An alternative splicing signature defines the basal-like phenotype and predicts worse clinical outcome in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by extremely poor prognosis. PDAC presents with molecularly distinct subtypes, with the basal-like one being associated with enhanced chemoresistance. Splicing dysregulation contributes to PDAC; however, its involvement in subtype specification remains elusive. Herein, we uncover a subtype-specific splicing signature associated with prognosis in PDAC and the splicing factor Quaking (QKI) as a determinant of the basal-like signature. Single-cell sequencing analyses highlight QKI as a marker of the basal-like phenotype. QKI represses splicing events associated with the classical subtype while promoting basal-like events associated with shorter survival. QKI favors a plastic, quasi-mesenchymal phenotype that supports migration and chemoresistance in PDAC organoids and cell lines, and its expression is elevated in high-grade primary tumors and metastatic lesions. These studies identify a splicing signature that defines PDAC subtypes and indicate that QKI promotes an undifferentiated, plastic phenotype, which renders PDAC cells chemoresistant and adaptable to environmental changes.

Sterile inflammation via TRPM8 RNA-dependent TLR3-NF-kB/IRF3 activation promotes antitumor immunity in prostate cancer.

Inflammation is a common condition of prostate tissue, whose impact on carcinogenesis is highly debated. Microbial colonization is a well-documented cause of a small percentage of prostatitis cases, but it remains unclear what underlies the majority of sterile inflammation reported. Here, androgen- independent fluctuations of PSA expression in prostate cells have lead us to identify a prominent function of the Transient Receptor Potential Cation Channel Subfamily M Member 8 (TRPM8) gene in sterile inflammation. Prostate cells secret TRPM8 RNA into extracellular vesicles (EVs), which primes TLR3/NF-kB-mediated inflammatory signaling after EV endocytosis by epithelial cancer cells. Furthermore, prostate cancer xenografts expressing a translation-defective form of TRPM8 RNA contain less collagen type I in the extracellular matrix, significantly more infiltrating NK cells, and larger necrotic areas as compared to control xenografts. These findings imply sustained, androgen-independent expression of TRPM8 constitutes as a promoter of anticancer innate immunity, which may constitute a clinically relevant condition affecting prostate cancer prognosis.

Same-session endoscopic diagnosis and symptom palliation in pancreato-biliary malignancies: Clinical impact of rapid on-site evaluation (ROSE).

Background and study aims Besides increasing adequacy, rapid on-site evaluation (ROSE) during endoscopic ultrasound (EUS) or endoscopic retrograde cholangiopancreatography (ERCP) may impact choices and timing of subsequent therapeutic procedures, yet has been unexplored. Patients and methods This was a retrospective evaluation of a prospectively maintained database of a tertiary, academic centre with availability of ROSE and hybrid EUS-ERCP suites. All consecutive patients referred for pathological confirmation of suspected malignancy and jaundice or gastric outlet obstruction (GOO) between Jan-2020 and Sep-2022 were included. Results Of 541 patients with underlying malignancy, 323 (59.7%) required same-session pathological diagnosis (male: 54.8%; age 70 [interquartile range 63-78]; pancreatic cancer: 76.8%, biliary tract adenocarcinoma 16.1%). ROSE adequacy was 96.6%, higher for EUS versus ERCP. Among 302 patients with jaundice, ERCP-guided stenting was successful in 83.1%, but final drainage was completed in 97.4% thanks to 43 EUS-guided biliary drainage procedures. Twenty-one patients with GOO were treated with 15 EUS-gastroenterostomies and six duodenal stents. All 58 therapeutic EUS procedures occurred after adequate ROSE. With ERCP-guided placement of stents, the use of plastic stents was significantly higher among patients with inadequate ROSE (10/11; 90.9%) versus adequate sampling (14/240; 5.8%) P <0.0001; OR 161; 95%CI 19-1352). Median hospital stay for diagnosis and palliation was 3 days (range, 2-7) and median time to chemotherapy was 33 days (range, 24-47). Conclusions Nearly two-thirds of oncological candidates for endoscopic palliation require contemporary pathological diagnosis. ROSE adequacy allows, since the index procedure, state-of-the-art therapeutics standardly restricted to pathologically confirmed malignancies (e.g. uncovered SEMS or therapeutic EUS), potentially reducing hospitalization and time to oncological treatments.

