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The treatment landscape of chronic lymphocytic leukemia (CLL), the most frequent leukemia in adults, is constantly changing. CLL patients can be divided into three risk categories, based on their IGHV mutational status and the occurrence of TP53 disruption and/or complex karyotype. For the first-line treatment of low- and intermediate-risk CLL, both the BCL2 inhibitor venetoclax plus obinutuzumab and the second generation BTK inhibitors (BTKi), namely acalabrutinib and zanubrutinib, are valuable and effective options. Conversely, venetoclax-based fixed duration therapies have not shown remarkable results in high-risk CLL patients, while continuous treatment with acalabrutinib and zanubrutinib displayed favorable outcomes, similar to those obtained in TP53 wild-type patients. The development of acquired resistance to pathway inhibitors is still a clinical challenge, and the optimal treatment sequencing of relapsed/refractory CLL is not completely established. Covalent BTKi-refractory patients should be treated with venetoclax plus rituximab, whereas venetoclax-refractory CLL may be treated with second generation BTKi in the case of early relapse, while venetoclax plus rituximab might be used if late relapse has occurred. On these grounds, here we provide an overview of the current state-of-the-art therapeutic algorithms for treatment-naïve patients, as well as for relapsed/refractory disease.

Abstract Artificial Intelligence has the potential to reshape the landscape of clinical trials through innovative applications, with a notable advancement being the emergence of synthetic patient generation. This process involves simulating cohorts of virtual patients that can either replace or supplement real individuals within trial settings. By leveraging synthetic patients, it becomes possible to eliminate the need for obtaining patient consent and creating control groups that mimic patients in active treatment arms. This method not only streamlines trial processes, reducing time and costs but also fortifies the protection of sensitive participant data. Furthermore, integrating synthetic patients amplifies trial efficiency by expanding the sample size. These straightforward and cost‐effective methods also enable the development of personalized subject‐specific models, enabling predictions of patient responses to interventions. Synthetic data holds great promise for generating real‐world evidence in clinical trials while upholding rigorous confidentiality standards throughout the process. Therefore, this study aims to demonstrate the applicability and performance of these methods in the context of onco‐hematological research, breaking through the theoretical and practical barriers associated with the implementation of artificial intelligence in medical trials.

Anemia is common in cancer patients and impacts on quality of life and prognosis. It is typically multifactorial, often involving different pathophysiological mechanisms, making treatment a difficult task. In patients undergoing active anticancer treatments like chemotherapy, decreased red blood cell (RBC) production due to myelosuppression generally predominates, but absolute or functional iron deficiency frequently coexists. Current treatments for chemotherapy-related anemia include blood transfusions, erythropoiesis-stimulating agents, and iron supplementation. Each option has limitations, and there is an urgent need for novel approaches. After decades of relative immobilism, several promising anti-anemic drugs are now entering the clinical scenario. Emerging novel classes of anti-anemic drugs recently introduced or in development for other types of anemia include activin receptor ligand traps, hypoxia-inducible factor-prolyl hydroxylase inhibitors, and hepcidin antagonists. Here, we discuss their possible role in the treatment of anemia observed in patients receiving anticancer therapies.

Introduction: In several fields, the process of fusing multiple two-dimensional (2D) closed lines is an important step. For instance, this is fundamental in histology and oncology in general. The treatment of a tumor consists of numerous steps and activities. Among them, segmenting the cancer area, that is, the correct identification of its spatial location by the segmentation technique, is one of the most important and at the same time complex and delicate steps. The difficulty in deriving reliable segmentations stems from the lack of a standard for identifying the edges and surrounding tissues of the tumor area. For this reason, the entire process is affected by considerable subjectivity. Given a tumor image, different practitioners can associate different segmentations with it, and the diagnoses produced may differ. Moreover, experimental data show that the analysis of the same area by the same physician at two separate timepoints may result in different lines being produced. Accordingly, it is challenging to establish which contour line is the ground truth.Methods: Starting from multiple segmentations related to the same tumor, statistical metrics and computational procedures could be exploited to combine them for determining the most reliable contour line. In particular, numerous algorithms have been developed over time for this procedure, but none of them is validated yet. Accordingly, in this field, there is no ground truth, and research is still active.Results: In this work, we developed the Two-Dimensional Segmentation Fusion Tool (TDSFT), a user-friendly tool distributed as a free-to-use standalone application for MAC, Linux, and Windows, which offers a simple and extensible interface where numerous algorithms are proposed to “compute the mean” (i.e., the process to fuse, combine, and “average”) multiple 2D lines.Conclusions: The TDSFT can support medical specialists, but it can also be used in other fields where it is required to combine 2D close lines. In addition, the TDSFT is designed to be easily extended with new algorithms thanks to a dedicated graphical interface for configuring new parameters. The TDSFT can be downloaded from the following link: https://sourceforge.net/p/tdsft.

