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Background: Pancreatic ductal adenocarcinoma (PDAC) features KRAS mutations in approximately 90% of human cases and excessive stromal response, termed desmoplastic reaction. Oncogenic KRAS drives pancreatic carcinogenesis by acting on both epithelial cells and tumor microenvironment (TME). We have previously shown that Homeodomain-Interacting Protein Kinase 2 (HIPK2) cooperates with KRAS in sustaining ERK1/2 phosphorylation in human colorectal cancers. Here, we investigated whether HIPK2 contributes to oncogenic KRAS-driven tumorigenesis in vivo, in the onset of pancreatic cancer., Methods: We employed an extensively characterized model of KRAS G12D -dependent preinvasive PDAC, the Pdx1-Cre;LSL-KRas G12D/+ (KC) mice. In these mice, HIPK2 was inhibited by genetic knockout in the pancreatic epithelial cells (KCH -/- ) or by pharmacologic inactivation with the small molecule 5-IodoTubercidin (5-ITu). The development of preneoplastic acinar-to-ductal metaplasia (ADM), intraepithelial neoplasia (PanIN), and their associated desmoplastic reaction were analyzed., Results: In Hipk2-KO mice (KCH -/- ), ERK phosphorylation was lowered, the appearance of ADM was slowed down, and both the number and pathologic grade of PanIN were reduced compared to Hipk2-WT KC mice. The pancreatic lesion phenotype in KCH -/- mice was characterized by abundant collagen fibers and reduced number of αSMA + and pSTAT3 + desmoplastic cells. These features were reminiscent of the recently described human "deserted" sub-TME, poor in cells, rich in matrix, and associated with tumor differentiation. In contrast, the desmoplastic reaction of KC mice resembled the "reactive" sub-TME, rich in stromal cells and associated with tumor progression. These observations were confirmed by the pharmacologic inhibition of HIPK2 in KC mice., Conclusion: This study demonstrates that HIPK2 inhibition weakens oncogenic KRAS activity and pancreatic tumorigenesis providing a rationale for testing HIPK2 inhibitors to mitigate the incidence of PDAC development in high-risk individuals., (© 2024. The Author(s).)

Colorectal cancer (CRC) patients with BRAF mutations develop resistance to BRAF inhibitors at a very early stage. Understanding the molecular mechanisms involved in BRAF inhibitor resistance is critical for the development of novel therapeutic opportunities for this subtype of CRC patients. CRC cells bearing BRAF mutations are mostly sensitive to the abrogation of Mitogen-Activated Protein Kinase Kinase 3 (MKK3), a specific activator of p38MAPKs signaling, suggesting that BRAF alterations might addict CRC cells to the MKK3/p38MAPK signaling. Interestingly, publicly available gene expression profiling data show significantly higher MKK3 transcript levels in CRC lines with acquired resistance to BRAF inhibitors. Herein, we investigated the roles of MKK3 in the response to BRAF targeting (dabrafenib) with COLO205 and HT29 BRAF V600E CRC lines and derived dabrafenib-resistant (DABR) sublines. Dabrafenib treatments reduce MKK3 activation by inducing autophagy in parental but not DABR cells. The MKK3 knockdown induces cell death in DABR cells, whereas ectopic MKK3 expression reduces dabrafenib sensitivity in parental cells. Mechanistically, activated MKK3 interacts and co-localizes with c-Myc oncoprotein (MYC), sustaining MYC protein stability and thus preventing the dabrafenib induced effects in CRC DABR cells both in vitro and in vivo. Overall, we identify a novel molecular mechanism beyond the dabrafenib resistance, shedding light on an uncovered vulnerability for the development of novel therapeutic opportunities in BRAF V600E CRC., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Hyperthermic intraperitoneal administration of chemotherapy (HIPEC) increases local drug concentrations and reduces systemic side effects associated with prolonged adjuvant intraperitoneal exposure in patients affected by either peritoneal malignancies or metastatic diseases originating from gastric, colon, kidney, and ovarian primary tumors. Mechanistically, the anticancer effects of HIPEC have been poorly explored. Herein we documented that HIPEC treatment promoted miR-145-5p expression paired with a significant downregulation of its oncogenic target genes c-MYC, EGFR, OCT4, and MUC1 in a pilot cohort of patients with ovarian peritoneal metastatic lesions. RNA sequencing analyses of ovarian peritoneal metastatic nodules from HIPEC treated patients unveils HSF-1 as a transcriptional regulator factor of miR-145-5p expression. Notably, either depletion of HSF-1 expression or chemical inhibition of its transcriptional activity impaired miR-145-5p tumor suppressor activity and the response to cisplatin in ovarian cancer cell lines incubated at 42 °C. In aggregate, our findings highlight a novel transcriptional network involving HSF-1, miR145-5p, MYC, EGFR, MUC1, and OCT4 whose proper activity contributes to HIPEC anticancer efficacy in the treatment of ovarian metastatic peritoneal lesions., (© 2023. The Author(s).)

Background: Approximately 20-50% of patients presenting with localized colorectal cancer progress to stage IV metastatic disease (mCRC) following initial treatment and this is a major prognostic determinant. Here, we have interrogated a heterogeneous set of primary colorectal cancer (CRC), liver CRC metastases and adjacent liver tissue to identify molecular determinants of the colon to liver spreading. Screening Food and Drug Administration (FDA) approved drugs for their ability to interfere with an identified colon to liver metastasis signature may help filling an unmet therapeutic need., Methods: RNA sequencing of primary colorectal cancer specimens vs adjacent liver tissue vs synchronous and asynchronous liver metastases. Pathways enrichment analyses. The Library of Integrated Network-based Cellular Signatures (LINCS)-based and Connectivity Map (CMAP)-mediated identification of FDA-approved compounds capable to interfere with a 22 gene signature from primary CRC and liver metastases. Testing the identified compounds on CRC-Patient Derived Organoid (PDO) cultures. Microscopy and Fluorescence Activated Cell Sorting (FACS) based analysis of the treated PDOs., Results: We have found that liver metastases acquire features of the adjacent liver tissue while partially losing those of the primary tumors they derived from. We have identified a 22-gene signature differentially expressed among primary tumors and metastases and validated in public databases. A pharmacogenomic screening for FDA-approved compounds capable of interfering with this signature has been performed. We have validated some of the identified representative compounds in CRC-Patient Derived Organoid cultures (PDOs) and found that pentoxyfilline and, to a minor extent, dexketoprofen and desloratadine, can variably interfere with number, size and viability of the CRC -PDOs in a patient-specific way. We explored the pentoxifylline mechanism of action and found that pentoxifylline treatment attenuated the 5-FU elicited increase of ALDHhigh cells by attenuating the IL-6 mediated STAT3 (tyr705) phosphorylation., Conclusions: Pentoxifylline synergizes with 5-Fluorouracil (5-FU) in attenuating organoid formation. It does so by interfering with an IL-6-STAT3 axis leading to the emergence of chemoresistant ALDHhigh cell subpopulations in 5-FU treated PDOs. A larger cohort of CRC-PDOs will be required to validate and expand on the findings of this proof-of-concept study., (© 2023. The Author(s).)

Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Homeodomain-interacting protein kinase 2 (HIPK2) is an evolutionary conserved kinase that has gained attention as a fine tuner of multiple signaling pathways, among which those commonly altered in colorectal cancer. The aim of this study was to evaluate the relationship of HIPK2 expression with progression markers and mutational pattern and gain insights into the contribution of HIPK2 activity in colorectal cancer. We evaluated a retrospective cohort of colorectal cancer samples by IHC for HIPK2 expression and by next-generation sequencing (NGS) for the detection of mutations of cancer associated genes. We show that the percentage of HIPK2-positive cells increases with tumor progression, significantly correlates with tumor-node-metastasis (TNM) staging and associates with a worse outcome. In addition, we observed that high HIPK2 expression significantly associates with KRAS mutations but not with other cancer-related genes. Functional characterization of the link between HIPK2 and KRAS show that activation of the RAS pathway either due to KRAS mutation or via upstream receptor stimulation, increases HIPK2 expression at the protein level. Of note, HIPK2 physically participates in the active RAS complex while HIPK2 depletion impairs ERK phosphorylation and the growth of tumors derived from KRAS mutated colorectal cancer cells. Overall, this study identifies HIPK2 as a prognostic biomarker candidate in patients with colorectal cancer and underscores a previously unknown functional link between HIPK2 and the KRAS signaling pathway., Implications: Our data indicate HIPK2 as a new player in the complex picture of the KRAS signaling network, providing rationales for future clinical studies and new treatment strategies for KRAS mutated colorectal cancer., (©2022 American Association for Cancer Research.)

Rosai-Dorfman disease (RDD) is also called sinus histiocytosis with massive lymphadenopathy, and it is caused by a histiocytic disorder with unclear etiology. It usually involves cervical lymph nodes, but it may also present with extranodal involvement. We report a rare condition of isolated hepatic RDD without nodal involvement, clinically manifested with three-month abdominal pain and tenderness of the right hypochondrium. CT- and PET-CT scans were compatible with a secondary lesion from an unknown primary tumor. Therefore, the patient underwent an atypical liver resection. Immunohistochemistry and histological results were compatible with a diagnosis of RDD. RDD is characterized by phenomena of emperipolesis, histiocytic proliferation and positive immunostaining for CD14, CD68 and S-100 protein. Cases of isolated gastrointestinal localization of RDD are particularly rare, especially in the liver. Instrumental exams might confuse RDD with other malignancies. RDD is a rare entity, which might be misdiagnosed using PET-CT due to its similarities with malignant tumors. An accurate multidisciplinary approach may help to clear diagnostic clues of this uncommon disease., Competing Interests: The authors have no conflicts of interest to disclose., (2022, International Research and Cooperation Association for Bio & Socio - Sciences Advancement.)

Background & Aims: About 15% of intrahepatic cholangiocarcinomas (iCCAs) express fibroblast growth factor receptor 2 (FGFR2) fusion proteins (FFs), usually alongside mutational inactivation of TP53, CDKN2A or BAP1. In FFs, FGFR2 residues 1-768 fuse to sequences encoded by a diverse array of partner genes (>60) causing oncogenic FF activation. While FGFR-specific tyrosine kinase inhibitors (F-TKI) provide clinical benefit in FF + iCCA, responses are partial and/or limited by resistance mechanisms, such as the V565F substitution in the FGFR2 gatekeeper residue. Improving on FF targeting in iCCA therefore remains a critical unmet need. Herein, we aimed to generate a murine model of FF-driven iCCA and use this to uncover actionable FF-associated dependencies., Methods: Four iCCA FFs carrying different fusion sequences were expressed in Tp53 -/- mouse liver organoids. Tumorigenic properties of genetically modified liver organoids were assessed by transplantation into immuno-deficient mice. Cellular models derived from neoplastic lesions were exploited for pre-clinical studies., Results: Transplantation of FF-expressing liver organoids yielded tumors diagnosed as CCA based on histological, phenotypic and transcriptomic analyses. The penetrance of this tumorigenic phenotype was influenced by FF identity. Tumor organoids and 2D cell lines derived from CCA lesions were addicted to FF signaling via Ras-Erk, regardless of FF identity or V565F mutation. Dual blockade of FF and the Ras-Erk pathway by concomitant pharmacological inhibition of FFs and Mek1/2 provided greater therapeutic efficacy than single agent F-TKI in vitro and in vivo., Conclusions: FF-driven iCCA pathogenesis was successfully modeled on a Tp53 -/- murine background, revealing biological heterogeneity among structurally different FFs. Double blockade of FF-ERK signaling deserves consideration for precision-based approaches against human FF + iCCA., Lay Summary: Intrahepatic cholangiocarcinoma (iCCA) is a rare cancer that is difficult to treat. A subtype of iCCA is caused by genomic alterations that generate oncogenic drivers known as FGFR2 fusions. Patients with FGFR2 fusions respond to FGFR inhibitors, but clinical responses are often of modest duration. We used animal and cellular models to show that FGFR2 fusions require the activity of a downstream effector named Mek1/2. We found that dual blockade of FGFR2 fusions and Mek1/2 was more effective than isolated inhibition of FGFR2 fusions, pointing to the potential clinical utility of dual FGFR2-MEK1/2 blockade in patients with iCCA., Competing Interests: Conflict of interest All Authors, except M.J.B., have no personal, professional or financial conflicts to disclose. M.J.B. disclosures: ADC Therapeutics – Consulting to self; Exelixis Pharmaceuticals – Consulting to self; Inspyr Therapeutics – Consulting to self; G1 Therapeutics – Consulting to self; Immunovative Therapies – Consulting to self; OncBioMune Pharmaceuticals – Consulting to self; Western Oncolytics – Consulting to self; Lynx Group – Consulting to self; Genentech – Consulting to self; Merck – Consulting to self; Huya – Consulting to self; Astra Zeneca – Travel Support to self. Please refer to the accompanying ICMJE disclosure forms for further details., (Copyright © 2021 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Inflammation might substantially contribute to the limited therapeutic success of current systemic therapies in colorectal cancer (CRC). Amongst cytokines involved in CRC biology, the proinflammatory chemokine IL-8 has recently emerged as a potential prognostic/predictive biomarker. Here, we show that BRAF mutations and PTEN-loss are associated with high IL-8 levels in CRC models in vitro and that BRAF/MEK/ERK, but not PI3K/mTOR, targeting controls its production in different genetic contexts. In particular, we identified a BRAF/ERK2/CHOP axis affecting IL-8 transcription, through regulation of CHOP subcellular localization, and response to targeted inhibitors. Moreover, RNA Pol II and an open chromatin status in the CHOP-binding region of the IL-8 gene promoter cooperate towards increased IL-8 expression, after a selective BRAF inhibition. Overall, our data show that IL-8 production is finely and differentially regulated depending on the tumor genetic context and might be targeted for therapeutic purposes in molecularly defined subgroups of CRC patients.