COVID-19. Biology, pathophysiology, and immunology: a pathologist view.

Even if the SARS-CoV-2 pandemic has been declared over, several risks and clinical problems remain to be faced, including long-COVID sequelae and possible outbreaks of pathogenic variants. Intense research on COVID-19 has provided in these few years a striking amount of data covering different fields and disciplines, which can help to provide a knowledge shield against new potential infective spreads, and may also potentially be applied to other fields of medicine, including oncology and neurology. Nevertheless, areas of uncertainty still remain regarding the pathogenic mechanisms that subtend the multifaceted manifestations of the disease. To better clarify the pathogenesis of the disease, a systematic multidisciplinary evaluation of the many mechanisms involved in COVID-19 is mandatory, including clinical, physiological, radiological, immunological and pathological studies. In COVID-19 syndrome the pathological studies have been mainly performed on autopsy cases, and only a few studies are available on biopsies. Nevertheless, these studies have provided relevant information that can substantially contribute to decipher the complex scenario characterizing the different forms of COVID-19 and long-COVID-19. In this review the data provided by pathological investigations are recapitulated and discussed, in the light of different hypothesis and data provided by clinical, physiological and immunological data.

Glucose and lactate levels are lower in EUS-aspirated cyst fluid of mucinous vs non-mucinous pancreatic cystic lesions.

BACKGROUND: Distinguishing mucinous (M) pancreatic cystic neoplasms (PCNs) from non-mucinous (NM) is challenging but crucial. Low intracystic glucose level has shown diagnostic tool promise, however further investigation is needed to understand metabolic processes. AIMS: To compare the diagnostic accuracy of intracystic glucose and CEA levels in a large cohort and explore lactate levels as potential marker. METHODS: PCNs≥15 mm which underwent EUS-fine needle aspiration were prospectively enrolled. Glucose, CEA and lactate levels were measured. Diagnostic accuracy for M-PCN diagnosis was evaluated using surgical/cytology reports or multidisciplinary evaluations. RESULTS: 169 PCNs were included (64 % M-PCNs). Median intracystic glucose was significantly lower in M-PCNs (1 mg/dL) compared to NM-PCNs (101 mg/dL); mean intracystic CEA was significantly higher in M-PCNs (152.5 ng/mL) compared to NM-PCNs (0.3 ng/mL). ROC curve analysis revealed best glucose cut-off ≤58 mg/dL (accuracy 93.5 %) and CEA cut-off >2.5 ng/mL (accuracy 90.5 %) for M-PCNs. Intracystic lactates were significantly lower in M-PCNs correlating directly with glucose. Single glucose dosage evidenced best diagnostic accuracy respect markers combination. CONCLUSION: Intracystic glucose demonstrated high diagnostic utility for M-PCNs differentiation, surpassing CEA. Lactate levels correlated with glucose, suggesting their uptake by M-PCNs cells. These findings contribute to a better metabolic landscape understanding glucose use as diagnostic marker.

IL-1ß+ macrophages fuel pathogenic inflammation in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease with high resistance to therapies1. Inflammatory and immunomodulatory signals co-exist in the pancreatic tumour microenvironment, leading to dysregulated repair and cytotoxic responses. Tumour-associated macrophages (TAMs) have key roles in PDAC2, but their diversity has prevented therapeutic exploitation. Here we combined single-cell and spatial genomics with functional experiments to unravel macrophage functions in pancreatic cancer. We uncovered an inflammatory loop between tumour cells and interleukin-1ß (IL-1ß)-expressing TAMs, a subset of macrophages elicited by a local synergy between prostaglandin E2 (PGE2) and tumour necrosis factor (TNF). Physical proximity with IL-1ß+ TAMs was associated with inflammatory reprogramming and acquisition of pathogenic properties by a subset of PDAC cells. This occurrence was an early event in pancreatic tumorigenesis and led to persistent transcriptional changes associated with disease progression and poor outcomes for patients. Blocking PGE2 or IL-1ß activity elicited TAM reprogramming and antagonized tumour cell-intrinsic and -extrinsic inflammation, leading to PDAC control in vivo. Targeting the PGE2-IL-1ß axis may enable preventive or therapeutic strategies for reprogramming of immune dynamics in pancreatic cancer.

