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BackgroundInfusion of second generation autologous CD19-targeted chimeric antigen receptor (CAR) T cells in patients with R/R relapsed/refractory B-cell lymphoma (BCL) is affected by inflammatory complications, such as Immune Effector Cell-Associated Neurotoxicity Syndrome (ICANS). Current literature suggests that the immune profile prior to CAR-T infusion modifies the chance to develop ICANS.MethodsThis is a monocenter prospective study on 53 patients receiving approved CAR T-cell products (29 axi-cel, 24 tisa-cel) for R/R-BCL. Clinical, biochemical, and hematological variables were analyzed at the time of pre-lymphodepletion (pre-LD). In a subset of 21 patients whose fresh peripheral blood sample was available, we performed cytofluorimetric analysis of leukocytes and extracellular vesicles (EVs). Moreover, we assessed a panel of soluble plasma biomarkers (IL-6/IL-10/GDF-15/IL-15/CXCL9/NfL) and microRNAs (miR-146a-5p, miR-21-5p, miR-126-3p, miR-150-5p) which are associated with senescence and inflammation.ResultsMultivariate analysis at the pre-LD time-point in the entire cohort (n=53) showed that a lower percentage of CD3+CD8+ lymphocytes (38.6% vs 46.8%, OR=0.937 [95% CI: 0.882-0.996], p=0.035) and higher levels of serum C-reactive protein (CRP, 4.52 mg/dl vs 1.00 mg/dl, OR=7.133 [95% CI: 1.796-28], p=0.005) are associated with ICANS. In the pre-LD samples of 21 patients, a significant increase in the percentage of CD8+CD45RA+CD57+ senescent cells (median % value: 16.50% vs 9.10%, p=0.009) and monocytic-myeloid derived suppressor cells (M-MDSC, median % value: 4.4 vs 1.8, p=0.020) was found in ICANS patients. These latter also showed increased levels of EVs carrying CD14+ and CD45+ myeloid markers, of the myeloid chemokine CXCL-9, as well of the MDSC-secreted cytokine IL-10. Notably, the serum levels of circulating neurofilament light chain, a marker of neuroaxonal injury, were positively correlated with the levels of senescent CD8+ T cells, M-MDSC, IL-10 and CXCL-9. No variation in the levels of the selected miRNAs was observed between ICANS and no-ICANS patients.DiscussionOur data support the notion that pre-CAR-T systemic inflammation is associated with ICANS. Higher proportion of senescence CD8+ T cells and M-MDSC correlate with early signs of neuroaxonal injury at pre-LD time-point, suggesting that ICANS may be the final event of a process that begins before CAR-T infusion, consequence to patient clinical history.

SummaryPatients with cancer of unknown primary (CUP) carry the double burden of an aggressive disease and reduced access to therapies. Experimental models summing up CUP features are pivotal for CUP biology investigation and drug testing. We derived two CUP cell lines (CUP#55 and #96), and corresponding patient-derived xenografts (PDXs), from ascites tumor cells. CUP cell lines and PDXs underwent histological, immune-phenotypical, molecular, and genomic characterization confirming the features of the original tumor. Genetic testing and FISH analysis identified FGFR2 amplification as therapeutic target in tumor tissues and patient-derived models. Drug-screening assays were performed to test the activity of FGFR2 targeting drug BGJ-398 (infigratinib) and the combination treatment with the MEK inhibitor trametinib, which proved to be synergic and exceptionally active, bothin vitroandin vivo. This study brings personalized therapy closer to CUP patients and paves the way to future applications of personalized medicine for metastatic patients with adverse prognosis.