Tumors with microsatellite instability (MSI) are caused by a defective DNA mismatch repair system that leads to the accumulation of mutations within microsatellite regions. Indels in microsatellites of coding genes can result in the synthesis of frameshift peptides (FSP). FSPs are tumor-specific neoantigens shared across patients with MSI. In this study, we developed a neoantigen-based vaccine for the treatment of MSI tumors. Genetic sequences from 320 MSI tumor biopsies and matched healthy tissues in The Cancer Genome Atlas database were analyzed to select shared FSPs. Two hundred nine FSPs were selected and cloned into nonhuman Great Ape Adenoviral and Modified Vaccinia Ankara vectors to generate a viral-vectored vaccine, referred to as Nous-209. Sequencing tumor biopsies of 20 independent patients with MSI colorectal cancer revealed that a median number of 31 FSPs out of the 209 encoded by the vaccine was detected both in DNA and mRNA extracted from each tumor biopsy. A relevant number of peptides encoded by the vaccine were predicted to bind patient HLA haplotypes. Vaccine immunogenicity was demonstrated in mice with potent and broad induction of FSP-specific CD8 and CD4 T-cell responses. Moreover, a vaccine-encoded FSP was processed in vitro by human antigen-presenting cells and was subsequently able to activate human CD8 T cells. Nous-209 is an "off-the-shelf" cancer vaccine encoding many neoantigens shared across sporadic and hereditary MSI tumors. These results indicate that Nous-209 can induce the optimal breadth of immune responses that might achieve clinical benefit to treat and prevent MSI tumors. SIGNIFICANCE: These findings demonstrate the feasibility of an "off-the-shelf" vaccine for treatment and prevention of tumors harboring frameshift mutations and neoantigenic peptides as a result of microsatellite instability., (©2020 American Association for Cancer Research.)

A 65-year-old Caucasian man was admitted to our department with progressive weakness, dyspepsia, and weight loss. Endoscopic biopsies revealed a gastric diffuse large B cell lymphoma. Computed tomography scan showed a locally advanced bulky gastric tumor with peritoneal involvement. Laparoscopic staging was performed and biopsies showed peritoneal localization of adenocarcinoma. A new endoscopy with multiple mapping biopsies confirmed the diagnosis of diffuse large B cell lymphoma. A multidisciplinary team evaluated the stage of both tumors and the patient started on a chemotherapy regimen with cisplatin, 5-fluorouracil, and trastuzumab (ToGA). He is currently alive with disease at 9 months after surgery. Synchronous occurrence of primary lymphoma and adenocarcinoma of the stomach is extremely rare. Symptomatology is similar and accurate diagnosis requires a combination of both morphological and immunohistochemical analyses. Treatment should be planned considering the stage of both diseases., Competing Interests: Conflict of InterestThe authors declare that they have no conflict of interest., (© Indian Association of Surgical Oncology 2020.)

HIPK2 is a DYRK-like kinase involved in cellular stress response pathways, development, and cell division. Two alternative splice variants of HIPK2, HIPK2-FL and HIPK2-Δe8, have been previously identified as having different protein stability but similar functional activity in the stress response. Here, we describe one additional HIPK2 splice variant with a distinct subcellular distribution and functional activity in cytokinesis. This novel splice variant lacks the last two exons and retains intron13 with a stop codon after 89 bp of the intron, generating a short isoform, HIPK2-S, that is detectable by 2D Western blots. RT-PCR analyses of tissue arrays and tumor samples show that HIPK2-FL and HIPK2-S are expressed in normal human tissues in a tissue-dependent manner and differentially expressed in human colorectal and pancreatic cancers. Gain- and loss-of-function experiments showed that in contrast to HIPK2-FL, HIPK2-S has a diffuse, non-speckled distribution and is not involved in the DNA damage response. Rather, we found that HIPK2-S, but not HIPK2-FL, localizes at the intercellular bridge, where it phosphorylates histone H2B and spastin, both required for faithful cell division. Altogether, these data show that distinct human HIPK2 splice variants are involved in distinct HIPK2-regulated functions like stress response and cytokinesis.

Background: To analyze long-term results and risk of relapse in the clinical TNM stages II and III, mid-low rectal cancer patients (RC pts), treated with transanal local excision (LE) after major response to neoadjuvant chemoradiation (n-CRT)., Methods: Thirty-two out of 345 extraperitoneal cT3-4 or N+ RC pts (9.3%) underwent LE., Inclusion Criteria: extraperitoneal RC, adenocarcinoma, ECOG Performance Status ≤2. Pts with distant metastases were excluded., Results: All pts showed histologically clear margins of resection and 81.2% were restaged ypT0/mic/1. Nine out of 32 (28.1%) pts relapsed: 7 (21.8%) showed a local recurrence, of which 5 (15.6%) at the endorectal suture, 1 (3.1%) pelvic and 1 (3.1%) mesorectal. Two pts (6.2%) relapsed distantly. Among the pT0/1, 11.5% relapsed vs 100% of the pT2 and pT4 ones. The six pts relapsing locally or in the mesorectal fat underwent a salvage total mesorectal excision surgery. The old patient with pelvic recurrence relapsed after 108 months and underwent a re-irradiation; the two pts with distant metastases were treated with chemotherapy followed by radical surgery., Conclusions: Presently combined approach seems a valid option in major responders, confirming its potential curative impact in the ypT0/mic/1 pts. A strict selection of pts is basic to obtain favourable results., (© 2019 Wiley Periodicals, Inc.)

Colorectal cancer (CRC) is one of the most common malignant tumors worldwide and understanding its underlying molecular mechanisms is crucial for the development of therapeutic strategies. The mitogen-activated protein kinase-kinase 3 (MKK3) is a specific activator of p38 MAP kinases (p38 MAPKs), which contributes to the regulation of several cellular functions, such as proliferation, differentiation, apoptosis as well as response to drugs. At present, the exact MKK3/p38 MAPK pathway contribution in cancer is heavily debated because of its pleiotropic function. In this work, we retrospectively explored the prognostic and pathobiologic relevance of MKK3 in a cohort of CRC patients and assessed MKK3 molecular functions in a panel of CRC lines and colonocytes primary cultures. We found increased MKK3 levels in late-stage CRC patients which correlated with shorter overall survival. Herein, we report that the MKK3 targeting by inducible RNA interference univocally exerts antitumor effects in CRC lines but not in primary colonocytes. While MKK3 depletion per se affects growth and survival by induction of sustained autophagy and death in some CRC lines, it potentiates response to chemotherapeutic drug 5-fluorouracil (5-FU) in all of the tested CRC lines in vitro. Here, we demonstrate for the first time that in CRC the MKK3 specifically activates p38delta MAPK isoform to sustain prosurvival signaling and that such effect is exacerbated upon 5-FU challenge. Indeed, p38delta MAPK silencing recapitulates MKK3 depletion effects in CRC cells in vitro and in vivo. Overall, our data identified a molecular mechanism through which MKK3 supports proliferation and survival signaling in CRC, further supporting MKK3 as a novel and extremely attractive therapeutic target for the development of promising strategies for the management of CRC patients.

