Supplementary Table 3. Results of the statistical analyses of SNPs from project 12 in BRCA-mutation carriers affected or non-affected with breast cancer and according to their estrogen receptor status.

Supplementary Table 2. Results of the statistical analyses in BRCA mutation carriers.

BACKGROUND The presence of Spigelman stage (SS) IV duodenal polyposis is considered the most significant risk factor for duodenal cancer in patients with MUTYH -associated polyposis (MAP). However, advanced SS disease is rarely reported in MAP patients, and no clear recommendations on small bowel (SB) surveillance have been proposed in this patient setting. AIM To research more because that case reports of duodenal cancers in MAP suggest that they may develop in the absence of advanced benign SS disease and often involve the distal portion of the duodenum. METHODS We describe a series of MAP patients followed up at the Regina Elena National Cancer Institute of Rome (Italy). A literature overview on previously reported SB cancers in MAP is also provided. RESULTS We identified two (6%) SB adenocarcinomas with no previous history of duodenal polyposis. Our observations, supported by literature evidence, suggest that the formula for staging duodenal polyposis and predicting risk factors for distal duodenum and jejunal cancer may need to be adjusted to take this into account rather than focusing solely on the presence or absence of SS IV disease. CONCLUSION Our study emphasizes the need for further studies to define appropriate upper gastrointestinal surveillance programs in MAP patients.

Supplementary Table 1. The 3,248 SNPs included in the study

Disruption in the HLA class I (HLA-I), β2-microglobulin (β2M) and/or Antigen Processing and Presentation Machinery (APPM) structural genes is believed to favor tumor immune escape, whereas purely regulatory mutants are few and poorly characterized. The endometrial carcinoma End9 was the extreme HLA-I/APPM-low outlier in a panel of >100 early-passage tumor cell lines established in culture by us from essentially all major tumor histotypes. The defect arose in vivo, was retained in culture, and remained stable for >100 passages. End9 was found to express no constitutive or IFN-responsive HLA-A, -B, -F, and-G, little HLA-C and β2m, ‘normal‘ HLA-E, and barely detectable TAP, tapasin, LMP2, LMP7, ERAP1 and ERAP2, as evaluated by protein and nucleic acid biochemistry. Lacking evidence for genomic damage, we used a reporter construct to assess the 3 major upstream HLA-I regulatory boxes (EnhA, ISRE and SXY), and attempted phenotypic rescue by transduction with missing trans-acting function(s). The NF-KB:EnhA interplay was apparently conserved, whereas the ISRE and SXY modules were simultaneously shut off due to (a) a complete JAK1 absence, (b) a physiological lack of CIITA, and (c) an epigenetic (5-Azacitidine-reversible) suppression of an intact (as per sequencing) NLRC5. The near-zero HLA-I/APPM phenotype of End9 highlighted several features of HLA-I regulation, including: (a) SXY-independent HLA-C transcription; (b) subordination of the EnhA and ISRE boxes (IFN and TNFα responsiveness) to promoter opening by CIITA/NLRC5; and (c) marginal HLA-I/APPM transactivation by residual NLRC5 at baseline and even upon >100X overexpression. End9 is the first regulatory mutant defective in a hierarchically high set of transacting functions operating upstream to most HLA-I/APPM members, resulting in a trophoblast-like, presumably immunoevasive phenotype. The defect involves NLRC5 but NLRC5 refractoriness implies a shortage of other transcriptional co-activators, making End9 a suitable recipient for complementation cloning to discover novel HLA-A/-B/APPM master coordinators.

Background and aim: SARS-CoV-2 is the agent responsible for the ongoing COVID-19 pandemic.In patients with coeliac disease (CD), there is evidence of an impaired response to certain vaccines, such as hepatitis B. Patients affected by chronic non-coeliac enteropathies characterized by villous atrophy (NCEs) are also vulnerable patients because of primary or acquired immune defects.Data in the literature is lacking on the immune response to vaccination against SARS-CoV-2 in patients affected by CD and NCEs.We aimed to assess response to SARS-CoV-2 vaccination in patients with chronic enteropathies.Materials and methods: Patients affected by CD and NCEs who attended our Centre for routine follow-up and were previously vaccinated against SARS-CoV-2 were prospectively enrolled starting from November 2021.Serologic testing for antibody response to vaccination was performed at time of enrolment.Controls were chosen among healthcare workers at our clinic who undergo serological surveillance matched by age ± 5 years, gender and ±1 months from first vaccination dose to sample collection.SARS-CoV-2 serologic testing was performed by means of ELISA (Anti-SARS-CoV-2 QuantiVac ELISA (IgG), BSN srl, Castellone, Italy) and expressed as BAU/mL (cutoffs: ≥ 35.2 BAU/mL positive, borderline 25.6-35.1 BAU/ mL, negative <25.6 BAU/mL).Demographics, months from first vaccine dose, and preliminary serology results were collected and statistically analysed.Results: 97 patients with CD (63 F, age 41±15 years), 12 patients with NCEs (9 Whipple's disease, 2 common variable immunodeficiency, 1 idiopathic villous atrophy; 4 F, 57±11) and 100 matched controls (69F, 42±12) were enrolled.At 3±1 months from vaccination no cases (22 CD, 2 NCE patients) nor controls (53) were found to have inadequate response to vaccination.At 6±1 months from vaccination 3/75 coeliac patients (4%) and 5/10 NCE patients (50%) had inadequate response to vaccination.Of the 47 controls enrolled at 6±1 months, none had inadequate response to vaccination.On Bonferroni corrected pairwise analysis no statistically significant differences were found between CD patients and controls (p=0.85);instead, patients with NCE had inadequate response to vaccination significantly more often than both controls (both p<0.01) and CD patients at 6±1 months from vaccination.Conclusions: Preliminary results of our ongoing study suggest an impaired response to vaccination against SARS-CoV-2 may occur in patients affected by NCE.