Chronic lymphocytic leukemia (CLL), is a hematologic malignancy characterized by the uncontrolled proliferation of mature B lymphocytes. CLL is the most prevalent leukemia in Western countries. Its presentation can range from asymptomatic with the incidental finding of absolute lymphocytosis on a routine blood test, to symptomatic disease requiring immediate intervention. Prognosis of the disease is defined by the presence or absence of specific mutations such as TP53, chromosomal abnormalities such as del(17p), a type of IGHV mutational status, and elevation of B2M and LDH. Treatment of CLL in the United States and Europe has evolved over the recent years thanks to the development of targeted therapies. The standard of care has shifted from traditional chemoimmunotherapy approaches to targeted therapies including Bruton tyrosine kinase inhibitors (BTKis) and BCL2 inhibitors, administered either as monotherapy or in combination with CD20 monoclonal antibodies. Several clinical trials have also recently evaluated combinations of BTKi and venetoclax and showed the combination to be well tolerated and able to induce deep remissions. Targeted therapies have a good safety profile overall; however, they also have unique toxicities that are important to recognize. Diarrhea, fatigue, arthralgia, infections, cytopenias, bleeding, and cardiovascular toxicities (including atrial fibrillation, ventricular arrhythmias, and hypertension) are the adverse events (AEs) commonly associated with BTKis. Initiation of therapy with venetoclax requires close monitoring because of the risk for tumor lysis syndrome associated with this agent, particularly in patients with a high disease burden. Development of newer target therapies is ongoing and the therapeutic landscape in CLL is expanding rapidly.

Abstract Half of the myelodysplastic syndromes (MDS) have normal karyotype by conventional banding analysis. The percentage of true normal karyotype cases can be reduced by 20–30% with the complementary application of genomic microarrays. We here present a multicenter collaborative study of 163 MDS cases with a normal karyotype (≥10 metaphases) at diagnosis. All cases were analyzed with the ThermoFisher® microarray (either SNP 6.0 or CytoScan HD) for the identification of both copy number alteration(CNA) and regions of homozygosity (ROH). Our series supports that 25 Mb cut‐off as having the most prognostic impact, even after adjustment by IPSS‐R. This study highlights the importance of microarrays in MDS patients, to detect CNAs and especially to detect acquired ROH which has demonstrated a high prognostic impact.

Abstract Background In older patients with acute myeloid leukemia (AML), the definition of fitness, prognosis, and risk of death represents an open question. Methods In the present study, we tested the impact on survival of disease‐ and patient‐related parameters in a large cohort of elderly AML patients homogeneously assigned to treatment with hypomethylating agents (HMAs). Results In 131 patients with a median age of 76 years, we confirmed that early response (

In recent decades, phenomenological methods known as Recognition of Earthquake-Prone Areas (REPA) were set up for identifying potential sites of powerful earthquakes. The information on potential earthquake sources provided by the REPA method is an essential part of seismic hazard assessment methodology. For the first time, we have combined global-scale information on the geographic occurrence of geofluids with global-scale information on earthquake occurrence, heat flow distribution, and S-wave dispersion, to gain insights into the evolution of local stress-strain fields. We focused on areas characterized by the occurrence of thermal waters and/or by the release of deep-seated gases, as traced by the isotope composition of associated helium. We noticed that the geographic distribution of these geofluids could serve as an indirect indicator of crustal permeability anomalies generated by crustal deformation procedures. This study proposes adding geofluids to the list of fundamental geological parameters to be considered in hazard assessment research.

Most of the time, the deep analysis of a biological sample requires the acquisition of images at different time points, using different modalities and/or different stainings. This information gives morphological, functional, and physiological insights, but the acquired images must be aligned to be able to proceed with the co-localisation analysis. Practically speaking, according to Aristotle’s principle, “The whole is greater than the sum of its parts”, multi-modal image registration is a challenging task that involves fusing complementary signals. In the past few years, several methods for image registration have been described in the literature, but unfortunately, there is not one method that works for all applications. In addition, there is currently no user-friendly solution for aligning images that does not require any computer skills. In this work, DS4H Image Alignment (DS4H-IA), an open-source ImageJ/Fiji plugin for aligning multimodality, immunohistochemistry (IHC), and/or immunofluorescence (IF) 2D microscopy images, designed with the goal of being extremely easy to use, is described. All of the available solutions for aligning 2D microscopy images have also been revised. The DS4H-IA source code; standalone applications for MAC, Linux, and Windows; video tutorials; manual documentation; and sample datasets are publicly available.

Extracellular vesicles (EVs) act as molecular mediators in the tumor microenvironment, by shuttling information contained within malignant cells and functioning as regulators of the immune system. Circular (circ)RNAs are characterized by a closed loop-like structure that makes them more stable in the extracellular milieu and suitable to be packaged inside EVs. circPVT1 (hsa_circ_0001821) showed an oncogenic role in several cancer types and immunosuppressive properties in myeloid and lymphoid cell subsets. In this study, we characterized EVs from acute myeloid leukemia (AML) patients in terms of size, concentrations, surface markers and circPVT1 cargo. We showed that circPVT1 is overexpressed by primary blast cells from newly-diagnosed AML patients compared with hematopoietic stem-progenitor cells and is released as cell-free RNA in the plasma. We isolated EVs from the plasma of AML patients and healthy subjects by size exclusion chromatography and characterized them by nanoparticle tracking analysis. EVs from patients’ plasma are larger compared with those from healthy subjects and their surface profile is characterized by higher levels of the leukemic cell markers CD133, CD105, CD49e and other immune-related epitopes, with differences according to AML molecular profile. Moreover, digital PCR analysis revealed that circPVT1 is more abundant inside EVs from the plasma of AML patients compared with healthy subjects. Our findings provide new insights on the features and content of AML EVs and suggest a role of circPVT1 in the crosstalk between AML cells and the tumor microenvironment.