Importance: Colorectal carcinomas in patients with Lynch syndrome (LS) arise in a background of mismatch repair (MMR) deficiency, display a unique immune profile with upregulation of immune checkpoints, and response to immunotherapy. However, there is still a gap in understanding the pathogenesis of MMR-deficient colorectal premalignant lesions, which is essential for the development of novel preventive strategies for LS., Objective: To characterize the immune profile of premalignant lesions from a cohort of patients with LS., Design, Setting, and Participants: Whole-genome transcriptomic analysis using next-generation sequencing was performed in colorectal polyps and carcinomas of patients with LS. As comparator and model of MMR-proficient colorectal carcinogenesis, we used samples from patients with familial adenomatous polyposis (FAP). In addition, a total of 47 colorectal carcinomas (6 hypermutants and 41 nonhypermutants) were obtained from The Cancer Genome Atlas (TCGA) for comparisons. Samples were obtained from the University of Texas MD Anderson Cancer Center and "Regina Elena" National Cancer Institute, Rome, Italy. All diagnoses were confirmed by genetic testing. Polyps were collected at the time of endoscopic surveillance and tumors were collected at the time of surgical resection. The data were analyzed from October 2016 to November 2017., Main Outcomes and Measures: Assessment of the immune profile, mutational signature, mutational and neoantigen rate, and pathway enrichment analysis of neoantigens in LS premalignant lesions and their comparison with FAP premalignant lesions, LS carcinoma, and sporadic colorectal cancers from TCGA., Results: The analysis was performed in a total of 28 polyps (26 tubular adenomas and 2 hyperplastic polyps) and 3 early-stage LS colorectal tumors from 24 patients (15 [62%] female; mean [SD] age, 48.12 [15.38] years) diagnosed with FAP (n = 10) and LS (n = 14). Overall, LS polyps presented with low mutational and neoantigen rates but displayed a striking immune activation profile characterized by CD4 T cells, proinflammatory (tumor necrosis factor, interleukin 12) and checkpoint molecules (LAG3 [lymphocyte activation gene 3] and PD-L1 [programmed cell death 1 ligand 1]). This immune profile was independent of mutational rate, neoantigen formation, and MMR status. In addition, we identified a small subset of LS polyps with high mutational and neoantigen rates that were comparable to hypermutant tumors and displayed additional checkpoint (CTLA4 [cytotoxic T-lymphocyte-associated protein 4]) and neoantigens involved in DNA damage response (ATM and BRCA1 signaling)., Conclusions and Relevance: These findings challenge the canonical model, based on the observations made in carcinomas, that emphasizes a dependency of immune activation on the acquisition of high levels of mutations and neoantigens, thus opening the door to the implementation of immune checkpoint inhibitors and vaccines for cancer prevention in LS.

Genomic technologies are reshaping the molecular landscape of colorectal cancer (CRC), revealing that oncogenic driver mutations (APC and TP53) coexist with still underappreciated genetic events. We hypothesized that mutational analysis of CRC-linked genes may provide novel information on the connection between genetically-deregulated pathways and clinical outcomes. We performed next-generation sequencing (NGS) analysis of 16 recurrently mutated genes in CRC exploiting tissue specimens from 98 advanced CRC patients. Multiple correspondence analysis (MCA) was used to identify gene sets characterizing negative and positive outliers (patients in the lowest and highest quartile of progression-free survival, PFS). Variables potentially affecting PFS and overall survival (OS) were tested in univariate and multivariate Cox proportional hazard models. Sensitivity analyses and resampling were used to assess the robustness of genomic predictors. MCA revealed that APC and TP53 mutations were close to the negative outlier group, whereas mutations in other WNT pathway genes were in proximity of the positive outliers. Reasoning that genetic alterations interact epistatically, producing greater or weaker consequences in combination than when individually considered, we tested whether patients whose tumors carried a genetic background characterized by APC and TP53 mutations without coexisting mutations in other WNT genes (AMER1, FBXW7, TCF7L2, CTNNB1, SOX9) had adverse survival outcomes. With this approach, we identified two oncodriver signatures (ODS1 and ODS2) associated with shorter PFS (ODS1 multivariate Cox for PFS: HR 2.16, 95%CI: 1.28-3.64, p = 0.004; ODS2 multivariate Cox for PFS: HR 2.61, 95%CI: 1.49-4.58, p = 0.001). Clinically-focused and molecularly-focused sensitivity analyses, resampling, and reclassification of mutations confirmed the stability of ODS1/2. Moreover, ODS1/2 negatively impacted OS. Collectively, our results point to co-occurring driver mutations as an adverse molecular factor in advanced CRC. This relationship depends on a broader genetic context highlighting the importance of genetic interactions.

Whether PD-L1 expression is associated with survival outcomes in gastric cancer (GC) is controversial. The inhibition of the PD-1/PD-L1 pathway is effective against genomically unstable tumors. Hypothesizing that also the clinical significance of PD-L1 might be dependent on the activation of molecular circuits ensuring genomic stability, we evaluated PD-L1 expression in tissue samples from 72 advanced GC patients treated with first-line chemotherapy. Samples were already characterized for DNA damage repair (DDR) component expression (pATM, pChk1, pWee1, γ-H2AX and pRPA2) along with mutations in DDR-linked genes ( TP53 and ARID1A ). Overall, PD-L1 expression was not associated with progression-free survival (PFS) and overall survival (OS), independently on whether we considered its expression in tumor cells (PD-L1-TCs) or in the immune infiltrate (PD-L1-TILs). In subgroup analysis, positive PD-L1-TC immunostaining was associated with better PFS in patients whose tumors did not carry DDR activation (multivariate Cox: HR 0.34, 95%CI: 0.15-0.76, p = 0.008). This subset (DDR off ) was characterized by negative pATM expression or the presence of ARID1A mutations. Conversely, the relationship between PD-L1-TC expression and PFS was lost in a molecular scenario denoting DDR activation (DDR on ), as defined by concomitant pATM expression and ARID1A wild-type form. Surprisingly, while PD-L1-TC expression was associated with better OS in the DDR off subset (multivariate Cox: HR 0.41, 95% CI: 0.17-0.96, p = 0.039), in the DDR on subgroup we observed an opposite impact on OS (multivariate Cox: HR 2.56, 95%CI: 1.06-6.16, p = 0.036). Thus, PD-L1-TC expression may impact survival outcomes in GC on the basis of the activation/inactivation of genome-safeguarding pathways.

