1 Avances en patología digestiva Jesús Javier Sola Hospital San Pedro, Logroño Barcelona, 11 Junio 2015

2  Resultados de la determinación sistemática de las alteraciones de MMRP en el adenocarcinoma de colon  ¿Qué hay de nuevo sobre el adenoma aserrado?  Patología molecular del Cáncer Colorectal  Marcadores inmunohistoquímicos para inmunoterapia en los adenocarcinomas del tracto gastrointestinal

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6  Resultados de la determinación sistemática de las alteraciones de MMRP en el adenocarcinoma de colon  Patrones IHQ inusuales o discordantes  Aplicación otras técnicas IHQ (BRAF)  Discordancias IHQ-Análisis línea germinal

7 [565] Unusual Immunohistochemistry Staining Patterns Encountered in Cancers Screened for Lynch Syndrome. Adams R, Geiersbach K, Tripp SR, Samowitz WS. University of Utah Health Sciences Center, ARUP Institute for Clinical & Experimental Pathology, Salt Lake City, UT; ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT 3564 casos de neoplasias asociadas a SL (colon, endometrio, otros) 20% de alteraciones en el patrón de IHQ de MMRP (757) 13% de patrones “anormales o inusuales” (93) Heterogeneidad intratumoral (Colon 22/57) Alteraciones en más de un gen (Colon 12/57) Tto previo, artefacto, desconocido (Colon 24/57) ¿Son tan frecuentes los patrones inusuales? ¿Cómo reflejar estos hallazgos en nuestros informes?

8 [746] Retained Colorectal Carcinoma MLH1 Expression in Lynch Syndrome Patients Carrying a Germline MLH1 Mutation. Rosty C, Clendenning M, Win AK, et al. Envoi Pathology, Brisbane, Australia; University of Melbourne, Melbourne, Australia; Sullivan Nicolaides Pathology, Brisbane, Australia; Colon Cancer Family Registry Investigators, Multiple Cities, Multiple States Registro de Cáncer Colon Familiar: tumores con pérdida exclusiva de PMS2, 72 casos Mutación en línea germinal de PMS2 en 78% (59) 12/17 casos secuenciación MLH1 7/12 mutación línea germinal MLH1 Mutaciones missense con retención de la antigenicidad de la proteína Pérdida aislada de PMS2 sin mutación en línea germinal, testar MLH1

9 [747] Discordant Mismatch Repair Protein Immunoreactivity in Lynch Syndrome-Associated Neoplasms: A Recommendation for Screening Synchronous/Metachronous Lesions. Roth RM, Hampel H, Arnold CA, Frankel WL. Ohio State University Wexner Medical Center, Columbus, OH 13 pacientes con 29 sinc/meta NASL 31% (4) con patrones discordantes, 3 con tumores sin pérdida MMRP Relevante testar todos los tumores si sospecha clínica [767] Comparison of Mismatch Repair Protein Immunohistochemistry in Matched Colorectal Adenocarcinoma Biopsy and Resection Specimens Steinbauer B, Findeis-Hosey B. University of Rochester Medical Center, Rochester, NY 43 pacientes con biopsia/pieza. 23 alteración MMRP 100% concordancia con MMRP intactas 87% concordancia total. 100% pérdida al menos de 1 proteína

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11 [624] Annexin A10 Is Useful in Screening for Lynch Syndrome By Decreasing the Need for MLH1 Promoter Hypermethylation: A Consecutive Analysis of 575 Colorectal Adenocarcinomas. Esnakula A, Carver P, Pai RK. Cleveland Clinic, Cleveland, OH 575 casos consecutivos ADC. 60 pérdida MLH-1. 53 IHQ AnA10 70% de mutación en BRAF, 33% AnA10 positivos 13/16 hipermetilación promotor MLH1, 46 % AnA10 positivos 3/16 sin hipermetilación promotor MLH1, 0% AnA10 positivos Los autores sugieren que la IHQ para Anexina A10 puede sustituir al estudio de hipermetilación del promotor MLH1 en un 40% casos