Graft versus host disease (GVHD) is a major complication of allogeneic hematopoietic stem cell transplantation (HSCT). Rabbit anti-T lymphocyte globulin (ATLG) in addition to calcineurin inhibitors and antimetabolites is a suitable strategy to prevent GVHD in several transplant settings. Randomized studies already demonstrated its efficacy in terms of GVHD prevention, although the effect on relapse remains the major concern for a wider use. Tailoring of ATLG dose on host characteristics is expected to minimize its side effects (immunological reconstitution, relapse, and infections). Here, day -6 to day +15 pharmacokinetics of active ATLG serum level was first assayed in an explorative cohort of 23 patients by testing the ability of the polyclonal serum to bind antigens on human leukocytes. Significantly lower levels of serum active ATLG were found in the patients who developed GVHD (ATLG_AUCCD45: 241.52 ± 152.16 vs. 766.63 +/- 283.52 (μg*day)/ml, p = 1.46e-5). Consistent results were obtained when the ATLG binding capacity was assessed on CD3+ and CD3+/CD4+ T lymphocytes (ATLG_AUCCD3: 335.83 ± 208.15 vs. 903.54 ± 378.78 (μg*day)/ml, p = 1.92e-4; ATLG_AUCCD4: 317.75 ± 170.70 vs. 910.54 ± 353.35 (μg*day)/ml, p = 3.78e-5. Concomitantly, at pre-infusion time points, increased concentrations of CD69+ extracellular vesicles (EVs) were found in patients who developed GVHD (mean fold 9.01 ± 1.33; p = 2.12e-5). Consistent results were obtained in a validation cohort of 12 additional ATLG-treated HSCT patients. Serum CD69+ EVs were mainly represented in the nano (i.e. 100 nm in diameter) EV compartment and expressed the leukocyte marker CD45, the EV markers CD9 and CD63, and CD103, a marker of tissue-resident memory T cells. The latter are expected to set up a host pro-inflammatory cell compartment that can survive in the recipient for years after conditioning regimen and contribute to GVHD pathogenesis. In summary, high levels of CD69+ EVs are significantly correlated with an increased risk of GVHD, and they may be proposed as a tool to tailor ATLG dose for personalized GVHD prevention.

BackgroundT cells engineered to target CD19 antigen on neoplastic B cells represent the most striking example of CAR-T cell therapy. The success rate of this therapy is affected by several limitations: target antigen loss, and/or acquisition of a senescent/exhausted phenotype by CAR and non-CAR T cells.Case presentationWe report on a patient affected by refractory Diffuse Large B-cell Lymphoma who was resistant to CAR T-cell therapy and to two cycles post CAR-T of pembrolizumab (PBZ) due to the evolution into a B-cell Hodgkin-like lymphoma. Owing to the CD30 expression and the Hodgkin-like phenotype, the patient was ultimately treated with Brentuximab-Vedotin and finally underwent remission. Upon PBZ treatment, 100% of circulating CAR-T+ cells showed a persistent CD8+ senescent/exhausted phenotype, while an increase in the percentage of senescent cells was found in the non-CAR CD8+ T cells compartment.ConclusionsPBZ is not able to reinvigorate exhausted CAR+ T cells and to confer durable clinical response. We hypothesize that the phenomenon is due to the senescent phenotype of CAR+ T cells, which did not allow PBZ-induced reactivation and proliferative rescue. The phenomenon, together with the loss of CAR-T target CD19 and the shift of non-CAR CD8+ T cells towards a senescent phenotype likely contributed to set up an immune landscape with poor antitumor capacity.

Small cell neuroendocrine carcinoma is most frequently found in the lung (SCLC), but it has been also reported, albeit with a very low incidence, in the ovary. Here, we analyze a case of primary small cell carcinoma of the ovary of pulmonary type (SCCOPT), a rare and aggressive tumor with poor prognosis, whose biology and molecular features have not yet been thoroughly investigated. The patient affected by SCCOPT had a residual tumor following chemotherapy which displayed pronounced similarity with neuroendocrine tumors and lung cancer in terms of its microRNA expression profile and mTOR-downstream activation. By analyzing the metabolic markers of the neoplastic lesion, we established a likely glycolytic signature. In conclusion, this in-depth characterization of SCCOPT could be useful for future diagnoses, possibly aided by microRNA profiling, allowing clinicians to adopt the most appropriate therapeutic strategy.

