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Aims: The nuclear proliferation marker Ki67 assayed by immunohistochemistry has multiple potential uses in breast cancer, but an unacceptable level of interlaboratory variability has hampered its clinical utility. The International Ki67 in Breast Cancer Working Group has undertaken a systematic programme to determine whether Ki67 measurement can be analytically validated and standardised among laboratories. This study addresses whether acceptable scoring reproducibility can be achieved on excision whole sections., Methods and Results: Adjacent sections from 30 primary ER + breast cancers were centrally stained for Ki67 and sections were circulated among 23 pathologists in 12 countries. All pathologists scored Ki67 by two methods: (i) global: four fields of 100 tumour cells each were selected to reflect observed heterogeneity in nuclear staining; (ii) hot-spot: the field with highest apparent Ki67 index was selected and up to 500 cells scored. The intraclass correlation coefficient (ICC) for the global method [confidence interval (CI) = 0.87; 95% CI = 0.799-0.93] marginally met the prespecified success criterion (lower 95% CI ≥ 0.8), while the ICC for the hot-spot method (0.83; 95% CI = 0.74-0.90) did not. Visually, interobserver concordance in location of selected hot-spots varies between cases. The median times for scoring were 9 and 6 min for global and hot-spot methods, respectively., Conclusions: The global scoring method demonstrates adequate reproducibility to warrant next steps towards evaluation for technical and clinical validity in appropriate cohorts of cases. The time taken for scoring by either method is practical using counting software we are making publicly available. Establishment of external quality assessment schemes is likely to improve the reproducibility between laboratories further., (© 2019 John Wiley & Sons Ltd.)

The analysis of estrogen receptor (ER) and progesterone receptor (PR) expression levels by immunohistochemistry is an important part of the initial evaluation of breast cancer and critically important in treatment planning. Anti-ERα (clone EP1) and anti-PR (clone PgR 1294) antibodies are in development for the Dako Omnis automated staining platform. These antibodies are not yet commercially available and are in performance evaluation, including the 4 international, multicenter studies reported here. For each antibody, a reproducibility study and a method comparison study was done in a randomized manner in order to test the antibodies under conditions closest to real-world user conditions. The reproducibility studies included 5 staining runs on the Dako Omnis with 20 formalin-fixed and paraffin-embedded human breast carcinoma specimens in 3 independent laboratories, and the method comparison studies included several hundred specimens stained on the Dako Omnis and on the Autostainer Link 48 platforms. Stained slides were evaluated for nuclear ER or PR expression according to American Society of Clinical Oncology/College of American Pathologists guidelines (≥1% cut-off for positive) by pathologists who were blinded from the staining method and specimen ID. For both anti-ERα (clone EP1) and anti-PR (clone PgR 1294) on the Dako Omnis, high reproducibility agreement rates were obtained on the interrun, interlaboratory, and interobserver endpoints. High concordance rates were observed between the specimens stained on the Dako Omnis platform and the Autostainer Link 48 platform. Staining quality was excellent for both anti-ERα (clone EP1) and anti-PR (clone PgR 1294) on the Dako Omnis. These results suggest that these antibodies are reliable and reproducible tools for immunohistochemistry analysis of ER and PR expression levels in formalin-fixed and paraffin-embedded breast carcinoma tissues on the Dako Omnis platform.

Importance: The Genomic Grade Index (GGI) was previously developed, evaluated on frozen tissue, and shown to be prognostic in early breast cancer. To test the GGI in formalin-fixed, paraffin-embedded breast cancer tumors, a quantitative reverse transcriptase polymerase chain reaction assay was developed and named the Genomic Grade (GG). The GG assay has the potential to increase the clinical application of the GGI, but robust demonstration of the clinical validity of the GG assay is required., Objective: To evaluate the prognostic ability of the GG assay to detect breast cancer recurrence compared with centrally reviewed immunohistochemical testing of Ki67 antigen proliferation., Design, Setting, and Participants: This is an internationally collaborative substudy of a large phase 3 4-arm adjuvant trial. Patients had endocrine receptor-positive, node-positive, or node-negative nonmetastatic primary breast cancer. Patients included in this study had available formalin-fixed, paraffin-embedded samples of their primary tumors and were randomized to either a 5-year tamoxifen monotherapy arm or a 5-year letrozole monotherapy arm. Associations between either GG assay results or log2-transformed Ki67 data and survival end points were evaluated using Cox regression models stratified for chemotherapy use; the 2 vs 4 arm randomization option; and endocrine therapy assignment with and without adjustment for clinicopathological parameters, including centrally reviewed histological grade, hormone receptors, and ERBB2 (formerly HER2 or HER2/neu). The likelihood ratio statistic was used to assess the added prognostic value., Interventions: Central evaluation and comparison, blinded for clinical information, of the GG assay, breast cancer histological grade, and Ki67., Main Outcomes and Measures: Distant recurrence-free interval (DRFI)., Results: Genomic Grade assay data were obtained in 883 breast cancer samples (62%). At a median follow-up of 8.1 years, 84 (10%) had distant recurrences. Increasing GG or Ki67 were both significantly associated with lower DRFI and added independent prognostic information to the clinicopathological prognostic factors. In patients with early node-negative breast cancer who were endocrine-only treated, 38% were GG1 with a 10-year DRFI of 99% (95% CI, 97%-100%), and 18% were histological grade 1 with a 10-year DRFI of 100% (95% CI, 100%-100%). For GG equivocal patients, the 10-year DRFI was 94% (95% CI, 90%-98%), and for GG3 patients, the 10-year DRFI was 87% (95% CI, 80%-94%)., Conclusions and Relevance: Either the GG assay or centrally reviewed Ki67 significantly improves clinicopathological models to determine distant recurrence of breast cancer. Compared with the histological grade, the GG assay can identify a higher proportion of endocrine-only treated patients with very low risk of distant recurrence at 10 years., Trial Registration: clinicaltrials.gov Identifiers: NCT00004205 and NCT00004205.