Background and purpose: In this study we want to evaluate the efficacy of yoga practice on dysfunctional stress, inflammation and QOL in breast cancer patients undergoing adjuvant radiotherapy. Patients and methods: Patients with stage 0 to III breast cancer were recruited before starting radiotherapy (XRT) and were randomly assigned to yoga group (YG) two times a week during XRT or control group (CG). Self-report measures of QOL, fatigue and sleep quality, and blood samples were collected at day 1 of treatment, day 15, end of treatment and 1, 3 and 6 months later. Cortisol blood level, IL6, IL10, IL1RA, TNFα and lymphocyte-to-monocyte ratio were analyzed as measures of dysfunctional stress and inflammation. Results: Patients started XRT and yoga classes in October 2019. Due to COVID-19 pandemic we closed the enrollment in March 2020. We analysed 24 patients, 12 YG and 12 CG. The analysis of blood cortisol levels revealed an interaction (p = 0.04) between yoga practice and time, in particular YG had lower cortisol levels at the end of XRT respect to CG (p-adj = 0.02). The analysis of IL-1RA revealed an interaction effect (p = 0.04) suggesting differences between groups at some time points that post-hoc tests were not able to detect. Conclusions: To our knowledge, this is the first study to evaluate the effects of yoga in a cancer population studying inflammation markers, cortisol trend and QOL during and until 6 months after XRT. This study suggests that yoga practice is able to reduce stress and inflammation levels over time. Besides including a larger number of patients to increase the power, future studies should consider other inflammatory or pro inflammatory factors and long-term yoga program to gain more evidence on yoga practice benefits.

High-risk relapsed/refractory adult Philadelphia-negative (Ph−) B-cell acute lymphoblastic leukemia (B-ALL) is a great challenge due to limited possibilities to achieve and maintain a complete response. This also applies to cases with extramedullary (EM) involvement that have poor outcomes and no accepted standard therapeutic approaches. The incidence of EM localization in relapsed/refractory B-ALL is poorly investigated: data on patients treated with blinatumomab reported a 40% rate. Some responses were reported in EM patients with relapsed/refractory B-ALL treated with inotuzumab ozogamicin or CAR-T. However, molecular mechanisms of response or refractoriness are usually investigated neither at the medullary nor at EM sites. In the complex scenario of pluri-relapsed/refractory B-ALL patients, new target therapies are needed. Our analysis started with the case of an adult pluri-relapsed Ph− B-ALL patient, poorly sensitive to inotuzumab ozogamicin, donor lymphocyte infusions, and blinatumomab in EM disease, who achieved a durable/complete response after treatment with the BCL2-inhibitor venetoclax. The molecular characterization of medullary and EM samples revealed a tyrosine kinase domain JAK1 mutation in the bone marrow and EM samples at relapse. By comparing the expression level of BCL2- and JAK/STAT pathway-related genes between the patient samples, 136 adult JAK1wt B-ALL, and 15 healthy controls, we identified differentially expressed genes, including LIFR, MTOR, SOCS1/2, and BCL2/BCL2L1, that are variably modulated at diverse time points and might explain the prolonged response to venetoclax (particularly in the EM site, which was only partially affected by previous therapies). Our results suggest that the deep molecular characterization of both medullary and EM samples is fundamental to identifying effective and personalized targeted therapies.

BackgroundAccording to Europe’s Beating Cancer Plan, the number of cancer survivors is growing every year and is now estimated at over 12 million in Europe. A main objective of the European Commission is to ensure that cancer survivors can enjoy a high quality of life, underlining the role of digital technology and eHealth apps and tools to achieve this. ObjectiveThe main objective of this study is the development of a user-centered artificial intelligence system to facilitate the input and integration of patient-related biopsychosocial data to improve posttreatment quality of life, well-being, and health outcomes and examine the feasibility of this digitally assisted workflow in a real-life setting in patients with colorectal cancer and acute myeloid leukemia. MethodsA total of 60 patients with colorectal cancer and 30 patients with acute myeloid leukemia will be recruited from 2 clinical centers: Universitätsmedizin der Johannes Gutenberg-Universität Mainz (Mainz, Germany) and IRCCS Istituto Romagnolo per lo Studio dei Tumori “Dino Amadori” (IRST, Italy). Psychosocial data (eg, emotional distress, fatigue, quality of life, subjective well-being, sleep problems, and appetite loss) will be collected by questionnaires via a smartphone app, and physiological data (eg, heart rate, skin temperature, and movement through step count) will be collected by a customizable smart wrist-worn sensor device. Each patient will be assessed every 2 weeks over their 3-month participation in the ONCORELIEF study. Inclusion criteria include patients with the diagnosis of acute myeloid leukemia or colorectal cancer, adult patients aged 18 years and older, life expectancy greater than 12 months, Eastern Cooperative Oncology Group performance status ≤2, and patients who have a smartphone and agree to use it for the purpose of the study. Exclusion criteria include patients with a reduced cognitive function (such as dementia) or technological illiteracy and other known active malignant neoplastic diseases (patients with a medical history of treated neoplastic disease are included). ResultsThe pilot study started on September 1, 2022. As of January 2023, we enrolled 33 patients with colorectal cancer and 7 patients with acute myeloid leukemia. As of January 2023, we have not yet started the data analysis. We expect to get all data in June 2023 and expect the results to be published in the second semester of 2023. ConclusionsWeb-based and mobile apps use methods from mathematical decision support and artificial intelligence through a closed-loop workflow that connects health professionals and patients. The ONCORELIEF system has the potential of continuously identifying, collecting, and processing data from diverse patient dimensions to offer health care recommendations, support patients with cancer to address their unmet needs, and optimize survivorship care. Trial RegistrationGerman Clinical Trials Register (DRKS) 00027808; https://drks.de/search/en/trial/DRKS00027808 International Registered Report Identifier (IRRID)DERR1-10.2196/45475

