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.

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 occasionally 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.

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BackgroundAltered 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).MethodsFirst, 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.ResultsCfDNA 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.ConclusionsOur 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.

Poor assembly of class I major histocompatibility HLA-C heavy chains results in their intracellular accumulation in two forms: free of and associated with their light chain subunit (␤ 2 -microglobulin). Both intermediates are retained in the endoplasmic reticulum by promiscuous and HLA-dedicated chaperones and are poorly associated with peptide antigens. In this study, the eight serologically defined HLA-C alleles and the interlocus recombinant HLA-B46 allele (sharing the HLA-Cspecific motif KYRV at residues 66 -76 of the ␣1-domain ␣-helix) were compared with a large series of HLA-B and HLA-A alleles. Pulse-labeling experiments with HLA-C transfectants and HLA homozygous cell lines demonstrated that KYRV alleles accumulate as free heavy chains because of both poor assembly and post-assembly instability. Reactivity with antibodies to mapped linear epitopes, co-immunoprecipitation experiments, and molecular dynamics simulation studies additionally showed that the KYRV motif confers association to the HLA-dedicated chaperones TAP and tapasin as well as reduced plasticity and unfolding in the peptide-binding groove. Finally, in vitro assembly experiments in cell extracts of the T2 and 721.220 mutant cell lines demonstrated that HLA-Cw1 retains the ability to form a peptide-receptive interface despite a lack of TAP and functional tapasin, respectively. In the context of the available literature, these results indicate that a single locusspecific biosynthetic bottleneck renders HLA-C peptide-selective (rather than peptide-unreceptive) and a preferential natural killer cell ligand.Class I human leukocyte antigens (called HLA) are cell-surface heterotrimers formed by a highly polymorphic heavy (44 kDa) chain, a non-polymorphic light (12 kDa) chain subunit (␤ 2 -microglobulin (␤ 2 m)3 ), and a short (8 -11-mer) peptide antigen derived from the degradation of intracellular proteins (1). The assembly pathway of most class I molecules involves an early interaction of the heavy chain, still free of ␤ 2 m, with calnexin, followed by association with ␤ 2 m and binding to the so-called peptide-loading complex. This is a supramolecular endoplasmic reticulum structure comprising, among others, two HLA-dedicated chaperones: TAP transporter associated with antigen processing) and the peptide editor/facilitator tapasin (1). Successful peptide loading results in tight association of the heavy chain with ␤ 2 m and the release of thermally stable, folded class I conformers (1-3). These are exported to the cell surface, where they activate and inhibit cytotoxic T lymphocytes expressing the rearranging T cell receptor and natural killer (NK) cells expressing non-rearranging receptors such as the killer immunoglobulin-like receptors, respectively (4).There are Ͼ1000 class I molecules, encoded by three highly polymorphic allelic series: HLA-A, -B, and -C (www. anthonynolan.org.UK/HIG/index.html). They share a conserved general architecture, a common peptide-loading pathway, and a similar set of functions, but also display a number of allel...

), all of the MEM Abs unexpectedly reacted with ␤ 2 -microglobulin (␤ 2 m)-free and denatured (but not ␤ 2 m-associated and folded) HLA-E H chains. Remarkably, other HLA-E-restricted Abs were also reactive with free H chains. Immunodepletion, in vitro assembly, flow cytometry, and three distinct surface-labeling methods, including a modified (conformation-independent) biotin-labeling assay, revealed the coexistence of HLA-E conformers with unusual and drastically antithetic features. MEM-reactive conformers were thermally unstable and poorly surface expressed, as expected, whereas ␤ 2 m-associated conformers were either unstable and weakly reactive with the prototypic conformational Ab W6/32, or exceptionally stable and strongly reactive with Abs to ␤ 2 m even in cells lacking permissive alleles (721.221), TAP (T2), or tapasin (721.220). Noncanonical, immature (endoglycosidase H-sensitive) HLA-E glycoforms were surface expressed in these cells, whereas mature glycoforms were exclusively expressed (and at much lower levels) in cells carrying permissive alleles. Thus, HLA-E is a good, and not a poor, ␤ 2 m assembler, and TAP/tapasin-assisted ligand donation is only one, and possibly not even the major, pathway leading to its stabilization and surface expression.

Lacking monospecific antibodies to HLA-C, the expression and synthesis of these molecules have been difficult to evaluate. Using biochemical and flow cytometry approaches, the present report demonstrates that the reactivity of the murine monoclonal antibody L31 is restricted to naturally occurring HLA-C (HLA-Cw1 through -Cw8), HLA-B8 and HLA-B51 heavy chains not associated with beta2-microglobin (beta2m). This is due to two properties of HLA-C heavy chains: (a) they share the L31 epitope which distinguishes them from all the HLA-A and most HLA-B molecules; (b) they accumulate intracellularly, in a beta2m-free form, in much greater amounts than most L31-reacting HLA-B heavy chains. On the basis of this restricted reactivity, a representative panel of normal and neoplastic human tissues and cells derived from HLA-B8- B51- individuals was selected and employed to assess the tissue distribution, surface expression and IFN-gamma responsiveness of beta2m-free HLA-C heavy chains. At variance from antibody W6/32 to beta2m-associated heavy chains, L31 stains normal and neoplastic tissues with a ground-glass pattern and weakly binds to the surface of viable cells, even after treatment with interferon gamma (IFN-gamma). Thus, beta2m-free HLA-C heavy chains are, for the most part, located intracellularly. In spite of their distinct cellular localization, L31- and W6/32-reacting molecules have an overlapping tissue distribution, undergo concordant changes upon transformation and are upregulated in their synthesis by IFN-gamma to a similar extent. These observations demonstrate a coordinate regulation of HLA-C with HLA-A and -B molecules. In addition, they indicate that the assembly of HLA-C is impaired in most body districts and IFN-gamma is unable to completely reverse this impairment. The present results are consistent with a low surface expression of HLA-C and with a privileged role of these molecules in signaling class I loss to cytotoxic effectors in pathological conditions.

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.