Delphi Initiative for Early-Onset Colorectal Cancer (DIRECt) International Management Guidelines

Background & aims: Patients with early-onset colorectal cancer (eoCRC) are managed according to guidelines that are not age-specific. A multidisciplinary international group (DIRECt), composed of 69 experts, was convened to develop the first evidence-based consensus recommendations for eoCRC. Methods: After reviewing the published literature, a Delphi methodology was used to draft and respond to clinically relevant questions. Each statement underwent 3 rounds of voting and reached a consensus level of agreement of ≥80%. Results: The DIRECt group produced 31 statements in 7 areas of interest: diagnosis, risk factors, genetics, pathology-oncology, endoscopy, therapy, and supportive care. There was strong consensus that all individuals younger than 50 should undergo CRC risk stratification and prompt symptom assessment. All newly diagnosed eoCRC patients should receive germline genetic testing, ideally before surgery. On the basis of current evidence, endoscopic, surgical, and oncologic treatment of eoCRC should not differ from later-onset CRC, except for individuals with pathogenic or likely pathogenic germline variants. The evidence on chemotherapy is not sufficient to recommend changes to established therapeutic protocols. Fertility preservation and sexual health are important to address in eoCRC survivors. The DIRECt group highlighted areas with knowledge gaps that should be prioritized in future research efforts, including age at first screening for the general population, use of fecal immunochemical tests, chemotherapy, endoscopic therapy, and post-treatment surveillance for eoCRC patients. Conclusions: The DIRECt group produced the first consensus recommendations on eoCRC. All statements should be considered together with the accompanying comments and literature reviews. We highlighted areas where research should be prioritized. These guidelines represent a useful tool for clinicians caring for patients with eoCRC

Delphi initiative for early-onset colorectal cancer (DIRECt). International Management Guidelines.

BACKGROUND AND AIMS: Patients with early-onset colorectal cancer (eoCRC) are managed according to guidelines that are not age-specific. A multidisciplinary international group (DIRECt), comprised of 69 experts, was convened to develop the first evidence-based consensus recommendations for eoCRC. METHODS: After reviewing the published literature, a Delphi methodology was employed to draft and respond to clinically relevant questions. Each statement underwent 3 rounds of voting and reached a consensus level of agreement of ≥80%. RESULTS: The DIRECt group produced 31 statements in 7 areas of interest: diagnosis, risk factors, genetics, pathology-oncology, endoscopy, therapy, and supportive care. There was strong consensus that all individuals younger than 50 should undergo CRC risk stratification and prompt symptom assessment. All newly diagnosed eoCRC patients should receive germline genetic testing, ideally before surgery. Based on current evidence, endoscopic, surgical, and oncologic treatment of eoCRC should not differ from later onset CRC, except for individuals with pathogenic or likely pathogenic germline variants. The evidence on chemotherapy is not sufficient to recommend changes to established therapeutic protocols. Fertility preservation and sexual health are important to address in eoCRC survivors.The DIRECt group highlighted areas with knowledge gaps that should be prioritized in future research efforts, including age at first screening for the general population, use of fecal immunochemical tests, chemotherapy, endoscopic therapy, and post-treatment surveillance for eoCRC patients. CONCLUSIONS: The DIRECt group produced the first consensus recommendations on eoCRC. All statements should be considered together with the accompanying comments and literature reviews. We highlighted areas where research should be prioritized. These guidelines represent a useful tool for clinicians caring for patients with eoCRC

The CASP8 rs3834129 polymorphism and breast cancer risk in BRCA1 mutation carriers