12 [631] Prospective BRAF V600E Mutation Testing of dMMR Colorectal Cancer: Detailed Correlation With Patholological and Clinical Features Geraghty R, Boyle L, McCarthy A, Mohan H, et al. St. Vincent's University Hospital and Centre for Colorectal Disease, Dublin, Ireland 226/2165 dMMRP (12%); 80 casos dMLH-1 60 % BRAF-mut 100% concordancia BRAF-mut entre IHQ específica y PCR [644] BRAF Mutation Frequency Is Fewer in Asian Colorectal Carcinomas (CRCs) Patients and Immunohistochemical Detection of BRAF V600E Mutant Protein Using the VE1 Antibody in Colorectal Carcinoma Is Highly Concordant With Molecular Testing Hang JF, Liang WY, Li AFY, Chang SC. Veterans General Hospital-Taipei, Taipei, Taiwan; National Yang-Ming University School of Medicine, Taipei, Taiwan 430 ADC BRAF-mut por PCR e IHQ VE1 100% sensibilidad y 98% especificidad para IHQ VE1

13 [704] Clinicopathologic Comparison of Colorectal Carcinoma in Patients With Suspected Lynch Syndrome without Germline Mutation (Lynch-Like Syndrome) versus Patients with Germline Mutation (Lynch Syndrome) Mas-Moya J, Dudley B, Brand RE, Pai RK. University of Pittsburgh Medical Center, Pittsburgh, PA, OH 3213 casos con 337 MSI-H; 114 sugestivos de SL, 48 estudio en línea germinal 33 (69%) SL: MSH2 (19), MLH1 (5), MSH6 (7), PMS2 (9) 15 sin alteraciones en línea germinal: MLH1/PMS2 (6), MSH2/MSH6 (7), PMS2 (3) [704] Lynch Syndrome Screening: Discordance in MMR and Germline Test Results Mafnas CT, Martin BA, Ford JM, Longacre TA. Stanford University, Stanford, CA 21 casos con resultados discordantes, 14 de colon. Discusión y explicación confusa

14  ¿Qué hay de nuevo sobre el adenoma aserrado?  Alteraciones genéticas en los diferentes subtipos morfológicos  IHQ y PM en la distinción PH y adenoma aserrado  Relación de la Enfermedad Inflamatoria Intestinal y pólipos aserrados

15 [571] Tracing Immunohistochemical and Molecular Markers of Serrated Carcinoma Along the Serrated and Conventional Polyp Pathways of Colorectal Carcinogenesis Alcaraz-Mateos E, Garcia-Solano J, Garcia-Solano M, Carbonell P, Torres-Moreno D, Trujillo-Santos J, Perez-Guillermo M, Conesa-Zamora P. Morales Meseguer University Hospital, Murcia, Spain; Santa Lucia University Hospital, Cartagena, Murcia, Spain; Virgen Arrixaca University Hospital, Murcia, Spain [584] A Clinicopathological and Molecular Analysis of 107 Sessile Serrated Adenomas With Dysplasia Bettington M, Walker NI, Rosty C, Brown I, Clouston AD, Pearson SA, McKeone DM, Leggett BA, Whitehall VLJ. QIMR Berghofer Medical Research Institute, Brisbane, Australia; University of Queensland, Brisbane, Australia; Envoi Pathology, Brisbane, Australia; Queensland Health, Brisbane, Australia; Royal Brisbane and Women's Hospital, Brisbane, Australia [722] Immunohistochemical and Molecular Findings in Sessile Serrated Polyps With Dysplasia Panarelli N, Samowitz WS, Pai RK, Vaughn CP, Qin L, Yantiss RK. Weill Cornell Medical College, New York, NY; University of Utah, Salt Lake City, UT; Cleveland Clinic, Cleveland, OH [734] Phenotype-Genotype Correlation in Serrated Precursor Lesions of the Colon based on Recent Classifications Rau T, Aust D, Baretton G, Eck M, Erlenbach-Wunsch K, Hartmann A, Lugli A, Stohr R, Vieth M, Zlobec I, Katzenberger T. Institute of Pathology, University Bern, Bern, Switzerland; Institute of Pathology, University Hospital, Dresden, Germany; Institute of Pathology, Hospital Aschaffenburg, Aschaffenburg, Germany; Institute of Pathology, Friedrich-Alexander-University Erlangen-Nürnberg, Erlangen, Germany

