p53 proteinaren eginkizuna kolon-ondesteko minbizian

[EUS] Zenbait minbizietan p53 proteinaren adierazpena eta TP53 genean gertatzen diren aldaera motaren arteko korrelazioa frogatu da. Zehazki, p53 adierazpen gabezia aldaera bukatzaileekin lotu da, eta aldiz, gainadierazpena zentzu-aldaketazko aldaerekin. Lan honetan kolon-ondesteko tumoreetan p53ren immunotindaketaren eta TP53ren egoeraren arteko korrelazioa analizatu dugu. Guztira, 99 lagin ikertu ziren.Azterketa immunohistokimikoa Roche Ventana (anti-P53 (DO-7)) protokoloa jarraituz burutu zen. TP53 genearen 5-8 exoiak analizatu ziren Sanger sekuentzazioaren bidez, Asai et al-k (Asian Pac J Cancer Prev, 2014) deskribatutako hasleak erabiliz. Mutazioa aurkitu zen pazienteen kasuan, ehun osasuntsuaren sekuentziazioa egin zen ere aldaera germinalen presentzia baztertzeko. 69 lagin immunotindaktea positiboa (zelulen %90 baino gehiago) aurkezten zuten, 30 laginetan tindaketaren gabezia zegoen bitartean (<%2). Guztira, 99 laginetatik 62tan (%62,7) aurkitu zen TP53 genean aldaeraren bat. Immunohistokimika positiboa zeukaten laginen artean, 48tan (%69,6) aurkitu zen zentzu-aldaketazko mutazioak. Horietako batean bi mutazio aurkitu ziren. Immunotindaketaren gabezia zeukaten laginen artean, 14k (%46,7) proteina motzagoa sortzen duten aldaerak (protein-truncating) zituzten. DNAn gertatzen diren baseen ordezkapenei dagokionez (guztira 61), 53 (%86,9) trantsizioak ziren eta 8 (%13,1) transbertzioak. Horrez gain, 38 aldaera (%62,3) CpG guneetan gertatu ziren. Gure ikerketako emaitzek aurreko literaturarekin bat egiten dute, p53 proteinaren immunotindaketa TP53 genearen egoera aurresateko tresna erabilgarria dela aditzera emanez. Hala ere, lagin askotan ez dira mutaziorik aurkitu. Horretaz aparte, kolon-ondesteko minbizian DNAren metilazioak izan dezakeen garrantzia azpimarratzen da, izan ere, ikerketa honetan maiztasun handiagoz aurkitu diren aldaerak CpG gune metilatuetan gertatzen da.[ES] En algunos cánceres se ha demostrado la correlación entre la expresión de la proteína p53 y el tipo de variante que se producen en el gen TP53. En concreto, la ausencia de expresión p53 se ha asociado a variantes terminantes, mientras que la sobreexpresión se ha asociado a variaciones de sentido. En este trabajo hemos analizado la correlación entre la inmunotindación de p53 en tumores colorrectal y la situación de TP53. En total, se investigaron 99 muestras. El estudio inmunohistoquímico se llevó a cabo siguiendo el protocolo Roche Ventana (anti-P53 (DO-7)). Los 5-8 exones del gen TP53 fueron analizados mediante la secuenciación Sanger utilizando los iniciadores descritos por Asai et al (Asian Pac J Cancer Prev, 2014). En el caso de los pacientes donde se descubrió la mutación, también se realizó la secuenciación del tejido sano para descartar la presencia de variantes germinales. 69 muestras presentaban inmunotindacte positiva (más del 90% de las células), mientras que 30 muestras presentaban ausencia de tintura (< 2%). En total, en 62 de las 99 muestras (62,7%) se encontró alguna variante en el gen TP53. Entre las muestras con inmunohistoquímica positiva, se encontraron mutaciones de sentido en 48 (69,6%). En una de ellas se encontraron dos mutaciones. Entre las muestras con ausencia de inmunotindación, 14 (46,7%) presentan variantes que producen proteína más corta (protein-truncating). En cuanto a las sustituciones de bases en el ADN (61 en total), 53 (86,9%) eran transiciones y 8 (13,1%) transiciones. Además, 38 variantes (62,3%) se produjeron en zonas CpG. Los resultados de nuestra investigación coinciden con la literatura anterior, dando a entender que la inmunotindación de la proteína p53 es una herramienta útil para predecir el estado del gen TP53. Sin embargo, en muchas muestras no se han encontrado mutaciones. Además, se destaca la importancia que puede tener la metilación del ADN en el cáncer colorrectal, ya que las variantes encontradas con mayor frecuencia en este estudio se dan en los sitios metilados CpG