The rs3834129 polymorphism, in the promoter of CASP8 gene, has been recently reported as associated with breast cancer risk in the general population, with the minor allele del having a protective effect. Some of the genetic variants found associated with breast cancer risk were reported as risk modifiers in individuals with mutations in BRCA1 and BRCA2 genes. Here, we tested the effect of the rs3834129 del allele on breast cancer risk in BRCA mutation carriers. The rs3834129 was genotyped in a total of 1,207 Italian female BRCA mutation carriers. Of these, 740 carried a BRCA1 mutation and 467 a BRCA2 mutation. Overall, 699 were affected with breast cancer and 508 were unaffected. When considering class 1 (loss-of-function) BRCA mutations, hazard ratios estimated by weighted multivariable Cox regression model, for individuals with at least one copy of the del allele, were 1.46 (95% confidence interval (CI): 1.08-1.99) for BRCA1 and BRCA2 mutation carriers combined, 1.74 (95% CI: 1.24-2.46) for BRCA1 mutation carriers, and 1.09 (95% CI: 0.66-1.80) for BRCA2 mutation carriers. These results suggest that the minor allele del of rs3834129 is associated under a dominant model with increased breast cancer risk in carriers of BRCA1 mutations but not in carriers of BRCA2 mutations

Functional analysis of BRCA2 p.Val2985_Thr3001del.

<p>(<b>A</b>) Schematic representation of GST-BRCA2 recombinant proteins. Wild-type and mutant <i>BRCA2</i> fragments, encoding the DBD and the N-terminal region, were cloned into pGEX4T1 vector to express GST-BRCA2 fusion proteins under the control of lacUV5 promoter. BRCA2 amino acid positions, helical domain (HD) and OB fold domains 1, 2, 3 (OB1, OB2, OB3) are indicated. (<b>B</b>) Interaction of wild-type and mutated BRCA2 DBD polypeptides with DSS1<b>.</b> Equivalent amounts of GST-tagged wild-type or mutated BRCA2 fusion proteins were immobilized on GSH-Sepharose beads and challenged with MCF7 lysates as a source of GFP-DSS1. Input (top panel) and pulled down (middle panel) GFP-DSS1 protein were visualized by Western blotting with anti-GFP antibody. GSH-Sepharose beads and GST protein were used as negative controls. GST-tagged recombinant proteins were visualized by Coomassie staining of the SDS-PAGE gel used in the pull-down experiment (bottom panel)<b>.</b> (<b>C</b>) Interaction of wild-type and mutated BRCA2 polypeptides with ssDNA. The mutated and wild-type peptides, removed from glutathione-agarose beads by thrombin digestion, were chromatographed on ssDNA agarose beads. A 200 amino acids N-terminal peptide was used as negative control. The free (F) and bound (B) fractions were separated, submitted to gel electrophoresis and visualized by Coomassie staining. Immunoblots were scanned using HP Scanjet G3010 Photo Scanner (Hewlett Packard).</p

Experimentally observed effects on mRNA splicing of group A variants and predicted protein change.

a<p>Protein change was predicted using ExPASy Proteomics Server (<a href="http://www.expasy.ch/" target="_blank">http://www.expasy.ch/</a>);</p>b<p>The classification as class 5 (pathogenic) or class 4 (likely pathogenic) was based on mono- or bi-allelic expression of the normal transcript <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057173#pone.0057173-Spurdle2" target="_blank">[23]</a>. Previously characterized variants are indicated;</p>c<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057173#pone.0057173-Houdayer1" target="_blank">[22]</a>;</p>d<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057173#pone.0057173-Machackova1" target="_blank">[43]</a>;</p>e<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057173#pone.0057173-Houdayer1" target="_blank">[22]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057173#pone.0057173-Colombo1" target="_blank">[44]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057173#pone.0057173-Pensabene1" target="_blank">[45]</a>;</p>f<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057173#pone.0057173-Machackova1" target="_blank">[43]</a>;</p>g<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057173#pone.0057173-Acedo1" target="_blank">[18]</a>. An asterisk indicates variants for which the observed transcript pattern differed from that reported by previous studies (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057173#pone.0057173.s006" target="_blank">Table S6</a>). Abbreviations: SS, splice Site (D, donor; A, acceptor); BIC, Breast Cancer Information Core (<a href="http://research.nhgri.nih.gov/bic/" target="_blank">http://research.nhgri.nih.gov/bic/</a>); HGVS, Human Genetic Variation Society (<a href="http://www.hgvs.org/mutnomen" target="_blank">http://www.hgvs.org/mutnomen</a>).</p

<i>In silico</i> predicted effect of group B variants and comparison with experimental results.