Background In the last years, several efforts have been made to classify colorectal cancer (CRC) into well-defined molecular subgroups, representing the intrinsic inter-patient heterogeneity, known as Consensus Molecular Subtypes (CMSs). Methods In this work, we performed a meta-analysis of CRC patients stratified into four CMSs. We identified a negative correlation between a high level of anaplastic lymphoma kinase (ALK) expression and relapse-free survival, exclusively in CMS1 subtype. Stemming from this observation, we tested cell lines, patient-derived organoids and mice with potent ALK inhibitors, already approved for clinical use. Results ALK interception strongly inhibits cell proliferation already at nanomolar doses, specifically in CMS1 cell lines, while no effect was found in CMS2/3/4 groups. Furthermore, in vivo imaging identified a role for ALK in the dynamic formation of 3D tumor spheroids. Consistently, ALK appeares constitutively phosphorylated in CMS1, and it signals mainly through the AKT axis. Mechanistically, we found that CMS1 cells display several copies of ALKAL2 ligand and ALK-mRNAs, suggesting an autocrine loop mediated by ALKAL2 in the activation of ALK pathway, responsible for the invasive phenotype. Consequently, disruption of ALK axis mediates the pro-apoptotic action of CMS1 cell lines, both in 2D and 3D and enhanced cell-cell adhesion and e-cadherin organization. In agreement with all these findings, the ALK signature encompassing 65 genes statistically associated with worse relapse-free survival in CMS1 subtype. Finally, as a proof of concept, the efficacy of ALK inhibition was demonstrated in both patient-derived organoids and in tumor xenografts in vivo. Conclusions Collectively, these findings suggest that ALK targeting may represent an attractive therapy for CRC, and CMS classification may provide a useful tool to identify patients who could benefit from this treatment. These findings offer rationale and pharmacological strategies for the treatment of CMS1 CRC.

BACKGROUND Rhabdomyolysis is a syndrome characterized by muscle necrosis and the subsequent release of intracellular muscle constituents into the bloodstream. Although the specific cause is frequently evident from the history or from the immediate events, such as a trauma, extraordinary physical exertion, or a recent infection, sometimes there are hidden risk factors that have to be identified. For instance, individuals with sickle cell trait (SCT) have been reported to be at increased risk for rare conditions, including rhabdomyolysis. Moreover, there have been a few case reports of SARS-CoV-2 infection-related rhabdomyolysis. CASE REPORT We present a case of a patient affected by unknown SCT and admitted with SARS-CoV-2 pneumonia, who suffered non-traumatic non-exertional rhabdomyolysis leading to acute kidney injury (AKI), requiring acute hemodialysis (HD). The patients underwent 13 dialysis session, of which 12 were carried out using an HFR-Supra H dialyzer. He underwent kidney biopsy, where rhabdomyolysis injury was ascertained. No viral traces were found on kidney biopsy samples. The muscle biopsy showed the presence of an "open nucleolus" in the muscle cell, which was consistent with virus-infected cells. After 40 days in the hospital, his serum creatinine was 1.62 mg/dL and CPK and Myoglobin were 188 U/L and 168 ng/mL, respectively; therefore, the patient was discharged. CONCLUSIONS SARS-CoV-2 infection resulted in severe rhabdomyolysis with AKI requiring acute HD. Since SARS-CoV-2 infection can trigger sickle-related complications like rhabdomyolysis, the presence of SCT needs to be ascertained in African patients.

Cancers of unknown primary (CUPs) comprise a heterogeneous group of rare metastatic tumors whose primary site cannot be identified after extensive clinical–pathological investigations. CUP patients are generally treated with empirical chemotherapy and have dismal prognosis. As recently reported, CUP genome presents potentially druggable alterations for which targeted therapies could be proposed. The paucity of tumor tissue, as well as the difficult DNA testing and the lack of dedicated panels for target gene sequencing are further relevant limitations. Here, we propose that circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) could be used to identify actionable mutations in CUP patients. Blood was longitudinally collected from two CUP patients. CTCs were isolated with CELLSEARCH® and DEPArrayTM NxT and Parsortix systems, immunophenotypically characterized and used for single-cell genomic characterization with Ampli1TM kits. Circulating cell-free DNA (ccfDNA), purified from plasma at different time points, was tested for tumor mutations with a CUP-dedicated, 92-gene custom panel using SureSelect Target Enrichment technology. In parallel, FFPE tumor tissue was analyzed with three different assays: FoundationOne CDx assay, DEPArray LibPrep and OncoSeek Panel, and the SureSelect custom panel. These approaches identified the same mutations, when the gene was covered by the panel, with the exception of an insertion in APC gene. which was detected by OncoSeek and SureSelect panels but not FoundationOne. FGFR2 and CCNE1 gene amplifications were detected in single CTCs, tumor tissue, and ccfDNAs in one patient. A somatic variant in ARID1A gene (p.R1276∗) was detected in the tumor tissue and ccfDNAs. The alterations were validated by Droplet Digital PCR in all ccfDNA samples collected during tumor evolution. CTCs from a second patient presented a pattern of recurrent amplifications in ASPM and SEPT9 genes and loss of FANCC. The 92-gene custom panel identified 16 non-synonymous somatic alterations in ccfDNA, including a deletion (I1485Rfs∗19) and a somatic mutation (p. A1487V) in ARID1A gene and a point mutation in FGFR2 gene (p.G384R). Our results support the feasibility of non-invasive liquid biopsy testing in CUP cases, either using ctDNA or CTCs, to identify CUP genetic alterations with broad NGS panels covering the most frequently mutated genes.

