Therapy with anti-PD-L1 immune check-point inhibitors is approved for several cancers, including advanced urothelial carcinomas. PD-L1 prevalence estimates vary widely in bladder cancer, and lack of ...correlation between expression and clinical outcomes and immunotherapy response may be attributed to methodological differences of the immunohistochemical reagents and procedures. We characterized PD-L1 expression in 235 urothelial carcinomas including 79 matched pairs of primary and metastatic cancers using a panel of four PD-L1 immunoassays in comparison with RNAscope assay using PD-L1-specific probe (CD274). The antibody panel included three FDA-approved clones (22C3 for pembrolizumab, 28.8 for nivolumab, SP142 for atezolizumab), and a commonly used clone E1L3N. Manual scoring of tissue microarrays was performed in each of 235 tumors (624 tissue cores) and compared to an automated image analysis. Expression of PD-L1 in tumor cells by ≥1 marker was detected in 41/142 (28.9%) primary tumors, 13/77 (16.9%) lymph nodes, and 2/16 (12.5%) distant metastases. In positive cases, high PD-L1 expression (>50% cells) was detected in 34.1% primary and 46.7% metastases. Concordant PD-L1 expression status was present in 71/79 (89.9%) cases of matched primary and metastatic urothelial carcinomas. PD-L1 sensitivity ranked from highest to lowest as follows: RNAscope, clone 28.8, 22C3, E1L3N, and SP142. Pairwise concordance correlation coefficients between the four antibodies in 624 tissue cores ranged from 0.76 to 0.9 for tumor cells and from 0.30 to 0.85 for immune cells. RNA and protein expression levels showed moderate to high agreement (0.72-0.87). Intra-tumor expression heterogeneity was low for both protein and RNA assays (interclass correlation coefficients: 0.86-0.94). Manual scores were highly concordant with automated Aperio scores (0.94-0.97). A significant subset of 56/235 (23.8%) urothelial carcinomas stained positive for PD-L1 with high concordance between all four antibodies and RNA ISH assay. Despite some heterogeneity in staining, the overall results are highly concordant suggesting diagnostic equivalence of tested assays.
Laboratories must validate all assays before they can be used to test patient specimens, but currently there are no evidence-based guidelines regarding validation of immunohistochemical assays.
To ...develop recommendations for initial analytic validation and revalidation of immunohistochemical assays.
The College of American Pathologists Pathology and Laboratory Quality Center convened a panel of pathologists and histotechnologists with expertise in immunohistochemistry to develop validation recommendations. A systematic evidence review was conducted to address key questions. Electronic searches identified 1463 publications, of which 126 met inclusion criteria and were extracted. Individual publications were graded for quality, and the key question findings for strength of evidence. Recommendations were derived from strength of evidence, open comment feedback, and expert panel consensus.
Fourteen guideline statements were established to help pathology laboratories comply with validation and revalidation requirements for immunohistochemical assays.
Laboratories must document successful analytic validation of all immunohistochemical tests before applying to patient specimens. The parameters for cases included in validation sets, including number, expression levels, fixative and processing methods, should take into account intended use and should be sufficient to ensure that the test accurately measures the analyte of interest in specimens tested in that laboratory. Recommendations are also provided for confirming assay performance when there are changes in test methods, reagents, or equipment.
The aim of this study was to describe breast tumor subtypes by common breast cancer risk factors and to determine correlates of subtypes using baseline data from two pooled prospective breast cancer ...studies within a large health maintenance organization.
Tumor data on 2544 invasive breast cancer cases subtyped by estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 (Her2) status were obtained (1868 luminal A tumors, 294 luminal B tumors, 288 triple-negative tumors and 94 Her2-overexpressing tumors). Demographic, reproductive and lifestyle information was collected either in person or by mailed questionnaires. Case-only odds ratios (ORs) and 95% confidence intervals (CIs) were estimated using logistic regression, adjusting for age at diagnosis, race/ethnicity, and study origin.