ETV6::ABL1 rearranged neoplasms are rare hematological diseases. To date, about 80 cases have been reported, including myeloid and lymphoid leukemias. The ETV6 gene codes for an ETS family transcription factor and several fusion partners have been described. When translocated, ETV6 causes the constitutive activation of the partner genes. Here, we report the case of a 54-year-old woman with a cryptic insertion of the 3′ region of ABL1 in the ETV6 gene. The patient was first diagnosed with idiopathic hypereosinophilic syndrome, according to the clinical history, conventional cytogenetics, standard molecular analyses and pathologist description. Next generation sequencing of diagnosis samples unexpectedly detected both ETV6::ABL1 type A and B fusion transcripts, which were then confirmed by FISH. The diagnosis was Myeloid/Lymphoid neoplasm with ETV6::ABL1 fusion, and the patient received imatinib mesylate treatment. In a follow-up after more than one year, the patient still maintained the molecular and complete hematological responses. This case highlights the importance of timely and proper diagnostics and prompt tyrosine kinase inhibitor treatment.

Abstract The fms-like tyrosine kinase 3 (FLT3) inhibitor gilteritinib is indicated for relapsed or refractory (R/R) FLT3-mutated acute myeloid leukemia (AML), based on its observed superior response and survival outcomes compared with salvage chemotherapy (SC). Frontline use of FLT3 tyrosine kinase inhibitors (TKIs) midostaurin and sorafenib may contribute to cross-resistance to single-agent gilteritinib in the R/R AML setting but has not been well characterized. To clarify the potential clinical impact of prior TKI use, we retrospectively compared clinical outcomes in patients with R/R FLT3-mutated AML in the CHRYSALIS and ADMIRAL trials who received prior midostaurin or sorafenib against those without prior FLT3 TKI exposure. Similarly high rates of composite complete remission (CRc) were observed in patients who received a FLT3 TKI before gilteritinib (CHRYSALIS, 42%; ADMIRAL, 52%) and those without prior FLT3 TKI therapy (CHRYSALIS, 43%; ADMIRAL, 55%). Among patients who received a prior FLT3 TKI in ADMIRAL, a higher CRc rate (52%) and trend toward longer median overall survival was observed in the gilteritinib arm versus the SC arm (CRc = 20%; overall survival, 5.1 months; HR = 0.602; 95% CI: 0.299, 1.210). Remission duration was shorter with prior FLT3 TKI exposure. These findings support gilteritinib for FLT3-mutated R/R AML after prior sorafenib or midostaurin.

Abstract Cell division cycle 20 homologue (CDC20) is a well-known regulator of cell cycle, as it controls the correct segregation of chromosomes during mitosis. Many studies have focused on the biological role of CDC20 in cancer development, as alterations of its functionality have been linked to genomic instability and evidence demonstrated that high CDC20 expression levels are associated with poor overall survival in solid cancers. More recently, novel CDC20 functions have been demonstrated or suggested, including the regulation of apoptosis and stemness properties and a correlation with immune cell infiltration. Here, we here summarize and discuss the role of CDC20 inside and outside mitosis, starting from its network of interacting proteins. In the last years, CDC20 has also attracted more interest in the blood cancer field, being overexpressed and showing an association with prognosis both in myeloid and lymphoid malignancies. Preclinical findings showed that selective CDC20 and APC/CCDC20/APC/CCDH1 inhibitors, namely Apcin and proTAME, are effective against lymphoma and multiple myeloma cells, resulting in mitotic arrest and apoptosis and synergizing with clinically-relevant drugs. The evidence and hypothesis presented in this review provide the input for further biological and chemical studies aiming to dissect novel potential CDC20 roles and targeting strategies in hematological malignancies.

Germ cell tumors (GCTs) represent a heterogeneous neoplasm family affecting gonads and rarely occurring in extragonadal areas. Most of patients have a good prognosis, often even in the presence of metastatic disease; however, in almost 15% of cases, tumor relapse and platinum resistance are the main challenges. Thus, novel treatment strategies with both improved antineoplastic activity and minor treatment-related adverse events compared with platinum are really expected. In this context, the development and the high activity demonstrated by immune checkpoint inhibitors in solid tumors and, subsequently, the interesting results obtained from the use of chimeric antigen receptor (CAR-) T cell therapy in hematological tumors, have stimulated research in this direction also in GCTs. In this article, we will analyze the molecular mechanisms underlying the immune action in the development of GCTs, and we will report the data from the studies that tested the new immunotherapeutic approaches in these neoplasms.