Background: The role of Mandard's tumor regression grade (TRG) classification is still controversial in defining the prognostic role of patients who have undergone neoadjuvant chemoradiation (CRT) and total mesorectal excision. The present study evaluated multiple correspondence analysis (MCA) as a tool to better cluster variables, including TRG, for a homogeneous prognosis., Patients and Methods: A total of 174 patients with a minimum follow-up period of 10 years were stratified into 2 groups: group A (TRG 1-3) and group B (TRG 4-5) using Mandard's classification. Overall survival and disease-free survival were analyzed using univariate and multivariate analysis. Subsequently, MCA was used to analyze TRG plus the other prognostic variables., Results: The overall response to CRT was 55.7%, including 13.2% with a pathologic complete response. TRG group A correlated strictly with pN status (P = .0001) and had better overall and disease-free survival than group B (85.1% and 75.6% vs. 71.1% and 67.3%; P = .06 and P = .04, respectively). The TRG 3 subset (about one third of our series) showed prognostically heterogeneous behavior. In addition to multivariate analysis, MCA separated TRG 1 and TRG 2 versus TRG 4 and TRG 5 well and also allocated TRG 3 patients close to the unfavorable prognostic variables., Conclusion: TRG classification should be used in all pathologic reports after neoadjuvant CRT and radical surgery to enrich the prognostic profile of patients with an intermediate risk of relapse and to identify patients eligible for more conservative treatment. Thus, MCA could provide added value., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background: An extensive crosstalk co-regulates the Hippo and Wnt pathway. Preclinical studies revealed that the Hippo transducers YAP/TAZ mediate a number of oncogenic functions in gastric cancer (GC). Moreover, comprehensive characterization of GC demonstrated that the Wnt pathway is targeted by oncogenic mutations. On this ground, we hypothesized that YAP/TAZ- and Wnt-related biomarkers may predict clinical outcomes in GC patients treated with chemotherapy., Methods: In the present study, we included 86 patients with advanced GC treated with first-line chemotherapy in prospective phase II trials or in routine clinical practice. Tissue samples were immunostained to evaluate the expression of YAP/TAZ. Mutational status of key Wnt pathway genes (CTNNB1, APC and FBXW7) was assessed by targeted DNA next-generation sequencing (NGS). Survival curves were estimated and compared by the Kaplan-Meier product-limit method and the log-rank test, respectively. Variables potentially affecting progression-free survival (PFS) were verified in univariate Cox proportional hazard models. The final multivariate Cox models were obtained with variables testing significant at the univariate analysis, and by adjusting for all plausible predictors of the outcome of interest (PFS)., Results: We observed a significant association between TAZ expression and Wnt mutations (Chi-squared p = 0.008). Combined TAZ expression and Wnt mutations (TAZ pos /WNT mut ) was more frequently observed in patients with the shortest progression-free survival (negative outliers) (Fisher p = 0.021). Uni-and multivariate Cox regression analyses revealed that patients whose tumors harbored the TAZ pos /WNT mut signature had an increased risk of disease progression (univariate Cox: HR 2.27, 95% CI 1.27-4.05, p = 0.006; multivariate Cox: HR 2.73, 95% CI 1.41-5.29, p = 0.003). Finally, the TAZ pos /WNT mut signature negatively impacted overall survival., Conclusions: Collectively, our findings indicate that the oncogenic YAP/TAZ-Wnt crosstalk may be active in GC, conferring chemoresistant traits that translate into adverse survival outcomes.

Introduction: Recent studies indicated tumors may be comprised of heterogeneous molecular subtypes and incongruent molecular portraits may emerge if different areas of the tumor are sampled. This study explored the impact of intra-tumoral heterogeneity in terms of activation/phosphorylation of FDA approved drug targets and downstream kinase substrates., Material and Methods: Two independent sets of liver metastases from colorectal cancer were used to evaluate protein kinase-driven signaling networks within different areas using laser capture microdissection and reverse phase protein array., Results: Unsupervised hierarchical clustering analysis indicated that the signaling architecture and activation of the MAPK and AKT-mTOR pathways were consistently maintained within different regions of the same biopsy. Intra-patient variability of the MAPK and AKT-mTOR pathway were <1.06 fold change, while inter-patients variability reached fold change values of 5.01., Conclusions: Protein pathway activation mapping of enriched tumor cells obtained from different regions of the same tumor indicated consistency and robustness independent of the region sampled. This suggests a dominant protein pathway network may be activated in a high percentage of the tumor cell population. Given the genomic intra-tumoral variability, our data suggest that protein/phosphoprotein signaling measurements should be integrated with genomic analysis for precision medicine based analysis.

8-Oxoguanine, a common mutagenic DNA lesion, generates G:C>T:A transversions via mispairing with adenine during DNA replication. When operating normally, the MUTYH DNA glycosylase prevents 8-oxoguanine-related mutagenesis by excising the incorporated adenine. Biallelic MUTYH mutations impair this enzymatic function and are associated with colorectal cancer (CRC) in MUTYH-Associated Polyposis (MAP) syndrome. Here, we perform whole-exome sequencing that reveals a modest mutator phenotype in MAP CRCs compared to sporadic CRC stem cell lines or bulk tumours. The excess G:C>T:A transversion mutations in MAP CRCs exhibits a novel mutational signature, termed Signature 36, with a strong sequence dependence. The MUTYH mutational signature reflecting persistent 8-oxoG:A mismatches occurs frequently in the APC, KRAS, PIK3CA, FAT4, TP53, FAT1, AMER1, KDM6A, SMAD4 and SMAD2 genes that are associated with CRC. The occurrence of Signature 36 in other types of human cancer indicates that DNA 8-oxoguanine-related mutations might contribute to the development of cancer in other organs., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

The DNA damage response (DDR) network is exploited by cancer cells to withstand chemotherapy. Gastric cancer (GC) carries deregulation of the DDR and harbors genetic defects that fuel its activation. The ATM-Chk2 and ATR-Chk1-Wee1 axes are deputed to initiate DNA repair. Overactivation of these pathways in cancer cells may represent an adaptive response for compensating genetic defects deregulating G 1 -S transition (e.g., TP53) and ATM/ATR-initiated DNA repair (e.g., ARID1A). We hypothesized that DDR-linked biomarkers may predict clinical outcomes in GC patients treated with chemotherapy. Immunohistochemical assessment of DDR kinases (pATM, pChk2, pChk1 and pWee1) and DNA damage markers (γ-H2AX and pRPA32) was performed in biological samples from 110 advanced GC patients treated with first-line chemotherapy, either in phase II trials or in routine clinical practice. In 90 patients, this characterization was integrated with targeted ultra-deep sequencing for evaluating the mutational status of TP53 and ARID1A. We recorded a positive association between the investigated biomarkers. The combination of two biomarkers (γ-H2AX high /pATM high ) was an adverse factor for both progression-free survival (multivariate Cox: HR 2.23, 95%CI: 1.47-3.40) and overall survival (multivariate Cox: HR: 2.07, 95%CI: 1.20-3.58). The relationship between the γ-H2AX high /pATM high model and progression-free survival was consistent across the different TP53 backgrounds and was maintained in the ARID1A wild-type setting. Conversely, this association was no longer observed in an ARID1A-mutated subgroup. The γ-H2AX high /pATM high model negatively impacted survival outcomes in GC patients treated with chemotherapy. The mutational status of ARID1A, but apparently not TP53 mutations, affects its predictive significance., (© 2017 UICC.)

Background & Aims: There is high need of novel diagnostic and prognostic tools for tumors of the digestive system, such as gastric cancer and cholangiocarcinoma. We recently found that miR-204 was deeply downregulated in gastric cancer tissues. Here we investigated whether this was common to other tumors of the digestive system and whether this elicited a miR-204-dependent gene target signature, diagnostically and therapeutically relevant. Finally, we assessed the contribution of the identified target genes to the cell cycle progression and clonogenicity of gastric cancer and cholangiocarcinoma cell lines., Methods: We employed quantitative PCR and Affymetrix profiling for gene expression studies. In silico analysis aided us to identifying a miR-204 target signature in publicly available databases (TGCA). We employed transient transfection experiments, clonogenic assays and cell cycle profiling to evaluate the biological consequences of miR-204 perturbation., Results: We identified a novel miR-204 gene target signature perturbed in gastric cancer and in cholangiocarcinoma specimens. We validated its prognostic relevance and mechanistically addressed its biological relevance in GC and CC cell lines., Conclusions: We suggest that restoring the physiological levels of miR-204 in some gastrointestinal cancers might be exploited therapeutically.

