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Immunotherapy including immune checkpoint inhibitors (ICIs) has become the backbone of treatment for most lung cancers with advanced or metastatic disease. In addition, they have increasingly been used for early stage tumors in neoadjuvant and adjuvant settings. Unfortunately, however, only a subset of patients experiences meaningful response to ICIs. Although programmed death-ligand 1 (PD-L1) protein expression by immunohistochemistry (IHC) has played a role as the principal predictive biomarker for immunotherapy, its performance may not be optimal, and it suffers multiple practical issues with different companion diagnostic assays approved. Similarly, tumor mutational burden (TMB) has multiple technical issues as a predictive biomarker for ICIs. Now, ongoing research on tumor- and host immune-specific factors has identified immunotherapy biomarkers that may provide better response and prognosis prediction, in particular in a multimodal approach. This review by the International Association for the Study of Lung Cancer Pathology Committee provides an overview of various immunotherapy biomarkers, including updated data on PD-L1 IHC and TMB, and assessments of neoantigens, genetic and epigenetic signatures, immune microenvironment by IHC and transcriptomics, and microbiome and pathologic response to neoadjuvant immunotherapies. The aim of this review is to underline the efficacy of new individual or combined predictive biomarkers beyond PD-L1 IHC and TMB.

Table with Details of Patient Characteristics

Methods for quantitative immunofluorescence and IFN gamma treatment of PDXs

Supplementary Figure Legends

Supplemental Fig 1. Epithelial and stromal PD-L1 expression. Supplemental Fig 2. Validation of specificity of PD-L1 antibody used in this study. Supplemental Fig 3. Correlation with PD-L1 expression and pCR in patient subsets. Supplemental Fig 4. Joinpoint analysis of PD-L1 quantitative scores in the tumor mask (A) and stromal compartment (B). Supplemental Fig 5. Contingency tables of PD-L1 in the epithelium (A) and stroma (B) dichotomized by Joinpoint into low and high with pCR.

Supplemental Table 1. Exclusion of Ki67 from Table 4 shows that both stromal and epithelial PD-L1 expressions are highly significant in multivariate analysis.

Programmed death 1 ligand 1 (PD-L1) is an immune regulatory molecule that limits antitumor immune activity. Targeting of PD-L1 and other immune checkpoint proteins has shown therapeutic activity in various tumor types. The expression of PD-L1 and its correlation with response to neoadjuvant chemotherapy in breast cancer has not been studied extensively. Our goal was to assess PD-L1 expression in a cohort of breast cancer patients treated with neoadjuvant chemotherapy. Pretreatment biopsies from 105 patients with breast cancer from Yale New Haven Hospital that subsequently received neoadjuvant chemotherapy were assessed for PD-L1 protein expression by automated quantitative analysis with a rabbit monoclonal antibody (E1L3N) to the cytoplasmic domain of PD-L1. In addition, tumor-infiltrating lymphocytes (TIL) were assessed on hematoxylin and eosin slides. PD-L1 expression was observed in 30% of patients, and it was positively associated with hormone-receptor–negative and triple-negative status and high levels of TILs. Both TILs and PD-L1 measured in the epithelium or stroma predicted pathologic complete response (pCR) to neoadjuvant chemotherapy in univariate and multivariate analyses. However, because they are strongly associated, TILs and PD-L1 cannot both be included in a significant multivariate model. PD-L1 expression is prevalent in breast cancer, particularly hormone-receptor–negative and triple-negative patients, indicating a subset of patients that may benefit from immune therapy. Furthermore, PD-L1 and TILs correlate with pCR, and high PD-L1 predicts pCR in multivariate analysis. Cancer Immunol Res; 3(4); 326–32. ©2014 AACR.

Supplementary Figures S1-S8

Supplemental Figure 3. PD-L1 in tumor by QIF does not correlate with OS or PFS in 1st line A-B and >1st line C-D of ITx treatment.