IntroductionNeuroendocrine neoplasms (NENs) are a rare group of tumors exceptionally heterogeneous, with clinical presentation ranging from well differentiated more indolent tumors to poorly differentiated very aggressive forms. Both are often diagnosed after the metastatic spread and require appropriate medical treatment. A high priority need in the management of this disease is the identification of effective therapeutic strategies for advanced and metastatic patients. The recent TALENT trial demonstrated the efficacy of lenvatinib, a multi-tyrosine kinase inhibitor, in patients with gastroenteropancreatic neuroendocrine tumors (GEP-NETs) with no other treatment indication. Further development of this drug in advanced NETs is warranted.MethodsWe investigated potential clinical and molecular determinants of lenvatinib response in human primary cultures derived from patients with GEP-NET of different grades and sites of origin. We correlated response to treatment with patient clinical characteristics, with the mutational status of 161-cancer associated genes and with the expression levels of MKI-related genes.ResultsLenvatinib exerted a significant antitumor activity in primary GEP-NET cells, with median survival inhibitions similar or higher than those of standard frontline treatments. Of the 11 primary cultures analyzed in our case series, 6 were classified as responder showing a significant survival inhibition, and 5 as non-responder. We observed that the overexpression of HRAS in the original tumor tissue compared to the matched healthy tissue significantly correlated with responsiveness of primary cells to lenvatinib (p=.048). All 5 non-responder cultures showed normal HRAS expression, while of the 6 responder cultures, 4 had HRAS overexpression. Overexpression of HRAS was not associated with gene mutation. None of the other evaluated clinical variables (grade, Ki67, site of origin and syndromic disease) or molecular markers correlated with response.DiscussionLenvatinib appears to be a highly effective drug for the treatment of NETs. The evaluation of HRAS expression in the tumor tissue might improve patient selection and optimize therapeutic outcome.

Abstract In adult patients, acute lymphoblastic leukemia (ALL) is a rare hematological cancer with a cure rate below 50% and frequent relapses. With traditional therapies, patients with relapsed or refractory (R/R) ALL have a survival that may be measured in months; in these patients, inotuzumab ozogamicin (IO) is an effective therapy. IO was linked to increased risk of veno‐occlusive disease/sinusoid obstruction syndrome (VOD/SOS), liver injury, and various grade of liver‐related complications during clinical trials and real‐life settings; however, hepatologic monitoring protocol is not established in this population. In our institution, 21 patients who received IO (median of 6 doses of IO administered) for R/R ALL were prospectively followed for hepatologic surveillance, including clinical evaluation, ultrasonography, and liver stiffness measurement (LSM) biochemistry. After a median follow‐up of 17.2 months, two SOS events were reported (both after allogeneic transplant) as IO potentially related clinically relevant adverse event. Mild alterations were reported in almost the totality of patients and moderate‐severe liver biochemical alterations in a quarter of patients. Within biochemicals value, AST and ALP showed an augment related to IO administration. LSM linearly augmented for each IO course administered. Baseline LSM was related to liver‐related changes, especially with the severity of portal hypertension (PH)‐related complications. Pre‐transplant LSM was higher in patients receiving IO when compared with a control cohort. PH‐related complications were discovered in nearly 77% of patients, with clinically significant PH occurrence and development of ascites in 38% and 14%, respectively. This prospective experience constitutes the rationale to design a hepatologic monitoring program in patients receiving IO. LSM may be of pivotal importance in this program, constituting a rapid and effective screening that quantitatively correlates with liver alterations.

To improve the success rate of current preclinical drug trials, there is a growing need for more complex and relevant models that can help predict clinical resistance to anticancer agents. Here, we present a three‐dimensional (3D) technology, based on biomimetic collagen scaffolds, that enables the modeling of the tumor hypoxic state and the prediction of in vivo chemotherapy responses in terms of efficacy, molecular alterations, and emergence of resistance mechanisms. The human breast cancer cell lines MDA‐MB‐231 (triple negative) and MCF‐7 (luminal A) were treated with scaling doses of doxorubicin in monolayer cultures, 3D collagen scaffolds, or orthotopically transplanted murine models. Lineage‐specific resistance mechanisms were revealed by the 3D tumor model. Reduced drug uptake, increased drug efflux, and drug lysosomal confinement were observed in triple‐negative MDA‐MB‐231 cells. In luminal A MCF‐7 cells, the selection of a drug‐resistant subline from parental cells with deregulation of p53 pathways occurred. These cells were demonstrated to be insensitive to DNA damage. Transcriptome analysis was carried out to identify differentially expressed genes (DEGs) in treated cells. DEG evaluation in breast cancer patients demonstrated their potential role as predictive biomarkers. High expression of the transporter associated with antigen processing 1 (TAP1) and the tumor protein p53‐inducible protein 3 (TP53I3) was associated with shorter relapse in patients affected by ER+ breast tumor. Likewise, the same clinical outcome was associated with high expression of the lysosomal‐associated membrane protein 1 LAMP1 in triple‐negative breast cancer. Hypoxia inhibition by resveratrol treatment was found to partially re‐sensitize cells to doxorubicin treatment. Our model might improve preclinical in vitro analysis for the translation of anticancer compounds as it provides: (a) more accurate data on drug efficacy and (b) enhanced understanding of resistance mechanisms and molecular drivers.