Metastasis is responsible for the majority of cancer-related deaths. Particularly challenging is the management of metastatic cancer of unknown primary site (CUP), whose tissue-of-origin (TOO) remains undetermined even after expensive investigations. CUP therapy is rather unspecific and poorly effective. Molecular approaches developed to identify CUPs’ potential tissue-of-origin, can overcome some of these issues. In this study, we applied a pre-determined set of microRNAs (miRNAs) to infer the TOO of 53 metastatic cancers of unknown or uncertain origin. We designed a molecular assay to quantify 89 miRNAs at the copy number level, using EvaGreen-based Droplet Digital PCR. We assessed miRNA expression in 159 samples including primary tumors from 17 tumor classes (reference set), metastases of known and unknown origin. We applied two different statistical models for class prediction to obtain CUP’s most probable TOOs. Specifically, we used the shrunken centroids using PAMR (Prediction Analysis of Microarrays for R) and the least absolute shrinkage and selection operator (LASSO) models. The molecular test was successfully applied to FFPE samples and provided a site-of-origin identification within one-week from the biopsy procedure. The most frequently predicted origins were gastrointestinal, pancreas, breast and lung. The assay was applied to multiple metastases from the same CUP, collected from different metastatic sites: the molecular prediction revealed an impressive agreement in site-of-origin prediction, intrinsically validating our assay. The final prediction was compared with the clinico-pathological hypothesis of primary site. Moreover, a panel of 14 miRNAs proved to have prognostic value and being associated with overall survival. Our study demonstrated that miRNA expression profiling in CUP samples could be employed as diagnostic and prognostic test. Our molecular analysis can be performed on-request, concomitantly with standard diagnostic workup and in association with genetic profiling, to offer valuable indication about the possible primary site, thereby supporting treatment decisions.

Highlights • Tocilizumab (TCZ) is currently being tested in COVID‐19‐induced cytokine storm. • COVID-19 patients responding to TCZ have higher post-treatment levels of circulating miR-146a. • Low levels of miR-146a are associated with death in COVID-19 patients not responding to TCZ. • MicroRNAs can represent biomarkers of response to anti-inflammatory interventions in COVID-19., Current COVID-19 pandemic poses an unprecedented threat to global health and healthcare systems. The most amount of the death toll is accounted by old people affected by age-related diseases that develop a hyper-inflammatory syndrome. In this regard, we hypothesized that COVID-19 severity may be linked to inflammaging. Here, we examined 30 serum samples from patients enrolled in the clinical trial NCT04315480 assessing the clinical response to a single-dose intravenous infusion of the anti-IL-6 receptor drug Tocilizumab (TCZ) in COVID-19 patients with multifocal interstitial pneumonia. In these serum samples, as well as in 29 age- and gender-matched healthy control subjects, we assessed a set of microRNAs that regulate inflammaging, i.e. miR-146a-5p, miR-21-5p, and miR-126-3p, which were quantified by RT-PCR and Droplet Digital PCR. We showed that COVID-19 patients who did not respond to TCZ have lower serum levels of miR-146a-5p after the treatment (p = 0.007). Among non-responders, those with the lowest serum levels of miR-146a-5p experienced the most adverse outcome (p = 0.008). Our data show that a blood-based biomarker, such as miR-146a-5p, can provide clues about the molecular link between inflammaging and COVID-19 clinical course, thus allowing to better understand the use of biologic drug armory against this worldwide health threat.

