Wang, Yihong

Abstract Background: Prior research on the associations of p16, COX-2, and Ki67 immunopositivity in ductal carcinoma in situ (DCIS) tissue with the risk of subsequent ipsilateral invasive breast cancer (IBC) is limited. Methods: In a case–control study nested in a cohort of women diagnosed with DCIS, immunostaining for p16, COX-2, and Ki67 was performed on DCIS tissue from those who developed subsequent ipsilateral IBC (cases; n = 146) and on matched subjects who did not develop IBC (controls; n = 273). Conditional logistic regression was used to estimate ORs and 95% confidence intervals for the associations between immunopositivity for p16, COX-2, and Ki67 and the risk of subsequent ipsilateral IBC. Results: There was no association between p16, COX-2, and Ki67 immunopositivity, examined either individually or in combination, and a risk of ipsilateral IBC. Compared with all other groups, the multivariable OR (95% confidence interval) for women who were triple positive for the three markers was 1.16 (0.38–3.54). Conclusions: p16, COX-2, and Ki67 immunopositivity was not associated with altered risk of ipsilateral IBC in women with DCIS. Impact: p16, COX-2, and Ki67 may not be prognostic for ipsilateral IBC in women with DCIS.

Context.— The Nottingham Grading System (NGS) developed by Elston and Ellis is used to grade invasive breast cancer (IBC). Glandular (acinar)/tubule formation is a component of NGS. Objective.— To investigate the ability of pathologists to identify individual structures that should be classified as glandular (acinar)/tubule formation. Design.— A total of 58 hematoxylin-eosin photographic images of IBC with 1 structure circled were classified as tubules (41 cases) or nontubules (17 cases) by Professor Ellis. Images were sent as a PowerPoint (Microsoft) file to breast pathologists, who were provided with the World Health Organization definition of a tubule and asked to determine if a circled structure represented a tubule. Results.— Among 35 pathologists, the κ statistic for assessing agreement in evaluating the 58 images was 0.324 (95% CI, 0.314–0.335). The median concordance rate between a participating pathologist and Professor Ellis was 94.1% for evaluating 17 nontubule cases and 53.7% for 41 tubule cases. A total of 41% of the tubule cases were classified correctly by less than 50% of pathologists. Structures classified as tubules by Professor Ellis but often not recognized as tubules by pathologists included glands with complex architecture, mucinous carcinoma, and the “inverted tubule” pattern of micropapillary carcinoma. A total of 80% of participants reported that they did not have clarity on what represented a tubule. Conclusions.— We identified structures that should be included as tubules but that were not readily identified by pathologists. Greater concordance for identification of tubules might be obtained by providing more detailed images and descriptions of the types of structures included as tubules.

Introduction Ductal carcinoma in situ (DCIS) of the breast is a non-obligate precursor of invasive breast cancer (IBC). Many DCIS patients are either undertreated or overtreated. The overarching goal of the study described here is to facilitate detection of patients with DCIS at risk of IBC development. Here, we propose to use risk factor data and formalin-fixed paraffin-embedded (FFPE) DCIS tissue from a large, ethnically diverse, population-based cohort of 8175 women with a first diagnosis of DCIS and followed for subsequent IBC to: identify/validate miRNA expression changes in DCIS tissue associated with risk of subsequent IBC; evaluate ipsilateral IBC risk in association with two previously identified marker sets (triple immunopositivity for p16, COX-2, Ki67; Oncotype DX Breast DCIS score); examine the association of risk factor data with IBC risk. Methods and analysis We are conducting a series of case–control studies nested within the cohort. Cases are women with DCIS who developed subsequent IBC; controls (2/case) are matched to cases on calendar year of and age at DCIS diagnosis. We project 485 cases/970 controls in the aim focused on risk factors. We estimate obtaining FFPE tissue for 320 cases/640 controls for the aim focused on miRNAs; of these, 173 cases/346 controls will be included in the aim focused on p16, COX-2 and Ki67 immunopositivity, and of the latter, 156 case–control pairs will be included in the aim focused on the Oncotype DX Breast DCIS score®. Multivariate conditional logistic regression will be used for statistical analyses. Ethics and dissemination Ethics approval was obtained from the Institutional Review Boards of Albert Einstein College of Medicine (IRB 2014-3611), Kaiser Permanente Colorado, Kaiser Permanente Hawaii, Henry Ford Health System, Mayo Clinic, Marshfield Clinic Research Institute and Hackensack Meridian Health, and from Lifespan Research Protection Office. The study results will be presented at meetings and published in peer-reviewed journals.

