New reimbursement models to promote better patient outcomes and overall value in laboratory medicine and healthcare.

The most widespread healthcare reimbursement models, including diagnostic laboratory services, are Fee-for-Service, Reference Pricing and Diagnosis-Related Groups. Within these models healthcare providers are remunerated for each specific service or procedure they operate. Healthcare payers are increasingly exploring alternative models, such as bundled payments or value-based reimbursement to encourage value of patient care rather than the simple amount of delivered services. These alternative models are advised, as they are more efficient in promoting cost-effective, high-quality laboratory testing, thereby improving patient health outcomes. If outcomes-based evaluation is a pillar in a new vision of "Value-Based Healthcare", an active policy of Value-Based Reimbursement in laboratory medicine will assure both an efficiency-based sustainability and a high-quality effectiveness-based diagnostic activity. This review aims to evaluate current and alternative reimbursement models, to support a wider agenda in encouraging more Value-Based Healthcare and Value-Based Reimbursement in laboratory medicine.

Point-of-care testing: state-of-the art and perspectives.

Point-of-care testing (POCT) is becoming an increasingly popular way to perform laboratory tests closer to the patient. This option has several recognized advantages, such as accessibility, portability, speed, convenience, ease of use, ever-growing test panels, lower cumulative healthcare costs when used within appropriate clinical pathways, better patient empowerment and engagement, and reduction of certain pre-analytical errors, especially those related to specimen transportation. On the other hand, POCT also poses some limitations and risks, namely the risk of lower accuracy and reliability compared to traditional laboratory tests, quality control and connectivity issues, high dependence on operators (with varying levels of expertise or training), challenges related to patient data management, higher costs per individual test, regulatory and compliance issues such as the need for appropriate validation prior to clinical use (especially for rapid diagnostic tests; RDTs), as well as additional preanalytical sources of error that may remain undetected in this type of testing, which is usually based on whole blood samples (i.e., presence of interfering substances, clotting, hemolysis, etc.). There is no doubt that POCT is a breakthrough innovation in laboratory medicine, but the discussion on its appropriate use requires further debate and initiatives. This collective opinion paper, composed of abstracts of the lectures presented at the two-day expert meeting "Point-Of-Care-Testing: State of the Art and Perspective" (Venice, April 4-5, 2024), aims to provide a thoughtful overview of the state-of-the-art in POCT, its current applications, advantages and potential limitations, as well as some interesting reflections on the future perspectives of this particular field of laboratory medicine.

LH increases the response to FSH in granulosa-lutein cells from sub/poor-responder patients in vitro.

STUDY QUESTION: Does LH addition to FSH in vitro recover the human primary granulosa lutein cell (hGLC) sub/poor-response? SUMMARY ANSWER: A picomolar concentration of LH may recover the FSH-induced cAMP and progesterone production of hGLC from sub/poor-responder women. WHAT IS KNOWN ALREADY: Clinical studies suggested that FSH and LH co-treatment may be beneficial for the ovarian response of sub/poor-responders undergoing ovarian stimulation during ART. STUDY DESIGN, SIZE, DURATION: hGLC samples from 286 anonymous women undergoing oocyte retrieval for ART were collected from October 2017 to February 2021. PARTICIPANTS/MATERIALS, SETTING, METHODS: hGLCs from women undergoing ovarian stimulation during ART were blindly purified, cultured, genotyped and treated in vitro by increasing concentrations of FSH (nM) ±0.5 nM LH. cAMP and progesterone levels produced after 3 and 24 h, respectively, were measured. In vitro data were stratified a posteriori, according to the donors' ovarian response, into normo-, sub- and poor-responder groups and statistically compared. The effects of LH addition to FSH were compared with those obtained by FSH alone in all the groups as well. MAIN RESULTS AND THE ROLE OF CHANCE: hGLCs from normo-responders were shown to have higher sensitivity to FSH treatment than sub-/poor-responders in vitro. Equimolar FSH concentrations induced higher cAMP (about 2.5- to 4.2-fold), and progesterone plateau levels (1.2- to 2.1-fold), in cells from normo-responder women than those from sub-/poor-responders (ANOVA; P < 0.05). The addition of LH to the cell treatment significantly increased overall FSH efficacy, indicated by cAMP and progesterone levels, within all groups (P > 0.05). Interestingly, these in vitro endpoints, collected from the normo-responder group treated with FSH alone, were similar to those obtained in the sub-/poor-responder group under FSH + LH treatment. No different allele frequencies and FSH receptor (FSHR) gene expression levels between groups were found, excluding genetics of gonadotropin and their receptors as a factor linked to the normo-, sub- and poor-response. In conclusion, FSH elicits phenotype-specific ovarian lutein cell response. Most importantly, LH addition may fill the gap between cAMP and steroid production patterns between normo- and sub/poor-responders. LIMITATIONS, REASONS FOR CAUTION: Although the number of experimental replicates is overall high for an in vitro study, clinical trials are required to demonstrate if the endpoints evaluated herein reflect parameters of successful ART. hGLC retrieved after ovarian stimulation may not fully reproduce the response to hormones of granulosa cells from the antral follicular stage. WIDER IMPLICATIONS OF THE FINDINGS: This in vitro assay may describe the individual response to personalize ART stimulation protocol, according to the normo-, sub- and poor-responder status. Moreover, this in vitro study supports the need to conduct optimally designed, randomized clinical trials exploring the personalized use of LH in assisted reproduction. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by Merck KGaA. M.L. and C.C. are employees of Merck KGaA or of the affiliate Merck Serono SpA. Other authors have no competing interests to declare. TRIAL REGISTRATION NUMBER: N/A.

