Exploratory pilot study to characterize the immune landscapes of malignant pleural effusions and their corresponding primary tumors from patients with breast carcinoma and lung adenocarcinoma.

INTRODUCTION: Malignant pleural effusion (MPE) is a frequent complication of advanced malignancies. In this pilot study, we characterized the immune landscapes of MPEs, compared them to their primary tumor (PT) samples from breast carcinoma (BC) and lung adenocarcinoma (LADC), and tested the utility of multiplexed image technology in cytological samples. MATERIALS AND METHODS: We evaluated the immune contexture of 6 BC and 5 LADC MPEs and their PTs using 3 multiplex immunofluorescence panels. We explored the associations between sample characteristics and pleural effusion-free survival. RESULTS: No MPE samples had positive programmed death-ligand 1 expression in malignant cells, although 3 of 11 PTs has positive programmed death-ligand 1 expression (more than 1% expression in malignant cells). Overall, in LADC samples, cluster of differentiation 3 (CD3)+ T cells and CD3+CD8+ cytotoxic T cells predominated (median percentages for MPEs versus PTs: 45.6% versus 40.7% and 4.7% versus 6.6%, respectively) compared with BC. CD68+ macrophages predominated in the BC samples (medians for MPEs 61.2% versus PTs for 57.1%) but not in the LADC samples. Generally in PTs, CD3+CD8+ forkhead box P3+ T cells and the median distances from the malignant cells to CD3+CD8+Ki67+ and CD3+ programmed cell death protein 1 + T cells correlated to earlier MPE after PT diagnosis. CONCLUSIONS: The immune cell phenotypes in the MPEs and PTs were similar within each cancer type but different between BC versus LADC. An MPE analysis can potentially be used as a substitute for a PT analysis, but an expanded study of this topic is essential.

Multi-omics Analysis Reveals Immune Features Associated with Immunotherapy Benefit in Patients with Squamous Cell Lung Cancer from Phase III Lung-MAP S1400I Trial.

PURPOSE: Identifying molecular and immune features to guide immune checkpoint inhibitor (ICI)-based regimens remains an unmet clinical need. EXPERIMENTAL DESIGN: Tissue and longitudinal blood specimens from phase III trial S1400I in patients with metastatic squamous non-small cell carcinoma (SqNSCLC) treated with nivolumab monotherapy (nivo) or nivolumab plus ipilimumab (nivo+ipi) were subjected to multi-omics analyses including multiplex immunofluorescence (mIF), nCounter PanCancer Immune Profiling Panel, whole-exome sequencing, and Olink. RESULTS: Higher immune scores from immune gene expression profiling or immune cell infiltration by mIF were associated with response to ICIs and improved survival, except regulatory T cells, which were associated with worse overall survival (OS) for patients receiving nivo+ipi. Immune cell density and closer proximity of CD8+GZB+ T cells to malignant cells were associated with superior progression-free survival and OS. The cold immune landscape of NSCLC was associated with a higher level of chromosomal copy-number variation (CNV) burden. Patients with LRP1B-mutant tumors had a shorter survival than patients with LRP1B-wild-type tumors. Olink assays revealed soluble proteins such as LAMP3 increased in responders while IL6 and CXCL13 increased in nonresponders. Upregulation of serum CXCL13, MMP12, CSF-1, and IL8 were associated with worse survival before radiologic progression. CONCLUSIONS: The frequency, distribution, and clustering of immune cells relative to malignant ones can impact ICI efficacy in patients with SqNSCLC. High CNV burden may contribute to the cold immune microenvironment. Soluble inflammation/immune-related proteins in the blood have the potential to monitor therapeutic benefit from ICI treatment in patients with SqNSCLC.

The Immunological Landscape of M1 and M2 Macrophages and Their Spatial Distribution in Patients with Malignant Pleural Mesothelioma.

BACKGROUND: Several tumor-associated macrophages (TAMs) have shown promise as prognosticators in cancer. Our aim was to validate the importance of TAMs in malignant pleural mesothelioma (MPM) using a two-stage design. METHODS: We explored The Cancer Genome Atlas (TCGA-MESO) to select immune-relevant macrophage genes in MPM, including M1/M2 markers, as a discovery cohort. This computational cohort was used to create a multiplex immunofluorescence panel. Moreover, a cohort of 68 samples of MPM in paraffin blocks was used to validate the macrophage phenotypes and the co-localization and spatial distribution of these immune cells within the TME and the stromal or tumor compartments. RESULTS: The discovery cohort revealed six immune-relevant macrophage genes (CD68, CD86, CD163, CD206, ARG1, CD274), and complementary genes were differentially expressed by M1 and M2 phenotypes with distinct roles in the tumor microenvironment and were associated with the prognosis. In addition, immune-suppressed MPMs with increased enrichment of CD68, CD86, and CD163 genes and high densities of M2 macrophages expressing CD163 and CD206 proteins were associated with worse overall survival (OS). Interestingly, below-median distances from malignant cells to specific M2a and M2c macrophages were associated with worse OS, suggesting an M2 macrophage-driven suppressive component in these tumors. CONCLUSIONS: The interactions between TAMs in situ and, particularly, CD206+ macrophages are highly relevant to patient outcomes. High-resolution technology is important for identifying the roles of macrophage populations in tissue specimens and identifying potential therapeutic candidates in MPM.