Supplemental Table 2. Co-location scores among clinical benefit and LTB subgroups. (LTB: long-term benefit)

Supplemental Figure 1. Multivariate analysis of co-location score, age, gender smoking and histologic subtype association with PFS and OS in total cohort population A-B and in >1st line of treatment subgroup C-D. (Cox PH: Cox proportional hazards model)

Supplemental Table 1. Cohort characteristics.

Purpose:Programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1) interaction suppresses local T-cell responses and promotes peripheral tolerance. In the current study, we focus on PD-1/PD-L1 co-location as a surrogate for this interaction and assess its association with immunotherapy outcomes in patients with non–small cell lung cancer (NSCLC).Experimental Design:Pretreatment biopsies from a retrospective cohort of 154 immunotherapy-treated patients with advanced NSCLC were analyzed. Expression of PD-1 and PD-L1 was assessed by multiplexed quantitative immunofluorescence (QIF) and PD-1 expression in the same pixels as PD-L1 (called a co-location score) was measured using an algorithm to define overlapping expression areas. Co-location scores were correlated with immunotherapy outcomes and PD-L1 tumor proportion score.Results:PD-1/PD-L1 co-location score was associated with best overall response (P = 0.0012), progression-free survival (P = 0.0341), and overall survival after immunotherapy (P = 0.0249). The association was driven by patients receiving immune checkpoint inhibitors in the second or subsequent line of treatment. PD-L1 tumor proportion score by IHC was also correlated with best overall response and progression-free survival. PD-L1 measured within the tumor compartment by QIF did not show any significant association with either best overall response or overall survival. Finally, co-location score was not associated with PD-L1 expression by either method.Conclusions:On the basis of our findings, co-location score shows promise as a biomarker associated with outcome after immunotherapy. With further validation, it could have value as a predictive biomarker for the selection of patients with NSCLC receiving treatment with immune checkpoint inhibitors.

Supplemental Figure 2. PD-L1 association with response and survival for cases that received 1stline ITx A-G and for cases that received >1st line ITx H-N.

Additional file 1: Table S1. CD8+ by study arm (natural scale). Aza, azacitidine plus pembrolizumab (Arm A); Rom, romidepsin plus pembrolizumab (arm B); Aza + Rom, azacitidine plus romidepsin plus embrolizumab (Arm C). Table S2. CD8+/CD4+ by study arm (natural scale). Aza, azacitidine plus pembrolizumab (Arm A); Rom, romidepsin plus pembrolizumab (arm B); Aza + Rom, azacitidine plus romidepsin plus pembrolizumab (Arm C). Table S3. ANOVA log(CD8+) and log(CD8+/CD4+) changes. Aza, azacitidine plus pembrolizumab (Arm A); Rom, romidepsin plus pembrolizumab (arm B); Aza + Rom, azacitidine plus romidepsin plus pembrolizumab (Arm C).