Background: Biomarkers are crucial for decisions regarding adjuvant therapy in primary breast cancer, and their correct assessment is therefore of the utmost importance., Aims: To investigate the concordance between Swedish pathology departments and a reference laboratory, for routine analysis of oestrogen receptor (ER), progesterone receptor (PR), Ki67, and human epidermal growth factor receptor 2 (HER2), alone, and in combination (St Gallen subtypes)., Methods: This survey included 27 of the 28 pathology laboratories in Sweden, covering 98% of cases of primary breast cancer surgery in Sweden. Paraffin-embedded tumour blocks (n = 270) were collected and sent to the central reference laboratory, together with the originally stained slides, for re-analysis. The primary evaluations were previously performed according to national Swedish guidelines, without any knowledge of the subsequent central assessment., Results: The agreement for ER, PR, and Ki67 was 99% [kappa value (κ) = 0.95], 95% (κ = 0.85), and 85% (κ = 0.70), respectively. The agreement for HER2 (0/1 + vs. 2+/3+) was 85% (κ = 0.64), but when equivocal tumours were further analysed with in situ hybridisation, only one discrepancy was observed. Discrepancies between results for ER and PR seem to be explained by analytical differences, whereas the interpretation of staining seems to be more critical for Ki67 and HER2 immunohistochemistry. The agreement between the results from the Swedish laboratories and the reference laboratory, based on the St Gallen subtypes, was 88% (κ = 0.81)., Conclusions: When applying national guidelines, highly reproducible results were obtained in routine assessment of breast cancer biomarkers, and the results of this study confirm the clinical utility of these markers for decisions regarding the treatment of primary breast cancer.

Although an important biomarker in breast cancer, Ki67 lacks scoring standardization, which has limited its clinical use. Our previous study found variability when laboratories used their own scoring methods on centrally stained tissue microarray slides. In this current study, 16 laboratories from eight countries calibrated to a specific Ki67 scoring method and then scored 50 centrally MIB-1 stained tissue microarray cases. Simple instructions prescribed scoring pattern and staining thresholds for determination of the percentage of stained tumor cells. To calibrate, laboratories scored 18 'training' and 'test' web-based images. Software tracked object selection and scoring. Success for the calibration was prespecified as Root Mean Square Error of scores compared with reference <0.6 and Maximum Absolute Deviation from reference <1.0 (log2-transformed data). Prespecified success criteria for tissue microarray scoring required intraclass correlation significantly >0.70 but aiming for observed intraclass correlation ≥0.90. Laboratory performance showed non-significant but promising trends of improvement through the calibration exercise (mean Root Mean Square Error decreased from 0.6 to 0.4, Maximum Absolute Deviation from 1.6 to 0.9; paired t-test: P=0.07 for Root Mean Square Error, 0.06 for Maximum Absolute Deviation). For tissue microarray scoring, the intraclass correlation estimate was 0.94 (95% credible interval: 0.90-0.97), markedly and significantly >0.70, the prespecified minimum target for success. Some discrepancies persisted, including around clinically relevant cutoffs. After calibrating to a common scoring method via a web-based tool, laboratories can achieve high inter-laboratory reproducibility in Ki67 scoring on centrally stained tissue microarray slides. Although these data are potentially encouraging, suggesting that it may be possible to standardize scoring of Ki67 among pathology laboratories, clinically important discrepancies persist. Before this biomarker could be recommended for clinical use, future research will need to extend this approach to biopsies and whole sections, account for staining variability, and link to outcomes.

Purpose: To develop a guideline to improve the accuracy of immunohistochemical (IHC) estrogen receptor (ER) and progesterone receptor (PgR) testing in breast cancer and the utility of these receptors as predictive markers., Methods: The American Society of Clinical Oncology and the College of American Pathologists convened an international Expert Panel that conducted a systematic review and evaluation of the literature in partnership with Cancer Care Ontario and developed recommendations for optimal IHC ER/PgR testing performance., Results: Up to 20% of current IHC determinations of ER and PgR testing worldwide may be inaccurate (false negative or false positive). Most of the issues with testing have occurred because of variation in pre-analytic variables, thresholds for positivity, and interpretation criteria., Recommendations: The Panel recommends that ER and PgR status be determined on all invasive breast cancers and breast cancer recurrences. A testing algorithm that relies on accurate, reproducible assay performance is proposed. Elements to reliably reduce assay variation are specified. It is recommended that ER and PgR assays be considered positive if there are at least 1% positive tumor nuclei in the sample on testing in the presence of expected reactivity of internal (normal epithelial elements) and external controls. The absence of benefit from endocrine therapy for women with ER-negative invasive breast cancers has been confirmed in large overviews of randomized clinical trials.

Background: Tumor levels of steroid hormone receptors, a factor used to select adjuvant treatment for early-stage breast cancer, are currently determined with immunohistochemical assays. These assays have a discordance of 10%-30% with previously used extraction assays. We assessed the concordance and predictive value of hormone receptor status as determined by immunohistochemical and extraction assays on specimens from International Breast Cancer Study Group Trials VIII and IX. These trials predominantly used extraction assays and compared adjuvant chemoendocrine therapy with endocrine therapy alone among pre- and postmenopausal patients with lymph node-negative breast cancer. Trial conclusions were that combination therapy provided a benefit to pre- and postmenopausal patients with estrogen receptor (ER)-negative tumors but not to ER-positive postmenopausal patients. ER-positive premenopausal patients required further study., Methods: Tumor specimens from 571 premenopausal and 976 postmenopausal patients on which extraction assays had determined ER and progesterone receptor (PgR) levels before randomization from October 1, 1988, through October 1, 1999, were re-evaluated with an immunohistochemical assay in a central pathology laboratory. The endpoint was disease-free survival. Hazard ratios of recurrence or death for treatment comparisons were estimated with Cox proportional hazards regression models, and discriminatory ability was evaluated with the c index. All statistical tests were two-sided., Results: Concordance of hormone receptor status determined by both assays ranged from 74% (kappa = 0.48) for PgR among postmenopausal patients to 88% (kappa = 0.66) for ER in postmenopausal patients. Hazard ratio estimates were similar for the association between disease-free survival and ER status (among all patients) or PgR status (among postmenopausal patients) as determined by the two methods. However, among premenopausal patients treated with endocrine therapy alone, the discriminatory ability of PgR status as determined by immunohistochemical assay was statistically significantly better (c index = 0.60 versus 0.51; P = .003) than that determined by extraction assay, and so immunohistochemically determined PgR status could predict disease-free survival., Conclusions: Trial conclusions in which ER status (for all patients) or PgR status (for postmenopausal patients) was determined by immunohistochemical assay supported those determined by extraction assays. However, among premenopausal patients, trial conclusions drawn from PgR status differed--immunohistochemically determined PgR status could predict response to endocrine therapy, unlike that determined by the extraction assay.