Variability of cardiac troponin levels in normal subjects and in patients with cardiovascular diseases: analytical considerations and clinical relevance.

In accordance with all the most recent international guidelines, the variation of circulating levels of cardiac troponins I and T, measured with high-sensitivity methods (hs-cTnI and hs-cTnT), should be used for the detection of acute myocardial injury. Recent experimental and clinical evidences have demonstrated that the evaluation of hs-cTnI and hs-cTnT variations is particularly relevant: a) for the differential diagnosis of Acute Coronary Syndromes (ACS) in patients admitted to the Emergency Department (ED); b) for the evaluation of cardiovascular risk in patients undergoing major cardiac or non-cardiac surgery, and in asymptomatic subjects of the general population aged >55 years and with co-morbidities; c) for the evaluation of cardiotoxicity caused by administration of some chemotherapy drugs in patients with malignant tumors. The aim of this document is to discuss the fundamental statistical and biological considerations on the intraindividual variability of hs-cTnI and hs-cTnT over time in the same individual. Firstly, it will be discussed in detail as the variations of circulating levels strictly depend not only on the analytical error of the method used but also on the intra-individual variability of the biomarker. Afterwards, the pathophysiological interpretation and the clinical relevance of the determination of the variability of the hs-cTnI and hs-cTnT values ​​ in patients with specific clinical conditions are discussed. Finally, the evaluation over time of the variation in circulating levels of hs-cTnI and hs-cTnT is proposed for a more accurate estimation of cardiovascular risk in asymptomatic subjects from the general population.

Fibrinogen Depletion Coagulopathy Predicts Major Bleeding After Thrombolysis for Ischemic Stroke: A Multicenter Study.

BACKGROUND: Symptomatic intracerebral hemorrhage (sICH) and major bleeding can be fatal complications of intravenous thrombolysis (IVT) for acute ischemic stroke. We investigated the impact of early fibrinogen depletion after IVT on major bleeding events. METHODS: This multicenter observational prospective cohort study enrolled 1678 consecutive patients receiving IVT for acute ischemic stroke at 6 Italian centers, undergoing fibrinogen concentration assessment at baseline, 2 hours and 6 hours after IVT. Fibrinogen depletion was defined as a reduction below 200 mg/dL after 2 hours from IVT, or as a reduction below 50% of baseline fibrinogen levels after 2 hours from IVT. Main outcomes were (1) sICH (National Institute of Neurological Disorders and Stroke criteria) and (2) major bleeding defined as fatal bleeding, decrease in the hemoglobin level>2 g/dL/>1 unit transfusion, or bleeding at critical site. Additional outcomes were (1) any ICH, (2) any bleeding, (3) fatal ICH, and (4) sICH according to ECASSII definition. Good functional recovery was defined as modified Rankin Scale score 0 to 2 at 3 months. RESULTS: Overall, 1678 patients were included (mean age 72 years, 46% female). sICH (n=116) and major bleeding (n=297) were associated with lower rate of good functional recovery (P<0.001). Despite similar fibrinogen levels at admission, fibrinogen depletion after 2 hours from IVT was more common in people with sICH, major bleeding and all additional bleeding outcomes. In the backward stepwise multivariable logistic regression model, fibrinogen depletion remained a significant predictor of sICH (OR, 1.55 [95% CI, 1.04-2.32]) and major bleeding (OR, 1.36 [95% CI, 1.03-1.8]). Thirty-one percent of sICH could be attributable to fibrinogen depletion. The association between fibrinogen depletion and worse clinical outcome at 3 months after stroke (P=0.012) was attributable to the higher risk of major bleeding/sICH. CONCLUSIONS: Fibrinogen depletion significantly increases the risk of sICH and major bleeding after IVT for acute ischemic stroke. Fibrinogen depletion represents an independent risk factor for bleeding, and routine assessment could be considered to stratify the risk of ICH. Trials on early fibrinogen repletion are needed to investigate mitigation of bleeding risk.