Phase II trial of neoadjuvant sitravatinib plus nivolumab in patients undergoing nephrectomy for locally advanced clear cell renal cell carcinoma.

Sitravatinib is an immunomodulatory tyrosine kinase inhibitor that can augment responses when combined with programmed death-1 inhibitors such as nivolumab. We report a single-arm, interventional, phase 2 study of neoadjuvant sitravatinib in combination with nivolumab in patients with locally advanced clear cell renal cell carcinoma (ccRCC) prior to curative nephrectomy (NCT03680521). The primary endpoint was objective response rate (ORR) prior to surgery with a null hypothesis ORR = 5% and the alternative hypothesis set at ORR = 30%. Secondary endpoints were safety; pharmacokinetics (PK) of sitravatinib; immune effects, including changes in programmed cell death-ligand 1 expression; time-to-surgery; and disease-free survival (DFS). Twenty patients were evaluable for safety and 17 for efficacy. The ORR was 11.8%, and 24-month DFS probability was 88·0% (95% CI 61.0 to 97.0). There were no grade 4/5 treatment-related adverse events. Sitravatinib PK did not change following the addition of nivolumab. Correlative blood and tissue analyses showed changes in the tumour microenvironment resulting in an immunologically active tumour by the time of surgery (median time-to-surgery: 50 days). The primary endpoint of this study was not met as short-term neoadjuvant sitravatinib and nivolumab did not substantially increase ORR.

Impact of Region-of-Interest Size on Immune Profiling Using Multiplex Immunofluorescence Tyramide Signal Amplification for Paraffin-Embedded Tumor Tissues.

INTRODUCTION: Representative regions of interest (ROIs) analysis from the whole slide images (WSI) are currently being used to study immune markers by multiplex immunofluorescence (mIF) and single immunohistochemistry (IHC). However, the amount of area needed to be analyzed to be representative of the entire tumor in a WSI has not been defined. METHODS: We labeled tumor-associated immune cells by mIF and single IHC in separate cohorts of non-small cell lung cancer (NSCLC) samples and we analyzed them as whole tumor area as well as using different number of ROIs to know how much area will be need to represent the entire tumor area. RESULTS: For mIF using the InForm software and ROI of 0.33 mm2 each, we observed that the cell density data from five randomly selected ROIs is enough to achieve, in 90% of our samples, more than 0.9 of Spearman correlation coefficient and for single IHC using ScanScope tool box from Aperio and ROIs of 1 mm2 each, we found that the correlation value of more than 0.9 was achieved using 5 ROIs in a similar cohort. Additionally, we also observed that each cell phenotype in mIF influence differently the correlation between the areas analyzed by the ROIs and the WSI. Tumor tissue with high intratumor epithelial and immune cells phenotype, quality, and spatial distribution heterogeneity need more area analyzed to represent better the whole tumor area. CONCLUSION: We found that at minimum 1.65 mm2 area is enough to represent the entire tumor areas in most of our NSCLC samples using mIF.

Phase II study of durvalumab (anti-PD-L1) and trametinib (MEKi) in microsatellite stable (MSS) metastatic colorectal cancer (mCRC).