* Purpose.--To update key recommendations of the American Society of Clinical Oncology/College of American Pathologists estrogen receptor (ER) and progesterone receptor (PgR) testing in breast cancer guideline. Methods.--A multidisciplinary international Expert Panel was convened to update the clinical practice guideline recommendations informed by a systematic review of the medical literature. Recommendations.--The Expert Panel continues to recommend ER testing of invasive breast cancers by validated immunohistochemistry as the standard for predicting which patients may benefit from endocrine therapy, and no other assays are recommended for this purpose. Breast cancer samples with 1% to 100% of tumor nuclei positive should be interpreted as ER positive. However, the Expert Panel acknowledges that there are limited data on endocrine therapy benefit for cancers with 1% to 10% of cells staining ER positive. Samples with these results should be reported using a new reporting category, ER Low Positive, with a recommended comment. A sample is considered ER negative if < 1% or 0% of tumor cell nuclei are immunoreactive. Additional strategies recommended to promote optimal performance, interpretation, and reporting of cases with an initial low to no ER staining result include establishing a laboratory-specific standard operating procedure describing additional steps used by the laboratory to confirm/adjudicate results. The status of controls should be reported for cases with 0% to 10% staining. Similar principles apply to PgR testing, which is used primarily for prognostic purposes in the setting of an ER-positive cancer. Testing of ductal carcinoma in situ (DCIS) for ER is recommended to determine potential benefit of endocrine therapies to reduce risk of future breast cancer, while testing DCIS for PgR is considered optional. Additional information can be found at www.asco.org/breast-cancer-guidelines. (Arch Pathol Lab Med. 2020;144:545-563; doi: 10.5858/arpa.2019-0904-SA), INTRODUCTION First released in 2010, the American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) estrogen receptor (ER) and progesterone receptor (PgR) testing guideline is aimed at improving the [...]

Triple-negative breast cancer (TNBC) accounts for approximately 12% to 17% of all breast cancers and has an aggressive clinical behavior. Increased tumor-infiltrating lymphocyte counts are prognostic for survival in TNBC, making this disease a potential target for cancer immunotherapy. Research on immunophenotyping of tumor-infiltrating lymphocytes is revealing molecular and structural organization in the tumor microenvironment that may predict patient prognosis. The anti-programmed death-ligand 1 (PD-L1) antibody atezolizumab plus nab-paclitaxel was the first cancer immunotherapy combination to demonstrate progression-free survival benefit and clinically meaningful overall survival benefit in the first-line treatment of metastatic TNBC (mTNBC) in patients with PD-L1-expressing tumor-infiltrating immune cells in 1% or more of the tumor area. This led to its United States and European Union approval for mTNBC and US approval of the VENTANA PD-L1 (SP142) assay as a companion diagnostic immunohistochemistry assay. Subsequently, the anti-programmed death-1 (PD-1 ) antibody pembrolizumab plus chemotherapy was approved by the US Food and Drug Administration for mTNBC based on progression-free survival benefit in patients with a combined positive score of at least 10 by its concurrently approved 22C3 companion diagnostic assay. Treatment guidelines now recommend PD-L1 testing for patients with mTNBC, and the testing landscape will likely become increasingly complex as new anti-PD-L1 and anti-PD-1 agents and diagnostics are approved for TNBC. Integrating PD-L1 testing into current diagnostic workflows for mTNBC may provide more treatment options for these patients. Therefore, it is critical for medical oncologists and pathologists to understand the available assays and their relevance to therapeutic options to develop an appropriate workflow for immunohistochemistry testing. [ABSTRACT FROM AUTHOR]

Background: In the phase III IMpassion130 study, atezolizumab plus nab-paclitaxel (A+nP) showed clinical benefit in advanced or metastatic triple-negative breast cancer patients who were programmed death-ligand 1 (PD-L1)+ (tumor-infiltrating immune cells [IC] ≥1%) using the SP142 immunohistochemistry assay. Here we evaluate 2 other PD-L1 assays for analytical concordance with SP142 and patient-associated clinical outcomes.Methods: Samples from 614 patients (68.1% of intention-to-treat population) were centrally evaluated by immunohistochemistry for PD-L1 status on IC (VENTANA SP142, SP263, Dako 22C3) or as a combined positive score (CPS; 22C3).Results: Using SP142, SP263, and 22C3 assays, PD-L1 IC ≥1% prevalence was 46.4% (95% confidence interval [CI] = 42.5% to 50.4%), 74.9% (95% CI = 71.5% to 78.3%), and 73.1% (95% CI = 69.6% to 76.6%), respectively; 80.9% were 22C3 CPS ≥1. At IC ≥1% (+), the analytical concordance between SP142 and SP263 and 22C3 was 69.2% and 68.7%, respectively. Almost all SP142+ cases were captured by other assays (double positive), but several SP263+ (29.6%) or 22C3+ (29.0%) cases were SP142- (single positive). A+nP clinical activity vs placebo+nP in SP263+ and 22C3+ patients (progression-free survival [PFS] hazard ratios [HRs] = 0.64 to 0.68; overall survival [OS] HRs = 0.75 to 0.79) was driven by double-positive cases (PFS HRs = 0.60 to 0.61; OS HRs = 0.71 to 0.75) rather than single-positive cases (PFS HRs = 0.68 to 0.81; OS HRs = 0.87 to 0.95). Concordance for harmonized cutoffs for SP263 (IC ≥4%) and 22C3 (CPS ≥10) to SP142 (IC ≥1%) was subpar (approximately 75%).Conclusions: 22C3 and SP263 assays identified more patients as PD-L1+ (IC ≥1%) than SP142. No inter-assay analytical equivalency was observed. Consistent improved A+nP efficacy was captured by the SP142 PD-L1 IC ≥1% subgroup nested within 22C3 and SP263 PD-L1+ (IC ≥1%) populations. [ABSTRACT FROM AUTHOR]