mTOR eosinophilic renal cell carcinoma: a distinctive tumor characterized by mTOR mutation, loss of chromosome 1, cathepsin-K expression, and response to target therapy.

In the spectrum of oncocytic renal neoplasms, a subset of tumors with high-grade-appearing histologic features harboring pathogenic mutations in mammalian target of rapamycin (mTOR) and hitherto clinical indolent behavior has been described. Three cases (2F,1 M) with histologically documented metastases (lymph node, skull, and liver) were retrieved and extensively investigated by immunohistochemistry, FISH, and next-generation sequencing. Tumors were composed of eosinophilic cells with prominent nucleoli (G3 by ISUP/WHO) arranged in solid to nested architecture. Additionally, there were larger cells with perinuclear cytoplasmic shrinkage and sparse basophilic Nissl-like granules, superficially resembling the so-called spider cells of cardiac rhabdomyomas. The renal tumors, including the skull and liver metastases, showed immunoexpression PAX8, CK8-18, and cathepsin-K, and negativity for vimentin. NGS identified mTOR genetic alterations in the three cases, including the skull and liver metastases. One patient was then treated with Everolimus (mTOR inhibitors) with clinical response (metastatic tumor shrinkage). We present a distinct renal tumor characterized by high-grade eosinophilic cells, cathepsin-K immunohistochemical expression, and harboring mTOR gene mutations demonstrating a malignant potential and showing responsiveness to mTOR inhibitors.

Epithelial ovarian cancer is infiltrated by activated effector T cells co-expressing CD39, PD-1, TIM-3, CD137 and interacting with cancer cells and myeloid cells.

Introduction: Despite predicted efficacy, immunotherapy in epithelial ovarian cancer (EOC) has limited clinical benefit and the prognosis of patients remains poor. There is thus a strong need for better identifying local immune dynamics and immune-suppressive pathways limiting T-cell mediated anti-tumor immunity. Methods: In this observational study we analyzed by immunohistochemistry, gene expression profiling and flow cytometry the antigenic landscape and immune composition of 48 EOC specimens, with a focus on tumor-infiltrating lymphocytes (TILs). Results: Activated T cells showing features of partial exhaustion with a CD137+CD39+PD-1+TIM-3+CD45RA-CD62L-CD95+ surface profile were exclusively present in EOC specimens but not in corresponding peripheral blood or ascitic fluid, indicating that the tumor microenvironment might sustain this peculiar phenotype. Interestingly, while neoplastic cells expressed several tumor-associated antigens possibly able to stimulate tumor-specific TILs, macrophages provided both co-stimulatory and inhibitory signals and were more abundant in TILs-enriched specimens harboring the CD137+CD39+PD-1+TIM-3+CD45RA-CD62L-CD95+ signature. Conclusion: These data demonstrate that EOC is enriched in CD137+CD39+PD-1+TIM-3+CD45RA-CD62L-CD95+ T lymphocytes, a phenotype possibly modulated by antigen recognition on neoplastic cells and by a combination of inhibitory and co-stimulatory signals largely provided by infiltrating myeloid cells. Furthermore, we have identified immunosuppressive pathways potentially hampering local immunity which might be targeted by immunotherapeutic approaches.

In vivo macrophage engineering reshapes the tumor microenvironment leading to eradication of liver metastases.