Abstract The members of the Poly(ADP‐ribose) polymerase (PARP) superfamily are involved in several biological processes and, in particular, in the DNA damage response (DDR). The most studied members, PARP1, PARP2 and PARP3, act as sensors of DNA damages, in order to activate different intracellular repair pathways, including single-strand repair, homologous recombination, conventional and alternative non-homologous end joining. This review recapitulates the functional role of PARPs in the DDR pathways, also in relationship with the cell cycle phases, which drives our knowledge of the mechanisms of action of PARP inhibitors (PARPi), encompassing inhibition of single-strand breaks and base excision repair, PARP trapping and sensitization to antileukemia immune responses. Several studies have demonstrated a preclinical activity of the current available PARPi, olaparib, rucaparib, niraparib, veliparib and talazoparib, as single agent and/or in combination with cytotoxic, hypomethylating or targeted drugs in acute leukemia, thus encouraging the development of clinical trials. We here summarize the most recent preclinical and clinical findings and discuss the synthetic lethal interactions of PARPi in acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). Despite the low frequency of genomic alterations of PARP and other DDR-related genes in acute leukemia, selective vulnerabilities have been reported in several disease subgroups, along with a “BRCAness phenotype.” AML carrying the RUNX1-RUNX1T1 or PML-RARA fusion genes or mutations in signaling genes (FLT3-ITD in combination with TET2 or TET2 and DNMT3A deficiency), cohesin complex members (STAG2), TP53 and BCOR as co-occurring lesions, IDH1/2 and ALL cases expressing the TCF3-HLF chimera or TET1 was highly sensitive to PARPi in preclinical studies. These data, along with the warning coming from the observation of cases of therapy-related myeloid malignancies among patients receiving PARPi for solid tumors treatment, indicate that PARPi represents a promising strategy in a personalized medicine setting. The characterization of the clonal and subclonal genetic background and of the DDR functionality is crucial to select acute leukemia patients that will likely benefit of PARPi-based therapeutic regimens.

Comet assay provides an easy solution to estimate DNA damage in single cells through microscopy assessment. It is widely used in the analysis of genotoxic damages induced by radiotherapy or chemotherapeutic agents. DNA damage is quantified at the single-cell level by computing the displacement between the genetic material within the nucleus, typically called “comet head”, and the genetic material in the surrounding part of the cell, considered as the “comet tail”. Today, the number of works based on Comet Assay analyses is really impressive. In this work, besides revising the solutions available to obtain reproducible and reliable quantitative data, we developed an easy-to-use tool named CometAnalyser. It is designed for the analysis of both fluorescent and silver-stained wide-field microscopy images and allows to automatically segment and classify the comets, besides extracting Tail Moment and several other intensity/morphological features for performing statistical analysis. CometAnalyser is an open-source deep-learning tool. It works with Windows, Macintosh, and UNIX-based systems. Source code, standalone versions, user manual, sample images, video tutorial and further documentation are freely available at: https://sourceforge.net/p/cometanalyser.

Bottled mineral waters originate from groundwater aquifers, their chemical composition being initially determined by geochemical water-rock interaction processes. The waters used for bottling originate from different parts of the hydrological cycle and have a unique hydro-geochemical fingerprint. As water moves through the water cycle, the isotopic composition of oxygen and hydrogen in the water molecule may change. Determining 18O and 2H can help to characterize the source of bottled water and the natural conditions of the parent water body, of the recharge area, and the influence of various processes during infiltration and water flow within the water body. Usually, the chemical composition is reported on the label of bottled waters, while stable isotopes data are often unreported and are sometimes available from scientific publications. Bottled waters from selected sites of Europe where chemical and stable isotopic composition were available have been considered and accompanying data reinterpreted. The available data have been reinterpreted by obtaining results comparable, within limitations, to traditional sampling and analytical procedures, demonstrating the usefulness of the adopted methodology in emergency cases. Therefore, the utilization of isotopic values of bottled waters should be limited to the observation of general trends in isotopic composition of feeding waters, while more local studies are advised for a better understanding of the hydro-geological circuits.

Genome wide association studies in cancer are used to understand the heritable genetic contribution to disease risk. Here, the authors perform a genome wide association study in European patients with acute myeloid leukemia and identify loci associated with risk of developing the disease.

Combining phenotypical and molecular characterization of rare cells is challenging due to their scarcity and difficult handling. In oncology, circulating tumor cells (CTCs) are considered among the most important rare cell populations. Their phenotypic and molecular characterization is necessary to define the molecular mechanisms underlying their metastatic potential. Several approaches that require cell fixation make difficult downstream molecular investigations on RNA. Conversely, the DEPArray technology allows phenotypic analysis and handling of both fixed and unfixed cells, enabling a wider range of applications. Here, we describe an experimental workflow that allows the transcriptomic investigation of single and pooled OE33 cells undergone to DEPArray analysis and recovery. In addition, cells were tested at different conditions (unfixed, CellSearch fixative (CSF)- and ethanol (EtOH)-fixed cells). In a forward-looking perspective, this workflow will pave the way for novel strategies to characterize gene expression profiles of rare cells, both single-cell and low-resolution input.

Non-small-cell lung cancer (NSCLC) is the primary cause of cancer-related death. Gene rearrangements involving the anaplastic lymphoma kinase (ALK) tyrosine kinase identify a clinical and molecular subset of NSCLC patients, who benefit from the monotherapy with ALK tyrosine kinase inhibitors. Nonetheless, responsiveness to TKIs and prognosis of these patients are influenced by several factors, including resistance mechanisms and mutations affecting genes involved in key molecular pathways of cancer cells. In a cohort of 98 NSCLC patients with ALK gene rearrangements, we investigated the role of Tumor Protein (TP53) gene mutations in predicting patients prognosis. TP53 mutations were evaluated in relation to disease control rate (DCR), objective response rate (ORR), progression-free survival (PFS) and overall survival (OS).Results: In patients with available clinical and TP53 mutation information, we found that 13 patients (20.3%) were affected by TP53 mutations. Considered together, even though showing a trend, TP53 mutations were not associated with PFS and OS. Considering the different TP53 mutations by functionality in terms of disruptive and non-disruptive mutations, we observed that TP53 non-disruptive mutations were able to predict worse OS in the overall case series. Moreover, a worse PFS was seen in the subgroup of patients with TP53 non-disruptive mutation, in first-, second-, and third line of treatment. Our results show that mutations affecting TP53 gene, especially non-disruptive mutations, are able to affect prognosis of ALK-rearranged NSCLC patients.