Recommendations for the classification of germline variants in the exonuclease domain of POLE and POLD1

BackgroundGermline variants affecting the proofreading activity of polymerases epsilon and delta cause a hereditary cancer and adenomatous polyposis syndrome characterized by tumors with a high mutational burden and a specific mutational spectrum. In addition to the implementation of multiple pieces of evidence for the classification of gene variants, POLE and POLD1 variant classification is particularly challenging given that non-disruptive variants affecting the proofreading activity of the corresponding polymerase are the ones associated with cancer. In response to an evident need in the field, we have developed gene-specific variant classification recommendations, based on the ACMG/AMP (American College of Medical Genetics and Genomics/Association for Molecular Pathology) criteria, for the assessment of non-disruptive variants located in the sequence coding for the exonuclease domain of the polymerases.MethodsA training set of 23 variants considered pathogenic or benign was used to define the usability and strength of the ACMG/AMP criteria. Population frequencies, computational predictions, co-segregation data, phenotypic and tumor data, and functional results, among other features, were considered.ResultsGene-specific variant classification recommendations for non-disruptive variants located in the exonuclease domain of POLE and POLD1 were defined. The resulting recommendations were applied to 128 exonuclease domain variants reported in the literature and/or public databases. A total of 17 variants were classified as pathogenic or likely pathogenic, and 17 as benign or likely benign.ConclusionsOur recommendations, with room for improvement in the coming years as more information become available on carrier families, tumor molecular characteristics and functional assays, are intended to serve the clinical and scientific communities and help improve diagnostic performance, avoiding variant misclassifications

Supplementary Table 4 from Exploring Co-occurring POLE Exonuclease and Non-exonuclease Domain Mutations and Their Impact on Tumor Mutagenicity

mTMB comparisons in TCGA dataset.</p

Supplementary Table 6 from Exploring Co-occurring POLE Exonuclease and Non-exonuclease Domain Mutations and Their Impact on Tumor Mutagenicity

Rosetta ΔΔG values for POLE variants and drivers with or without DNA</p

Supplementary Table 2 from Exploring Co-occurring POLE Exonuclease and Non-exonuclease Domain Mutations and Their Impact on Tumor Mutagenicity

Age Distribution of CRC, EC, and OC patients with POLE-mutated tumors.</p

Supplementary Table 3 from Exploring Co-occurring POLE Exonuclease and Non-exonuclease Domain Mutations and Their Impact on Tumor Mutagenicity

mTMB comparisons in the Caris Life Sciences dataset.</p

FIGURE 1 from Exploring Co-occurring POLE Exonuclease and Non-exonuclease Domain Mutations and Their Impact on Tumor Mutagenicity