Assessment of tumor-infiltrating lymphocytes (TILs) in histopathologic specimens can provide important prognostic information in diverse solid tumor types, and may also be of value in predicting response to treatments. However, implementation as a routine clinical biomarker has not yet been achieved. As successful use of immune checkpoint inhibitors and other forms of immunotherapy become a clinical reality, the need for widely applicable, accessible, and reliable immunooncology biomarkers is clear. In part 1 of this review we briefly discuss the host immune response to tumors and different approaches to TIL assessment. We propose a standardized methodology to assess TILs in solid tumors on hematoxylin and eosin sections, in both primary and metastatic settings, based on the International Immuno-Oncology Biomarker Working Group guidelines for TIL assessment in invasive breast carcinoma. A review of the literature regarding the value of TIL assessment in different solid tumor types follows in part 2. The method we propose is reproducible, affordable, easily applied, and has demonstrated prognostic and predictive significance in invasive breast carcinoma. This standardized methodology may be used as a reference against which other methods are compared, and should be evaluated for clinical validity and utility. Standardization of TIL assessment will help to improve consistency and reproducibility in this field, enrich both the quality and quantity of comparable evidence, and help to thoroughly evaluate the utility of TILs assessment in this era of immunotherapy.

Assessment of the immune response to tumors is growing in importance as the prognostic implications of this response are increasingly recognized, and as immunotherapies are evaluated and implemented in different tumor types. However, many different approaches can be used to assess and describe the immune response, which limits efforts at implementation as a routine clinical biomarker. In part 1 of this review, we have proposed a standardized methodology to assess tumor-infiltrating lymphocytes (TILs) in solid tumors, based on the International Immuno-Oncology Biomarkers Working Group guidelines for invasive breast carcinoma. In part 2 of this review, we discuss the available evidence for the prognostic and predictive value of TILs in common solid tumors, including carcinomas of the lung, gastrointestinal tract, genitourinary system, gynecologic system, and head and neck, as well as primary brain tumors, mesothelioma and melanoma. The particularities and different emphases in TIL assessment in different tumor types are discussed. The standardized methodology we propose can be adapted to different tumor types and may be used as a standard against which other approaches can be compared. Standardization of TIL assessment will help clinicians, researchers and pathologists to conclusively evaluate the utility of this simple biomarker in the current era of immunotherapy.

Formalin-fixed paraffin-embedded (FFPE) specimens, when used in conjunction with patient clinical data history, represent an invaluable resource for molecular studies of cancer. Even though nucleic acids extracted from archived FFPE tissues are degraded, their molecular analysis has become possible. In this study, we optimized a laboratory-based next-generation sequencing barcoded cDNA library preparation protocol for analysis of small RNAs recovered from archived FFPE tissues. Using matched fresh and FFPE specimens, we evaluated the robustness and reproducibility of our optimized approach, as well as its applicability to archived clinical specimens stored for up to 35 years. We then evaluated this cDNA library preparation protocol by performing a miRNA expression analysis of archived breast ductal carcinoma in situ (DCIS) specimens, selected for their relation to the risk of subsequent breast cancer development and obtained from six different institutions. Our analyses identified six miRNAs (miR-29a, miR-221, miR-375, miR-184, miR-363, miR-455-5p) differentially expressed between DCIS lesions from women who subsequently developed an invasive breast cancer (cases) and women who did not develop invasive breast cancer within the same time interval (control). Our thorough evaluation and application of this laboratory-based miRNA sequencing analysis indicates that the preparation of small RNA cDNA libraries can reliably be performed on older, archived, clinically-classified specimens.