Estimate false-negative RT-PCR rates for SARS-CoV-2. A systematic review and meta-analysis.

BACKGROUND: Molecular-based tests used to identify symptomatic or asymptomatic patients infected by SARS-CoV-2 are characterized by high specificity but scarce sensitivity, generating false-negative results. We aimed to estimate, through a systematic review of the literature, the rate of RT-PCR false negatives at initial testing for COVID-19. METHODS: We systematically searched Pubmed, Embase and CENTRAL as well as a list of reference literature. We included observational studies that collected samples from respiratory tract to detect SARS-CoV-2 RNA using RT-PCR, reporting the number of false-negative subjects and the number of final patients with a COVID-19 diagnosis. Reported rates of false negatives were pooled in a meta-analysis as appropriate. We assessed the risk of bias of included studies and graded the quality of evidence according to the GRADE method. All information in this article is current up to February 2021. RESULTS: We included 32 studies, enrolling more than 18,000 patients infected by SARS-CoV-2. The overall false-negative rate was 0.12 (95%CI from 0.10 to 0.14) with very low certainty of evidence. The impact of misdiagnoses was estimated according to disease prevalence; a range between 2 and 58/1,000 subjects could be misdiagnosed with a disease prevalence of 10%, increasing to 290/1,000 misdiagnosed subjects with a disease prevalence of 50%. CONCLUSIONS: This systematic review showed that up to 58% of COVID-19 patients may have initial false-negative RT-PCR results, suggesting the need to implement a correct diagnostic strategy to correctly identify suspected cases, thereby reducing false-negative results and decreasing the disease burden among the population.

Primary, secondary and compensated male biochemical hypogonadism in people living with HIV (PLWH): relevance of sex hormone-binding globulin (SHBG) measurement and comparison between liquid chromatography-tandem mass spectrometry (LC-MS/MS) and chemiluminescent immunoassay for sex steroids assay.

BACKGROUND: Data about classification of hypogonadism and estrogen deficiency in male people living with HIV (PLWH) are scanty. AIM: To investigate the prevalence and characterization of biochemical hypogonadism and relative estrogen deficiency in male PLWH aged < 50 comparing liquid chromatography-tandem mass spectrometry (LC-MS/MS) with chemiluminescent immunoassay (CI), and combining gonadotropin, sex hormone-binding globulin (SHBG) and serum estradiol (E2) measurements. METHODS: Prospective, cross-sectional, observational study. Serum total testosterone (TT), E2, gonadotropins, SHBG were measured by CI. TT and E2 were also assessed by LC-MS/MS. Free testosterone (cFT) was calculated by Vermeulen equation. RESULTS: A total of 316 PLWH (45.3 ± 5.3 years) were enrolled. TT and cFT by LC-MS/MS were lower compared to CI (p < 0.0001). The prevalence of biochemical hypogonadism was higher with LC-MS/MS than CI, both for TT (5.1% vs 3.2%, p < 0.0001) or cFT (9.5% vs 7%, p < 0.0001). The prevalence of hypogonadism (overt + compensated) was 17.1% for cFT using LC-MS/MS. Secondary form of hypogonadism was more prevalent than primary. The prevalence of relative estrogen deficiency was of 30.0% among hypogonadal patients and 15.5% among eugonadal. CONCLUSIONS: The prevalence of male hypogonadism results underestimated by CI compared to LC-MS/MS in PLWH, both for TT and cFT. SHBG and gonadotropins are essential for detecting T deficiency.