BACKGROUND: Monotherapy with immune checkpoint blockade is ineffective for patients (pts) with microsatellite stable (MSS) metastatic colorectal cancer (mCRC). This study investigates whether the combination of trametinib (T) with durvalumab (D) can alter the immune tumor microenvironment (TME) by successfully priming and activating T-cells. METHODS: Open-label, single-center, phase II trial with primary endpoint of immune-related response rate for combination of T+D in refractory MSS mCRC pts (NCT03428126). T is 2 mg/day orally starting 1 week prior to D, which is given 1500 mg intravenously every 4 weeks. Simon 2-stage design used to enroll 29 pts into first stage, requiring a response in two or more pts to proceed to stage 2. Tumor biopsies were collected at baseline (BL) and early on-treatment (OT) at week 4. RESULTS: Twenty nine treated pts include 48% females, median age 48 years (range 28-75), and median prior therapies 2 (range 1-5). No grade (G) 4 or 5 treatment-related adverse events (TRAE). The most common TRAE of any grade was acneiform rash, 17% being G3. One of 29 pts had confirmed partial response (PR) lasting 9.3 months (mo) for an overall response rate of 3.4%. Seven pts had stable disease (SD) and five pts (1 PR, 4 SD) demonstrated decrease in total carcinoembryonic antigen ng/mL (best percentage reduction: 94%, 95%, 42%, 34%, and 22%, respectively). Median progression-free survival was 3.2 mo (range 1.1-9.3 months). Three pts with both liver and lung metastases demonstrated discrepant responses in which clinical benefit was present in the lung metastases but not liver metastases. Comparison of BL and 4-week OT tumor tissue flow cytometry demonstrated no changes in T-cell infiltration but upregulation expression of PD-1 and Tim3 on CD8 T cells. However, expression of PD-1 and Tim3 as single markers and as coexpressed markers was observed to increase OT relative to BL (p=0.03, p=0.06 and p=0.06, respectively). CONCLUSIONS: T+D demonstrated acceptable tolerability in pts with refractory MSS mCRC. The response rate in the first stage of the study did not meet efficacy criteria to proceed to the second stage. Specific site of metastatic disease may impact outcomes in novel immunotherapy combination trials. TRIAL REGISTRATION NUMBER: NCT03428126.

Multiplex Immunofluorescence and the Digital Image Analysis Workflow for Evaluation of the Tumor Immune Environment in Translational Research.

A robust understanding of the tumor immune environment has important implications for cancer diagnosis, prognosis, research, and immunotherapy. Traditionally, immunohistochemistry (IHC) has been regarded as the standard method for detecting proteins in situ, but this technique allows for the evaluation of only one cell marker per tissue sample at a time. However, multiplexed imaging technologies enable the multiparametric analysis of a tissue section at the same time. Also, through the curation of specific antibody panels, these technologies enable researchers to study the cell subpopulations within a single immunological cell group. Thus, multiplexed imaging gives investigators the opportunity to better understand tumor cells, immune cells, and the interactions between them. In the multiplexed imaging technology workflow, once the protocol for a tumor immune micro environment study has been defined, histological slides are digitized to produce high-resolution images in which regions of interest are selected for the interrogation of simultaneously expressed immunomarkers (including those co-expressed by the same cell) by using an image analysis software and algorithm. Most currently available image analysis software packages use similar machine learning approaches in which tissue segmentation first defines the different components that make up the regions of interest and cell segmentation, then defines the different parameters, such as the nucleus and cytoplasm, that the software must utilize to segment single cells. Image analysis tools have driven dramatic evolution in the field of digital pathology over the past several decades and provided the data necessary for translational research and the discovery of new therapeutic targets. The next step in the growth of digital pathology is optimization and standardization of the different tasks in cancer research, including image analysis algorithm creation, to increase the amount of data generated and their accuracy in a short time as described herein. The aim of this review is to describe this process, including an image analysis algorithm creation for multiplex immunofluorescence analysis, as an essential part of the optimization and standardization of the different processes in cancer research, to increase the amount of data generated and their accuracy in a short time.

Genotype-to-Phenotype Associations in the Aggressive Variant Prostate Cancer Molecular Profile (AVPC-m) Components.

The aggressive variant prostate cancer molecular profile (AVPC-m), composed of combined defects in TP53, RB1 and PTEN, characterizes a subset of prostate cancers linked to androgen indifference and platinum sensitivity. To contribute to the optimization of the AVPC-m assessment for inclusion in prospective clinical trials, we investigated the status of the AVPC-m components in 28 patient tumor-derived xenografts (PDXs) developed at MDACC. We subjected single formalin-fixed, paraffin-embedded (FFPE) blocks from each PDX to immunohistochemistry (IHC), targeted next-generation genomic sequencing (NGS) and Clariom-S Affymetrix human microarray expression profiling. Standard validated IHC assays and a 10% labeling index cutoff resulted in high reproducibility across three separate laboratories and three independent readers for all tumor suppressors, as well as strong correlations with loss-of-function transcriptional scores (LOF-TS). Adding intensity assessment to labeling indices strengthened the association between IHC results and LOF-TS for TP53 and RB1, but not for PTEN. For TP53, genomic alterations determined by NGS had slightly higher agreement scores with LOF-TS than aberrant IHC, while for RB1 and PTEN, NGS and IHC determinations resulted in similar agreement scores with LOF-TS. Nonetheless, our results indicate that the AVPC-m components can be assessed reproducibly by IHC using various widely available standardized assays.