Liver metastases are associated with poor response to current pharmacological treatments, including immunotherapy. We describe a lentiviral vector (LV) platform to selectively engineer liver macrophages, including Kupffer cells and tumor-associated macrophages (TAMs), to deliver type I interferon (IFNα) to liver metastases. Gene-based IFNα delivery delays the growth of colorectal and pancreatic ductal adenocarcinoma liver metastases in mice. Response to IFNα is associated with TAM immune activation, enhanced MHC-II-restricted antigen presentation and reduced exhaustion of CD8+ T cells. Conversely, increased IL-10 signaling, expansion of Eomes CD4+ T cells, a cell type displaying features of type I regulatory T (Tr1) cells, and CTLA-4 expression are associated with resistance to therapy. Targeting regulatory T cell functions by combinatorial CTLA-4 immune checkpoint blockade and IFNα LV delivery expands tumor-reactive T cells, attaining complete response in most mice. These findings support a promising therapeutic strategy with feasible translation to patients with unmet medical need.

Pro-tumor Tfh2 cells induce detrimental IgG4 production and PGE2-dependent IgE inhibition in pancreatic cancer.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis and it is characterized by predominant pro-tumor Th2-type inflammation. T follicular helper (Tfh) cells are relevant immunoregulators in cancer, and often correlate with better survival. How the Th2-skewed microenvironment in PDAC modulates the differentiation of Tfh cells and their immunoregulatory function is unknown. METHODS: We carried out high-dimensional flow cytometry and T-cell receptor- and RNA-sequencing, as well as bioinformatics, immunohistochemistry and in vitro mechanistic studies. FINDINGS: We identified Tfh1-, Tfh2-, and Tfh17-like cell clusters in the blood, tumors and tumor-draining lymph-nodes (TDLNs) of chemo-naïve PDAC patients and showed that high percentages of Tfh2 cells within the tumor tissue and TDLNs correlated with reduced patient survival. Moreover, only Tfh2 cells were highly activated and were reduced in frequency in patients who responded to neoadjuvant chemotherapy. RNA-sequencing analysis of immunoglobulin expression showed that tumor and TDLN samples expressed all immunoglobulin (IGH) isotypes apart from IGHE. Consistent with these findings, Tfh2 cells differentiated in vitro by tumor microenvironment-conditioned dendritic cells promoted the production of anti-inflammatory IgG4 antibodies by co-cultured B cells, dependent on IL-13. Moreover, unexpectedly, Tfh2 cells inhibited the secretion of pro-inflammatory IgE, dependent on prostaglandin E2. INTERPRETATION: Our results indicate that in PDAC, highly activated pro-tumor Tfh2 favor anti-inflammatory IgG4 production, while inhibit pro-inflammatory IgE. Thus, targeting the circuits that drive Tfh2 cells, in combination with chemotherapy, may re-establish beneficial anti-tumor Tfh-B cell interactions and facilitate more effective treatment. FUNDING: Research grants from the Italian Association for Cancer Research (AIRC) IG-19119 to MPP and the AIRC Special Program in Metastatic disease: the key unmet need in oncology, 5 per Mille no. 22737 to CB, MF, CD, MR and MPP; the ERA-NET EuroNanoMed III (a collaborative european grant financed by the Italian Ministry of Health, Italy) project PANIPAC (JTC2018/041) to MPP; the Fondazione Valsecchi to SC.

Diagnostic concordance between traditional and digital workflows. A study on 1427 prostate biopsies.