Background: Poly-ADP ribose polymerase inhibitors (PARPi) work by impairing DNA damage repair and have demonstrated activity in patients with BRCAmt breast cancer. Their mechanism of action leads to high mutational burden and increased neoantigens suggesting tumors exposed to PARPi may have increased response to immunotherapy. Preclinical and phase I/II trials have shown evidence of antitumor effect of PARPi + PD-L1 inhibition. In this study, we assessed the efficacy of the PARPi olaparib as monotherapy vs. in combination with PD-L1 inhibitor atezolizumab in patients with BRCAmt advanced breast cancer. Methods: A multicenter, randomized, open-label, phase II study was conducted. Patients were enrolled at 20 sites in the US. Patients were allowed prior hormone or chemotherapy and randomized to receive olaparib 300 mg twice daily (O) +/- atezolizumab 1200 mg IV every 21 days (O+A) until disease progression or intolerable side effect. Patients with progression on O were allowed to crossover to O+A arm. The primary endpoint was comparison of progression free survival (PFS) between arms. Overall survival (OS) was also assessed. Safety and activity analyses were done in patients who received at least one dose of study treatment. Serial tumor biopsies were collected at baseline, 6 weeks, and progression to evaluate the effect of therapy on tumor infiltrating lymphocyte populations, whole exome DNA sequencing, and correlate with outcome to assess predictive value. Enrollment has completed and the study is ongoing. The trial is registered with ClinicalTrials.gov, NCT02849496. Results: 78 patients with BRCAmt advanced breast cancer were enrolled between 5/3/2018 and 3/17/2022 and available for analysis. Groups were generally well matched on baseline characteristics. PFS was 7.0 months (95% CI 5.5-11.5) in O arm and 7.67 months (95% CI 5.6-10) in O+A arm (p=0.92). Median OS was 26.5 months (95% CI 19.2- NR) in O arm and 22.4 months (95% CI 16.6-31.3) in O+A arm (p=0.3). In triple negative breast cancer (TNBC) subgroup (n=23), there was no significant difference in PFS (p=0.92) or OS (p=0.6). Treatment was generally well tolerated with expected side effects, with O+A group experiencing more adverse events of all grades. Analysis of tumor samples from serial biopsies will be presented separately. Conclusion: Olaparib or combination olaparib + atezolizumab resulted in clinically meaningful PFS in both treatment arms. Addition of atezolizumab did not significantly increase PFS. Treatment in both arms was generally well tolerated with expected side effects based on prior drug profiles. This is the first randomized trial to show that the addition of immunotherapy (atezolizumab) to PARPi (olaparib) in patients with advanced BRCAmt breast cancer does not add to therapeutic response but carries with it additional toxicities. Citation Format: Kristina A. Fanucci, Mary Jo Pilat, Derek Shyr, Yu Shyr, Scott A. Boerner, Diane Durecki, Anne Noonan, Vandana Abramson, Cesar Santa-Maria, Hyo Han, Rita Nanda, Elizabeth Claire Dees, Haeseong Park, Saundra Buys, Namrata Peswani, Helen Chew, Hadeel Assad, Gerburg Wulf, Angelique Richardson, Meghna S. Trivedi, Adam Brufsky, James Abbruzzese, Anosheh Afghahi, Elad Sharon, Kurt Schalper, Patricia LoRusso. Olaparib +/- atezolizumab in patients with BRCA-mutated (BRCAmt) locally advanced unresectable or metastatic (advanced) breast cancer: an open-label, multicenter, randomized phase II trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT145.

Responders to checkpoint blockade in Non Small Cell Lung Cancer (NSCLC) often feature an inflamed microenvironment prior to therapy. However, the complete set of molecular drivers connecting this histologic observation to enhanced tumor clearance remain enigmatic. In updated analysis of the Stand Up 2 Cancer-Mark Foundation (SU2C-MARK) Cohort - a collection of 393 patients with whole exome and/or RNA sequencing along with matched checkpoint blockade response annotation - we identify a prominent predictive role for inducible components of the immunoproteasome, a non-canonical peptide processing complex upstream of antigen presentation. Notably, these subunits are enriched as predictors relative to interferon-inducible genes as well as proteasome components in general, and are consistently associated with objective response, progression-free survival and overall survival. Expression of Immunoproteasome subunits associates positively with TCR (but not BCR) burden, supporting a mechanistic model in which enhanced immunoproteasome processivity leads to superior T-cell recognition. Furthermore, although they are known to be targets of interferon gamma (IFNɣ), we demonstrate that their expression is better modeled via a combination of IFNɣ and tumor necrosis factor-α (TNFα) levels, suggesting they may act as integrators of multiple cytokine cascades. Given the fact that the immunoproteasome can alter both antigen quantity as well as quality (including peptide cleavage site preference), the enhanced expression of this complex in the setting of checkpoint blockade response may have important implications for modeling of antigen presentation. These data also suggest novel strategies to enhance immune checkpoint blockade. Citation Format: Vivek Naranbhai, Arvind Ravi, Matthew Hellmann, Monica Arniella, Mark Holton, Samuel Freeman, Chip Stewart, Ignaty Leshchiner, Jaegil Kim, Yo Akiyama, Aaron Griffin, Natalie Vokes, Mustafa Sakhi, Vashine Kamesan, Hira Rizvi, Biagio Ricciuti, Patrick Forde, Valsamo Anagnostou, Jonathan Riess, Don Gibbons, Nathan Pennell, Vamsidhar Velcheti, Subba Digumarthy, Mari Mino-Kenudson, Andrea Califano, John Heymach, Roy Herbst, Julie Brahmer, Kurt Schalper, Victor Velculescu, Brian Henick, Naiyer Rizvi, Pasi Janne, Mark Awad, Andrew Chow, Benjamin Greenbaum, Marta Luksza, Alice Shaw, Jedd Wolchok, Nir Hacohen, Gad Getz, Justin Gainor. Immunoproteasome expression and checkpoint blockade response in advanced non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3468.

