class: center, middle, inverse, title-slide .title[ # Pathology ] --- ## What we discuss * Gross * Histology * Immunohistochemistry * Cell of origin * BRCA variant significance * Homologous recombination * PARP inhibitor --- class: center ![:scale 80%](data:image/png;base64,#img/S21-28967^2021-07-19_15-00-19.jpg) --- ![:scale 80%](data:image/png;base64,#img/S21-28967_2.jpg) --- ![:scale 80%](data:image/png;base64,#img/S21-28967_3.jpg) --- ![:scale 80%](data:image/png;base64,#img/S21-28967_4.jpg) --- ![:scale 80%](data:image/png;base64,#img/S21-28967_5.jpg) --- ![:scale 80%](data:image/png;base64,#img/S21-28967_6.jpg) --- ![:scale 80%](data:image/png;base64,#img/he1.png) --- ![](data:image/png;base64,#img/he2.png) --- ![:scale 70%](data:image/png;base64,#img/he3.png) --- # p53 Immunohistochemistry ![:scale 60%](data:image/png;base64,#img/Image_4911.jpg) --- ### p53 * DNA damage and repair + Regulation or progression through the cell cycle + Apoptosis + Genomic stability * Mutation + Null-type + Gain-of-function + 95% of high-grade serous carcinoma --- ## **BRCA1/2** gene analysis * BRCA1 + p.Glu1148Argfs*7 + c.3442del + pathogenic --- ### The stem cell niche hypothesis ![:scale 90%](data:image/png;base64,#img/mfig002.jpg) ??? The stem cell niche hypothesis. (a) G6PD-stained frozen section of colonic mucosa from a CH3 mouse 14 days after a single dose of DMH showing a single wholly mutated crypt in which all cells have been replaced by a mutant phenotype (from ref.25, with permission). (b) Diagrammatic representation of the stem cell niche. An active multipotent stem cell (SC) produces a daughter cell (Co) that gives rise to enterocytic lineages, and another which generates goblet, Paneth, and enteroendocrine cells (although it is not known whether all derive from Mo). GLP-2 produced by a subset of enteroendocrine cells stimulates proliferation of the Co daughter via an interaction with enteric nervous system neurons that express the GLP-2 receptor (GLP-2R). The nature of the neuronal signal that affects Co is unknown (from ref.100 with permission) --- ### Monoclonal origin of colonic crypts in an XO/XY patient ![](data:image/png;base64,#img/mfig004.jpg) ??? Monoclonal origin of colonic crypts in an XO/XY patient. Normal colonic mucosa in an XO/XY mosaic individual stained by in situ hybridization for a Y-chromosome-specific probe showing an XO crypt (central) surrounded by two XY crypts (courtesy of M. Novelli) --- ## Robbins definition > A neoplasm is defined as a genetic disorder of cell growth that is triggered by acquired or less commonly inherited mutations affecting a single cell and its clonal progeny. --- class: center ![:scale 40%](data:image/png;base64,#img/41467_2017_962_Fig1_HTML.webp) .footnote[Nature Communications volume 8, Article number: 1093 (2017)] --- ![:scale 80%](data:image/png;base64,#img/41467_2017_962_Fig2_HTML.webp) --- class: middle ![](data:image/png;base64,#img/41467_2017_962_Fig4_HTML.webp) --- ![](data:image/png;base64,#img/image--000.png) --- # Double Strand Break (DSB) - The most deleterious form of DNA damage - Generated by * IR radiation * Free radicals * Topoisomerase II inhibitor * VDJ recombination * Meiotic recombination - Repaired by two major pathways * Homologous recombination (HR) * Nonhomologous end-joining (NHEJ) --- # Double Strand Break (DSB) ![:scale 70%](data:image/png;base64,#img/image--036.jpg) --- class: center #Nonhomologous end-joining ![:scale 50%](data:image/png;base64,#img/image--037.jpg) --- # Homologous recombination .center[ ![:scale 30%](data:image/png;base64,#img/image--038.jpg) ] --- # Diseases Due to Defects in DNA Repair ![:scale 80%](data:image/png;base64,#img/image--053.jpg) --- # Why is BRCA1/2 special? * High prevalence in population * Frequent benign variant --- # What about hereditary breast and ovarian cancer syndrome (HBOCS) * BRCA1/2 and other genes * Breast, ovarian cancer and other cancers * Prevalence (between 1 in 400 to 1 in 800 people) * Penetration rate (40-90%) --- # Categories of interpretation of variants * Pathogenic * Likely-pathogenic * Uncertain (VUS) * Likely-benign * Benign --- # Let's guess the evidences --- # Famly pedigree --- # Segregation data (BS1, PP1) * Caveat: linkage disequilibrium * Penetration rate * Difficult statistical evaluation --- # Population data * 5%: benign stand alone (BA1) * 0.5-5% (BS1) * Wow! The first time observed variant! (Absent in population DB, PM2) --- # Null variant * Frameshift, Nonsense, canonical +-1 or 2 splicing site, initiation codon * Caveat: LOF variants at the extreme 3′ end of a gene * Caveat: presence of multiple transcripts --- # Computational (in silico) data * PolyPhen2, SIFT, MutationTaster, etc * Mutational hot spot and/or critical and wellestablished (PM1) * Protein length changes due to in-frame deletions/insertions and stop losses functional domain (PM4 BP3) * Novel missense at the same position (PM5) --- # Other evidence * de novo variants (PS2 PM6) * Functional studies (PS3 BS3) * Allelic data (BP2 PM3) --- # Evidences of interpretation * Population data * Computational data * Functional data * Segregation data * De novo data * Allele data * Other databases * Other data --- class: small-font # Characteristics of BRCA1/2 * LOF known mechanism of disease (for PVS1) * Mode of inheritance (for PM3/BP2) * AD/AR (BRCA2) * Missense pathogenic (for PP2/BP1) * BRCA2 1% * Hot spot or critical/well-established functional domain (for PM1) * BRCA2, Helical (2479-2667), OB (2670-2799 and 3052-3190), Tower (2831-2872) --- ## **BRCA1/2** gene analysis * BRCA1 + p.Glu1148Argfs*7 + c.3442del --- class: center ![](data:image/png;base64,#img/parp2.webp) .footnote[Nature Reviews Clinical Oncology volume 18, pages773–791 (2021)] ??? Box 1 PARP enzymes and PARylation Poly(ADP-ribose) polymerase 1 (PARP1) is the most abundant of a 17-member family of enzymes that share a common ADP-ribosyltransferase motif. By hydrolysing nicotinamide adenine dinucleotide (NAD+), PARP1 post-translationally modifies itself and/or other proteins with negatively charged poly(ADP-ribose) (PAR) moieties, a process known as PARylation249. Following DNA damage, PARP1 is rapidly recruited to single-stranded DNA breaks, where it initiates a series of PARylation events, serving as a cellular sensor of DNA breaks and as a platform for the recruitment of downstream repair factors. In this process, PARP1 also autoPARylates, promoting its release from DNA250. In addition to PARP1, PARP2 and PARP3 are also activated by binding to DNA breaks251,252,253,254,255. PARP1 is responsible for more than 80% of PAR synthesis, while PARP2 accounts for the remainder251,252. Unlike PARP1 and PARP2, PARP3 modifies proteins primarily with mono(ADP-ribose)253,256. PARylation is a highly dynamic and reversible modification as its rapid turnover is mediated by PAR glycohydrolase (PARG), which degrades PAR249,250. ADP-ribosylhydrolase 3 (ARH3) is another PAR-degrading enzyme257,258. PARG has both endoglycosidase and exoglycosidase activities, while ARH3 seems to exert only exoglycosidase activity257,259. The eviction of PARP1 from sites of DNA damage is additionally regulated by the E3 ubiquitin ligase CHFR, which has been proposed to ubiquitylate the PARylated form of PARP1, resulting in PARP1 targeting for proteasomal degradation260. The removal of PARP1 from DNA is crucial for successful DNA repair and to prevent the collapse of replication forks owing to PARP1 trapping. Further studies attempting to understand which factors mediate the removal of PARylated PARP1 from DNA will be vital for optimizing the efficacy of PARP inhibitors and to uncover additional genetic vulnerabilities that could be exploited therapeutically. --- class: center ![](data:image/png;base64,#img/parp3.webp) ??? DSB, double-strand break; MRE11, meiotic recombination 11; MRN, MRE11–RAD50–NBS1 complex; RAD51, RAD51 recombinase; RPA1, replication protein A1. BRCA1 and BRCA2 do not share homology and, although acting via a common pathway, also have several different additional functions. Both factors are involved in homologous recombination (HR) and prevent replication-associated DNA damage, although BRCA1 is also known to regulate cell-cycle checkpoint activation as well as transcription261. BRCA1 and BRCA2 act at different levels during HR, resulting in functionally distinct mechanisms of resistance to PARP inhibitors. Resistance to PARP inhibitors via restoration of replication fork protection is observed in both BRCA1-deficient and BRCA2-deficient tumours, whereas the reactivation of HR owing to loss of 53BP1 and its downstream factors is only reported in BRCA1-deficient tumours. The mutational signatures of BRCA1-deficient tumours and those of BRCA2-deficient tumours feature notable differences10. Small tandem duplications (<10 kb) are exclusively found in BRCA1-mutated tumours, although large deletions (beyond 100 kb) are shared between tumours harbouring mutations in BRCA1 or BRCA2, thus further emphasizing the different implications of the loss of BRCA1 versus BRCA2 function for DNA repair. BRCA1-deficient tumours and BRCA2-deficient tumours also differ on a pathological level. BRCA1-deficient tumours are usually of a basal-like or triple-negative subtype, while BRCA2-mutated cancers are not biased towards a specific subtype262. Partner and localizer of BRCA2 (PALB2) has been reported to interact with BRCA2 and is required for its recruitment and DNA strand invasion during HR263. In line with its biological function, PALB2 mutations have been shown to confer an increased risk of developing breast cancer74. --- class: center ![](data:image/png;base64,#img/parp_1.webp) --- ## What we discuss * Gross * Histology * Immunohistochemistry * Cell of origin * BRCA variant significance * Homologous recombination * PARP inhibitor