IntroductionIn immunocompromised patients, SARS-CoV-2 mRNA vaccine has been used in Italy from the beginning of the vaccination campaign, but several studies have shown that the serological response of onco-hematological patients was reduced compared to healthy subjects, due to the state of immunosuppression because of both underlying disease and administered therapy.MethodsWe evaluated the association of anti-SARS-CoV-2 spike IgG titers in 215 hematological patients with clinical and demographic variables to verify if it was possible to identify predictive parameters of serological response, as well as using a control group, consisting of healthy health workers of San Carlo Hospital in Potenza. Anti-SARS-CoV2 IgG titers were evaluated after 30–45 days post second dose vaccine using chemiluminescent microparticle immunoassay technology.ResultsPatients with hematological malignancies, compared with the control arm, had both a mean concentration of anti-SARS-CoV-2 IgG significantly lower and a seroconversion rate numerically lower. All chronic lymphatic leukemia patients showed levels of antibody titer below the mean concentration, also in only clinical surveillance patients. Comparing serological response in hematological malignancies, only acute leukemia patients who were off therapy had the highest seroconversion rate among the patients’ cohorts and a mean antibody concentration greater than the control arm. Patients treated with steroids and rituximab showed a lower level of anti-SARS-CoV-2 spike IgG. Differences in anti-spike IgG levels among chronic myeloid leukemia patients stratified according to tyrosine kinase inhibitor therapy and molecular response were observed, and they could have interesting implications on the evaluation of the effects of these drugs on the immune system, but having not reached statistical significance at the moment. The cohort of patients who received a stem cell transplant was very heterogeneous because it included different hematological malignancies and different types of transplant; however, a mean concentration of anti-SARS-CoV2 IgG greater than the control arm was reported. Indeed, among patients who performed a transplant for over 6 months only one had a spike IgG concentration below the cutoff.ConclusionsOur data confirm reduced serological response in hematological patients after anti-SARS-CoV-2 vaccination. However, we found a great diversity of SARS-CoV-2 antibody response according to types of pathologies and therapies.

Risk stratification in acute myeloid leukemia (AML) has been extensively improved thanks to the incorporation of recurrent cytogenomic alterations into risk stratification guidelines. However, mortality rates among fit patients assigned to low or intermediate risk groups are still high. Therefore, significant room exists for the improvement of AML prognostication. In a previous work, we presented the Stellae-123 gene expression signature, which achieved a high accuracy in the prognostication of adult patients with AML. Stellae-123 was particularly accurate to restratify patients bearing high-risk mutations, such as ASXL1, RUNX1 and TP53. The intention of the present work was to evaluate the prognostic performance of Stellae-123 in external cohorts using RNAseq technology. For this, we evaluated the signature in 3 different AML cohorts (2 adult and 1 pediatric). Our results indicate that the prognostic performance of the Stellae-123 signature is reproducible in the 3 cohorts of patients. Additionally, we evidenced that the signature was superior to the European LeukemiaNet 2017 and the pediatric clinical risk scores in the prediction of survival at most of the evaluated time points. Furthermore, integration with age substantially enhanced the accuracy of the model. In conclusion, Stellae-123 is a reproducible machine learning algorithm based on a gene expression signature with promising utility in the field of AML.

Lung cancer (LC) is the second most common neoplasm in men and the third most common in women. In the last decade, LC therapies have undergone significant improvements with the advent of immunotherapy. However, the effectiveness of the available treatments remains insufficient due to the presence of therapy-resistant cancer cells. For decades, chemotherapy and radiotherapy have dominated the treatment strategy for LC; however, relapses occur rapidly and result in poor survival. Malignant lung tumors are classified as either small- or non-small-cell lung carcinoma (SCLC and NSCLC). Despite improvements in the treatment of LC in recent decades, the benefits of surgery, radiotherapy, and chemotherapy are limited, although they have improved the prognosis of LC despite the persistent low survival rate due to distant metastasis in the late stage. The identification of novel prognostic molecular markers is crucial to understand the underlying mechanisms of LC initiation and progression. The potential role of phosphatidylinositol in tumor growth and the metastatic process has recently been suggested by some researchers. Phosphatidylinositols are lipid molecules and key players in the inositol signaling pathway that have a pivotal role in cell cycle regulation, proliferation, differentiation, membrane trafficking, and gene expression. In this review, we discuss the current understanding of phosphoinositide-specific phospholipase enzymes and their emerging roles in LC.

Adenocarcinoma of the esophagus (EAC) and gastroesophageal junction (GEJ-AC) is associated with poor prognosis, treatment resistance and limited systemic therapeutic options. To deeply understand the genomic landscape of this cancer type, and potentially identify a therapeutic target in a neoadjuvant chemotherapy non-responder 48-year-old man, we adopted a multi-omic approach. We simultaneously evaluated gene rearrangements, mutations, copy number status, microsatellite instability and tumor mutation burden. The patient displayed pathogenic mutations of the TP53 and ATM genes and variants of uncertain significance of three kinases genes (ERBB3, CSNK1A1 and RPS6KB2), along with FGFR2 and KRAS high copy number amplification. Interestingly, transcriptomic analysis revealed the Musashi-2 (MSI2)-C17orf64 fusion that has never been reported before. Rearrangements of the RNA-binding protein MSI2 with a number of partner genes have been described across solid and hematological tumors. MSI2 regulates several biological processes involved in cancer initiation, development and resistance to treatment, and deserves further investigation as a potential therapeutic target. In conclusion, our extensive genomic characterization of a gastroesophageal tumor refractory to all therapeutic approaches led to the discovery of the MSI2-C17orf64 fusion. The results underlie the importance of deep molecular analyses enabling the identification of novel patient-specific markers to be monitored during therapy or even targeted at disease evolution.