Metastasis is responsible for the majority of cancer‐related deaths. Particularly, challenging is the management of metastatic cancer of unknown primary site (CUP), whose tissue of origin (TOO) remains undetermined even after extensive investigations and whose therapy is rather unspecific and poorly effective. Molecular approaches to identify the most probable TOO of CUPs can overcome some of these issues. In this study, we applied a predetermined set of 89 microRNAs (miRNAs) to infer the TOO of 53 metastatic cancers of unknown or uncertain origin. The miRNA expression was assessed with droplet digital PCR in 159 samples, including primary tumors from 17 tumor classes (reference set) and metastases of known and unknown origin (test set). We combined two different statistical models for class prediction to obtain the most probable TOOs: the nearest shrunken centroids approach of Prediction Analysis of Microarrays (PAMR) and the least absolute shrinkage and selection operator (LASSO) models. The molecular test was successful for all formalin‐fixed paraffin‐embedded samples and provided a TOO identification within 1 week from the biopsy procedure. The most frequently predicted origins were gastrointestinal, pancreas, breast, lung, and bile duct. The assay was applied also to multiple metastases from the same CUP, collected from different metastatic sites: The predictions showed a strong agreement, intrinsically validating our assay. The final CUPs' TOO prediction was compared with the clinicopathological hypothesis of primary site. Moreover, a panel of 13 miRNAs proved to have prognostic value and be associated with overall survival in CUP patients. Our study demonstrated that miRNA expression profiling in CUP samples could be employed as diagnostic and prognostic test. Our molecular analysis can be performed on request, concomitantly with standard diagnostic workup and in association with genetic profiling, to offer valuable indications about the possible primary site, thereby supporting treatment decisions., Cancer of unknown primary site (CUP) patients suffer the burden of a metastatic disease whose site of origin is unidentifiable, even after intensive clinical investigations. The lack of a recognized primary site limits patients' treatment options. In this manuscript, we present a molecular assay, based on digital miRNA profiling, that allowed the prediction of the primary site of 53 CUPs.

Simple Summary Patients with cancer of unknown primary site suffer the burden of an uncertain disease, which is characterized by the impossibility to identify the tissue where the tumor has originated. The identification of the primary site of a tumor is of great importance for the patient to have access to site-specific treatments and be enrolled in clinical trials. Therefore, patients with cancer of unknown primary have reduced therapeutic opportunities and poor prognosis. Advancements have been made in the molecular characterization of this tumor, which could be used to infer the tumor site-of-origin and thus broaden the diagnostic outcome. Moreover, we describe here the novel therapeutic opportunities that are based on the genetic and immunophenotypic characterization of the tumor, and thus independent from the tumor type, which could provide most benefit to patients with cancer of unknown primary. Abstract Distant metastases are the main cause of cancer-related deaths in patients with advanced tumors. A standard diagnostic workup usually contains the identification of the tissue-of-origin of metastatic tumors, although under certain circumstances, it remains elusive. This disease setting is defined as cancer of unknown primary (CUP). Accounting for approximately 3–5% of all cancer diagnoses, CUPs are characterized by an aggressive clinical behavior and represent a real therapeutic challenge. The lack of determination of a tissue of origin precludes CUP patients from specific evidence-based therapeutic options or access to clinical trial, which significantly impacts their life expectancy. In the era of precision medicine, it is essential to characterize CUP molecular features, including the expression profile of non-coding RNAs, to improve our understanding of CUP biology and identify novel therapeutic strategies. This review article sheds light on this enigmatic disease by summarizing the current knowledge on CUPs focusing on recent discoveries and emerging diagnostic strategies.

BackgroundCurrent COVID-19 pandemic poses an unprecedented threat to global health and healthcare systems. At least in western countries, the most amount of the death toll is accounted by old people affected by age-related diseases. In this regard, we proposed that COVID-19 severity may be tightly related to inflammaging, i.e. the age-related onset of inflammation, which is responsible for age-related diseases. It has been reported that systemic hyper-inflammation may turn to be detrimental in COVID-19 patients.ObjectiveHere, we exploited a recently closed clinical trial (NCT04315480) on the anti-IL-6 drug tocilizumab to assess whether microRNAs regulating inflammaging can be assessed as biomarkers of drug response and outcome.MethodsSerum levels of miR-146a-5p, −21-5p, and −126-3p were quantified by RT-PCR and Droplet Digital PCR by two independent laboratories on 30 patients with virologically confirmed COVID-19, characterized by multifocal interstitial pneumonia confirmed by CT-scan and requiring oxygen therapy, and 29 age- and gender-matched healthy control subjects. COVID-19 patients were treated with a single-dose intravenous infusion of 8 mg/kg tocilizumab and categorized into responders and non-responders.ResultsWe showed that COVID-19 patients who did not respond to tocilizumab have lower serum levels of miR-146a-5p after the treatment (p=0.007). Moreover, among non-responders, those with the lowest serum levels of miR-146a-5p experienced the most adverse outcome (p=0.008).ConclusionOur data show that blood-based biomarkers, such as miR-146a-5p, can provide a molecular link between inflammaging and COVID-19 clinical course, thus allowing to enlarge the drug armory against this worldwide health threat.