Analysis of cryoproteins with a focus on cryofibrinogen: a study on 103 patients.

OBJECTIVES: Cryofibrinogen (CF) is an abnormal protein in plasma that precipitates at 4 °C and dissolves at 37 °C. Whilst serum cryoglobulins (CGs) analysis is common practice, CF investigation is rarely performed. This study aims to describe the testing methodology developed at our laboratory, potential pitfalls for all analytical phases, the distribution among hospital wards and clinical conditions underlying test requests and clinical conditions in which to order CF analysis is useful. METHODS: Retrospective analysis of laboratory samples received between January 2019 and June 2021 with CF testing requests. RESULTS: A complete protocol for CF pre-analytical, analytical and post-analytical phases are supplied. Most test requests were received from the rheumatology department for systemic sclerosis or liver transplant screening. Among the 103 in-patients included, CF+ was confirmed in 68 patients (66%). Of observed CF+ patients (n=68) most cases were CGs- (n=44, 67%). Isolated CF was found in 43% of the cases. Among CF- patients (n=35; 34%) only 2 patients had positive CGs (CGs+). Among rheumatology patients (n=66), isolated CF+ was observed in 45% (n=30/66), whilst among patients with systemic sclerosis with CF+ (n=19), isolated CF+ was detected in 79% (n=15/19). CONCLUSIONS: Described analytical procedures may be used for the creation of harmonized recommendations and indications for CF analysis. Isolated CF positivity among hospitalized patients, predominantly rheumatology and systemic sclerosis patients, appears higher than rates previously reported in literature. We propose CF test recommendations should be included in investigation protocols for diseases where cryofibrinogenemia may occur.

Evaluation of the cardiovascular risk in patients undergoing major non-cardiac surgery: role of cardiac-specific biomarkers.

Major adverse cardiovascular events are frequently observed in patients undergoing major non-cardiac surgery during the peri-operative period. At this time, the possibility to predict cardiovascular events remains limited, despite the introduction of several algorithms to calculate the risk of adverse events, mainly death and major adverse cardiovascular events (MACE) based on the clinical history, risk factors (sex, age, lipid profile, serum creatinine) and non-invasive cardiac exams (electrocardiogram, echocardiogram, stress tests). The cardiac-specific biomarkers natriuretic peptides (NPs) and cardiac troponins (cTn) have been proposed as additional tools for risk prediction in the peri-operative period, particularly for the identification of myocardial injury in patients undergoing major non-cardiac surgery. The prognostic information from the measurement of BNP/NT-proBNP and hs-cTn is independent and complementary to other important indicators of risk, also including ECG and imaging techniques. Elevated levels of cardiac-specific biomarkers before surgery are associated with a markedly higher risk of MACE during the peri-operative period. BNP/NT-proBNP and hs-cTn should be measured in all patients during the clinical evaluation before surgery, particularly during intermediate- or high-risk surgery, in patients aged >65 years and/or with comorbidities. Several questions remain to be assessed in dedicated clinical studies, such as how to optimize the management of patients with raised cardiac specific biomarkers before surgery, and whether a strategy based on biomarker measurement improves patient outcomes and is cost-effective.

Use of high-sensitivity cardiac troponins in the emergency department for the early rule-in and rule-out of acute myocardial infarction without persistent ST-segment elevation (NSTEMI) in Italy.

Serial measurements of cardiac troponin are recommended by international guidelines to diagnose myocardial infarction (MI) since 2000. However, some relevant differences exist between the three different international guidelines published between 2020 and 2021 for the management of patients with chest pain and no ST-segment elevation. In particular, there is no agreement on the cut-offs or absolute change values to diagnose non-ST-segment elevation MI (NSTEMI). Other controversial issues concern the diagnostic accuracy and cost-effectiveness of cut-off values for the most rapid algorithms (0 h/1 h or 0 h/2 h) to rule-in and rule-out NSTEMI. Finally, another important point is the possible differences between demographic and clinical characteristics of patients enrolled in multicenter trials compared to those routinely admitted to the Emergency Department in Italy. The Study Group of Cardiac Biomarkers, supported by the Italian Scientific Societies Società Italiana di Biochimica Clinica, Italian Society of the European Ligand Assay Society, and Società Italiana di Patolgia Clinica e Medicina di Laboratorio decided to revise the document previously published in 2013 about the management of patients with suspected NSTEMI, and to provide some suggestions for the use of these biomarkers in clinical practice, with a particular focus on the Italian setting.