16 El adenoma serrado sésil (SSA) es el precursor más probable del adenocarcinoma serrado, con el que comparte perfil inmunohistoquímico y molecular, seguido del adenoma velloso convencional El 72% de los SSA con displasia son dMMRP, con 95% de mutaciones en BRAF y expresión aberrante de CK7 y CK20 Hay diferencias en las alteraciones moleculares e inmuno- histoquímicas entre las zonas de displasia, dependiendo de de su morfología (serrado, convencional o mixto), y el resto del pólipo sin displasia (BRAFmut, Anexina A10), coexistiendo ambas vías en la misma lesión El adenoma serrado tradicional (TSA) puede mostrar tanto alteraciones propias de la vía serrada (BRAFmut) como de la vía convencional (KRASmut), lo que sugiere la posibilidad de establecer subgrupos de este tipo de lesiones

17 [574] DNA Methylation Array Shows Overlapping Methylation Profiles in Hyperplastic Polyps (HP) and Sessile Serrated Adenoma/Polyps (SSA/P) of the Colorectum Andrew AS, Li J, Koestler D, Tsongalis GJ, Marsit CJ, Butterly L, Amos CI, Srivastava A. Dartmouth-Hitchcock Medical Center, Lebanon, NH; Brigham & Women's Hospital, Boston, MA [610] Detection of the Loss of Hes1 Expression Differentiate Sessile Serrated Adenoma/polyp From Hyperplastic Polyp Cui M, Awadallah A, Liu W, Xin W. University Hospital Case Medical Center, Cleveland, OH; Case Western Reserve University, Cleveland, OH [634] Co-Expression of MUC5AC and TFF1 Distinguishes Sessile Serrated Adenomas/Polyps From Hyperplastic Polyps Hannah E Goyne, Magomed Khaidakov, Curt H Hagedorn, Laura W Lamps, Keith Lai. University of Arkansas for Medical Sciences, Little Rock, AR

18 35 pólipos hiperplásicos (PH) y 42 adenomas serrados sesiles (SSA) no difieren significativamente en cuanto a la hipermetilación de >485000 islas CpG a lo largo de todo el genoma, lo que sugiere mecanismos epigenéticos de desarrollo similares La pérdida de la expresión de Hes1 tiene una sensibilidad del 91% y una especificidad del 100% para el diagnóstico diferencial entre PH y SSA. Hes1 interviene en la diferenciación de las células intestinales por la vía Notch En 5 PH y 12 SSA la coexpresión intensa de MUC5AC y TFF1, permite diferenciar SSA de PH. Ámbos se expresan en mucosa gástrica y no en mucosa de colon normal, y esta expresión se correlaciona con estudios de expresión previa de RNA

19 [625] BRAF Mutations in Sessile Serrated Polyps Associated With Inflammatory Bowel Disease and Comparison With Sporadic Counterparts Esnakula A, Pai RK, Shadrach B, Liu X, Allende A. Cleveland Clinic, Cleveland, OH [670] Serrated Colorectal Polyps in Inflammatory Bowel Disease Ko HM, McBride RB, Cui M, Ye F, Zhang D, Ullman TA, Harpaz N, Polydorides AD. Mount Sinai Hospital, NY; Icahn School of Medicine at Mount Sinai, New York, NY [772] Clinical, Histologic, and Immunophenotypic Features of Serrated Polyps in Patients With Inflammatory Bowel Disease Tarabishy Y, Nalbantoglu I. Washington University, St. Louis, MO

20 Entre 20 SSP esporádicos y 30 SSP en el contexto de EII no hay diferencias significativas en ningún dato clinicopatológico, ni en el porcentaje de casos con BRAFmut (70% vs 86%) En 78 SPs de 6602 pacientes con EII, la progresión a displasia o carcinoma es muy inferior en los casos de SSA de tipo esporádico sin displasia con respecto a TSA con displasia de tipo convencional Los pólipos serrados en pacientes con EII tiene una distribución topográfica, morfología y perfil inmunohistoquímico (ki67, BRAF y beta-catenina) completamente superponibles a los de los pacientes sin EII

21  Patología molecular del Cáncer Colorectal  Resultados de NGS en CCR  Heterogeneidad Intratumoral  Ampliación opciones terapeúticas  Diferencias en metástasis sincrónicas y metacrónicas  Resistencia a tratamientos antiEGFR