Objective: To evaluate intra-observer diagnostic reproducibility using traditional slides (TS) versus whole slide images (WSI). Methods: TS and WSI of 1427 prostatic biopsies (107 consecutive patients) were evaluated by a single pathologist. Agreement between readings was evaluated with Gwet's Agreement coefficient (AC) and Landis and Koch benchmark scale. Results: The positive/negative agreement between the readings was almost perfect (AC1= 0.962; 95% CI[0.949,0.974]), with method independent distribution of discrepancies. Among positive biopsies, 212 had identical Gleason score (GS) on TS and WSI and discordant GS in 69 cases (AC2 = 0.932; 95% CI[0.907, 0.956]). Concordant negative and positive patient classification was observed in 39 and 64 cases, respectively; two cases were assigned to the positive group on TS and 2 on WSI configuring an almost perfect agreement (AC1=0.929; 95% C1[0.860, 0.998]). ISUP Grade group (ISUP GG) agreement was evaluated in the 60 concordantly positive cases: in 45 cases it was identical on TS and WSI; in 10 biopsies the discrepancy implied a modification of the assigned ISUP GG of ≤ 1 class and in 5 the discrepancy implied a modification of 2 classes. Gwet's agreement coefficient was (95% CI [0.834, 0.962]), i.e.: almost perfect agreement. Conclusions: Our data show almost perfect agreement between digital and traditional diagnostic activity in a routine setting, confirming that digital pathology can be safely introduced into routine workflows.

Loose Tumor Cells in Pulmonary Arteries of Lung Adenocarcinoma Resection Specimen: No Correlation With Survival, Despite High Prevalence.

CONTEXT.­: Loose tumor cells and tumor cell clusters can be recognized in the lumen of intratumoral pulmonary arteries of resected non-small cell lung cancer specimens. It is unclear whether these should be considered tumor-emboli, and as such could predict a worsened prognosis. OBJECTIVE.­: To investigate the nature and prognostic impact of pulmonary artery intraluminal tumor cells. DESIGN.­: This multicenter study involved an exploratory pilot study and a validation study from 3 institutions. For the exploratory pilot, a retrospective pulmonary resection cohort of primary adenocarcinomas, diagnosed between November 2007 and November 2010, were scored for the presence of tumor cells, as well as potentially other cells in the intravascular spaces using hematoxylin-eosin, and cytokeratin 7 (CK7) stains. In the validation part, 2 retrospective cohorts of resected pulmonary adenocarcinomas, between January 2011 and December 2016, were included. Recurrence-free survival (RFS) and overall survival (OS) data were collected. RESULTS.­: In the pilot study, CK7+ intravascular cells, mainly tumor cells, were present in 23 of 33 patients (69.7%). The 5-year OS for patients with intravascular tumor cells was 61%, compared with 40% for patients without intravascular tumor cells (P = .19). In the validation study, CK7+ intravascular tumor cells were present in 41 of 70 patients (58.6%). The 5-year RFS for patients with intravascular tumor cells was 80.0%, compared with 80.6% in patients without intravascular tumor cells (P = .52). The 5-year OS rates were, respectively, 82.8% and 71.6% (P = .16). CONCLUSIONS.­: Loose tumor cells in pulmonary arterial lumina were found in most non-small cell lung cancer resection specimens and were not associated with a worse RFS or OS. Therefore, most probably they represent an artifact.

Revealing and harnessing CD39 for the treatment of colorectal cancer and liver metastases by engineered T cells.

OBJECTIVE: Colorectal tumours are often densely infiltrated by immune cells that have a role in surveillance and modulation of tumour progression but are burdened by immunosuppressive signals, which might vary from primary to metastatic stages. Here, we deployed a multidimensional approach to unravel the T-cell functional landscape in primary colorectal cancers (CRC) and liver metastases, and genome editing tools to develop CRC-specific engineered T cells. DESIGN: We paired high-dimensional flow cytometry, RNA sequencing and immunohistochemistry to describe the functional phenotype of T cells from healthy and neoplastic tissue of patients with primary and metastatic CRC and we applied lentiviral vectors (LV) and CRISPR/Cas9 genome editing technologies to develop CRC-specific cellular products. RESULTS: We found that T cells are mainly localised at the front edge and that tumor-infiltrating T cells co-express multiple inhibitory receptors, which largely differ from primary to metastatic sites. Our data highlighted CD39 as the major driver of exhaustion in both primary and metastatic colorectal tumours. We thus simultaneously redirected T-cell specificity employing a novel T-cell receptor targeting HER-2 and disrupted the endogenous TCR genes (TCR editing (TCRED)) and the CD39 encoding gene (ENTPD1), thus generating TCREDENTPD1KOHER-2-redirected lymphocytes. We showed that the absence of CD39 confers to HER-2-specific T cells a functional advantage in eliminating HER-2+ patient-derived organoids in vitro and in vivo. CONCLUSION: HER-2-specific CD39 disrupted engineered T cells are promising advanced medicinal products for primary and metastatic CRC.