Although the medical treatment of colorectal cancer has evolved greatly in the last years, a significant portion of early-stage patients develops recurrence after therapies. The current clinical trials are directed to evaluate new drug combinations and treatment schedules. By the use of patient-derived or established colon cancer cell lines, we found that the tyrosine kinase receptor HER3 is involved in the mechanisms of resistance to therapies. In agreement, the immunohistochemical analysis of total and phospho-HER3 expression in 185 colorectal cancer specimens revealed a significant correlation with lower disease-free survival. Targeting HER3 by the use of the monoclonal antibody patritumab we found induction of growth arrest in all cell lines. Despite the high efficiency of patritumab in abrogating the HER3-dependent activation of PI3K pathway, the HER2 and EGFR-dependent MAPK pathway is activated as a compensatory mechanism. Interestingly, we found that the MEK-inhibitor trametinib inhibits, as expected, the MAPK pathway but induces the HER3-dependent activation of PI3K pathway. The combined treatment results in the abrogation of both PI3K and MAPK pathways and in a significant reduction of cell proliferation and survival. These data suggest a new strategy of therapy for HER3-overexpressing colon cancers., Competing Interests: CONFLICTS OF INTEREST No potential conflicts of interest were disclosed

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease in need of prognostic markers to address therapeutic choices. We have previously shown that alternative splicing of the actin regulator, hMENA, generates hMENA 11a , and hMENAΔv6 isoforms with opposite roles in cell invasion. We examined the expression pattern of hMENA isoforms by immunohistochemistry, using anti-pan hMENA and specific anti-hMENA 11a antibodies, in 285 PDACs, 15 PanINs, 10 pancreatitis, and normal pancreas. Pan hMENA immunostaining, absent in normal pancreas and low-grade PanINs, was weak in PanIN-3 and had higher levels in virtually all PDACs with 64% of cases showing strong staining. Conversely, the anti-invasive hMENA 11a isoform only showed strong staining in 26% of PDAC. The absence of hMENA 11a in a subset (34%) of pan-hMENA-positive tumors significantly correlated with poor outcome. The functional effects of hMENA isoforms were analyzed by loss and gain of function experiments in TGF-β1-treated PDAC cell lines. hMENA 11a knock-down in PDAC cell lines affected cell-cell adhesion but not invasion. TGF-β1 cooperated with β-catenin signaling to upregulate hMENA and hMENAΔv6 expression but not hMENA 11a In the absence of hMENA 11a , the hMENA/hMENAΔv6 up-regulation is crucial for SMAD2-mediated TGF-β1 signaling and TGF-β1-induced EMT. Since the hMENA isoform expression pattern correlates with patient outcome, the data suggest that hMENA splicing and related pathways are novel key players in pancreatic tumor microenvironment and may represent promising targets for the development of new prognostic and therapeutic tools in PDAC.

We previously identified EMICORON as a novel G-quadruplex (G4) ligand showing high selectivity for G4 structures over the duplex DNA, causing telomere damage and inhibition of cell proliferation in transformed and tumor cells. Here, we evaluated the antitumoral effect of EMICORON on advanced models of human colon cancer that could adequately predict human clinical outcomes. Our results showed that EMICORON was well tolerated in mice, as no adverse effects were reported, and a low ratio of sensitivity across human and mouse bone marrow cells was observed, indicating a good potential for reaching similar blood levels in humans. Moreover, EMICORON showed a marked therapeutic efficacy, as it inhibited the growth of patient-derived xenografts (PDX) and orthotopic colon cancer and strongly reduced the dissemination of tumor cells to lymph nodes, intestine, stomach, and liver. Finally, activation of DNA damage and impairment of proliferation and angiogenesis are proved to be key determinants of EMICORON antitumoral activity. Altogether, our results, performed on advanced experimental models of human colon cancer that bridge the translational gap between preclinical and clinical studies, demonstrated that EMICORON had an unprecedented antitumor activity warranting further studies of EMICORON-based combination treatments., (©2015 American Association for Cancer Research.)

Colorectal carcinoma remains among the most frequent causes of cancer death. Besides the well-known genetic predisposition, a key role in colorectal adenoma and adenocarcinoma etio-pathogenesis, mainly in sporadic cases, is played by definite risk factors, such as obesity, type 2 diabetes, insulin resistance, hyper-insulinemia, and insulin therapy. These epidemiological data motivated us to determine, by means of immunohistochemistry, the amount of activated (phosphorylated) insulin receptor in archival samples from 22 colorectal adenoma and 117 adenocarcinoma patients, with the objective to estimate the role of this factor in colorectal epithelium transformation and cancer progression. Statistical analysis of the results clearly showed that positive staining for phosphorylated insulin receptor was significantly more frequent in adenomas than adenocarcinomas (P < 0.0001) and, within the adenocarcinoma cohort, it was more frequent in low-grade tumors (P = 0.005). In adenomas, staining was exclusively cytoplasmic, while in adenocarcinomas it was cytoplasmic and/or nuclear (P < 0.0001). Interestingly, disease-free survival in colorectal adenocarcinoma patients pointed out a significantly better prognosis for those bearing a positive staining for phosphorylated insulin receptor (P = 0.02). From these data, we can argue that activated insulin receptor plays a fundamental role at the early stages of tumorigenesis, where late stages could be characterized by a shift toward more active oncogenic drivers. Determining the amount of phosphorylated insulin receptor could thus represent a novel prognostic/predictive tool in colorectal adenocarcinoma patients., (© 2014 Wiley Periodicals, Inc., A Wiley Company.)