Aim: To investigate the genome-wide methylation of genetically characterized colorectal cancer stem cell (CR-CSC) lines. Materials & methods: Eight CR-CSC lines were isolated from primary colorectal cancer (CRC) tissues, cultured and characterized for aneuploidy, mutational status of CRC-related genes and microsatellite instability (MSI). Genome-wide DNA methylation was assessed by MethylationEPIC microarray. Results: We describe a distinctive methylation pattern that is maintained following in vivo passages in immune-compromised mice. We identified an epigenetic CR-CSC signature associated with MSI. We noticed that the preponderance of the differentially methylated positions do not reside at CpG islands, but spread to shelf and open sea regions. Conclusion: Given that CRCs with MSI-high status have a lower metastatic potential, the identification of a MSI-related methylation signature could provide new insights and possible targets into metastatic CRC.

Patients with occult primary cancer or cancer of unknown primary (CUP) carry the double burden of an aggressive disease and reduced access to therapies, which are typically tumor-type oriented. Moreover, experimental in-vitro and in-vivo models summing up CUP features and heterogeneity are currently unavailable. We derived and expanded two CUP cell lines, and corresponding patient-derived xenografts (PDXs), from ascites tumor cells. CUP cell lines, one spontaneously growing as organoid, and PDXs were subjected to histological, immune-phenotypical, molecular and genomic (CNV and target sequencing) characterization to show their ability to recapitulate the original tumor. MicroRNA profile and genomic information were used to identify a possible primary site and patient-specific therapeutic approaches. Data on CUP most frequently altered genes were used to design a custom CUP-specific, 92-gene panel for target NGS analysis with Sure Select XS technology (Agilent Technology). Drug-screening assays were performed in vitro and in vivo on two models targeting the most promising actionable pathways. We obtained unexpected and promising results on drug combinations efficacy. This study brings personalized therapy closer to CUP patients and paves the way to future applications of personalized medicine for metastatic patients with adverse prognosis. This work was supported by grants from the Italian Association for Cancer Research (AIRC) to MF (IG 18464). Citation Format: Noemi Laprovitera, Claudia Fumarola, Elisa Porcellini, Stefano Cairo, Giulia Gallerani, Francesco Gelsomino, Irene Salamon, Maria Naddeo, Andrea Ardizzoni, Andrea Cavazzoni, Manuela Ferracin. Cancer of Unknown Primary: novel therapeutic opportunities from patient-derived cell cultures and in vivo models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3011.

Background Cancer of Unknown Primary is a rare syndrome of metastatic cancers for which the primary site cannot be recognized after detailed physical examinations, blood analyses, imaging and immunohistochemical (IHC) testing. CUPs make up 3-5% of newly diagnosed cancers worldwide and represent an important diagnostic and clinical problem. Treatment failure to empirical chemotherapy is common, resulting in progressive disease and poor prognosis. We and others proposed that a molecular inference of the tissue of origin could be feasible for most CUP tumors (Ferracin et al. J Pathol, 2011), thus giving access to more effective therapeutic agents and potentially extending CUP patient life-expectancy. Methods We optimized a cancer-type classifier based on microRNA (miRNA) expression for the prediction of CUP primary tumor site (Laprovitera et al., Front Oncol. 2018). Specifically, we designed a molecular assay to quantify the absolute copy number of 89 miRNAs in formalin-fixed paraffin-embedded (FFPE) samples using EvaGreen-based Droplet Digital PCR (BioRad). We tested 153 samples from three groups: primary tumors (N=95), metastases of known origin (N=10) and cancers of unknown origin (N=48). CUP diagnosis was obtained after a detailed histo-pathological investigation as per CUP diagnostic guidelines (NCCN occult primary v.2-2019 and ESMO guidelines). The study was approved by the local ethical committee. Results We investigated a pre-determined set of 89 miRNAs to infer the site-of origin of metastatic cancers of unknown or uncertain primary. Primary tumors from 16 different sites (lung, pancreas, liver, bile duct, kidney, bowel, testis, ovary, endometrium, stomach, bladder, breast, prostate, melanoma, gastrointestinal neuroendocrine, head and neck) were tested for absolute miRNA expression and used to train our classifier (Tibshirani et al. PNAS, 2002). We used the metastases of known origin as a test-set for our molecular prediction. We applied the shrunken centroids approach as predictive algorithm to obtain the most probable CUP site(s)-of- origin. Our results were judged against the hypothesized primary site suggested by standard diagnostic workup and pathological assessment. The molecular test was successfully applied to all CUP samples and provided a site-of-origin identification (with a probability > 50%) for all samples, potentially within a one-week time frame from sample inclusion. Conclusions Our study demonstrated that miRNA expression profiling in CUP samples have the potential to be employed in a clinical setting. Our molecular analysis can be performed on-request, concomitantly with the standard diagnostic workup and in association with genetic profiling, to offer valuable indication about the possible primary site thereby supporting treatment decisions. This work was supported by grants from the Italian Association for Cancer Research (AIRC) to MF (IG 18464). Citation Format: Noemi Laprovitera, Elisa Porcellini, Mattia Riefolo, Elisabetta Broseghini, Ingrid Garajova, Silvia Sabbioni, Martin Pichler, Andrea Ardizzoni, Antonia D'Errico, Manuela Ferracin. Cancer of unknown primary site-of-origin: An enigma ready to be miR-solved [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5432.