Detecting Somatic Mutations for Well-Differentiated Pancreatic Neuroendocrine Tumors in Endoscopic Ultrasound-Guided Fine Needle Aspiration with Next-Generation Sequencing.

BACKGROUND: Pancreatic neuroendocrine tumors (PanNETs) exhibit heterogenous behavior, whereby some small tumors are aggressive with a propensity for metastasis. Detection of somatic mutations associated with aggressive biology may help with patient stratification and surgical decision-making in patients with well-differentiated PanNETs. Using next-generation sequencing (NGS), we investigated the feasibility of detecting somatic mutations in endoscopic ultrasound-guided, fine-needle aspiration (EUS-FNA) specimens and determining the mutational concordance between the EUS-FNA specimens and the primary tumors. METHODS: Thirty-eight patients with well-differentiated, nonfunctioning PanNETs were obtained from two tertiary referral centers. Patient demographic characteristics and tumor, clinicopathologic features were collected. Tissue from both the EUS-FNA specimen and the primary tumor was extracted from archival tissue blocks. NGS using a panel of ten genes was performed on both samples. RESULTS: In our series, the median age was 61.1 years. Tumors were predominantly left-sided (60.5%) and unifocal (94.7%). The median tumor size was 2.2 cm. NGS detected somatic mutations in 29% of primary tumors and 36.8% of EUS-FNA specimens. In primary tumors, DAXX/ATRX mutations were predominantly detected (63.6%). In EUS-FNA specimens, MEN1 mutations were predominantly detected (64.3%). Among non-wild-type specimens, mutational concordance was achieved in 31.6% of cases. In 11 patients with a detectable mutation in the primary tumor, a mutation was detected in the EUS-FNA specimen in 45.5% of cases, with a mutational concordance of 54.5%. CONCLUSIONS: NGS can detect somatic mutations in EUS-FNA specimens of well-differentiated PanNETs. Efforts to improve detection sensitivity and mutational concordance are required to overcome current technical limitations.

Pathological staging in postneoadjuvant pancreatectomy for pancreatic cancer: implications for adjuvant therapy.

BACKGROUND: It is unclear whether pathological staging is significant prognostically and can inform the delivery of adjuvant therapy after pancreatectomy preceded by neoadjuvant therapy. METHODS: This multicentre retrospective study included patients who underwent pancreatectomy for pancreatic ductal adenocarcinoma after neoadjuvant treatment at two Italian centres between 2013 and 2017. T and N status were assigned in accordance with the seventh and eighth editions of the AJCC staging system, as well as according to a modified system with T status definition combining extrapancreatic invasion and tumour size. Patients were then stratified by receipt of adjuvant therapy. Survival analysis and multivariable interaction analysis of adjuvant therapy with pathological parameters were performed. The results were validated in an external cohort from the USA. RESULTS: The developmental set consisted of 389 patients, with a median survival of 34.6 months. The modified staging system displayed the best prognostic stratification and the highest discrimination (C-index 0.763; 1-, 2- and 3-year time-dependent area under the curve (AUC) 0.746, 0.722, and 0.705; Uno's AUC 0.710). Overall, 67.0 per cent of patients received adjuvant therapy. There was no survival difference by receipt of adjuvant therapy (35.0 versus 36.0 months; P = 0.772). After multivariable adjustment, interaction analysis suggested a benefit of adjuvant therapy for patients with nodal metastases or with tumours larger than 2 cm with extrapancreatic extension, regardless of nodal status. These results were confirmed in the external cohort of 216 patients. CONCLUSION: Modified staging with a T status definition combining extrapancreatic invasion and tumour size is associated with better prognostic segregation after postneoadjuvant pancreatectomy. This system allows identification of patients who might benefit from adjuvant therapy.