Purpose. To update the American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) guideline recommendations for human epidermal growth factor receptor 2 (HER2) testing in breast cancer to improve the accuracy of HER2 testing and its utility as a predictive marker in invasive breast cancer. Methods. ASCO/CAP convened an Update Committee that included coauthors of the 2007 guideline to conduct a systematic literature review and update recommendations for optimal HER2 testing. Results. The Update Committee identified criteria and areas requiring clarification to improve the accuracy of HER2 testing by immunohistochemistry (IHC) or in situ hybridization (ISH). The guideline was reviewed and approved by both organizations. Recommendations. The Update Committee recommends that HER2 status (HER2 negative or positive) be determined in all patients with invasive (early stage or recurrence) breast cancer on the basis of one or more HER2 test results (negative, equivocal, or positive). Testing criteria define HER2-positive status when (on observing within an area of tumor that amounts to >10% of contiguous and homogeneous tumor cells) there is evidence of protein overexpression (IHC) or gene amplification (HER2 copy number or HER2/CEP17 ratio by ISH based on counting at least 20 cells within the area). If results are equivocal (revised criteria), reflex testing should be performed using an alternative assay (IHC or ISH). Repeat testing should be considered if results seem discordant with other histopathologic findings. Laboratories should demonstrate high concordance with a validated HER2 test on a sufficiently large and representative set of specimens. Testing must be performed in a laboratory accredited by CAP or another accrediting entity. The Update Committee urges providers and health systems to cooperate to ensure the highest quality testing. (Arch Pathol Lab Med. 2014; 138:241-256; doi: 10.5858/arpa.2013-0953-SA), In 2007, a joint Expert Panel convened by the American Society of Clinical Oncology (ASCO) and the College of American Pathologists (CAP) met to develop guidelines for when and how [...]

Background and Aims: Hepatobiliary phase (HBP) Gd‐EOB‐DTPA‐enhanced magnetic resonance imaging (MRI) has increased the accuracy in differentiating focal nodular hyperplasia (FNH) and hepatocellular adenoma (HCA). However, the ability of this technique to distinguish HCA subtypes remains controversial. The aim of this study was to investigate the expression of hepatocyte transporters (OATPB1/B3, MRP2, MRP3) in HCA subtypes, hence to understand their MRI signal intensity on HBP Gd‐EOB‐DTPA‐enhanced MRI. Methods: By means of immunohistochemistry (IHC), we scored the expression of OATPB1/B3, MRP2 and MRP3, in resected specimens of FNH (n = 40), subtyped HCA (n = 58) and HCA with focal malignant transformation (HCA‐HCC, n = 4). Results were validated on a supplementary set of FNH (n = 6), subtyped HCA (n = 17) and HCA‐HCC (n = 1) with Gd‐EOB‐DTPA MR images. Results: All FNH showed a preserved expression of hepatocytes transporters. Beta‐catenin‐activated HCA (at highest risk of malignant transformation) and HCA‐HCC were characterized by preserved/increased OATPB1/B3 expression (predictor of hyperintensity on HBP), as opposed to other HCA subtypes (P < 0.01) that mostly showed OATPB1/B3 absence (predictor of hypointensity on HBP). HCA‐HCC showed an additional MRP3 overexpressed profile (P < 0.01). On HBP Gd‐EOB‐DTPA‐enhanced MRI, FNH and HCA signal intensity reflected the profile predicted by their specific OATPB1/B3 tissue expression. The hyperintense vs hypointense HBP signal criterion was able to distinguish all higher risk HCA and HCA‐HCC (100% accuracy). Conclusions: OATPB1/B3 and MRP3 IHC and signal intensity on HBP Gd‐EOB‐DTPA‐enhanced MRI can help to stratify HCA according to their risk of malignant transformation. [ABSTRACT FROM AUTHOR]

BACKGROUND: The primary objectives of this study on carcinomas with equivocal HER2 expression were to assess the impact of distinct recommendations with regard to identifying patients eligible for anti-HER2 agents by fluorescence in situ hybridization (FISH) and to elucidate whether multiplex ligation-dependent probe amplification (MLPA) may be of support in assessing HER2 gene status. METHODS: A cohort of 957 immunohistochemistry-evaluated HER2-equivocal cases was analyzed by dual-color FISH. The results were assessed according to U.S. Food and Drug Administration (FDA) and European Medicines Agency (EMA) guidelines and American Society of Clinical Oncology (ASCO) and College of American Pathologists (CAP) 2007 and 2013 guidelines for dual- and single-signal in situ hybridization (ISH) assays. A subgroup of 112 cases was subjected to MLPA. RESULTS: HER2 amplification varied from 15% (ASCO/CAP 2007 HER2/CEP17 ratio) to 29.5% (FDA/EMA HER2 copy number). According to the ASCO/CAP 2013 interpretation of the dual-signal HER2 assay, ISH-positive carcinomas accounted for 19.7%. In contrast with the ASCO/CAP 2007 ratio, this approach labeled as positive all 32 cases (3.34%) with a HER2/CEP17 ratio <2 and an average HER2 copy number ≥6.0 signals per cell. In contrast, only one case showing a HER2 copy number <4 but a ratio ≥2 was diagnosed as positive. MLPA data correlated poorly with FISH results because of the presence of heterogeneous HER2 amplification in 33.9% of all amplified carcinomas; however, MLPA ruled out HER2 amplification in 75% of ISH-evaluated HER2-equivocal carcinomas. CONCLUSION: The ASCO/CAP 2013 guidelines seem to improve the identification of HER2-positive carcinomas. Polymerase chain reaction-based methods such as MLPA can be of help, provided that heterogeneous amplification has been ruled out by ISH. [ABSTRACT FROM AUTHOR]