Compared with luminal A cases, luminal B cases were more likely to be younger at diagnosis (P = 0.0001) and were less likely to consume alcohol (OR = 0.74, 95% CI = 0.56 to 0.98), use hormone replacement therapy (HRT) (OR = 0.66, 95% CI = 0.46 to 0.94), and oral contraceptives (OR = 0.73, 95% CI = 0.55 to 0.96). Compared with luminal A cases, triple-negative cases tended to be younger at diagnosis (P < or = 0.0001) and African American (OR = 3.14, 95% CI = 2.12 to 4.16), were more likely to have not breastfed if they had parity greater than or equal to three (OR = 1.68, 95% CI = 1.00 to 2.81), and were more likely to be overweight (OR = 1.82, 95% CI = 1.03 to 3.24) or obese (OR = 1.97, 95% CI = 1.03 to 3.77) if premenopausal. Her2-overexpressing cases were more likely to be younger at diagnosis (P = 0.03) and Hispanic (OR = 2.19, 95% CI = 1.16 to 4.13) or Asian (OR = 2.02, 95% CI = 1.05 to 3.88), and less likely to use HRT (OR = 0.45, 95% CI = 0.26 to 0.79).
These observations suggest that investigators should consider tumor heterogeneity in associations with traditional breast cancer risk factors. Important modifiable lifestyle factors that may be related to the development of a specific tumor subtype, but not all subtypes, include obesity, breastfeeding, and alcohol consumption. Future work that will further categorize triple-negative cases into basal and non-basal tumors may help to elucidate these associations further.
This review summarizes the three major breast‐associated markers that can be of assistance in evaluating metastatic carcinomas for which a breast primary diagnosis is entertained. These markers ...include gross cystic disease fluid protein‐15 (GCDFP‐15), mammaglobin, and GATA3. The first two are cytoplasmic markers that show comparable sensitivities for breast cancer, although relatively few of the published studies have employed the same antibodies against the target molecule, making direct comparisons challenging. GATA3 is a nuclear transcription factor that shows superior sensitivity to GCDFP‐15 and mammaglobin. However, the specificity of GATA3 can pose challenges, inasmuch as carcinomas of the bladder and other sites can show significant levels of positivity. Determination of the optimal panel of antibodies employed in a given clinical setting will thus depend on the non‐breast tumours included in the differential diagnosis.
The aim was to identify clinical parameters and immunohistochemical markers predictive of recurrence and overall survival (OS) in a community cohort of patients with primary uterine leiomyosarcoma ...(ULMS).
All patients with new diagnosis of ULMS from 1999 to 2007 were identified from the Kaiser Permanente Northern California pathology database. A retrospective chart review was performed to gather demographic and clinical data. The primary outcomes were recurrence-free survival and OS. In addition, a subset of tumor samples was available to analyze 3 immunohistochemical markers using tissue microarray techniques; these are as follows: estrogen receptor (ER) alpha, epidermal growth factor receptor (EGFR), and Ki-67.
Seventy-five patients with ULMS were identified, of which 63 had adequate tumor tissue available for immunohistochemical evaluation. The median follow-up for all stages was 28 months. The rate of recurrence or progressive disease was 76% for stage I patients compared with 85% for stage II to IV patients. At 3 years, 37% of stage I patients were recurrence free compared with 27% of stage II to IV patients. Overall survival for stage I patients declined from 64% to 38% between 3 and 5 years while remaining stable at 30% for stage II to IV patients. In multivariable analysis, increasing mitotic counts were associated with increased risk of recurrence (hazards ratio HR, 3.2; P = 0.013) and a trend toward decreased OS (HR, 2.2; P = 0.10). Expression of ER (HR, 1.0), EGFR expression (HR, 1.0), and Ki-67 expression (HR, 1.0) were not predictive of recurrence or OS.
Recurrence rate of 76% for patients with stage I ULMS was higher than previously published cohorts. Mitotic counts were associated with increased recurrence and decreased OS. Expressions of ER, EGFR, and Ki-67 were not useful for predicting overall recurrence or survival.