Abstract Background Pancreatic cancer (PC) is a major cause of cancer death. In an effort to improve treatment strategies and outcomes, DNA damage repair (DDR) pathways have been introduced as a new target in PC and in other cancers, through the exploitation of synthetic lethality. Furthermore, genes involved in DDR are among the major determinants of cancer susceptibility. In addition to the well-known BRCA1 and BRCA2 genes, a plethora of other targets in the same pathways are now emerging. Methods We analyzed samples from 60 patients, affected by PC and already tested for BRCA, using a panel with 24 other cancer susceptibility genes. Results We detected 8 pathogenic or likely pathogenic mutations (13.3% of samples analyzed), 4 of which were found in non-BRCA genes (2 in ATM, 1 each in PALB2 and RAD50). Furthermore, 4 pathogenic or likely pathogenic mutations were found in patients without a personal or familial history of cancer. Conclusions Our results suggest that genetic testing with a comprehensive gene panel should be perfomed in all patients with PC, in order to allow screening for PC and other gene-related cancers in all at risk family members and to assess patients’ eligibility for emerging therapeutic options.

Abstract Background Blinatumomab is a BiTE® immuno‐oncology therapy indicated for the treatment of patients with relapsed or refractory (r/r) B‐cell precursor (BCP) acute lymphoblastic leukemia (ALL). Aims To assess the efficacy and safety of blinatumomab as first salvage versus second or later salvage in patients with r/r BCP ALL. Materials & Methods Patient‐level pooled data were used for this analysis. In total, 532 adults with r/r BCP ALL treated with blinatumomab were included (first salvage, n = 165; second or later salvage, n = 367). Results Compared with patients who received blinatumomab as second or later salvage, those who received blinatumomab as first salvage had a longer median overall survival (OS; 10.4 vs. 5.7 months; HR, 1.58; 95% CI, 1.26–1.97; P < .001) and relapse‐free survival (10.1 vs. 7.3 months; HR, 1.38; 95% CI, 0.98–1.93; P = .061), and higher rates of remission (n = 89 [54%] vs. n = 150 [41%]; odds ratio, 0.59; 95% CI, 0.41–0.85; P = .005), minimal residual disease response (n = 68 [41%] vs. n = 118 [32%]), and allogeneic hematopoietic stem cell transplant (alloHSCT) realization (n = 60 [36%] vs. n = 88 [24%]), and alloHSCT in continuous remission (n = 33 [20%] vs. n = 52 (14%]). In a subgroup analysis, there was no apparent effect of prior alloHSCT on median OS in either salvage group. The safety profile of blinatumomab was generally similar between the groups; however, cytokine release syndrome, febrile neutropenia, and infection were more frequent with second or later salvage than with first salvage. Discussion In this pooled analysis, the logistic regression analyses indicated greater benefit with blinatumomab as first salvage than as second or later salvage, as evident by the longer median OS, longer median RFS, and higher rates of remission. Conclusion Overall, blinatumomab was beneficial as first salvage and as second or later salvage, but the effects were favorable as first salvage.

Abstract Background During the COVID-19 emergency, IRST IRCCS, an Italian cancer research institute and promoter of no profit clinical studies, adapted its activities and procedures as per European and national guidelines to maintain a high standard of clinical trials, uphold participant safety and guarantee the robustness and reliability of the data collected. This study presents the measures adopted by our institute with the aim of providing information that could be useful to other academic centers promoting clinical trials during the pandemic. Main text After an in-depth analysis of European and Italian guidelines and consultation and analysis of publications regarding the actions implemented by international no profit clinical trial promoters during the emergency, we monitored the way in which the institute managed clinical trials, verifying compliance with regulatory guidelines and clinical procedures, and evaluating screening and recruitment trends in studies. During the pandemic, our center activated a new clinical trial for the treatment of patients with COVID-19. A number of procedural changes in clinical trials were also authorized through notified amendments, in accordance with Italian Medicines Agency (AIFA) guidelines. Patient screening and enrolment was not interrupted in any site participating in multicenter interventional clinical trials on drugs. The institute provided clear indications about essential procedures to be followed, identifying those that could be postponed or carried out by telephone/teleconference. All external sites were monitored remotely, avoiding on-site visits. Although home-working was encouraged, the presence of staff in the central office was also guaranteed to ensure the continuity of promoter activities. Conclusions Some measures adopted by IRST could also be effective outside of the COVID-19 period, e.g. numerous activities relating to clinical trial management could be performed on a home-working basis, using suitable digital technologies. In the future, electronic medical records and shared guidelines will be essential for the correct identification and management of trial risks, including the protection of the rights and privacy of subjects taking part. Promoter supervision could be increased by implementing centralized monitoring tools to guarantee data quality. Closer collaboration between promoters and local study staff is needed to optimize trial management.