Patients Recovering from Severe COVID-19 Develop a Polyfunctional Antigen-Specific CD4+ T Cell Response.

Specific T cells are crucial to control SARS-CoV-2 infection, avoid reinfection and confer protection after vaccination. We have studied patients with severe or moderate COVID-19 pneumonia, compared to patients who recovered from a severe or moderate infection that had occurred about 4 months before the analyses. In all these subjects, we assessed the polyfunctionality of virus-specific CD4+ and CD8+ T cells by quantifying cytokine production after in vitro stimulation with different SARS-CoV-2 peptide pools covering different proteins (M, N and S). In particular, we quantified the percentage of CD4+ and CD8+ T cells simultaneously producing interferon-γ, tumor necrosis factor, interleukin (IL)-2, IL-17, granzyme B, and expressing CD107a. Recovered patients who experienced a severe disease display high proportions of antigen-specific CD4+ T cells producing Th1 and Th17 cytokines and are characterized by polyfunctional SARS-CoV-2-specific CD4+ T cells. A similar profile was found in patients experiencing a moderate form of COVID-19 pneumonia. No main differences in polyfunctionality were observed among the CD8+ T cell compartments, even if the proportion of responding cells was higher during the infection. The identification of those functional cell subsets that might influence protection can thus help in better understanding the complexity of immune response to SARS-CoV-2.

The Role of T Cell Immunity in Monoclonal Gammopathy and Multiple Myeloma: From Immunopathogenesis to Novel Therapeutic Approaches.

Multiple Myeloma (MM) is a malignant growth of clonal plasma cells, typically arising from asymptomatic precursor conditions, namely monoclonal gammopathy of undetermined significance (MGUS) and smoldering MM (SMM). Profound immunological dysfunctions and cytokine deregulation are known to characterize the evolution of the disease, allowing immune escape and proliferation of neoplastic plasma cells. In the past decades, several studies have shown that the immune system can recognize MGUS and MM clonal cells, suggesting that anti-myeloma T cell immunity could be harnessed for therapeutic purposes. In line with this notion, chimeric antigen receptor T cell (CAR-T) therapy is emerging as a novel treatment in MM, especially in the relapsed/refractory disease setting. In this review, we focus on the pivotal contribution of T cell impairment in the immunopathogenesis of plasma cell dyscrasias and, in particular, in the disease progression from MGUS to SMM and MM, highlighting the potentials of T cell-based immunotherapeutic approaches in these settings.

Cytomegalovirus reactivation after hematopoietic stem cell transplant with CMV-IG prophylaxis: A monocentric retrospective analysis.

Human cytomegalovirus (CMV) represents the most common viral infection after hematopoietic stem cell transplant (HSCT), mainly occurring as reactivation from latency in seropositive patients, with a different prevalence based on the extent and timing of seroconversion in a specific population. Here, we retrospectively analyzed a cohort of patients who underwent HSCT at our Institution between 2013 and 2018, all of whom were prophylactically treated with CMV-IG (Megalotect Biotest®), to define the incidence and clinical outcomes of CMV reactivation and clinically significant infection. CMV infection occurred in 69% of our patient series, mainly resulting from reactivation, and CMV clinically significant infection (CS-CMVi) occurred in 48% of prophylactically treated patients. CMV infection and CS-CMVi impacted neither on relapse incidence nor on overall survival nor on relapse-free survival. Moreover, a very low incidence of CMV end-organ disease was documented. CMV-IG used alone as prophylactic therapy after HSCT does not effectively prevent CMV reactivation.

Accuracy of the serological detection of IgG and IgM to SARS-Cov-2: a prospective, cross-sectional study.