Highlights from the 7th Oncological Pathology Conference 'Pathological Anatomy in the context of the National Cancer Law: An overview of the Latin American experience', 15, 22 and 23 July 2022, Trujillo, Peru.

The seventh session of the Oncological Pathology Conference (JoPaO) entitled 'Pathological Anatomy in the context of the National Cancer Law: An overview of the Latin American experience', was held virtually on July 15, 22 and 23. Peru was the headquarters for this event, where 17 national and international professors of high academic standing participated. They interacted in a multidisciplinary context through talks with national panellists and the general public. The recent promulgation of the 'National Cancer Law' fosters the development of discussion forums to analyse the national realities and uphold continuous learning about experiences in other Latin American countries with successful cancer programmes, in which pathology holds a principal role. The topics addressed during this JoPaO included the exchange of Latin American cancer management experiences, an emphasis on investments in and the development of strategic plans to improve care, the use of new technologies, laboratory quality control, and the need to advance scientific research.

Best Practices for Technical Reproducibility Assessment of Multiplex Immunofluorescence.

Multiplex immunofluorescence (mIF) tyramide signal amplification is a new and useful tool for the study of cancer that combines the staining of multiple markers in a single slide. Several technical requirements are important to performing high-quality staining and analysis and to obtaining high internal and external reproducibility of the results. This review manuscript aimed to describe the mIF panel workflow and discuss the challenges and solutions for ensuring that mIF panels have the highest reproducibility possible. Although this platform has shown high flexibility in cancer studies, it presents several challenges in pre-analytic, analytic, and post-analytic evaluation, as well as with external comparisons. Adequate antibody selection, antibody optimization and validation, panel design, staining optimization and validation, analysis strategies, and correct data generation are important for reproducibility and to minimize or identify possible issues during the mIF staining process that sometimes are not completely under our control, such as the tissue fixation process, storage, and cutting procedures.

Estado del tamizaje del cáncer de cérvix en países de América Latina desde la perspectiva de los profesionales de salud / Cervical cancer screening in Latin American countries from the perspective of health professionals

RESUMEN Objetivos . Determinar el estado del tamizaje del cáncer de cérvix en países de América Latina desde la perspectiva del personal de salud. Método . Estudio tipo encuesta online a grupos cerrados de patólogos y citotecnólogos. Resultados . Se obtuvo 155 cuestionarios llenados. La mayoría considera que existen deficiencias en el tamizaje del cáncer de cérvix, tales como mal abordaje y falta de entrenamiento en el personal. Conclusiones . Es necesario invertir económicamente en las políticas creadas contra el cáncer de cérvix.

ABSTRACT Objectives : To determine the status of cervical cancer screening in Latin American countries from the perspective of health personnel. Method : Online survey study of closed groups of pathologists and cytotechnologists. Results : 155 completed questionnaires were obtained. Most professionals surveyed considered that there are deficiencies in the screening of cervical cancer, such as poor approach and lack of staff training. Conclusion : It is necessary to invest financially in the policies created against cervical cancer.

Correlación citohistológica de ASC-H en el Hospital Nacional Guillermo Almenara Irigoyen durante el período enero 2013 a julio 2015 / Cyto-histological correlation of ASC-H at Hospital Nacional Guillermo Almenara Irigoyen, January 2013 - July 2015

En nuestro país, el cáncer de cuello uterino es aún la neoplasia maligna más frecuente en mujeres. Como prueba de tamizaje se utiliza el examen de Papanicolaou o citología cérvico-vaginal, el cual es informado utilizando el Sistema de Bethesda. En dicho sistema, la categoría ASC-H (atypícal squamous cells - cannot exclude HSIL-high grade squamous epithelial lesions, por sus siglas en inglés) designa los casos con presencia de células escamosas atípicas en las cuales los cambios son sugestivos de una lesión intraepitelial escamosa de alto grado pero insuficientes para una interpretación citopatológica definitiva, por lo que es importante determinar la correlación citohistológica de esta categoría. Objetivo. Correlacionar los resultados citopatológicos informados como ASC-H con los diagnósticos histopatológicos. Diseño. Estudio de tipo descriptivo, transversal y retrospectivo. Material. Citología cérvico-vaginal e histopatología. Métodos. Se revisó los casos con estudio citopatológico realizado entre enero de 2013 y julio de 2015, y con estudio histopatológico hasta un año después. Mediante la base de datos se determinó la cantidad poblacional y los diagnósticos. Se consideró como prueba de oro el diagnóstico histopatológico. Principales medidas de resultados. Correlación cito-histológica en casos de ASC-H. Resultados. Durante el período de estudio se realizaron 53 716 estudios de citología cérvico-vaginal convencional; de estos, 119 fueron catalogados como ASC-H; finalmente, 43 casos (0,07%) cumplieron los criterios de inclusión. El rango de edad de las pacientes fue de 22 a 70 años, siendo la media 43,8 años. El 42% de casos de ASC-H tuvo el diagnóstico de NIC2 y NIC3 en el estudio histopatológico. Conclusión. Se encontró una correlación entre los resultados de ASC-H y las lesiones intraepiteliales de alto grado (NIC II y NIC III), que concuerda con la encontrada en la bibliografía.