An expert consensus panel convened by the Italian Association for Inherited and Familial Gastrointestinal Tumors (Associazione Italiana per lo Studio della Familiarità ed Ereditarietà dei Tumori Gastrointestinali, AIFEG) reviewed the literature and agreed on a number of position statements regarding the definition and management of polyposis coli without an identified pathogenic mutation on the APC or MUTYH genes, defined in the document as NAMP (non-APC/MUTYH polyposis).

(1) Background: desmoid tumors (DTs) are common in patients with familial adenomatous polyposis (FAP). An active surveillance approach has been recently proposed as a valuable alternative to immediate treatment in some patients. However, no clear indication exists on which patients are suitable for active surveillance, how to establish the cut-off for an active treatment, and which imaging technique or predictive factors should be used during the surveillance period. (2) Results: we retrospectively analyzed 13 FAP patients with DTs. A surveillance protocol consisting of scheduled follow-up evaluations depending on tumor location and tissue thickening, abdominal computed tomography (CT) scan/Magnetic resonance imaging (MRI) allowed prompt intervention in 3/11 aggressive intra-abdominal DTs, while sparing further interventions in the remaining cases, despite worrisome features detected in three patients. Moreover, we identified a possible predictive marker of tumor aggressiveness, i.e., the "average monthly growth rate" (AMGR), which could distinguish patients with very aggressive/life-threatening tumor behavior (AMGR > 0.5) who need immediate active treatment, from those with stable DTs (AMGR < 0.1) in whom follow-up assessments could be delayed. (3) Conclusion: surveillance protocols may be a useful approach for DTs. Further studies on larger series are needed to confirm the usefulness of periodic CT scan/MRI and the value of AMGR as a prognostic tool to guide treatment strategies.

The United States Food and Drug Administration (FDA) recently approved the clinical use of two comprehensive 'mid-size' Next Generation Sequencing (NGS) panels calling actionable genomic aberrations in cancer. This is the first endorsement, by a regulatory body, of a new standard of care in oncology. Herein, we argue that besides its many practice-changing implications, this approval tears down the conceptual walls dividing system biology from clinical practice, diagnosis from research, prevention from therapy, cancer genetics from cancer genomics, and computational biology from empirical therapy assignment.

Background Altered circulating cell-free DNA (cfDNA) levels are related to cancer development and aggressiveness. Up to now, very few studies have been performed for evaluating cfDNA content in endometrial cancer (EC). Methods First, we measured cfDNA release in blood serum of EC cancer patients collected before surgery and before the beginning of any treatment by SYBR Gold assay and correlated it with tumor aggressiveness. We also assessed the relative mitochondrial cell-free DNA (cfmtDNA) content by qRT-PCR. Next, we correlated cfDNA levels with BMI, age, hypertension and inflammation markers. Results CfDNA levels are higher in G2 and G3 compared with G1 EC sera. A significant modulation of cfDNA content was detected in sera from patients with BMI>30 compared with those with BMI<30. We observed a further and significant alteration in cfDNA level in hypertensive patients with G2-G3, but not in G1 EC. Analysis of preoperative neutrophil-to-lymphocyte (NLR) and monocyte-to-lymphocyte (MLR) ratios suggests a contribution of the host response in the altered cfDNA levels in EC. Conclusions Our data indicate that assessment of total and mitochondrial cfDNA levels in blood sera and the relative NLR and MLR in blood obtained from preoperative patients may help clinical management and prognosis in EC.

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.

To determine prevalence, spectrum and genotype–phenotype correlations of MUTYH variants in Italian patients with suspected MAP (MUTYH-associated polyposis), a retrospective analysis was conducted to identify patients who had undergone MUTYH genetic testing from September 2002 to February 2014. Results of genetic testing and patient clinical characteristics were collected (gender, number of polyps, age at polyp diagnosis, presence of colorectal cancer (CRC) and/or other cancers, family data). The presence of large rearrangements of the MUTYH gene was evaluated by Multiplex Ligation-dependent Probe Amplification analysis. In all, 299 patients with colorectal neoplasia were evaluated: 61.2% were males, the median age at polyps or cancer diagnosis was 50 years (16–80 years), 65.2% had <100 polyps and 51.8% had CRC. A total of 36 different MUTYH variants were identified: 13 (36.1%) were classified as pathogenetic, whereas 23 (63.9%) were variants of unknown significance (VUS). Two pathogenetic variants were observed in 78 patients (26.1%). A large homozygous deletion of exon 15 was found in one patient (<1.0%). MAP patients were younger than those with negative MUTYH testing at polyps diagnosis (P<0.0001) and at first cancer diagnosis (P=0.007). MAP patients carrying the p.Glu480del variant presented with a younger age at polyp diagnosis as compared to patients carrying p.Gly396Asp and p.Tyr179Cys variants. A high heterogeneity of MUTYH variants and a high rate of VUS were identified in a cohort of Italian patients with suspected MAP. Genotype–phenotype analysis suggests that the p.Glu480del variant is associated with a severe phenotype.