Accurate subtyping of NSCLC into lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) is the cornerstone of NSCLC diagnosis. Cytology samples reveal higher rates of classification failures, that is, subtyping as non-small cell carcinoma-not otherwise specified (NSCC-NOS), as compared with histology specimens. This study aims to identify specific algorithms on the basis of known cytomorphologic features that aid accurate and successful subtyping of NSCLC on cytology.A total of 13 expert cytopathologists participated anonymously in an online survey to subtype 119 NSCLC cytology cases (gold standard diagnoses being LUAD in 80 and LUSC in 39) enriched for nonkeratinizing LUSC. They selected from 23 predefined cytomorphologic features that they used in subtyping. Data were analyzed using machine learning algorithms on the basis of random forest method and regression trees.From 1474 responses recorded, concordant cytology typing was achieved in 53.7% (792 of 1474) responses. NSCC-NOS rates on cytology were similar among gold standard LUAD (36%) and LUSC (38%) cases. Misclassification rates were higher in gold standard LUSC (17.6%) than gold standard LUAD (5.5%; p0.0001). Keratinization, when present, recognized LUSC with high accuracy. In its absence, the machine learning algorithms developed on the basis of experts' choices were unable to reduce cytology NSCC-NOS rates without increasing misclassification rates.Suboptimal recognition of LUSC in the absence of keratinization remains the major hurdle in improving cytology subtyping accuracy with such cases either failing classification (NSCC-NOS) or misclassifying as LUAD. NSCC-NOS seems to be an inevitable morphologic diagnosis emphasizing that ancillary immunochemistry is necessary to achieve accurate subtyping on cytology.

Despite known histological, biological, and clinical differences between lung adenocarcinoma (LUAD) and squamous cell carcinoma (LUSC), relatively little is known about the spatial differences in their corresponding immune contextures. Our study of over 1000 LUAD and LUSC tumors revealed that computationally derived patterns of tumor-infiltrating lymphocytes (TILs) on H&E images were different between LUAD (N = 421) and LUSC (N = 438), with TIL density being prognostic of overall survival in LUAD and spatial arrangement being more prognostically relevant in LUSC. In addition, the LUAD-specific TIL signature was associated with OS in an external validation set of 100 NSCLC treated with more than six different neoadjuvant chemotherapy regimens, and predictive of response to therapy in the clinical trial CA209-057 (n = 303). In LUAD, the prognostic TIL signature was primarily comprised of CD4+ T and CD8+ T cells, whereas in LUSC, the immune patterns were comprised of CD4+ T, CD8+ T, and CD20+ B cells. In both subtypes, prognostic TIL features were associated with transcriptomics-derived immune scores and biological pathways implicated in immune recognition, response, and evasion. Our results suggest the need for histologic subtype-specific TIL-based models for stratifying survival risk and predicting response to therapy. Our findings suggest that predictive models for response to therapy will need to account for the unique morphologic and molecular immune patterns as a function of histologic subtype of NSCLC.