Background: HPV is one of the most common sexually transmitted infections. However little is known about its prevalence in the male population and concordance with female partners., Objectives: This cross-sectional study aimed to: (a) investigate HPV prevalence and genotype distribution among a series of stable male sexual partners of CIN/HPV positive women and (b) assess HPV infection and type-specific concordance between partners., Study Design: 378 stable and monogamous male partners of CIN/HPV positive women were selected. Of these, 238 cases were enrolled at the same time as their female partner. All the subjects were tested by the Linear Array HPV genotyping assay., Results: Overall, 153/378 men (40.5%) and 122/238 women (51.3%) were positive for at least one of the 37 HPV types detectable by the assay used. Among the HPV-positive participants, 69 of the 378 men (18.2%) and 54 of the 238 women (22.7%) harboured multiple genotypes. 75 couples (31.5%) were concordantly HPV positive, while 102 couples (42.9%) were concordantly negative (Kappa value: 0.491, p<0.0001). Among the couples in which both partners were HPV positive, 68% harboured at least one genotype in common. Results from a GEE model evidenced that when the male partner tested HPV positive for at least one genotype, this had a significant effect on the positivity of their relative female partner (p<0.0001)., Conclusions: We evidenced a high prevalence of HPV male infections and a moderate concordance between partners. However, we observed a significant HPV type-specific correlation between partners, which is unlikely to be coincidental., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background: Adjuvant chemotherapy (AC) in Stage II Colon Cancer (CC) is still under debate. Choice should be based on patients and disease characteristics. According to guidelines AC should be considered in high-risk T3N0 patients. No data are available for better option in low-risk patients. The aim of the study is to retrospectively evaluate relapse-free survival (RFS) and disease-free survival (DFS) according to treatment received in T3N0 CC., Methods: RFS and DFS are evaluated with Kaplan-Meier method. Multivariate Cox proportional hazard model was developed using stepwise regression, enter limit and remove limit were p = 0.10 and p = 0.15, respectively., Results: 834 patients with T3N0 CC were recruited. Median age was 69 (29-93), M/F 463/371, 335 low-risk patients (40.2%), 387 high-risk (46.4%), 112 unknown (13.4%); 127 (15.2%) patients showed symptoms at diagnosis. Median sampled lymph nodes were 15 (1-76); 353 (42.3%) patients were treated with AC. Median follow up was 5 years (range 3-24). The 5-years RFS was 78.4% and the 5-years DFS was 76.7%. At multivariate analysis symptoms, lymph nodes, and adjuvant chemotherapy were prognostic factors for RFS. AC is prognostic factor for all endpoints. In low-risk group 5-years RFS was 87.3% in treated patients and 74.7% in non-treated patients (p 0.03); in high-risk group was respectively 82.7% and 71.4% (p 0.005)., Conclusions: Data confirmed the role of known prognostic factors and suggest the relevance of adjuvant chemotherapy also in low-risk stage II T3N0 CC patients. However, the highest risk in low-risk subgroup should be identified to be submitted to AC.

In a prospective multicenter phase II trial of radioembolization with yttrium-90 ((90)Y-RE) in chemorefractory liver-dominant metastatic colorectal cancer (mCRC), we showed that median survival was 12.6 months (95% CI 7.0-18.3) with 48% of 50 patients achieving disease control. In this extension retrospective study, we analyzed whether a panel of biomarkers, known to be associated to an adverse clinical outcome, underwent variations in CRC liver metastases pre and post (90)Y-RE.Of the 50 patients included in the study, 29 pre-(90)Y-RE therapy and 15 post-(90)Y-RE had liver biopsy specimens available. In these series we investigated survivin, p53, Bcl-2 and Ki-67 expression pre- and post-(90)Y-RE by immuhistochemistry (IHC). Our findings evidenced a decrease of survivin (77% vs 33%), p53 (93% vs 73%), Bcl-2 (37% vs 26%) expression as well as of Ki-67 proliferation index (62.5% vs 40%) on liver biopsies collected post-(90)Y-RE as compared to pre-(90)Y-RE. In the subset of 13 matched liver metastases we further confirmed the reduction of survivin (92.3% vs 53.8%; p = 0.06), p53 (100% vs 69.2%; p = 0.05) and Bcl-2 (69.2% vs 53.8%; p = 0.05) expression post-(90)Y-RE. This biomarker modulation was accompanied by morphological changes as steatohepatitis, hepatocyte necrosis, collagen deposition, proliferating and/or bile duct ectasia, focal sinusoidal dilatation and fibrosis.Although our analysis was conducted in a very limited number cases, these changes appear strictly related to the response to (90)Y-RE therapy and may deserve further investigation on a larger series of patients.

Background: Human Leukocyte Antigen (HLA)-E is a non-classical class I HLA molecule that can be stabilized by ligands donated by other classical (HLA-A, -B, -C) and non-classical (HLA-G) family members. HLA-E engages a variety of immune receptors expressed by cytotoxic T lymphocytes (CTLs), Natural killer (NK) cells and NK-CTLs. In view of the opposing outcomes (activation or inhibition) of the different HLA-E receptors, the preferred role (if any) of HLA-E expressed in vivo on tumor cells remains to be established., Methods: Taking advantage of MEM-E/02, a recently characterized antibody to denatured HLA-E molecules, HLA-E expression was assessed by immunohistochemistry on an archival collection (formalin-fixed paraffin-embedded) of 149 colorectal primary carcinoma lesions paired with their morphologically normal mucosae. Lymphoid infiltrates were assessed for the expression of the HLA-E-specific, inhibitory, non-rearranging receptor NKG2A., Results: High HLA-E expression did not significantly correlate with the expression of classical HLA-B and HLA-C molecules, but it did correlate with high expression of its preferential ligand donor HLA-A. In addition, it correlated with lymphoid cell infiltrates expressing the inhibitory NKG2A receptor, and was an independent predictor of good prognosis, particularly in a subset of patients whose tumors express HLA-A levels resembling those of their paired normal counterparts (HLA-A). Thus, combination phenotypes (HLA-Elo-int/HLA-AE and HLA-Ehi/HLA-AE) of classical and non-classical class I HLA molecules mark two graded levels of good prognosis., Conclusions: These results suggest that HLA-E favors activating immune responses to colorectal carcinoma. They also provide evidence in humans that tumor cells entertain extensive negotiation with the immune system until a compromise between recognition and escape is reached. It is implied that this process occurs stepwise, as predicted by the widely accepted 'immunoediting' model.

Background: This multi-centre phase II clinical trial is the first prospective evaluation of radioembolisation of patients with colorectal liver metastases (mCRC) who failed previous oxaliplatin- and irinotecan-based systemic chemotherapy regimens., Methods: Eligible patients had adequate hepatic, haemopoietic and renal function, and an absence of major hepatic vascular anomalies and hepato-pulmonary shunting. Gastroduodenal and right gastric arteries were embolised before hepatic arterial administration of yttrium-90 resin microspheres (median activity, 1.7 GBq; range, 0.9-2.2)., Results: Of 50 eligible patients, 38 (76%) had received > or =4 lines of chemotherapy. Most presented with synchronous disease (72%), >4 hepatic metastases (58%), 25-50% replacement of total liver volume (60%) and bilateral spread (70%). Early and intermediate (>48 h) WHO G1-2 adverse events (mostly fever and pain) were observed in 16 and 22% of patients respectively. Two died due to renal failure at 40 days or liver failure at 60 days respectively. By intention-to-treat analysis using Response Evaluation Criteria in Solid Tumours, 1 patient (2%) had a complete response, 11 (22%) partial response, 12 (24%) stable disease, 22 (44%) progressive disease; 4 (8%) were non-evaluable. Median overall survival was 12.6 months (95% CI, 7.0-18.3); 2-year survival was 19.6%., Conclusion: Radioembolisation produced meaningful response and disease stabilisation in patients with advanced, unresectable and chemorefractory mCRC.

