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Purpose:Preclinical data suggest that antiprogestins inhibit the growth of luminal breast carcinomas that express higher levels of progesterone receptor isoform A (PRA) than isoform B (PRB). Thus, we designed a presurgical window of opportunity trial to determine the therapeutic effects of mifepristone in patients with breast cancer, based on their high PRA/PRB isoform ratio (MIPRA; NCT02651844).Patients and Methods:Twenty patients with luminal breast carcinomas with PRA/PRB > 1.5 (determined by Western blots), and PR ≥ 50%, naïve from previous treatment, were included for mifepristone treatment (200 mg/day orally; 14 days). Core needle biopsies and surgical samples were formalin fixed for IHC studies, while others were snap-frozen to perform RNA sequencing (RNA-seq), proteomics, and/or Western blot studies. Plasma mifepristone levels were determined using mass spectrometry. The primary endpoint was the comparison of Ki67 expression pretreatment and posttreatment.Results:A 49.62% decrease in Ki67 staining was observed in all surgical specimens compared with baseline (P = 0.0003). Using the prespecified response parameter (30% relative reduction), we identified 14 of 20 responders. Mifepristone induced an increase in tumor-infiltrating lymphocytes; a decrease in hormone receptor and pSer118ER expression; and an increase in calregulin, p21, p15, and activated caspase 3 expression. RNA-seq and proteomic studies identified downregulated pathways related to cell proliferation and upregulated pathways related to immune bioprocesses and extracellular matrix remodeling.Conclusions:Our results support the use of mifepristone in patients with luminal breast cancer with high PRA/PRB ratios. The combined effects of mifepristone and estrogen receptor modulators warrant clinical evaluation to improve endocrine treatment responsiveness in these patients.See related commentary by Ronchi and Brisken, p. 833

RNA-Seq DEG and GSEA

RNA-Seq and Proteomics analysis. A and B, Dot plot of relevant enriched pathways from GSEA results (Reactome and Hallmark databases) separating responsive (A) and unresponsive tumors (B). C, Immune cell deconvolution (xcell) in the different tumors analyzed by RNA-Seq. Plots with significant p values analyzed by Wilcoxon test and the T cell CD8+ plot are shown. D, Kegg diagram of the Cell cycle pathway, that may explain mifepristone therapeutics effects. A colored code was used: the first half of the box is colored according to the RNA-Seq data (8 tumors) and the last half of the box is colored according to the proteomic data (n=10 tumors; nuclear fraction).

Purpose:Preclinical data suggest that antiprogestins inhibit the growth of luminal breast carcinomas that express higher levels of progesterone receptor isoform A (PRA) than isoform B (PRB). Thus, we designed a presurgical window of opportunity trial to determine the therapeutic effects of mifepristone in patients with breast cancer, based on their high PRA/PRB isoform ratio (MIPRA; NCT02651844).Patients and Methods:Twenty patients with luminal breast carcinomas with PRA/PRB > 1.5 (determined by Western blots), and PR ≥ 50%, naïve from previous treatment, were included for mifepristone treatment (200 mg/day orally; 14 days). Core needle biopsies and surgical samples were formalin fixed for IHC studies, while others were snap-frozen to perform RNA sequencing (RNA-seq), proteomics, and/or Western blot studies. Plasma mifepristone levels were determined using mass spectrometry. The primary endpoint was the comparison of Ki67 expression pretreatment and posttreatment.Results:A 49.62% decrease in Ki67 staining was observed in all surgical specimens compared with baseline (P = 0.0003). Using the prespecified response parameter (30% relative reduction), we identified 14 of 20 responders. Mifepristone induced an increase in tumor-infiltrating lymphocytes; a decrease in hormone receptor and pSer118ER expression; and an increase in calregulin, p21, p15, and activated caspase 3 expression. RNA-seq and proteomic studies identified downregulated pathways related to cell proliferation and upregulated pathways related to immune bioprocesses and extracellular matrix remodeling.Conclusions:Our results support the use of mifepristone in patients with luminal breast cancer with high PRA/PRB ratios. The combined effects of mifepristone and estrogen receptor modulators warrant clinical evaluation to improve endocrine treatment responsiveness in these patients.See related commentary by Ronchi and Brisken, p. 833

Morphological evaluation of CNBs and matched surgical samples; RNA-Seq analysis; Proteomics