22 [591] Gastrointestinal Tract Adenocarcinomas Display Intratumoral Heterogeneity Detectable By 47 Gene Next Generation Sequencing Panel Braxton DR, Morrissette JJD, Furth EE. University of Pennsylvania, Philadelphia, PA DNA was extracted from FFPE samples(Qiagen), enriched for 47 genes (TruSeq cancer panel), and sequenced on a MiSeq (Illumina, CA). Variant calls were made by an in-house bioinformatic pipeline. AI and SC were called based on allele frequencies and percent tumor. Results: A total of 75 cases were studied with 166 variants detected for a mean of 2.2 (stdev 1.2) per case and an average read depth of 6759. AI was detected in 49/166(30%) variants in 36/75(48%) cases. TP53, KRAS, and APC were the most frequently mutated genes showing AI. SC was detected in 11/75(15%) cases with PIK3CA(3/11), FBXW7(2/11), and TP53(2/11) being the most frequent genes defining SC. Evolving subclones which harbored actionable mutations were only found in resection/excisions and cases with greater than 25% tumor.

23 [710] Genotype Driven Anti-Cancer Therapies for Colon Cancer Mockus BS, Stafford G, Potter C, Ananda G, Hinerfeld D, Tsongalis GJ. The Jackson Laboratory for Genomic Medicine, Farmington, CT; The Jackson Laboratory for Mammalian Genomics, Bar Harbor, ME; Geisel School of Medicine at Dartmouth and Dartmouth-Hitchcock Medical Center, Lebanon, NH DNA from ten colon adenocarcinoma FFPE samples was sequenced on Illumina HiSeq 2500 or MiSeq sequencers using a CLIA-certified 358-gene targeted sequencing assay. Actionable variants were defined as: 1) gene variant associated with an approved drug in colon cancer, 2) gene variant associated with an approved drug in another tumor type, 3) gene variant associated with a drug that is in an active colon or solid tumor clinical trial, and/or 4) gene variant confers resistance to a drug. An average of 2.7 actionable variants were identified per sample with KRAS mutations in exons 12 and 13 being the most common. The average number of therapeutic options was 1.5, 10.7, and 15.4 for FDA approved drugs in colon cancer, FDA approved drugs in other tumor types, and investigational drugs in clinical trials, respectively.

24 [728] Next Generation Sequencing (NGS) Identifies Mutational Distinction Between Synchronous and Metachronous Distant Metastases of Colorectal Carcinoma (CRC) Patel S, Farahani N, Chevarie-Davis M, Andy P, Aguiluz A, Riley C, Hodge J, Alkan S, Lopategui JR. Cedars-Sinai Medical Center, Los Angeles, CA Thirty-two samples from eleven patients with metastatic CRC were selected from institutional archives. Seven patients had synchronous distant metastases and 4 patients developed metachronous distant metastases. Samples were evaluated using the AmpliSeq cancer panel v2 with aberrations filtered to include only non-synonymous amino acid changes in 2855 "hotspots" frequently mutated in 50 cancer genes. Across all patients, 25 different hotspot mutations were identified involving TP53, APC, BRAF, PTEN, PIK3CA, KRAS, SMAD4, FBXW7, and KDR. Mutations per case ranged from 1-4. About half of synchronous DM demonstrated completely divergent mutations from the PT. This is in contrast to metachronous DM in which all had the same or overlapping mutations as the PT.

25 [583] Molecular Characterization of Acquired Resistance To Anti-EGFR Monoclonal Antibodies in Colorectal Cancer Patients Bellosillo B, Dalmases A, Martinez A, Iglesias M, Gonzalez I, Salido M, Busto M, Sanchez J, Vidal J, Clave S, Moragon E, Pairet S, Bellmunt J, Serrano S, Rovira A, Albanell J, Montagut C. Hospital del Mar, Barcelona, Spain To increase our understanding on mechanisms of acquired resistance to anti-EGFR treatment, we evaluated molecular changes before and after receiving anti-EGFR treatment in paired tumor samples from CRC patients. 37 colorectal cancer patients were included in the study. Mutations in EGFR, KRAS, NRAS, BRAF and PIK3CA genes were evaluated by next- generation pyrosequencing. KRAS, EGFR and HER2 gene amplification was evaluated by FISH. Molecular analysis of samples at the time of progression showed the emergence of novel alterations in 81% of patients. RAS (40% of patients) mostly affected exons 3 and 4, PIK3CA (19%), BRAF (11%), EGFRS492R (8%) and novel mutations in EGFR ectodomain (5%). FISH showed EGFR and HER2 gene amplification in 57% and 18%. In 9 patients the mutations were already detected by next-generation sequencing at diagnosis as well as in 8 patients EGFR and HER2 amplification.

