despite daily EPO (600 UI/kg) and iron supplementation. Epoetin pharmacokinetics revealed a urinary loss of 27% of the given dose within the first 24 h after IV injection. However, plasma levels remained increased after 24 h (228 UI/l). Plasma transferrin and transcobalamin levels were undetectable. Atransferrinaemia and atranscobalaminaemia seem to be responsible for disturbed erythropoiesis.

Successful Treatment of Differentiated Thyroid Carcinoma with Transaxillary Robotic Surgery and Radioiodine: The First European Experience.

OBJECTIVES: Transaxillary robotic thyroidectomy surgery (TARS) has been reported to be a safe approach in patients with differentiated thyroid carcinoma, and oncological responses are promising. STUDY DESIGN: This study aimed to evaluate the oncological outcomes of TARS followed by radioiodine (RAI) therapy in patients with differentiated thyroid carcinoma. Between 2011 and 2016, patients treated for differentiated thyroid carcinoma by TARS in a single institution, followed by RAI, were retrospectively included. The oncological response was performed according to the 2015 American Thyroid Association (ATA) guidelines 6-12 months later and at the last available visit. RESULTS: A total of 42 patients (30 females) were included, with a median tumor size of 20 mm (12 cases of N1a and 5 cases of N1b on initial pathology report). According to ATA classification of recurrence risk after surgery, 17 and 25 patients were classified as low and intermediate risk, respectively. After RAI, all patients had a normal posttherapeutic whole body scan (except 1 patient, who had pathological lymph node uptake), but no unusual uptake was seen. At the 6- to 12-month evaluation (n = 37), 24 patients had excellent response, 8 had indeterminate response, and 5 had incomplete response (2 biological and 3 structural); no distant metastasis was found. At the last evaluation (median follow-up 15.9 months), 35 patients had no evidence of disease and 1 patient had a structural incomplete response. In total, a second open surgery was necessary for 3 patients to treat persistent lymph nodes (all intermediate risk). CONCLUSION: In this study, TARS followed by RAI therapy seems to be curative, even for patients with lymph node metastases, after good preoperative staging. More studies are required to confirm the findings.