Supplementary Table S2. Synergy between AV25CDC and GEM therapy on pancreatic cancer xenograft

Purpose: We decided to construct a novel oncolytic adenovirus whose replication was driven by the CDC25B promoter for its use in preclinical models of pancreatic cancer.Experimental Design: We placed the essential E1A gene under control of the CDC25B promoter. Based on preliminary data, we pseudotyped the adenovirus with a chimeric fiber of serotypes 5/3. We investigated the in vitro lytic effect and the in vivo therapeutic efficacy in combination with gemcitabine on human pancreatic tumor xenografts orthotopically growing in nude mice and in tumors growing in Syrian hamsters. We also assessed biochemical markers of hepatic toxicity and CA19.9 levels.Results: AV25CDC exhibited a strong in vitro lytic effect on pancreatic cancer cells. In vivo administration of AV25CDC combined with gemcitabine in mice harboring subcutaneously growing SW1990 pancreatic tumors almost abrogated tumor growth. Nude mice harboring 15-day-old orthotopic tumors, treated intratumorally or systemically with AV25CDC combined with gemcitabine, exhibited 70% to 80% reduction in tumor size compared with control mice that lasted for at least 60 days. Chemovirotherapy treatment induced a return to normal levels of biochemical parameters of hepatic toxicity; these mice exhibited more than 90% reduction in CA19.9 serum levels compared with control. Chemovirotherapy efficacy was confirmed in mice harboring Mia PaCa-2 tumors and in Syrian hamster harboring HaP-T1 tumors. We observed that viral treatment disrupted tumor architecture and induced an increase in MMP-9 activity that might facilitate gemcitabine penetrability.Conclusion: These data demonstrate that AV25CDC is an effective oncolytic agent candidate for pancreatic cancer chemovirotherapy combination. Clin Cancer Res; 21(7); 1665–74. ©2015 AACR.

Supplementary Figure S1. Schematic representation of fragment of the human CDC25 promoter and In vitro and in vivo efficacy of AV25CDC in combination or not with gemcitabine.

Supplementary Materials and Methods

Preclinical data suggest that antiprogestins inhibit the growth of luminal breast carcinomas that express higher levels of progesterone receptor isoform A (PRA) than isoform B (PRB). Thus, we designed a pre-surgical window of opportunity trial to determine the therapeutic effects of mifepristone in patients with breast cancer based on their high PRA/PRB isoform ratio (MIPRA; NCT02651844).Twenty patients with luminal breast carcinomas with PRA/PRB1.5 (determined by western blots), and PR ≥50%, naive from previous treatment, were included for mifepristone treatment (200 mg/day p.o.; 14 days). Core needle biopsies (CNB) and surgical samples were formalin-fixed for immunohistochemical studies, while others were snap-frozen to perform RNA-Seq, proteomics, and/or western blot studies. Plasma mifepristone levels were determined using mass spectrometry. The primary endpoint was the comparison of Ki67 expression pre- and post-treatment.A 49.62% decrease in Ki67 staining was observed in all surgical specimens compared to baseline (p=0.0003). Using the prespecified response parameter (30% relative reduction), we identified 14/20 responders. Mifepristone induced an increase in tumor-infiltrating lymphocytes, a decrease in hormone receptor and pSer118ER expression, and an increase in calregulin, p21, p15, and activated caspase3 expression. RNA-Seq and proteomics studies identified downregulated pathways related to cell proliferation and upregulated pathways related to immune bioprocesses and extracellular matrix remodeling.Our results support the use of mifepristone in patients with luminal breast cancer with high PRA/PRB ratios. The combined effects of mifepristone and estrogen receptor modulators warrant clinical evaluation to improve endocrine treatment responsiveness in these patients.