26  Marcadores inmunohistoquímicos para inmunoterapia en los adenocarcinomas del tracto gastrointestinal  Determinación de PDL-1 por IHQ en adenocarcinomas de colon y estómago  Inmunofenotipado de TIL’s en adenocarcinomas de colon y estómago

27 [743] Programmed Cell Death Ligand 1 Expression Is Associated With BRAF Mutation, Microsatellite Instability, Medullary Morphology, and Helps To Characterize Tumor Behavior in Colorectal Carcinoma Rosenbaum M, Bledsoe J, Huynh TG, Mino-Kenudson M. Massachusetts General Hospital, Boston, MA We performed immunohistochemistry for PD-L1 (clone: E1L3N #13684, 1:200, Cell Signaling Technology, MA) on tissue microarrays (TMAs) consisting of 336 tissue cores from 164 CRC cases. Mutational and microsatellite instability (MSI) status was known in most cases. The same TMAs were stained with anti-CD8 (clone: IgG2b 4B11, RTU, Leica, IL). Fifteen cases (9%) showed positive membranous staining for PD-L1 (PD- L1-P). PD-L1 expression correlated with abundant TILs (P

28 [776] Expression of PDL1 in Gastric Adenocarcinomas and Associated Tumor Infiltrating Lymphocytes Thompson E, Abdelfatah E, Taube J, Duncan MD, Ahuja N, Kelly R, Anders R. Johns Hopkins, Baltimore, MD 34 cases of invasive gastric adenocarcinoma were stained for PDL1 and the tumors and associated TILs were scored for PDL1 expression. Tumors with greater than 5% membranous staining were considered PDL1 positive. TILs were scored as no significant staining (0), less than 50% (focal) or greater than 50% (high). 4/34 tumors (12%) showed membranous PDL1 expression. Overall, 45% of gastric adenocarcinomas showed some level of PDL1 expression among TILs with 27% showing focal interface expression and 18% showing high expression. 55% of TILs in had no expression of PDL1. High PDL1 expression on TILs correlated with lower tumor stage at presentation, while deaths occurred in a higher percentage of patients with PDL1 positive tumors than negative tumors.

29 [642] Tumor-Infiltrating Lymphocytes in Colorectal Adenocarcinomas: Tracking the Immune Response Through Different Tumor Stages Hallas C, Stamm M, Falk M, Tiemann M. Hematopathology Hamburg, Hamburg, Germany; Semmelweis University Medical Faculty, Asclepios Campus Hamburg, Hamburg, Germany B-cells (CD19+ and CD20+, resp.), T-cells (CD3+, CD4+, and CD8+), activated T-cells (CD25+), Treg (FoxP3+) and tissue macrophages (histiocytes, CD68+) were stained by immunohistochemistry and counted in 131 colorectal adenocarcinomas of different stages. The immune response in colorectal tumors was dominated by T cells in all tumor stages. CD4+ cells were more abundant than CD8+ cells throughout all TNM stages except for pT2. In pT2, CD8+ T cells were significantly more abundant.The high CD8+ count in pT2 correlated with increased numbers of activated CD25+ T cells and histiocytes. The strongest CD8+ cytotoxic T cell response was found in pT2, pointing towards a functional immune response in early stage disease.

30 [755] Characterization and Clinical Value of Tumor Infiltrating Lymphocytes (TILs) in Gastric Cancer Schalper K, Carvajal-Hausdorf D, Syrigos KN, Rimm DL. Yale University, New Haven, CT; University of Athens, Sotiria General Hospital, Athens, Greece Using multiplexed quantitative fluorescence (QIF), we simultaneously measured the levels of CD3 (T cells), CD8 (cytotoxic T cells), CD20 (B- lymphocytes), cytokeratin (tumor cells) and DAPI (all nuclei) in a retrospective collection of 302 GCs represented in tissue microarray format. The level of TILs was scored using the AQUA method. Measurements were performed in two cores obtained from different tumor areas to assess heterogeneity of the markers. GCs with diffuse histology showed higher levels of CD20 than intestinal neoplasms (P=0.04). All markers showed a trend for lower levels in stage IV tumors. Using the median score as cutpoint, elevated CD8 but not CD3 or CD20 signal was significantly associated with longer overall survival (log- rank P=0.004).

31 ¡¡¡Gracias por su atención!!!