Background In breast cancer, immunohistochemical assessment of proliferation using the marker Ki67 has potential use in both research and clinical management. However, lack of consistency across laboratories has limited Ki67’s value. A working group was assembled to devise a strategy to harmonize Ki67 analysis and increase scoring concordance. Toward that goal, we conducted a Ki67 reproducibility study. Methods Eight laboratories received 100 breast cancer cases arranged into 1-mm core tissue microarrays—one set stained by the participating laboratory and one set stained by the central laboratory, both using antibody MIB-1. Each laboratory scored Ki67 as percentage of positively stained invasive tumor cells using its own method. Six laboratories repeated scoring of 50 locally stained cases on 3 different days. Sources of variation were analyzed using random effects models with log2-transformed measurements. Reproducibility was quantified by intraclass correlation coefficient (ICC), and the approximate two-sided 95% confidence intervals (CIs) for the true intraclass correlation coefficients in these experiments were provided. Results Intralaboratory reproducibility was high (ICC = 0.94; 95% CI = 0.93 to 0.97). Interlaboratory reproducibility was only moderate (central staining: ICC = 0.71, 95% CI = 0.47 to 0.78; local staining: ICC = 0.59, 95% CI = 0.37 to 0.68). Geometric mean of Ki67 values for each laboratory across the 100 cases ranged 7.1% to 23.9% with central staining and 6.1% to 30.1% with local staining. Factors contributing to interlaboratory discordance included tumor region selection, counting method, and subjective assessment of staining positivity. Formal counting methods gave more consistent results than visual estimation. Conclusions Substantial variability in Ki67 scoring was observed among some of the world’s most experienced laboratories. Ki67 values and cutoffs for clinical decision-making cannot be transferred between laboratories without standardizing scoring methodology because analytical validity is limited. [ABSTRACT FROM PUBLISHER]

Abstract: We collected information on 497 consecutive breast cancer patients aged less than 35 years operated at the European Institute of Oncology. The main aim of the study is to compare biological and clinical features dividing the population by age: <25 years, 25–29 and 30–34 years old. Pattern of recurrence and survival were also analyzed. Patients aged <25 years had 81.8% poorly differentiated tumors compared with 66.7% and 56.5% in the 25–29 and 30-34 groups, respectively; no other significant difference were found in the distribution of clinical and immunohistochemical features The distribution of Luminal A and B, Triple Negative and HER2 subtypes (immunohistochemically defined) was not statistically different among the three age groups. No difference was found in the incidence of loco-regional relapses, distant metastases, disease-free survival (p = 0.79) and overall survival (p = 0.99) between the three age groups. This latter findings was confirmed using age as a continuous variable assuming a linear association between age and the outcomes considered, too. In conclusion, our data indicate that the group of patients with breast cancer below 35 years is essentially a homogenous group when classical clinical and immunohistochemical features were considered. [Copyright &y& Elsevier]

Aims To ascertain the prognostic relevance of micropapillary carcinoma, a specific type of breast tumour. Methods and results We interrogated the clinical records of a series of 49 pure micropapillary carcinoma patients and 13 487 invasive ductal carcinoma patients, diagnosed and treated consecutively in our institution over a 9-year time-frame. Compared with invasive ductal carcinoma, patients with micropapillary carcinoma more frequently had moderately differentiated tumours ( P = 0.02) with extensive peritumoral vascular invasion ( P < 0.0001), associated with a significantly higher rate of axillary lymph node involvement ( P < 0.0001). Survival data obtained by comparing 49 micropapillary carcinoma patients with a set of 98 invasive ductal carcinoma patients matched for age, tumour size and grade, peritumoral vascular invasion, immunohistochemically defined molecular subtype, number of positive lymph nodes and year of surgery showed that the micropapillary histotype did not add any independent information to the risk of locoregional ( P = 0.48) or distant ( P = 0.79) relapse, or overall survival ( P = 0.60). Conclusions Our data reinforce the notion that micropapillary carcinoma usually arises as a locally advanced disease, and provide evidence that micropapillary histology does not add any additional information on clinical outcome independent of clinicopathological characteristics such as lymph node status and immunohistochemically defined molecular subtype. [ABSTRACT FROM AUTHOR]

Summary: Gastric cancer shows intratumoral heterogeneity for human epidermal growth factor receptor 2 expression. We evaluated whether the number of tissue blocks analyzed or the antibodies used may influence the immunohistochemical results in gastrectomy specimens. Clinicopathologic data from 148 patients receiving gastric surgery for cancer were collected. One tissue block for each of 88 primary tumors and 60 paired primary tumors and metastases was examined for human epidermal growth factor receptor 2 status by immunohistochemistry using 3 different antibodies (HercepTest, CB11, and 4B5) and by fluorescent in situ hybridization. Two additional tissue blocks of the primary tumor were tested by immunohistochemistry if the results were negative on the first tissue block. The concordance among the 3 antibodies was 94.5% (testing 1 tissue block). Two cases showed a clinically significant discrepancy between primary tumor (score 0) and lymph nodes metastases (score 3+). Additional block analysis increased both the sensitivity (from 63% to 83%) and the accuracy (from 91% to 94%) of immunohistochemistry as compared with fluorescent in situ hybridization. The multiblock approach could potentially identify a greater number of human epidermal growth factor receptor 2–positive gastric cancers, particularly those with higher levels of intratumor heterogeneity. In turn, human epidermal growth factor receptor 2 positivity correlated with a worse prognosis (P = .011) and was an independent variable in multivariate analysis (hazard ratio, 1.57). In conclusion, testing more than 1 tissue block of cancer from specimens of gastric resection provides a more reliable human epidermal growth factor receptor 2 assessment regardless of the antibody used. [ABSTRACT FROM AUTHOR]

Background and aims: Probiotics and their metabolic products, here called postbiotics, have been proposed as food supplements for a healthier intestinal homeostasis, but also as therapeutic aids in inflammatory bowel disease (IBD) with, however, very little clinical benefit. This may be due to the lack of reliable preclinical models for testing the efficacy of different strains. Methods: The activity of three probiotic strains of Lactobacillus (or a postbiotic) was analysed and compared with a pathogenic strain of Salmonella on a novel organ culture system of human healthy and IBD intestinal mucosa developed in our laboratory. The system maintains an apical to basolateral polarity during stimulation due to the presence of a glued cave cylinder. The cylinder is detached at the end of the experiment and the tissue is processed for histology and immunohistochemistry. Cytokines released from the basolateral side are analysed. Results: The model system provides several physiological characteristics typical of a mucosal microenvironment including the presence of an organised mucus layer and an apical to basolateral polarity. Polarised administration of bacteria is critical to control the ensuing immune response as it mimics the physiological entrance of bacteria. The authors show that probiotics are not always beneficial for the healthy host and can also be detrimental in inflamed IBD. This study shows that a potent postbiotic can protect against the inflammatory properties of invasive Salmonella on healthy tissue and also downregulate ongoing inflammatory processes in IBD tissue. Conclusions: Probiotics can have inflammatory activities in both healthy and IBD tissue. Valid preclinical data on proper model systems should therefore be obtained before specific probiotic strains enter the clinics, especially if administered during acute inflammatory responses. Postbiotics may be a safe alternative for the treatment of patients with IBD in the acute inflammatory phase. INSET: Significance of this study. [ABSTRACT FROM AUTHOR]