The introduction of checkpoint blockade therapy, specifically anti-PD-1/PD-L1 agents, has transformed the treatment landscape of advanced Non-Small Cell Lung Cancer (NSCLC). While our understanding of the biology underlying immunotherapy in NSCLC is still incomplete, studies to date have established central roles for Tumor Mutation Burden (TMB) and PD-L1 Tumor Proportion Score (PDL1-TPS). In order to expand our understanding of the molecular features underlying response in NSCLC, we describe here the first joint analysis of the Stand Up 2 Cancer-Mark Foundation (SU2C-MARK) Cohort, a collection of 393 patients with whole exome and/or RNA sequencing along with matched checkpoint blockade response annotation. We identify a number of significant associations between molecular features and response, including: 1) favorable and unfavorable genomic subgroups; 2) distinct immune infiltration signatures associated with wound healing (unfavorable) and immune activated (favorable) microenvironments; and 3) a novel de-differentiated tumor-intrinsic subtype characterized by high TMB, immune activation, and enhanced response rate. Taken together, results from this cohort extend our understanding of NSCLC-specific predictors, providing a rich set of molecular and immunologic hypotheses with which to further our understanding of the biology of checkpoint blockade in NSCLC. Citation Format: Arvind Ravi, Justin Gainor, Monica Arniella, Chip Stewart, Sam Freeman, Mark M. Awad, Patrick Forde, Valsamo Anagnostou, Brian Henick, Jonathan W. Riess, Don Gibbons, Nathan Pennell, Vamisdhar Velcheti, Ignaty Leshchiner, Jaegil Kim, Subba Digumarthy, Mari Mino-Kenudson, John Heymach, Natalie Vokes, Andrew Griffin, Biagio Ricciuti, Naiyer Rizvi, Roy Herbst, Victor Velculescu, Julie Brahmer, Kurt Schalper, Pasi Janne, Jedd Wolchok, Alice Shaw, Nir Hacohen, Gad Getz, Matthew D. Hellmann. Integrative genomics of checkpoint blockade response in advanced non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3580.

Introduction: Most patients with advanced NSCLC fail to develop durable responses from PD-1 axis blockade, and more robust predictive biomarkers are needed. In this study, we aimed to discover and validate spatially resolved protein markers associated with sensitivity to PD-1 axis inhibition across multiple NSCLC cohorts. Methods: We first used the GeoMx Digital Spatial Profiling (DSP) system in a discovery cohort of 56 NSCLC patients treated with PD-1 axis inhibitors at Yale (USA). Pre-treatment tumors were represented in a tissue microarray (YTMA471) and analyzed using a 71-plex primary antibody panel. Proteins were measured from three molecularly defined tissue compartments: tumor (CK+), leukocytes (CD45+/CD68-), and macrophages (CD68+). We used quantitative immunofluorescence (QIF) to orthogonally validate candidate biomarkers. For external validation of identified predictors, we assessed pre-treatment whole tissue sections from a cohort of 128 NSCLC patients treated with single-agent PD-1 axis inhibitors at the Hospital 12 de Octubre (Madrid, Spain) using DSP (39-plex protein panel, measured from CK+ and CD45+ compartments). In addition, we analyzed two immunotherapy untreated cohorts to address prognostic significance: YTMA423 (Yale, USA; n = 252) and CIMA-CUN (UNAV, Spain; n = 124), using QIF and DSP respectively. Results: Using continuous log-scaled data, we found 5 markers independently associated with progression-free survival (PFS) in the tumor compartment (including PD-L1, p = 0.002). Among the novel candidate predictors, tumor cell CD44, a marker of pluripotency and stemness, was associated with longer PFS (multivariate HR = 0.63, p= 0.002). Using QIF, we orthogonally validated that CD44 expression in the tumor compartment was associated with longer PFS (p 1.5, p Conclusion: This work highlights CD44 as a novel indicative biomarker of sensitivity to PD-1 axis blockade in NSCLC. NSCLCs with high CD44 expression in epithelial cells may be associated with an immune contexture primed for higher response to immune checkpoint blockade. Further studies are needed to understand the interplay between CD44+ cancer stem cell phenotype and mechanisms of immune evasion. Citation Format: Myrto Moutafi, Magdalena Molero, Sandra Martinez-Morilla, Javier Baena, Ioannis Vathiotis, Niki Gavrielatou, Laura Castro, Gorka Ruiz de Garibay, Vera Adradas, Daniel Orive, Karmele Valencia, Alfonso Galvo, Luis M. Montuenga, Santiago Ponce, Kurt Schalper, Luis Paz-Ares, David L. Rimm, Jon Zugazagoitia. Spatially resolved proteomic profiling identifies tumor cell CD44 as a novel indicative biomarker of sensitivity to PD-1 axis blockade in advanced non-small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2028.