Background Cancer of unknown primary (CUP) is a heterogeneous group of metastatic cancers whose primary site cannot be determined after standard clinical and pathological evaluation. CUP patients are generally treated with empirical chemotherapy and have poor prognosis. As reported in recent studies, CUPs present an average of 4 genetic alterations per tumor and these genetic alterations can be detected in circulating tumor DNA (Kato 2017). Thus, a potential improvement in CUP outcomes could derive from targeted therapies directed toward actionable mutations, and Circulating tumor cells (CTCs) can provide valuable information. Here, we describe a case of a 51-yr-old Caucasian female (Pt#71) with metastatic CUP. CTCs were detectable in her blood and analyzed for genetic alterations. Methods Using CellSearch and DEPArray NxT, n=3 CTCs and n=1 leukocyte as a control were isolated from patient's peripheral blood. Single-cell whole genome amplification was obtained with Ampli1 WGA Kit and libraries were generated with Ampli1 OncoSeek Panel and Ampli1 LowPass kits. In parallel, formalin-fixed paraffin embedded (FFPE) tissue derived from a lymph node biopsy was analyzed with two different methods. One curl was sent for comprehensive genomic profile to FoundationOne assay testing (Roche). From another curl, DNA was extracted and directly processed with DEPArray LibPrep and DEPArray OncoSeek Panel kits. After sequencing on Illumina MiSeq platform, raw data were analyzed on MSBiosuite platform (Menarini Silicon Biosystems). Results CTCs genome-wide characterization allowed the detection of sub-chromosomal losses, including a LOH region comprising the APC gene, and patterns of extensive gains. Moreover, amplification signals were detected in correspondence of FGFR2 and CCNE1 genes. Targeted sequencing on both CTCs and bulk tumor DNA confirmed the FGFR2 amplification in all the samples and detected a somatic variant in APC gene (APC:p.T1556Nfs*3). FoundationOne assay on FFPE biopsy reported the same amplification on FGFR2 and CCNE1 genes, along with a somatic variant in ARID1A gene (ARID1A:p.R1276*), a gene not present in the OncoSeek Panel. On the other hand, APC somatic variant was not identified by FoundationOne, probably due to contamination by normal cells and/or tumor heterogeneity. Conclusions The comprehensive genomic profile of tumor FFPE tissue and CTCs was highly overlapping and allowed the characterization of genetic alterations in this CUP case, revealing potentially actionable mutations and copy number alterations. The advantage of isolating and analyzing single CTCs is clearly shown by the non-invasive procedure combined with a precise detection of druggable alterations due to cell purity. Citation Format: Elisa Porcellini, Francesco Gelsomino, Noemi Laprovitera, Mattia Riefolo, Marianna Garonzi, Paola Tononi, Francesca Fontana, Antonia D'Errico, Maria Pantaleo, Nicolò Manaresi, Andrea Ardizzoni, Manuela Ferracin. Isolation and genetic characterization of circulating tumor cells from cancer of unknown primary [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5361.

We are delighted to share with you our seventh Journal Club and highlight some of the most interesting papers published recently [...]

Archival formalin-fixed paraffin-embedded (FFPE) tissues represent an extraordinary source of smallRNAs, including microRNAs (miRNAs). Contrary to other RNA molecules, miRNAs are stable, nuclease-resistant and quantifiable even in low quality samples. The accurate assessment of miRNA levels in archival samples is of great interest for many pathological conditions, including cancer. In human tumors, microRNA expression is type-specific and can be used as diagnostic, prognostic or response-to-treatment biomarker. In this study, we provide a method for multiple miRNA quantification in 96-well plates, using EvaGreen-based droplet digital PCR technology and miRCURY LNA miRNA assays. This approach allows the absolute quantification of a customizable panel of miRNAs at the same time and under identical experimental conditions, to be used for diagnostic or prognostic applications.