Adjuvant chemotherapy decisions in breast cancer are increasingly based on the pathologist's assessment of tumor proliferation. The Swiss Working Group of Gyneco- and Breast Pathologists has surveyed inter- and intraobserver consistency of Ki-67-based proliferative fraction in breast carcinomas. Methods: Five pathologists evaluated MIB-1-labeling index (LI) in ten breast carcinomas (G1, G2, G3) by counting and eyeballing. In the same way, 15 pathologists all over Switzerland then assessed MIB-1-LI on three G2 carcinomas, in selfselected or pre-defined areas of the tumors, comparing centrally immunostained slides with slides immunostained in the different laboratoires. To study intra-observer variability, the same tumors were re-examined 4 months later. Results: The Kappa values for the first series of ten carcinomas of various degrees of differentiation showed good to very good agreement for MIB-1-LI (Kappa 0.56-0.72). However, we found very high inter-observer variabilities (Kappa 0.04-0.14) in the read-outs of the G2 carcinomas. It was not possible to explain the inconsistencies exclusively by any of the following factors: (i) pathologists' divergent definitions of what counts as a positive nucleus (ii) the mode of assessment (counting vs. eyeballing), (iii) immunostaining technique, and (iv) the selection of the tumor area in which to count. Despite intensive confrontation of all participating pathologists with the problem, inter-observer agreement did not improve when the same slides were reexamined 4 months later (Kappa 0.01-0.04) and intra-observer agreement was likewise poor (Kappa 0.00-0.35). Conclusion: Assessment of mid-range Ki-67-LI suffers from high inter- and intra-observer variability. Oncologists should be aware of this caveat when using Ki-67-LI as a basis for treatment decisions in moderately differentiated breast carcinomas. [ABSTRACT FROM AUTHOR]

Background. Breast cancer during pregnancy (BCP) is relatively rare and is associated with controversies about its biology and prognosis. Hence, we designed a case-control study to examine tumor features and outcome in a series of BCP patients diagnosed and treated in a single institution. Material and methods. We identified 65 patients diagnosed with BCP and for each; we selected two non-pregnant breast cancer patients, who were matched for age, year of surgery, stage, and neoadjuvant chemotherapy. We then compared the differences in pathology, immunohistochemical features (ER, PR, HER2 and ki-67), disease-free (DFS) and overall survival (OS). Results. We did not find any significant differences in tumor characteristics between the two groups. However, at a median follow-up of four years, BCP patients had an inferior DFS (HR 2.3; 95% CI 1.3-4.2), after adjustment for possible confounding covariates. No difference in OS was observed. However, upon restricting the analysis to patients who did not receive neoadjuvant chemotherapy, patients with BCP had inferior OS as well (HR 2.6; 95% CI 1.0-6.5). No association between induction of abortion and prognosis was observed. Conclusions. While we did not observe any differences in tumor features, BCP patients have poorer prognosis compared to age and stage-matched control. Further studies should try to elucidate reasons for such poor outcome. [ABSTRACT FROM AUTHOR]

Abstract: Triple negative breast cancer (TNBC) accounts for approximately 15% of breast cancer cases. TNBC is an immunohistochemically defined subtype, with significant diversity within the subtype. Generally TNBC occurs in younger women and is marked by high rates of relapse, visceral and CNS metastases, and early death. Current therapy fails to curtail the innate aggressive behaviour of TNBC in the majority of patients. The poor prognosis coupled with a lack of targeted use of therapies is reflected in the high mortality. In a minority of patients with highly chemosensitive disease, no robust clinical evidence exists to guide use of current cytotoxics. Critical to optimal future management are accurate identification of truly triple negative disease and adequately powered prospective TNBC trials to establish treatment efficacy and define predictive biomarkers. [ABSTRACT FROM AUTHOR]

Morphology still remains the cornerstone in lung cancer classification and always has been accompanying pathologists in their daily activity, even though several ancillary techniques have been incorporated over time to improve diagnostic, prognostic, and predictive capabilities in lung cancer. Currently, we are also faced with a global rethinking of lung cancer care, especially once novel therapy strategies have been made available on the basis of the diverse characteristics of tumors. Although morphology still remains a not easily replaceable tool for lung cancer classification, we are now challenged by the need of offering clinicians more detailed subtyping of non-small-cell lung cancer especially in event of limited diagnostic material, poorly differentiated tumors, or unresectable lesions. Close integration of improved morphology, immunohistochemistry, and molecular tests will be able to not only sharpen our diagnostic algorithms and prognostic and predictive potentialities but also get insights into several lung cancer biology issues, such as histogenesis and new classification schemes. [ABSTRACT FROM AUTHOR]