Background: Responsiveness to Cetuximab alone can be mediated by an increase of Epidermal Growth factor Receptor (EGFR) Gene Copy Number (GCN). Aim of this study was to assess the role of EGFR-GCN in advanced colorectal cancer (CRC) patients receiving chemotherapy plus Cetuximab., Methods: One hundred and one advanced CRC patients (43 untreated- and 58 pre-treated) were retrospectively studied by fluorescence in situ hybridization (FISH) to assess EGFR-GCN and by immunohistochemistry (IHC) to determine EGFR expression. Sixty-one out of 101 patients were evaluated also for k-ras status by direct sequencing. Clinical end-points were response rate (RR), progression-free survival (PFS) and overall survival (OS)., Results: Increased EGFR-GCN was found in 60/101 (59%) tumor samples. There was no correlation between intensity of EGFR-IHC and EGFR-GCN (p = 0.43). Patients receiving chemotherapy plus Cetuximab as first line treatment had a RR of 70% (30/43) while it was 18% (10/56) in the group with previous lines of therapy (p < 0.0001). RR was observed in 29/60 (48%) of patients with increased EGFR-GCN and in 6/28 (21%) in those without (p = 0.02). At multivariate analyses, number of chemotherapy lines and increased EGFR-GCN were predictive of response; EGFR-IHC score, increased EGFR-GCN and number of chemotherapy lines were significantly associated with a significant better PFS. Response to therapy was the only prognostic predictive factor for OS. In the 60 patients analyzed for k-ras mutations, number of chemotherapy lines, increased EGFR-GCN and k-ras wild type status predicted a better PFS., Conclusion: In metastatic CRC patients treated with chemotherapy plus Cetuximab number of chemotherapy lines and increased EGFR-GCN were significantly associated with a better clinical outcome, independent of k-ras status.

Multiple primary malignant neoplasms (MPMN) are not uncommon, however, finding more than three primary malignancies in one individual is unusual. Surviving five malignancies is considered exceptional. Two patients surviving five primary malignant neoplasms for 12 and 18 years are reported: a 55-year-old woman with a squamous cell carcinoma of the larynx, two carcinomas of the breast, a carcinoma of the kidney and an adenocarcinoma of the colon, and a 75-year-old woman with a sarcoma of the myometrium, a carcinoma of the thyroid, an adenocarcinoma of the rectum, a leiomyosarcoma of the colon and a bronchial carcinoid. Only twelve other reported cases with five or more primary infiltrating malignancies involving more than three sites, diagnosed while the patient was alive have been found. Relevant features were that colon cancer was quite often present more than once and survival was longer than expected for the stage (median overall survival, 20 years; 95% confidence interval: 12-28 years).

Genital human papillomavirus (HPV) is the causative agent of cervical cancer and is the most common sexually transmitted infection. Only limited and controversial data are available regarding HPV transmission in male sexual partners of women with cervical intraepithelial neoplasia (CIN). The aim of this study was to investigate the prevalence and the genotype distribution of HPV in penile scrapings of a series of Italian men, who had no visible penile lesions and were partners of women who were affected, or had been affected previously by cervical intraepithelial neoplasia or who were infected with HPV. The concordance of the viral group in the infected partners was determined. A total of 77 penile scrapings were screened for HPV infection by the polymerase chain reaction, while 59 cervicovaginal brushings of their female partners were tested. 35% of evaluable male samples and 64% of female sexual partners were found to be HPV positive. In the 55 simultaneously evaluable couples, a concordance of 45% was found, 11 couples (20%) with both partners being HPV negative and 14 couples (25%) with both partners HPV positive (P=0.001). Six out of the 14 couples (43%), where both partners were HPV positive, harbored the same HPV genotype group. These data, although preliminary, could support further the hypothesis that male HPV infection is more frequent in sexual partners of HPV positive or women with cervical intraepithelial neoplasia indicating that men could represent an important source of HPV transmission between sex partners.

Young people (40 years of age) with colorectal cancer (CRC) represent a distinct subgroup with more aggressive disease behaviour compared to older patients. We evaluate whether p53 and bcl-2 could be useful in identifying young patients at higher risk of tumour progression. We reviewed 1340 CRC patients with 58 patients 40 years (4.2%). They had more frequent moderately or poorly differentiated mucinous adenocarcinomas (26% versus 12.3%, p=0.03); higher advanced stage at diagnosis; shorter 5-year overall survival (49.8% versus 71%; p=0.02); more frequent p53 positive (89.8% versus 72.6%, p<0.05) and bcl-2 negative (88.0% versus 66.2%, p<0.05) tumours; no difference in DNA content or proliferation indexes. Moreover, p53+ and bcl-2- resulted in being independent predictors of survival with shorter survival for the p53+/bcl-2- patients. Combining p53 and bcl-2, we could identify young CRC patients at higher risk of progression, who probably require development of a more sophisticated therapeutic approach based on identification of predictive factors.

Aim: Survivin is a member of the inhibitors of apoptosis (IAP) gene family that acts through pathways different from those involving the bcl-2 family. Largely undetectable in normal adult tissues, survivin is deregulated in most human cancers including non-small-cell lung cancer (NSCLC) and may represent a tumor marker with prognostic and therapeutic implications. Aim of our study was to determine the prognostic role of survivin as an apoptosis-related biomarker in a series of resected NSCLC patients., Methods: A retrospective series of resected NSCLC patients were retrieved from the files of the Regina Elena National Cancer Institute. Survivin was detected by immunohistochemistry (IHC) using a polyclonal antibody. Survivin displayed two kinds of immunoreactivity: (i) a diffuse cytoplasmic staining and (ii) a distinct nuclear staining. A score-scale to distinguish positive (score 1-2) vs. negative (score 0) pattern was applied. Clinical and biological (nuclear and cytoplasmic survivin staining) covariables were screened for a prognostic relationship with overall survival (OS) and disease-free survival (DFS) into the univariate and multivariate analyses., Results: Data referring to 116 NSCLC patients who underwent surgery for stage I-IIIA NSCLC were collected. Multivariate analyses identified tumor size, nodal status and nuclear, but not cytoplasmic, expression of survivin as significant independent predictors of OS, with a hazard ratio of 2.40 (95% CI 1.44, 3.99, p=0.001), 2.03 (95% CI 1.26, 3.26, p=0.003) and 1.83 (95% CI 1.01, 3.30, p=0.044), respectively. Median OS for nuclear survivin positive (score 1-2) and negative (score 0) patients were 23 months (95% CI 15, 31) and 36 months (95% CI 1, 76), respectively (p=0.01); five-year survival for score 1-2 and score 0 patients were 20% and 44.5%, respectively. Conversely, no significant impact on survival is found when patients are stratified according to cytoplasmic survivin expression., Conclusions: Data presented herein open the issue that prognosis of stage I-IIIA NSCLC can be linked to the cellular pattern of distribution of survivin.

The HPV genotype concordance in the sexual couples could support the sexual viral transmission of HPV infection. The present study contains a case-report of a stable Italian sex couple harbouring the same five HPV genotypes in their genital samples. The female partner, affected by vulvar condilomatosis, evidenced positivity in her cervicovaginal scraping with high risk HPV DNA Hybrid Capture 2 test and was negative at liquid-based performed Pap Test and at colposcopic examination. The male partner was clinically healthy regarding his external genitalia. In both male and female genital scrapings, the following HPV genotypes were detected by means of a PCR-based assay: 6, 16, 53, 73 and 84. This considerably high genotype concordance does not appear to be casual and supports, in our opinion, the hypothesis that genital HPV types are sexually transmitted agents