Background: Different antiprogestins have been clinically evaluated in gynecological andbreast cancers. Mifepristone (MFP), as well as onapristone and telapristone acetate, showedpartial responses in breast cancer clinical trials. Preclinical data indicates that antiprogestinsinhibit cell proliferation of luminal breast carcinomas expressing higher levels of progesteronereceptor isoform A (PRA) than those of isoform B (PRB) evaluated by western blots (WB). Thus,we designed a pre-surgical window trial to determine the therapeutic effects of oral MFP oncell proliferation and on differential gene expression in 20 breast cancer patients selected bytheir high PRA/PRB isoform ratio.Methods. MIPRA is an open-label, one-arm, prospective interventional study (NCT02651844).We interviewed 140 naive breast cancer patients and 133 accepted to participate. Four coreultrasound-guided biopsies were performed, two were formalin-fixed for diagnosis, ER, PR,HER2, and Ki67 evaluation and two were snap-frozen for WB and molecular studies. Patientsthat met the inclusion criteria, with ER+, PRA/PRB>1.5 and total PR ≥50% determined by WBand immunohistochemistry (IHC), respectively, were included for MFP treatment. Plasma wasobtained before and after treatment for future studies. Patients were treated with oral MFP(200 mg/day) for 14 days before surgery which was performed on day 15. Clinical examinationwas performed at days 7 and 14 to register possible adverse effects and to measure tumorsize. During surgery, samples were formalin-fixed for IHC studies, and others were snap-frozenfor further molecular studies. One patient had a bilateral breast cancer, and both tumorsmatched with the inclusion criteria and were included. The primary endpoint was Ki67labeling, comparing diagnostic core needle biopsy to post-therapy surgical specimens.Considering previous studies performed with tamoxifen, we pre-specified that 30% of relativereduction in Ki67 would be considered as a positive response. Differences in Ki67 expressionwere quantitated by an expert pathologist counting at least ten 40x fields per slide. Theseresults are currently being validated by a second pathologist. One patient, with a core biopsywith less than 500 total cells, was excluded. Ongoing experiments include secondary and otherendpoints: comparison of apoptotic, proliferative and hormone receptor markers by IHC,measurement of MFP plasma levels and, RNAseq analysis in samples pre- and post-treatment. Ki67 changes from baseline were tested with paired Wilcoxon matched-pairssigned-rank test.Results: The median (range) Ki67 value of biopsies was 11.87% (2.70- 34.56) and for surgicalspecimens was 6.45% (0.48-23.77). A 45.67% of decrease in the median % Ki67 (41.63%comparing the arithmetic mean values and 50.83% comparing the geometric mean values) wasregistered in all surgical specimens compared to baseline (p= 0.003). Using the pre-specifiedresponse parameter (30% relative reduction in Ki67), we identified 15/20 (75%) responders.Considering only responsive tumors, a 49.87% decrease in the median % Ki67 (50.83%,arithmetic mean; 62.34% geometric mean) was observed (p Citation Format: Andres Elia, Silvia I Vanzulli, Hugo Gass, Caroline A Lamb, Victoria T Fabris, Paula Martinez Vazquez, Javier Burruchaga, Eunice Spengler, Ines Caillet Bois, Alejandra Castets, Silvia Lovisi, Marcos Liguori, Gabriela Pataccini, M Florencia Abascal, Virginia Novaro, Gabriela Acosta Haab, Alfredo Molinolo, Paola Rojas, Claudia Lanari. Mipra, a window of opportunity study evaluating mifepristone treatment for postmenopausal breast cancer patients with higher levels of progesterone receptor isoform a than b [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS11-35.