In response to the rapidly evolving of SARS-CoV-2 infection, numerous serological tests have been developed but their sensitivity and specificity are unclear. We collected serum samples of patients and health-care professionals to assess the accuracy of chemiluminescent (CLIA) and two lateral flow immunochromatographic assays (LFIA) to determine IgG and IgM antibodies to SARS-CoV-2 virus. We calculated the φ correlation for qualitative results and test accuracy, adopting the following case definition: either real-time-PCR positivity or serological positivity with at least two different tests. We analyzed 259 samples, obtaining strong correlation between CLIA and both LFIA for IgG (φ=0.9), and moderate correlation for IgM (φ=0.6). For patients, the sensitivity was suboptimal for all methods (CLIA 81%, LFIA A 85%, LFIA B 78%), while it was poor in asymptomatic health-care workers (CLIA 50%, LFIA A 50%, LFIA B 33%). Overall, CLIA is more sensitive and specific for the determination of both IgG and IgM, whilst both LFIA methods reported good sensitivity and specificity for IgG, but scarce sensitivity for the IgM determination. The determination of SARS-CoV-2-specific IgG is useful to detect infection 6 days from symptom onset.

High-sensitivity cardiac troponin I and T methods for the early detection of myocardial injury in patients on chemotherapy.

Important advances achieved in pharmacological cancer treatment have led progressively to a reduction in mortality from many forms of cancer, and increasing numbers of previously incurable patients can now hope to become cancer-free. Yet, to achieve these improved outcomes a high price has been paid in terms of untoward side effects associated with treatment, cardio-toxicity in particular. Several recent studies have reported that cardiac troponin assay using high-sensitivity methods (hs-cTn) can enable the early detection of myocardial injury related to chemotherapy or abuse of drugs that are potentially cardiotoxic. Several authors have recently suggested that changes in hs-cTn values enable the early diagnosis of cardiac injury from chemotherapy, thus potentially benefitting cancer patients with increased troponin values by initiating early cardioprotective therapy. However, large randomised clinical trials are needed in order to evaluate the cost/benefit ratio of standardised protocols for the early detection of cardiotoxicity using the hs-cTn assay in patients treated with chemotherapy.

Hemoadsorption cartridge and coronavirus disease 2019 infections: A case report and brief literature review.

The cytokine storm has been frequently reported to occur in patients with severe coronavirus disease 2019 (COVID-19). Data from the literature suggest that elevated levels of inflammatory mediators, such as interleukin (IL)-6, IL-8, and tumor necrosis factor, indicate a severe course or the fatality of the disease. Several therapeutic options have been employed to treat critically ill patients, including hemoadsorption of inflammatory mediators. We here present a case of severe acute respiratory syndrome caused by COVID-19 and acute renal failure. The patient was admitted to our intensive care unit and treated with mechanical ventilation, renal replacement therapy, and hemoadsorption to reduce the cytokine release syndrome, which plays a fundamental role in the clinical presentation of COVID-19 patients. We also discuss the potential advantages of reducing cytokine plasma levels using a hemoadsorption cartridge.

Neoantigen-Specific T-Cell Immune Responses: The Paradigm of NPM1-Mutated Acute Myeloid Leukemia.

The C-terminal aminoacidic sequence from NPM1-mutated protein, absent in normal human tissues, may serve as a leukemia-specific antigen and can be considered an ideal target for NPM1-mutated acute myeloid leukemia (AML) immunotherapy. Different in silico instruments and in vitro/ex vivo immunological platforms have identified the most immunogenic epitopes from NPM1-mutated protein. Spontaneous development of endogenous NPM1-mutated-specific cytotoxic T cells has been observed in patients, potentially contributing to remission maintenance and prolonged survival. Genetically engineered T cells, namely CAR-T or TCR-transduced T cells, directed against NPM1-mutated peptides bound to HLA could prospectively represent a promising therapeutic approach. Although either adoptive or vaccine-based immunotherapies are unlikely to be highly effective in patients with full-blown leukemia, these strategies, potentially in combination with immune-checkpoint inhibitors, could be promising in maintaining remission or preemptively eradicating persistent measurable residual disease, mainly in patients ineligible for allogeneic hematopoietic stem cell transplant (HSCT). Alternatively, neoantigen-specific donor lymphocyte infusion derived from healthy donors and targeting NPM1-mutated protein to selectively elicit graft-versus-leukemia effect may represent an attractive option in subjects experiencing post-HSCT relapse. Future studies are warranted to further investigate dynamics of NPM1-mutated-specific immunity and explore whether novel individualized immunotherapies may have potential clinical utility in NPM1-mutated AML patients.

Inflammatory Microenvironment and Specific T Cells in Myeloproliferative Neoplasms: Immunopathogenesis and Novel Immunotherapies.