A cancer cell is the final product of a complex mixture of genetic, epigenetic and epitranscriptomic alterations, whose final interplay contribute to cancer onset and progression. This is specifically true for colorectal cancer, a tumor with a strong epigenetic component, which acts earlier than any other genetic alteration in promoting cancer cell malignant transformation. The pattern of progressive, and usually subtype-specific, DNA and histone modifications that occur in colorectal cancer has been extensively studied in the last decade, providing plenty of data to explore. For this tumor, it became recently evident that also RNA modifications play a relevant role in the activation of oncogenes or repression of tumor suppressor genes. In this review we provide a brief overview of all epigenetic and epitranscriptomic changes that have been found associated to colorectal cancer till now. We explore the impact of these alterations in cancer prognosis and response to treatment and discuss their potential use as cancer biomarkers.

Background: Programmed cell death 1 (PD-1) and PD-ligand 1 (PD-L1) inhibitors represent novel therapeutic options for advanced non-small cell lung cancer (NSCLC). However, approximately 50% of patients do not benefit from therapy and experience rapid disease progression. PD-L1 expression is the only approved biomarker of benefit to anti-PD-1/PD-L1 therapy. However, its weakness has been evidenced in many studies. More recently, tumor mutational burden (TMB) has proved to be a suitable biomarker, but its calculation is difficult to obtain for all patients. Methods: We tested specific NSCLC genetic alterations as potential immunotherapy biomarkers. Tumor DNA was obtained from advanced NSCLC patients treated with anti-PD-1 monoclonal antibody nivolumab ( n = 44) or pembrolizumab ( n = 3). The mutational status of 22 genes was assessed by targeted next-generation sequencing and the association with survival was tested in uni- and multivariate models. The association between gene mutations and clinical benefit was also investigated. Results: The most frequently mutated genes were TP53 (49%), KRAS (43%), ERBB2 (13%), SMAD4 (13%), DDR2 (13%), STK11 (9%), ERBB4 (6%), EGFR (6%), BRAF (6%), and MET (6%). We confirmed that KRASmut patients have a better response to PD-1 inhibitors, showing a longer progression-free survival (PFS) and overall survival (OS) than KRASwt patients. In addition, we observed that patients with ERBB-family mutations, including EGFR, ERBB2, and ERBB4 all failed to respond to PD-1 antibodies, independently of KRAS status. Conclusions: This study suggests that the analysis of KRAS and ERBB-family gene mutational status is valuable when assessing the clinical practice for the selection of NSCLC patients to treat with PD-1 inhibitors.

Adjuvant therapy is a systemic treatment administered after primary tumor resection to reduce the risk of relapse and death in colorectal cancer (CRC) patients. Generally, adjuvant treatment is recommended for stage III and ‘high-risk' stage II CRC (NCCN guidelines). Nonetheless, adjuvant treatment is still debated for stage IIA (T3N0) patients, whose estimated recurrence rate is 15-20% in the absence of any further therapy after resection of the primary tumor. With the aim to find additional prognostic biomarkers that could improve current patient selection, we analyzed the global methylation profile of a discovery group of 10 relapsing/non-relapsing stage IIA colon cancer samples, without any other known risk factor (low-risk), by Illumina Epic 850K array. We identified a panel of 25 Illumina probes that were able to predict recurrence in chemotherapy-naïve patients. Then, we assessed the methylation of the most predictive Cytosine-Guanine (CpG) dinucleotides in 107 stage IIA colon cancer patients from two different cohorts, by EpiTYPER technology. We validated several CpGs as differentially methylated in relapsing vs. non-relapsing CRCs (p Citation Format: Sara Pagotto, Elisa Porcellini, Maria G. Bacalini, Diletta Sarti, Ingrid Garajova, Elisabetta Broseghini, Noemi Laprovitera, Francesco Vasuri, Paolo Garagnani, Renato Mariani-Costantini, Michele Basso, Rosa Visone, Luigi M. Larocca, Manuela Ferracin, Angelo Veronese. Epigenetic biomarkers of prognosis in stage IIA colon cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3313.