The pathological diagnosis of diffuse pleural mesothelioma (DPM) contributes to treatment selection and clinical trials interpretation. To know its characteristics and evaluate the viability of comprehensive pathological diagnosis of DPM in Argentina we did a retrospective descriptive study of DPM cases reported from 2009 to 2018. We analyzed 398 cases corresponding to 238 (60%) men and 160 (40%) women, median age 66 years, from surgical biopsies (78%), small biopsies (16.5%) and surgical resections (5.5%). The 77% were epithelioid (E-DPM), 12% biphasic, 10% sarcomatoid, and 4 cases transitional variant. In E-DPM the main pattern was tubular in 36% and solid in 33%. There was a second pattern in 179 cases. Considering the main pattern and the second together, 48% presented tubular subtype and 48% solid subtype. Stroma, necrosis, and nuclear score showed significant differences between E-DPM and non-epithelioid mesotheliomas. Overall tumor grade was predominantly low in E-DPM, except for 42% of the solid main pattern. We recognized the transitional variant extensively in 4 cases and focally in 8. The immunohistochemical antibody panel used included pan-cytokeratin, calretinin, WT-1, cytokeratin 5, CEA and TTF-1. The expression of cytokeratin 5, calretinin and WT-1 was lower in the sarcomatoid type (43%, 87 and 37%) than in the epithelioid type (92%, 98% and 93%). This study highlights the tumor heterogeneity of DPM that shows the diagnostic difficulty, and the feasibility of evaluating histological aggressiveness in E-DPM, B-DPM and S-DPM in our country.El diagnóstico patológico del mesotelioma pleural difuso (MPD) contribuye a la selección del tratamiento y a la interpretación de los ensayos clínicos. Para conocer sus características y evaluar la viabilidad del diagnóstico patológico de MPD en Argentina se realizó un estudio descriptivo retrospectivo de los casos de MPD informados de 2009 a 2018. Se analizaron 398 casos correspondientes a 238 (60%) hombres y 160 (40%) mujeres, mediana de edad de 66 años, a partir de biopsias quirúrgicas (78%), biopsias pequeñas (16.5%) y resecciones quirúrgicas (5.5%). El 77% fue epitelioide (E-MPD), 12% bifásicos, 10% sarcomatoides y 4 casos variante transicional. En E-MPD se encontró como patrón principal el tubular en 36% y el sólido en 33%. Hubo un segundo patrón en 179 casos. Considerando el principal y el segundo patrón en conjunto, el 48% presentó subtipo tubular y el 48% subtipo sólido. El estroma, la necrosis y el score nuclear mostraron diferencias significativas entre E-MPD y mesoteliomas no epitelioides. El grado general del tumor fue predominantemente bajo en E-MPD, a excepción del 42% del patrón principal sólido. Reconocimos la variante transicional en forma extensa en 4 casos y focalmente en 8. La expresión de citoqueratina 5, calretinina y WT-1 fue menor en el tipo sarcomatoide (43%, 87 y 37%) que en el tipo epitelioide (92%, 98% y 93%). Este estudio destaca la heterogeneidad tumoral de MPD que evidencia la dificu ltad en el diagnóstico y la viabilidad de evaluar la agresividad histológica en E-MPD, B-MPD y S-MPD en nuestro país.

BackgroundThe reported rates of HER2 positivity in uterine cervical cancer (CC) range from 0–87%. The organization of clinical studies of HER2 targeting agents in CC requires a better knowledge of HER2 positivity prevalence. We aimed to provide precise estimates of HER2 overexpression and amplification in CC.MethodsWe conducted a PRISMA compliant meta-analytic systematic review. We searched electronic databases for articles reporting the proportion of HER2 positivity by immunohistochemistry or in situ hybridization in CC. We performed descriptive analyses of all 65 included studies. Of these, we selected 26 studies that used standardized American Society of Clinical Oncology / College of American Pathologists (ASCO/CAP) Guidelines compliant methodology. We conducted several meta-analyses of proportions to estimate the pooled prevalence of HER2 positivity and subgroup analyses using geographic region, histology, tumor stage, primary antibody brand, study size, and publication year as moderators.ResultsThe estimated pooled prevalence of HER2 overexpression was 5.7% (CI 95%: 1.5–11.7%) in ASCO/CAP compliant studies and 27.0%, (CI 95%: 19.9–34.8%) in ASCO/CAP non-compliant ones, p

The reported rates of HER2 positivity in cervical cancer (CC) range from 0% to 87%. The importance of HER2 as an actionable target in CC would depend on HER2 positivity prevalence. Our aim was to provide precise estimates of HER2 overexpression and amplification in CC, globally and by relevant subgroups. We conducted a PRISMA compliant meta-analytic systematic review. We searched Medline, EMBASE, Cochrane database, and grey literature for articles reporting the proportion of HER2 positivity in CC. Studies assessing HER2 status by immunohistochemistry or in situ hybridization in invasive disease were eligible. We performed descriptive analyses of all 65 included studies. Out of these, we selected 26 studies that used standardized American Society of Clinical Oncology / College of American Pathologists (ASCO/CAP) Guidelines compliant methodology. We conducted several meta-analyses of proportions to estimate the pooled prevalence of HER2 positivity and subgroup analyses using geographic region, histology, tumor stage, primary antibody brand, study size, and publication year as moderators. The estimated pooled prevalence of HER2 overexpression was 5.7% (CI 95%: 1.5% to 11.7%) I2 = 87% in ASCO/CAP compliant studies and 27.0%, (CI 95%: 19.9% to 34.8%) I2 = 96% in ASCO/CAP non-compliant ones, p < 0.001. The estimated pooled prevalence of HER2 amplification was 1.2% (CI 95%: 0.0% to 5.8%) I2 = 0% and 24.9% (CI 95%: 12.6% to 39.6%) I2 = 86%, respectively, p = 0.004. No other factor was significantly associated with HER2 positivity rates. Our results suggest that a small, but still meaningful proportion of CC is expected to be HER2-positive. High heterogeneity was the main limitation of the study. Variations in previously reported HER2 positivity rates are mainly related to methodological issues.