<p>For all computational program except ASSA, the relative percent differences of the splice site prediction scores (SSPSs) in the wild-type and the mutated sequences are reported. For ASSA, which uses the information theory-base values (Ri), the percent differences of binding affinity in the mutated compared to the wild-type sequences are reported. Empty cells indicates natural splice site not recognized by the indicated programs, <i>In silico</i> analyses predicting spliceogenic (S) or non spliceogenic (NS) variants according to the described procedure (see text) are indicated. (C) indicates <i>in silico</i> predictions concordant with <i>in vitro</i> data; (D), discordant predictions. Abbreviations: HGVS, Human Genetic Variation Society (<a href="http://www.hgvs.org/mutnomen/" target="_blank">http://www.hgvs.org/mutnomen/</a>).</p

RT-PCR analyses of group B variants.

<p>For each variant, the RT-PCR products were characterized by agarose gel electrophoresis and sequencing. Gel images: lane 1, no template; lane 2, genomic DNA used as negative control of the RT-PCR reaction; lane 3, cDNA from the <i>BRCA1/BRCA2</i> wild-type LCL used as positive control; lane 4, cDNA from LCL carrying the UV. M, molecular marker (ΦX-174 HaeIII digest). The size of the full-length (FL) and aberrant transcripts are reported. Sequencing electropherogram data: (<b>B–G</b>) the RT-PCR products were directly sequenced; (<b>A, H</b>) the sequencing was performed after band excision or cloning step. (<b>H</b>) An additional band due to improper annealing of full-length and aberrant transcripts is shown by the asterisk. The Ex5del, visible in both sample and control is a naturally occurring isoform lacking exon 5. (<b>A</b>) In addition to the full-length and the Ex14del aberrant transcript, the naturally occurring isoform lacking the first 3 bp of exon 14 (Ex14_3 bp del) was observed. Ex, exon; I, intron.</p

Comparative <em>In Vitro</em> and <em>In Silico</em> Analyses of Variants in Splicing Regions of <em>BRCA1</em> and <em>BRCA2</em> Genes and Characterization of Novel Pathogenic Mutations

<div><p>Several unclassified variants (UVs) have been identified in splicing regions of disease-associated genes and their characterization as pathogenic mutations or benign polymorphisms is crucial for the understanding of their role in disease development. In this study, 24 UVs located at <i>BRCA1</i> and <i>BRCA2</i> splice sites were characterized by transcripts analysis. These results were used to evaluate the ability of nine bioinformatics programs in predicting genetic variants causing aberrant splicing (spliceogenic variants) and the nature of aberrant transcripts. Eleven variants in <i>BRCA1</i> and 8 in <i>BRCA2</i>, including 8 not previously characterized at transcript level, were ascertained to affect mRNA splicing. Of these, 16 led to the synthesis of aberrant transcripts containing premature termination codons (PTCs), 2 to the up-regulation of naturally occurring alternative transcripts containing PTCs, and one to an in-frame deletion within the region coding for the DNA binding domain of BRCA2, causing the loss of the ability to bind the partner protein DSS1 and ssDNA. For each computational program, we evaluated the rate of non-informative analyses, i.e. those that did not recognize the natural splice sites in the wild-type sequence, and the rate of false positive predictions, i.e., variants incorrectly classified as spliceogenic, as a measure of their specificity, under conditions setting sensitivity of predictions to 100%. The programs that performed better were Human Splicing Finder and Automated Splice Site Analyses, both exhibiting 100% informativeness and specificity. For 10 mutations the activation of cryptic splice sites was observed, but we were unable to derive simple criteria to select, among the different cryptic sites predicted by the bioinformatics analyses, those actually used. Consistent with previous reports, our study provides evidences that <i>in silico</i> tools can be used for selecting splice site variants for <i>in vitro</i> analyses. However, the latter remain mandatory for the characterization of the nature of aberrant transcripts.</p> </div