The Philadelphia-negative myeloproliferative neoplasms (MPNs) are malignancies of the hematopoietic stem cell (HSC) arising as a consequence of clonal proliferation driven by somatically acquired driver mutations in discrete genes (JAK2, CALR, MPL). In recent years, along with the advances in molecular characterization, the role of immune dysregulation has been achieving increasing relevance in the pathogenesis and evolution of MPNs. In particular, a growing number of studies have shown that MPNs are often associated with detrimental cytokine milieu, expansion of the monocyte/macrophage compartment and myeloid-derived suppressor cells, as well as altered functions of T cells, dendritic cells and NK cells. Moreover, akin to solid tumors and other hematological malignancies, MPNs are able to evade T cell immune surveillance by engaging the PD-1/PD-L1 axis, whose pharmacological blockade with checkpoint inhibitors can successfully restore effective antitumor responses. A further interesting cue is provided by the recent discovery of the high immunogenic potential of JAK2V617F and CALR exon 9 mutations, that could be harnessed as intriguing targets for innovative adoptive immunotherapies. This review focuses on the recent insights in the immunological dysfunctions contributing to the pathogenesis of MPNs and outlines the potential impact of related immunotherapeutic approaches.

Energy metabolism and cell motility defect in NK-cells from patients with hepatocellular carcinoma.

Functional rescue of NK-cells in solid tumors represents a central aim for new immunotherapeutic strategies. We have conducted a genomic, phenotypic and functional analysis of circulating NK-cells from patients with HCV-related liver cirrhosis and hepatocellular carcinoma. NK-cells were sorted from patients with HCC or liver cirrhosis and from healthy donors. Comparative mRNA gene expression profiling by whole-human-genome microarrays of sorted NK-cells was followed by phenotypic and functional characterization. To further identify possible mediators of NK-cell dysfunction, an in vitro model using media conditioned with patients' and controls' plasma was set up. Metabolic and cell motility defects were identified at the genomic level. Dysregulated gene expression profile has been translated into reduced cytokine production and degranulation despite a prevalent phenotype of terminally differentiated NK-cells. NKG2D-downregulation, high SMAD2 phosphorylation and other phenotypic and molecular alterations suggested TGF-ß as possible mediator of this dysfunction. Blocking TGF-ß could partially restore functional defects of NK-cells from healthy donors, exposed to TGF-ß rich HCC patients' plasma, suggesting that TGF-ß among other molecules may represent a suitable target for immunotherapeutic intervention aimed at NK-cell functional restoration. By an unbiased approach, we have identified energy metabolism and cell motility defects of circulating NK-cells as main mechanisms responsible for functional NK-cell impairment in patients with hepatocellular carcinoma. This opens the way to test different approaches to restore NK-cell response in these patients.

Two human menopausal gonadotrophin (hMG) preparations display different early signaling in vitro.

Commercial hMG drugs are marketed for the treatment of infertility and consist of highly purified hormones acting on receptors expressed in target gonadal cells. Menopur® and Meriofert® are combined preparation of FSH and hCG and are compared in vitro herein. To this purpose, the molecular composition of the two drugs was analyzed by immunoassay. The formation of FSH receptor and LH/hCG receptor (FSHR; LHCGR) heteromer, intracellular Ca2+ and cAMP activation, ß-arrestin 2 recruitment and the synthesis of progesterone and estradiol were evaluated in transfected HEK293 and human primary granulosa lutein cells treated by drugs administered within the pg-mg/ml concentration range. Molecular characterization revealed that Meriofert® has a higher FSH:hCG ratio than Menopur® which, in turn, displays the presence of LH molecules. While both drugs induced similar FSHR-LHCGR heteromeric formations and intracellular Ca2+ increase, Meriofert® had a higher potency than Menopur® in inducing a cAMP increase. Moreover, Meriofert® revealed a higher potency than Menopur® in recruiting ß-arrestin 2, likely due to different FSH content modulating the tridimensional structure of FSHR-LHCGR-ß-arrestin 2 complexes, as evidenced by a decrease in bioluminescence resonance energy transfer signal. This drug-specific activation of intracellular signaling pathways is consistent with the molecular composition of these preparations and impacts downstream progesterone and estradiol production, with Menopur® more potent than Meriofert® in inducing the synthesis of both the steroids. These findings are suggestive of distinct in-vivo activities of these preparations, but require cautious interpretation and further validation from clinical studies.