Cervical cancer is the most prevalent neoplasm in women in our country. The Papanicolaou test is used as a screening test, and is reported using the Bethesda System. In this system, the ASC-H (atypical squamous cells - cannot exclude HSIL-high grade squamous epithelial lesions) category designates cases with atypical squamous cells, where the changes are suggestive of a high grade squamous intraepithelial lesion but insufficient for a definitive cytopathologic interpretation. It becomes important to determine the cyto-histological correlation in this group. Objective: To correlate cytopathologic results reported as ASC-H with histopathological diagnoses. Designs: Descriptive, cross sectional retrospective study. Material: Cervicovaginal cytology and histology. Method: We reviewed the cases with a cytologicalpathological study between January 2013 and July 2015 and with a histopathological study until a year later. The population and diagnoses were determined using the database. Histopathological diagnosis was considered as the gold standard. Main outcome measures: Cyto-histological correlation in ASC-H cases. Results: Out of the total of 53 716 cervical cytology studies performed during the study period, 119 were classified as ASC-H; 43 (0.07%) cases met the inclusion criteria. The age ranged between 22 and 70 years, with an average of 43.8 years; 42% of ASC-H cases were diagnosed as presenting CIN2 and CIN3 in the histopathological study. Conclusion: This study showed correlation between ASC-H results and high-grade intraepithelial lesions (CIN2 and CIN3), in line with findings in the literature.

Carcinoma papilar y medular de tiroides simultáneo / Medullary carcinoma thyroid papillary and simultaneous

El sincronismo del Carcinoma Papilar y Medular es infrecuente. No se sabe la etiología clara sobre esta patología. El pronóstico está dado por el Carcinoma Medular ya que este es el más agresivo. A continuación se presenta el caso de una paciente mujer de 76 años con Carcinoma Medular y focos de Carcinoma y microcarcinoma Papilar de Tiroides.

Angiosarcoma primario de mama / Primary angiosarcoma of the breast

Introducción: Angiosarcoma primario de mama es sumamente infrecuente. Los métodos radiológicos tienen a ser muy inespecíficos para su diagnóstico, siendo la anatomía patológica la que da el diagnóstico definitivo. Se reporta el caso de una paciente de 18 años, que inicia con sintomatología inespecífica luego de su último embarazo. La tumoración a la histología presenta áreas sólidas, siendo catalogado con grado histológico II, reactiva a factor VIII, CD34, CD 31 y Ki67 alto.

Historia del tumi: símbolo de la medicina peruana y del Colegio Médico del Perú / History of the tumi: a symbol of ancient peruvian medicine adopted by the Peruvian College of physicians

El Tumi es un instrumento ceremonial semejante a un cuchillo con mango de forma rectangular o trapezoidal utilizado antiguamente en el Perú Precolombino. En la actualidad es un símbolo que forma parte del logotipo del Colegio Médico del Perú. Anteriormente se creía generalmente que el Tumi era utilizado principalmente para la realización de trepanaciones craneales, pero, posiblemente, su uso principal era para decapitar a los prisioneros de guerra. Nos enteramos de su existencia cuando un tumi se encontró por primera vez en Huaca Las Ventanas (un sitio arqueológico), ubicado en Batán Grande, comunidad de Poma, en Lambayeque, a finales de 1936 por el profesor Julio C. Tello. estos restos proceden desde 700- 1300 d.C.

The tumi (too-mee) is a ceremonial instrument resembling a knife with a rectangular or trapezoid handle used in ancient times in Peru. Now it is a symbol in the logo of the Peruvian Colege of Physicians. It was usually thought that the tumi was mainly used for performing cranial surgery, but possibly its main use was for beheading war prisioners. We learned of its existence when a tumi was first found in Huaca Las Ventanas (an archeological site), located in Batan Grande, Poma community in Lambayeque, at the end of 1936 bye Professor Julio C. Tello. The first tumi was dated somewhere between 700 to 1300 A.D.