Simple Summary: Four biomarkers [estrogen receptor (ER), progesterone receptor (PgR), Ki-67, and HER2], are used to stratify breast cancer (BC) into subtypes predictive of therapy response. In a Europe-wide external quality assessment, we compared performance of an mRNA-based method [Xpert® Breast Cancer STRAT4 (CE-IVD)] for determining ESR1, PGR, ERBB2, and MKI67 expression against the gold standard [immunohistochemistry (IHC)/HER2 in situ hybridization (ISH)]. The coordinating center (CC) and five European laboratories tested ten breast cancer samples. STRAT4 binary (positive or negative) results of each marker were compared with the gold standard. ESR1 and ERBB2 mRNA results were concordant with IHC/ISH in all single analyses. In contrast, PGR and MKI67 results were discordant in a few cases, which had STRAT4 expression values close to assay cut-offs and immunohistochemically presented heterogeneous low positive PgR and heterogeneous Ki-67. STRAT4 assay may be a reproducible method. However, cases with expression values close to cut-offs should be carefully reviewed. Estrogen receptor (ER), progesterone receptor (PgR), Ki-67, and HER2 immunohistochemistry (IHC) together with HER2 in situ hybridization (ISH) are utilized to classify invasive breast cancer (IBC) into predictive molecular subtypes. As IHC evaluation may be hampered by analytical errors, gene expression assays could offer a reliable alternative. In this first Europe-wide external quality assessment (EQA) study, we investigated performance of mRNA-based Xpert® Breast Cancer STRAT4 (CE-IVD) in five European laboratories. The cohort comprised ten pre-therapy IBC core biopsies diagnosed in the coordinating center (CC). STRAT4 binary (positive or negative) mRNA results of each marker (ESR1, PGR, ERBB2, MKI67) were compared with the gold standard IHC/ISH performed by the CC. Sensitivity, specificity, and accuracy of ESR1 and ERBB2 mRNA were 100% for all samples. In contrast, PGR expression was falsely negative for one case by two sites and MKI67 falsely negative for two cases (respectively by four and one sites). These cases had STRAT4 expression values close to assay cut-offs and immunohistochemically presented heterogeneous low positive PgR and heterogeneous Ki-67. Our EQA shows that STRAT4 mRNA assay may be a reproducible method to evaluate ER, PgR, HER2, and Ki-67 status. However, cases with expression values close to assay cut-offs should be carefully reviewed. [ABSTRACT FROM AUTHOR]

p63, a gene that shares structural and functional homologies with p53, codes for different isoforms, with (TA) and without (ΔN) transactivating properties. The anti-apoptotic ΔN isoform is often expressed in breast cancer (BC). DNA damaging drugs such as cisplatin (C) induce its degradation and stabilization of the TA, proapoptotic isoform. This supports the role of these drugs in the treatment of tumors expressing p63. The aim of the present study was to ascertain the predictive value of p63 immunoreactivity in patients treated preoperatively with regimens including cisplatin and/or anthracyclines. We reviewed the pretreatment biopsies of 189 patients with large or locally advanced BC (cT1–4d, N0-2, M0) treated with preoperative chemotherapy, performing p63 immunohistochemistry. The rate of pathological complete remission (pCR) at final surgery was assessed with respect to cisplatin administration and p63 immunoreaction. pCR was identified in 20 patients (11%); 147 patients (78%) had an objective response, 39 (21%) stable disease, and 3 (1%) disease progression. One hundred forty seven patients (78%) received a cisplatin-containing regimen. Only regimens including cisplatin without anthracyclines yielded a higher rate of pCR in p63-positive compared with p63-negative tumors (23 vs. 0%, P = 0.048). No significant difference in the pCR rate was observed for regimens containing anthracycline without cisplatin. Administration of cisplatin without anthracyclines correlates with a high rate of pCR after primary chemotherapy in patients with p63-positive BC. The role of cisplatin-based chemotherapy should be further studied in these patients. [ABSTRACT FROM AUTHOR]

The caudal-related homeobox transcription factor CDX2 regulates the differentiation of intestinal epithelial cells. Recent studies have suggested that CDX-2 immunoreactivity is strictly confined to benign and malignant intestinal epithelium. In the present study, we evaluated the prevalence of CDX2 immunoreactivity in a series of benign, borderline and malignant primary ovarian mucinous neoplasms. We tested 62 mucinous tumours of the ovary, including 28 benign cystadenomas, 18 borderline tumours, 16 adenocarcinomas, 35 serous and endometrioid ovarian lesions and 10 ovarian metastases of colonic adenocarcinoma. Overall, the CDX2 prevalence in primary mucinous tumours was 79%, including 20 of 28 (71.5%) cystadenomas, 14 of 18 (77.7%) borderline tumours and 15 of 16 (93.5%) adenocarcinomas. Immunoreactivity usually correlated with intestinal differentiation of tumour cells, although wide heterogeneity in the distribution of immunolabelled cells was noted. No immunoreactivity was observed in serous lesions; whereas, 1 of 13 (7.7%) endometrioid adenocarcinomas and all of the 10 metastatic colonic adenocarcinomas were immunostained. These results indicate that CDX2 is detectable in the majority of benign, borderline and malignant ovarian mucinous tumours and, therefore, makes this marker unsuitable when distinguishing primary from metastatic ovarian mucinous adenocarcinomas. However, CDX2 immunoreactivity could be useful in the distinction between endocervical and intestinal-type mucinous tumours of the ovary, which may have clinical relevance. [ABSTRACT FROM AUTHOR]

Most granular cell tumours (GCTs) are benign proliferations of purported Schwannian derivation, showing immunoreactivity for Schwann cell-related antigens. Due to incomplete agreement on the precise nature of GCTs (reactive vs neoplastic), we performed an immunohistochemical study with the alkaline phosphatase/anti-alkaline phosphatase (APAAP) technique on 30 GCTs. The aim was to evaluate their growth patterns and the possible relationships of granular cells with other nerve sheath-related cell types (i.e., Schwann and perineurial cells, and dendritic cells displaying CD34/vimentin immunoreactivity). An expansive growth pattern was detected in five cases, a pseudoinfiltrative growth pattern in nine cases and a mixture of the above in the remaining 16 cases. Besides immunoreactivity for S-100 protein, neuron-specific enolase, vimentin, CD57, CD68, MAC 387, alpha-1-antitrypsin and alpha-1-antichymotrypsin in granular cells, we documented intimate architectural relationships between granular cells, Schwann and perineurial cells, and a third type of CD34/vimentin-positive nerve sheath-related cell in most GCTs. These results suggest that GCTs are heterogeneous lesions. Some of them show a pseudoinfiltrative growth pattern and retain close relationships with the normal components of the nerve sheath. In other lesions, granular cells grow in an expansive fashion and constitute the predominant cell component of the tumour. These architectural and immunophenotypic differences may reflect a different nature of GCTs: they may initially represent reactive or hamartomatous lesions that subsequently acquire truly neoplastic potential. [ABSTRACT FROM AUTHOR]