PURPOSE: We decided to construct a novel oncolytic adenovirus whose replication was driven by the CDC25B promoter for its use in preclinical models of pancreatic cancer. EXPERIMENTAL DESIGN: We placed the essential E1A gene under control of the CDC25B promoter. Based on preliminary data, we pseudotyped the adenovirus with a chimeric fiber of serotypes 5/3. We investigated the in vitro lytic effect and the in vivo therapeutic efficacy in combination with gemcitabine on human pancreatic tumor xenografts orthotopically growing in nude mice and in tumors growing in Syrian hamsters. We also assessed biochemical markers of hepatic toxicity and CA19.9 levels. RESULTS: AV25CDC exhibited a strong in vitro lytic effect on pancreatic cancer cells. In vivo administration of AV25CDC combined with gemcitabine in mice harboring subcutaneously growing SW1990 pancreatic tumors almost abrogated tumor growth. Nude mice harboring 15-day-old orthotopic tumors, treated intratumorally or systemically with AV25CDC combined with gemcitabine, exhibited 70% to 80% reduction in tumor size compared with control mice that lasted for at least 60 days. Chemovirotherapy treatment induced a return to normal levels of biochemical parameters of hepatic toxicity; these mice exhibited more than 90% reduction in CA19.9 serum levels compared with control. Chemovirotherapy efficacy was confirmed in mice harboring Mia PaCa-2 tumors and in Syrian hamster harboring HaP-T1 tumors. We observed that viral treatment disrupted tumor architecture and induced an increase in MMP-9 activity that might facilitate gemcitabine penetrability. CONCLUSION: These data demonstrate that AV25CDC is an effective oncolytic agent candidate for pancreatic cancer chemovirotherapy combination. Fil: Weber, Helga L.. Fundación Instituto Leloir; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; Argentina Fil: Gidekel, Manuel. Universidad de la Frontera. Núcleo Científico y Tecnológico en Recursos Naturales; Chile Fil: Werbajh, Santiago Enrique. Fundación Instituto Leloir; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; Argentina Fil: Salvatierra, Edgardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; Argentina. Fundación Instituto Leloir; Argentina Fil: Rotondaro, Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; Argentina. Fundación Instituto Leloir; Argentina Fil: Sganga, Leonardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; Argentina. Fundación Instituto Leloir; Argentina Fil: Acosta Haab, Gabriela. Hospital de Oncología Marie Curie; Argentina Fil: Curiel, David T.. Washington University School of Medicine; Estados Unidos Fil: Cafferata, Eduardo Gustavo Alfredo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; Argentina. Fundación Instituto Leloir; Argentina Fil: Podhajcer, Osvaldo Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; Argentina. Fundación Instituto Leloir; Argentina

Introduction The proliferation factor Ki67 is prognostic in breast cancer and included in international therapy guidelines. In fact, a useful surrogate definition of Luminal A-like as distinct from Luminal B-like disease could be made using a combination of ER, PgR and Ki-67, without requiring molecular diagnostics. Furthermore, Ki67 is a predictive tool for hormonotherapy response in the neoadjuvant setting. Objective To compare two monoclonal antibodies MIBland SP6 for assessment of Ki67 in primary breast cancer with regard to reproducibility. Methods We used TMAs constructed from 82 archival FFPE blocks of primary breast. Staining was performed using Ventana Benchmark Autostainer and MIB-1 antibody (Novocastra) was done at a dilution of 1:100, whereas 1:100 dilution was used for SP6 antibody (Cellmarque). Results Tumor characteristics: ductal carcinomas GH1, 19 cases; GH2, 32 cases; GH3, 21 cases; and lobular 10 cases (classic, 8 and pleomorphic, 2). Criterion standard (Ki67-count) was obtained by counting positive and negative tumor cell profiles. Proliferation index between the two antibodies was compared by 3 independent observers. Proliferation index results were similar from both antibodies and ranged from 3% to 85%. Conclusion Both antibodies can be safely used to determine Ki67 proliferation index. SP6 and MIB1 provide highly comparable measures of Ki67 that predict prognostic in breast cancer.