Abstract Background:BRCA1 and BRCA2 mutation carriers are at substantially increased risk for developing breast and ovarian cancer. The incomplete penetrance coupled with the variable age at diagnosis in carriers of the same mutation suggests the existence of genetic and nongenetic modifying factors. In this study, we evaluated the putative role of variants in many candidate modifier genes. Methods: Genotyping data from 15,252 BRCA1 and 8,211 BRCA2 mutation carriers, for known variants (n = 3,248) located within or around 445 candidate genes, were available through the iCOGS custom-designed array. Breast and ovarian cancer association analysis was performed within a retrospective cohort approach. Results: The observed P values of association ranged between 0.005 and 1.000. None of the variants was significantly associated with breast or ovarian cancer risk in either BRCA1 or BRCA2 mutation carriers, after multiple testing adjustments. Conclusion: There is little evidence that any of the evaluated candidate variants act as modifiers of breast and/or ovarian cancer risk in BRCA1 or BRCA2 mutation carriers. Impact: Genome-wide association studies have been more successful at identifying genetic modifiers of BRCA1/2 penetrance than candidate gene studies. Cancer Epidemiol Biomarkers Prev; 24(1); 308–16. ©2014 AACR.

Introduction Several studies evaluated the prevalence of Lynch Syndrome (LS) in young onset colorectal cancer (CRC) patients and the results were extremely variable (5%-20%). Immunohistochemistry (IHC) for MMR proteins and/or MSI analysis are screening tests that are done, either by themselves or in conjunction, on colon cancer tissue to identify individuals at risk for LS. The primary aim of our study was to evaluate the prevalence of LS in a large series of early-onset CRC without family history compared with those with family history. The secondary aim was to assess the diagnostic accuracy of IHC and MSI analysis as pre-screening tools for LS. Methods Early-onset CRC patients (≤ 50 years) were prospectively recruited in the study. IHC and MSI analysis were performed in all the patients. Germ-line mutation analysis (GMA) was carried out in all MMR deficient tumors. A logistic regression model was performed to identify clinical features predictive of MSI-H. Results 117 early onset CRC cases were categorized in three groups (A, B, C) according with family history of CRC. IHC and MSI analysis showed MMR deficiency in 6/70 patients (8.6%) of group A, 24/40 patients (60%) of group B and none of group C. GMA showed a deleterious mutation in 19 (47.5%) patients of group B. MSI analysis had a diagnostic accuracy of 95.7% (CI 92.1-99.4) and IHC of 83.8% (CI 77.1-90.4). The logistic regression model revealed that by using a combination of the two features “No Amsterdam Criteria” and ”left sided CRC” to exclude MSI-H, accuracy was 89.7% (84.2-95.2). Conclusions Early-onset CRC patients, with left sided CRC and without family history are “at very low risk” for Lynch syndrome. The two simple criteria of family history and CRC site could be used as a pre-screening tool to evaluate whether or not patients should undergo tissue molecular screening. In the few cases of suspected LS (right sided CRC and/or Amsterdam Criteria), a reasonable approach could be to perform MSI analysis first and IHC afterwards only in MSI-H patients.

Colorectal cancer is the third most common cancer diagnosed worldwide.Although epidemiology data show a marked variability around the world, its overall incidence rate shows a slow but steady decrease, mainly in developed countries.Conversely, early-onset colorectal cancer appears to display an opposite trend with an overall prevalence in United States and European Union ranging from 3.0% and 8.6%.Colorectal cancer has a substantial proportion of familial cases.In particular, early age at onset is especially suggestive of hereditary predisposition.The clinicopathological and molecular features of colorectal cancer cases show a marked heterogeneity not only between early-and late-onset cases but also within the early-onset group.Two distinct subtypes of early-onset colorectal cancers can be identified: a "sporadic" subtype, usually without family history, and an inherited subtype arising in the context of well defined hereditary syndromes.The pathogenesis of the early-onset disease is substantially well characterized in the inherited subtype, which is mainly associated to the Lynch syndrome and occa-sionally to other rare mendelian diseases, whereas in the "sporadic" subtype the origin of the disease may be attributed to the presence of various common/rare genetic variants, so far largely unidentified, displaying variable penetrance.These variants are thought to act cumulatively to increase the risk of colorectal cancer, and presumably to also anticipate its onset.Efforts are ongoing in the attempt to unravel the intricate genetic basis of this "sporadic" early-onset disease.A better knowledge of molecular entities and pathways may impact on family-tailored prevention and clinical management strategies.