Characterization of POLE mutations in the CLS and TCGA dataset. A, Flowchart and analysis tree for colorectal cancer (CRC), endometrial cancer (EC), and ovarian cancer (OC) tumors by POLE mutations, TMB, and MSI/MSS status. Among 1,870 colorectal cancer, 4,481 endometrial cancers, and 8,910 ovarian cancer tumor genomic profiles, a total of 447 carried POLE mutations. Clinically relevant TMB cut-off points were used to define the TMB-H (≥10 mut/Mb) and TMB-L (POLE mutation cohorts along with TMB and MSI/MSS status were defined. TMB-L tumors with POLE variants but no established POLE ExoD driver are referred to as “POLE Variants TMB-L” (Group 1, MSS or MSI). TMB-H tumors with known POLE ExoD driver only were referred to as “POLE ExoD Driver” (Group 2, MSS or MSI). TMB-H tumors with co-occurring POLE ExoD driver and POLE variant(s) were referred to as “POLE ExoD Driver + POLE ExoD Variant” (Group 3, MSS or MSI). TMB-H tumors with only POLE variant(s) and no POLE ExoD driver were referred to as “POLE Variant TMB-H” (Group 4, MSS or MSI). B, Age distribution of patients in the CLS cohort with POLE-mutated tumors (n = 447) designated as Group 1 (green), Group 2 (red), Group 3 (purple), and Group 4 (blue). mTMB comparisons between Group 2 and 3 colorectal cancers (C), endometrial cancers (D), and ovarian cancers (E). mTMB comparisons between Group 2 and 3 genomic profiles of colorectal cancer (F), endometrial cancer (G), and ovarian cancer (H). MSI-H tumor profiles were removed from this analysis. TCGA cohort mTMB comparisons between Group 2 and 3 tumors, in I MSI-H or MSS tumor profiles were included and in J only MSS tumor profiles were included. Because of smaller sample size per tumor type, analyses were pooled. A Mann–Whitney test was performed and ***, P P < 0.05; NS, nonsignificant.</p

TABLE 1 from Exploring Co-occurring POLE Exonuclease and Non-exonuclease Domain Mutations and Their Impact on Tumor Mutagenicity

POLE variant groups, patient clinical and demographic characteristics for the CARIS cohort</p

Supplementary Table 5 from Exploring Co-occurring POLE Exonuclease and Non-exonuclease Domain Mutations and Their Impact on Tumor Mutagenicity

Neoantigens predictions for Group 3 tumor mutations by DeepNeo (sheet 1 for CRC; sheet 2 for EC) and Neodb (sheet 3 for CRC, and EC).</p

FIGURE 4 from Exploring Co-occurring POLE Exonuclease and Non-exonuclease Domain Mutations and Their Impact on Tumor Mutagenicity

Comparison of mTMB and ΔΔG values in Group 2 and 3 tumors in the CLS dataset. With AF2 DNA unbound model and Rosetta ddG_monomer, we generated 25 repacked decoys for each mutation and compared the average energy score for these decoys with an average for 25 decoys of the WT protein. For mutations in the NTL, we performed calculations on both models (with and without DN). For those in the CTL, we performed calculations only on the DNA unbound model. We used a cutoff of ±1.45 kcal/mol for significant ΔΔG, corresponding to approximately 3 SDs of the differences of the mean Rosetta scores for WT and mutant structures. A, Comparison of mTMB and ΔΔG values in Group 2 and Group 3 tumors by the number of POLE variants. Data for colorectal cancer, endometrial cancer, and ovarian cancer genomic profiles were combined, and ΔΔG values were plotted against the mTMB. For Group 2 or 3 data with + 1 POLE variant plots, each filled round circle represents a single tumor genomic profile. B, Comparison of mTMB and ΔΔG values in Group 2 and Group 3 tumors by POLE ExoD driver. Data for colorectal cancer, endometrial cancer, and ovarian cancer genomic profiles were combined, and ΔΔG values were plotted against the mTMB. A and B, Group 3 tumors with multiple variants, a circle next to another circle (without any space) represents a single tumor. For clarity, ΔΔG values for ExoD drivers in Group 3 tumors are not shown (they are same as in Group 2). Color in each filled circle—green and shades of green, structure-destabilizing variants (positive ΔΔG); white, variants that are within the SDs of ±1.45 kcal/mol and are structure neutral; red and shades of red, structure-stabilizing variants (negative ΔΔG). Yellow, nonsense, or frameshift variants.</p