Inhibitors of the programmed cell death 1 (PD-1) signaling axis have recently demonstrated efficacy and are rapidly being incorporated into the treatment of non–small cell lung cancers (NSCLCs). ...Despite clear benefits to certain patients, the association of these responses with a predictive biomarker remains uncertain. Several different biomarkers have been proposed, with differing results and conclusions. This study compares multiple methods of biomarker testing for treatment of NSCLCs with PD1-axis inhibitors. Tissue microarrays of matched primary and metastatic NSCLCs were used to compare four different PD-1 ligand (PD-L1) IHC techniques, as well as RNA ISH. Additional cases with whole genome and transcriptome data were assessed for molecular correlates of PD-L1 overexpression. Eighty cases were included in the IHC study. Multiple IHC methodologies showed a high rate of agreement (Kappa = 0.67). When calibrated to RNA expression, agreement improved significantly (Kappa = 0.90, p=0.0049). PD-L1 status of primary and metastatic tumors was discordant in 17 (22%) cases. This study suggests that different IHC methodologies for PD-L1 assessment provide slightly different results. There is significant discordance between the PD-L1 status of primary tumors and lymph node metastases. RNA ISH may be a useful adjunct to complement PD-L1 IHC testing.
The efficacy of the antibody drug conjugate (ADC) Trastuzumab deruxtecan (T-DXd) in HER2 low breast cancer patients suggests that the historical/conventional assays for HER2 may need revision for ...optimal patient care. Specifically, the conventional assay is designed to distinguish amplified HER2 from unamplified cases but is not sensitive enough to stratify the lower ranges of HER2 expression. Here we determine the optimal dynamic range for unamplified HER2 detection in breast cancer and then redesign an assay to increase the resolution of the assay to stratify HER2 expression in unamplified cases. We used the AQUA™ method of quantitative immunofluorescence to test a range of antibody concentrations to maximize the sensitivity within the lower range of HER2 expression. Then, using a cell line microarray with HER2 protein measured by mass spectrometry we determined the amount of HER2 protein in units of attomols/mm
. Then by calculation of the limits of detection, quantification, and linearity of this assay we determined that low HER2 range expression in unamplified cell lines is between 2 and 20 attomol/mm
. Finally, application of this assay to a serial collection of 364 breast cancer cases from Yale shows 67% of the population has HER2 expression above the limit of quantification and below the levels seen in HER2 amplified breast cancer. In the future, this assay could be used to determine the levels of HER2 required for response to T-DXd or similar HER2 conjugated ADCs.
To determine the invasive recurrence (IR) risk among patients with small, node-negative human epidermal growth factor receptor 2 (HER2) -positive breast cancer.
Among 16,975 consecutive patients with ...invasive breast cancer diagnosed from January 1, 2000, to December 31, 2006, in a large, integrated health care system, we identified a cohort of 234 patients with HER2-positive T1aN0M0 or T1bN0M0 (T1abN0M0) disease with a median follow-up of 5.8 years. Kaplan-Meier methods were used to estimate the percentage of patients who were free of invasive recurrence (recurrence-free interval RFI) at 5 years for both distant (DRFI) and local (LRFI) recurrences.
Of 15 IRs, 47% were locoregional only. Among T1ab patients not treated with adjuvant trastuzumab or chemotherapy (n = 171), the 5-year invasive DRFI was 98.2% (95% CI, 94.5% to 99.4%); it was 99.0% (95% CI, 93.0% to 99.9%) for T1a patients, and 97.0% (95% CI, 88.6% to 99.2%) for T1b patients. Locoregional plus distant 5-year invasive RFI was 97.0% (95% CI, 90.9% to 99.0%) for T1a and 91.9% (95% CI, 81.5% to 96.6%) for T1b patients; it was 89.4% (95% CI, 70.6% to 96.5%) for T1b tumors reported at 1.0 cm. T1b tumors reported at 1.0 cm accounted for 24% of the T1ab cohort, 61% of the cohort total tumor volume, and 75% of distant recurrences. Invasive RFI for T1b 1.0 cm tumors was lower than that for T1a tumors: 84.5% versus 97.4% (P = .009).
The distant IR risk of T1a HER2-positive breast cancer appears quite low. The distant IR risk in T1b patients, particularly those with 1.0-cm tumors, is higher. Potential risk differences for T1a and T1b, including the 1.0-cm tumors, should be considered when making treatment decisions.
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