El cancer de colon y recto es una importante causa de muerte en nuestro pais. El gen KRAS esta involucrado en la carcinogenesis entre 22-45% de los casos. El objetivo fue determinar la frecuencia y tipos de mutaciones del gen KRAS en el cancer de colo-rectal. Se realizo estudio de mutaciones del gen KRAS en tumores primarios y metastasis de canceres de colon y recto, en tejidos fijados en formalina e incluidos en parafina, mediante RFLP (Restriction Fragment Length Polimorphism) y secuenciacion directa. El grupo estaba constituido por 56 mujeres y 53 hombres con una edad promedio de 60 y 61,4 anos (DS ±13,7 y 10,7 anos respectivamente). Los tumores primarios estaban localizados en colon en 82 casos (75,2%) y 24 casos (20%). En tres casos no se logro precisar el sitio de extraccion de la muestra. En 46 casos (42,2%) se demostro mutacion del gen KRAS. El codon 12 en 37 casos (80,4%); G12D (39,1%); G12V(24,2%); G12S(6,5%); G12A(4,3%); G12C(4,3%); G12R(2,1%) y 9 casos (19,6%) en el codon 13 todas G13D. No se demostraron diferencias en la frecuencia y distribucion de las mutaciones por genero, edad, tumores primario versus metastasis, o localizacion del tumor. El 42% de los canceres colo-rectales presentan mutacion del gen KRAS con una frecuencia y distribucion similar a la reportada en la literatura en series extranjeras, a excepcion de la mutacion G12C. Ensayos clinicos son requeridos para determinar el valor predictivo y pronostico de estas mutaciones en nuestra poblacion.

Recent success of immunotherapy strategies such as immune checkpoint blockade in several malignancies has established the role of immunotherapy in the treatment of cancer. Cancers use multiple mechanisms to co-opt the host-tumor immune interactions, leading to immune evasion. Our understanding of the host-tumor interactions has evolved over the past few years and led to various promising new therapeutic strategies. This article will focus on the basic principles of immunotherapy, novel pathways/agents, and combinatorial immunotherapies.

Background: The quality of the archival samples stored at pathology services could be a limiting factor for molecular biology studies. Aim: To determine the quality of DNA extracted from gallbladder cancer samples at different institutions. Material and Methods: One hundred ninety four samples coming from fve medical centers in Chile, were analyzed. DNA extraction was quantifed determining genomic DNA concentration. The integrity of DNA was determined by polymerase chain reaction amplification of different length fragments of a constitutive gene (β-globin products of 110, 268 and 501 base pairs). Results: The mean DNA concentration obtained in 194 gallbladder cancer samples was 48 ± 43.1 ng/µl. In 22% of samples, no amplification was achieved despite obtaining a mean DNA concentration of 58.3 ng/ul. In 81, 67 and 22% of samples, a DNA amplification of at least 110, 268 or 501 base pairs was obtained, respectively. No differences in DNA concentration according to the source of the samples were demonstrated. However, there were marked differences in DNA integrity among participating centers. Samples from public hospitals were of lower quality than those from private clinics. Conclusions: Despite some limitations, in 80% of cases, the integrity of DNA in archival samples from pathology services in our country would allow the use of molecular biology techniques.