Quantification of perineural invasion in pancreatic ductal adenocarcinoma: proposal of a severity score system.

Perineural invasion (PNI) is a common feature in pancreatic ductal adenocarcinoma (PDAC) and correlates with an aggressive tumor behavior already at early stages of disease. PNI is currently considered as a "present vs. absent" feature, and a severity score system has not yet been established. The aim of the present study was thus to develop and validate a score system for PNI and to correlate it with other prognostic features. In this monocentric retrospective study, 356 consecutive PDAC patients (61.8% upfront surgery patients, 38.2% received neoadjuvant therapy) were analyzed. PNI was scored as follows: 0: absent; 1: the presence of neoplasia along nerves < 3 mm in caliber; and 2: neoplastic infiltration of nerve fibers ≥ 3 mm and/or massive perineural infiltration and/or the presence of necrosis of the infiltrated nerve bundle. For every PNI grade, the correlation with other pathological features, disease-free survival (DFS), and disease-specific survival (DSS) were analyzed. Uni- and multivariate analysis for DFS and DSS were also performed. PNI was found in 72.5% of the patients. Relevant trends between PNI score and tumor differentiation grade, lymph node metastases, vascular invasion, and surgical margins status were found. The latter was the only parameter statistically correlated with the proposed score. The agreement between pathologists was substantial (Cohen's K 0.61). PNI severity score significantly correlated also with decreased DFS and DSS at univariate analysis (p < 0.001). At multivariate analysis, only the presence of lymph node metastases was an independent predictor of DFS (HR 2.235 p < 0.001). Lymph node metastases (HR 2.902, p < 0.001) and tumor differentiation grade (HR 1.677, p = 0.002) were independent predictors of DSS. Our newly developed PNI score correlates with other features of PDAC aggressiveness and proved to have a prognostic role though less robust than lymph nodes metastases and tumor differentiation grade. A prospective validation is needed.

Gene Fusion Detection in NSCLC Routine Clinical Practice: Targeted-NGS or FISH?

The ability to identify the broadest range of targetable gene fusions is crucial to facilitate personalized therapy selection for advanced lung adenocarcinoma (LuADs) patients harboring targetable receptor tyrosine kinase (RTK) genomic alterations. In order to evaluate the most effective testing approach for LuAD targetable gene fusion detection, we analyzed 210 NSCLC selected clinical samples, comparing in situ (Fluorescence In Situ Hybridization, FISH, and ImmunoHistoChemistry, IHC) and molecular (targeted RNA Next-Generation Sequencing, NGS, and RealTime-PCR, RT-PCR) approaches. The overall concordance among these methods was high (>90%), and targeted RNA NGS was confirmed to be the most efficient technique for gene fusion identification in clinical practice, allowing the simultaneous analysis of a large set of genomic rearrangements at the RNA level. However, we observed that FISH was useful to detect targetable fusions in those samples with inadequate tissue material for molecular testing as well as in those few cases whose fusions were not identified by the RNA NGS panel. We conclude that the targeted RNA NGS analysis of LuADs allows accurate RTK fusion detection; nevertheless, standard methods such as FISH should not be dismissed, as they can crucially contribute to the completion of the molecular characterization of LuADs and, most importantly, the identification of patients as candidates for targeted therapies.

Giardia lamblia Infection in a Duodenal Duplication Cyst as a Potential Cause of Recurrent Acute Pancreatitis.

Giardia lamblia is a known etiological factor of a common infectious diarrhea. In rare cases, this parasite was found to be involved in the development of pancreatic and biliary tract diseases, both inflammatory and neoplastic. We present a case of a 64-year-old man known for a duodenal duplication cyst since 2013, with episodes of recurrent acute pancreatitis since 2017. He underwent endoscopic ultrasound-guided fine-needle aspiration of the duplication cyst, with evidence of G. lamblia infection. After treatment of giardiasis and evidence of parasite eradication, the episodes of recurrent acute pancreatitis ceased, following an ex adiuvantibus criterium.