The HER2/neu gene is a proto-oncogene that can predict the response to treatment with trastuzumab, pertuzumab, and lapatinib. This study was conducted to determine the frequency of HER2/neu overexpression and to identify a subgroup of patients with gallbladder cancer who would benefit from targeted therapy.Patients with gallbladder cancer (n = 187; 165 women and 22 men) with a recorded follow-up of at least 5 years were included, along with control subjects (n = 75). An automated immunohistochemical technique was used with an anti-ErbB2 antibody. Scoring was conducted according to the CAP/ASCO (College of American Pathologists/American Society of Clinical Oncology) criteria for breast cancer.Overexpression of HER2/neu was observed in 12.8% of the cases. Of those, 0% were mucosal, 14.3% muscular, 12.8% subserosal, and 10.6% serosal. In 20% of the cases, equivocal staining was observed. Overexpression was more frequent in the advanced cancers and in the better differentiated tumors (13.8% and 17.4%, respectively), but the difference was nonsignificant. The patients with overexpression of HER2/neu had a worse overall survival, when compared with those who had no expression at 5 years (34% vs. 41%).This is the single largest study of HER2/neu expression in gallbladder cancer to use commonly accepted scoring criteria. The results indicate that HER2/neu overexpression occurred in 14% of the advanced gallbladder cancer cases. This subgroup may benefit from inhibitors of the HER2/neu pathway.

Overexpression/amplification of the HER2 gene in advanced gastric cancer is a predictor of response to adjuvant therapy with monoclonal antibodies.To determine the frequency of HER2 gene overexpression and amplification in advanced gastric cancer.One hundred nine advanced gastric cancer biopsy specimens, from 76 men and 33 women aged 67 ± 14 and 62 ± 12 years respectively, were selected. Three histological patterns (diffuse, intestinal and mixed) were recognized. Automated immunohistochemistry was performed with monoclonal c-erbB-2 (NCL-356) Novocastra. Fluorescent in situ hybridization (FISH) for HER2 was performed in positive cases.In 39% of cases, immunohistochemical staining was negative. It was 1+, 2+ and 3+ positive in 15, 36 and 11% of cases, respectively. It was positive in 16% and 3% of intestinal type and mixed carcinomas, respectively. It was negative in all diffuse carcinomas. FISH was performed in 39 (2 +) cases and in 11 (3 +) cases. The gene amplification was positive in two (2 +) and 11 (3 +) cases (11.9%). The overall concordance between immunohistochemical staining and in situ hybridization was 85%.In advanced gastric cancer, HER2 gene overexpression or amplification was observed in 11% and 12% of cases, respectively.

In colorectal cancer, BRAF and KRAS mutation are mutually exclusive, but both are independent prognostic factors for the disease.To determine the frequency of BRAF V600E mutation in colorectal cancer.A KRAS mutation study was carried out in 100 tissue samples of primary and metastatic adenocarcinomas of colon and rectum from patients aged 61.1 ± 62 years (56 women). Negative KRAS mutation cases underwent study of BRAF V600E mutation by restriction fragment length polymorphism (RFLP) and direct sequencing.Primary tumors were located in the colon and rectum in 88 and six cases respectively. Five were liver metastases and in one case, the sample location was undetermined. Forty two samples were KRAS positive (mutated). In 12 of the 58 KRAS negative (wild type) samples, the V600E mutation in codon 15 of the BRAF gene was demonstrated. No differences in the frequency and distribution of mutations, stratified by gender, age, primary tumor versus metastasis, or tumor location were observed.Twelve percent of KRAS negative colorectal cancer samples showed BRAF gene mutation. Considering that 42% of samples have a KRAS mutation, 54% of patients should not respond to therapies with monoclonal antibodies directed against epidermic growth factor (EGFR) pathway.

KRAS oncogene is involved in colorectal carcinogenesis in 22 to 45% of cases.To determine the frequency, types and distribution of KRAS mutations in colorectal cancer.KRAS mutations studies were carried out in primary tumors and metastases of colo-rectal cancer from 56 women aged 60 ± 14 years and 53 men aged 61 ± 11 years. Formalin fixed and paraffin embedded tissue samples were evaluated using RFLP (Restriction Fragment Length Polymorphism) and direct sequencing.Primary tumors were located in the colon and rectum in 82 (75.2%) and 24 cases (20%), respectively. In three cases the extraction site of the tumor sample was unknown. In 46 cases (42.2%) KRAS mutations were demonstrated. The main point mutations were located in codon 12 (80.4%), G12D (39.1%), G12V (24.2%), G12S (6.5%), G12A (4.3%); G12C (4.3%), G12R (2.1%) and 19.6% at codon 13 (G13D). No differences were demonstrated in the frequency and distribution of mutations by gender, age, primary versus metastatic tumors or tumor location.In this series, 42% of colorectal cancer tissue samples had KRAS mutations. Their frequency and distribution are similar to those reported in the literature, except for G12C mutation.

The quality of the archival samples stored at pathology services could be a limiting factor for molecular biology studies.To determine the quality of DNA extracted from gallbladder cancer samples at different institutions.One hundred ninety four samples coming from five medical centers in Chile, were analyzed. DNA extraction was quantified determining genomic DNA concentration. The integrity of DNA was determined by polymerase chain reaction amplification of different length fragments of a constitutive gene (β-globin products of 110, 268 and 501 base pairs).The mean DNA concentration obtained in 194 gallbladder cancer samples was 48 ± 43.1 ng/µl. In 22% of samples, no amplification was achieved despite obtaining a mean DNA concentration of 58.3 ng/ul. In 81, 67 and 22% of samples, a DNA amplification of at least 110, 268 or 501 base pairs was obtained, respectively. No differences in DNA concentration according to the source of the samples were demonstrated. However, there were marked differences in DNA integrity among participating centers. Samples from public hospitals were of lower quality than those from private clinics.Despite some limitations, in 80% of cases, the integrity of DNA in archival samples from pathology services in our country would allow the use of molecular biology techniques.

RESUMENActinomicosis es una enfermedad infecciosa poco frecuente. La forma pelvica es rara y de dificildiagnostico. Se ha mencionado que su aparicion en la pelvis podria estar relacionada con el uso de DIU.Se busca asociacion entre Actinomyces y el uso de DIU en nuestro medio, asi como otros factoresrelacionados. Se incluyo 1.774 informes de PAP del Hospital de Florida entre 1996 y 2001. Se consideraronmetodo anticonceptivo y edad. Se utilizo docima no parametrica, Chi cuadrado de homogeneidad eindependencia. Los casos positivos para Actinomyces fueron 15, correspondientes al 0,84%. Un 86,7%eran usuarias de DIU. La edad media de presentacion fue de 33,2 anos. En 9 casos hubo presencia deActinomyces sin respuesta inflamatoria a diferencia de los 6 casos restantes, dondo hubo inflamacionespecifica. Actinomyces es un patogeno poco habitual en los genitales femeninos. Existe asociacionpositiva con el uso de DIU (p < 0,0001). No se encontro asociacion con edad.PALABRAS CLAVES: Actinomicosis, DIUSUMMARYActinomycosis is an infrequent infecious disease. The pelvic form is rare and of difficult diagnosis. It hasbeen mentioned that its appearance in female genital tract could be related to the use of IUD. Associationbetween Actinomyces and use of IUD is assesed in our environment, as well as other related factors. 1774reports of PAP of the Florida Hospital between 1996 and 2001 were included. It were considered birth-control method and age. It was used non parametric test, Chi test for homogeneity and independence ofMann-Whitney. The Actinomyces positive cases were 15, corresponding to 0.84%; 86.7% were IUD users.The median age of presentation was of 33.2 years. In 9 cases there was presence of Actinomyces withoutinflammatory reaction and the other six presented specific inflammation. Actinomyces is a very uncommonbacteria in the female genital tract. There is a positive association with IUD use (p < 0.0001). We found noassociation with age.KEY WORDS: Actinomyces, IUD