The search for novel prognostic and predictive parameters in breast carcinoma is relentless. The new technological advances in gene expression profiling and in mutational analysis will hopefully prove to be clinically useful in informing the choice for the systemic therapy. For the time being, we are still relying in established immunohistochemical markers, namely estrogen and progesterone receptors, HER2 and Ki67. Advances in the harmonization of pre-analytical, analytical and interpretative variables may improve accuracy and reproducibility of the results. [ABSTRACT FROM AUTHOR]

Abstract: One of the most important lessons learned from trials of neoadjuvant chemotherapy (NACT) is that achievement of pathological complete response (pCR) is a powerful prognostic predictor of long-term outcome, with significantly better disease-free and overall survival for patients achieving pCR, as compared with patients having residual tumour after NACT. The pathologists' role in the neoadjuvant setting is: (i) to ensure an accurate assessment of pCR, and (ii) to evaluate burden and biological characteristics of residual tumour if pCR has not been achieved. A conversion of receptor status from the core biopsy to the post-NACT surgical specimen may cause uncertainty in the choice of the post-surgical systemic treatment for the patients. It is therefore imperative to ensure accuracy in the assessment of ER, PgR and HER2, and to double check any apparent conversion by re-staining the previous core biopsy and the residual tumour in the same run, thus minimizing the technical artifacts, and to use both immunohistochemical and in situ hybridization assays to evaluate HER2 status. It is essential that protocols for evaluation of tumour response and for assessment of prognostic/predictive parameters of residual disease after NACT be eventually harmonized. [Copyright &y& Elsevier]

Summary: The primary tumour of patients with early breast cancer is the main source of information to assess the risk of disease recurrence and to inform the choice of the most appropriate systemic treatment. Accordingly, it is the main responsibility of the pathologists to ensure the patients and treating physicians that all the relevant information is derived from the primary tumour with the highest accuracy and reproducibility. The morphological changes of the tumour cells reflect the aggregate effects of changes occurring in hundreds of genes and may be a very faithful mirror of the biological and clinical behaviour of breast cancer. According to the 2009 St. Gallen Consensus, the systemic therapy of early breast cancer is mainly informed by the expression of hormone receptors and by the HER2 status, and the assessment of Ki67 has been included among the useful parameters to inform the choice of adding chemotherapy to endocrine therapies for patients with ER-positive and HER2-negative disease. A comprehensive approach that includes the accurate evaluation of the morphological features of the tumour, with special reference to the histological type and grade, and the assessment of the main prognostic and predictive parameters (ER, PgR, HER2 and Ki67) should offer to the patients and the treating physicians a robust background upon which the final therapeutic decisions can be safely taken. [ABSTRACT FROM AUTHOR]

Summary: The landscape of prognostication and prediction of responsiveness to systemic therapy for breast cancer patients has been recently enriched by the development of molecular assays, which enable to explore the whole universe of gene expression in the tumour cells and to unveil new prognostic and predictive markers. These molecular markers might well be used in combination with the established ones to address the many open questions that still pave the way to a truly personalized treatment. The actual clinical utility of the molecular assays is being tested in randomized clinical trials that require an unprecedented coordination of the activity of clinical investigators, pathologists and translational researchers worldwide. [Copyright &y& Elsevier]

Focuses on the status of optimal pathology reporting in breast cancer. Features of the Nordic model for quality assurance; Internal quality control implemented in Great Britain related to breast cancer; Countries that have adopted the immunohistochemistry breast markers quality assurance program.

The promising effect of antibody–drug conjugates on breast cancer with low expression of HER2 (HER2-low) raises many questions regarding the optimal selection of patients for this treatment. A key question is whether HER2 immunohistochemistry, an assay optimised to detect HER2 amplification, is reliable enough to assess HER2 protein levels to select patients with HER2-low breast cancer in daily pathology practices worldwide. Moreover, whether this assessment can be performed with sufficient reproducibility between pathologists in daily practices is debatable. Herein, we address the historical track record of the CAP-ASCO HER2 Guidelines, the reported limited reproducibility by pathologists of HER2 immunohistochemistry in the non-amplified cases, and the performance variation of different antibodies. Based on this summary, we propose solutions to improve the robustness to enable reliable identification of patients with HER2-low breast cancer. • The definitions of HER2 0 and 1+ by IHC need to be redefined by the ASCO/CAP. • HER2 IHC assays are not calibrated to detect low-levels of HER2. • Other issues include reproducibility, different HER2-assay performance, etc. • The errors of the PDL1-saga are currently being repeated in the HER2-low narrative. • Solutions are proposed to industry, regulators, and academia (Table 1). [ABSTRACT FROM AUTHOR]

Low human epidermal growth factor receptor 2 (HER2) expression is emerging as an actionable biomarker for the treatment of breast cancer (BC) with novel anti-HER2 drugs. However, the evolution of this biomarker during the course of disease is still poorly characterised, and controversial data exist on its prognostic implications. We reviewed data of patients with HER2-negative BC according to the latest ASCO/CAP guidelines referred between January 2014 and December 2020. We grouped patients based on the immunohistochemistry (IHC) expression of HER2, HER2-zero (IHC 0) and HER2-low subgroup (IHC 1+ or 2+/ISH-negative) and evaluated the evolution of HER2 expression between the primary tumour and the first biopsy collected in the advanced setting. Disease-free survival, overall survival and progression-free survival were compared between patients with HER2-zero and HER2-low expression on the primary tumour. 232 patients were included in the analysis. Among the overall population, there was a relevant discordance in HER2 expression between the primary tumour and the matched biopsy (K = 0.33, 95%CI 0.21–0.44): 44% of the HER2-zero primary tumour showed an increased HER2 score on biopsy, and 22% of the HER2-low primary tumours turned into HER2-IHC 0. The findings in the sub-populations of hormone-receptors positive (K = 0.32, 95%CI 0.19–0.45) and triple-negative tumours (K = 0.18, 95%CI -0.09-0.46) were consistent with the primary analysis. No difference in survival outcomes was observed between HER2-low and HER2-zero primary tumours. HER2-low expression is dynamic in BC and may be enriched in the advanced-stage setting. No prognostic significance was demonstrated for HER2-low expression. • HER2-low expression is emerging as an actionable biomarker in breast cancer. • HER2 IHC score increased from primary to metastatic tumour in 44% of the patients. • HER2-low to HER2-zero evolution was observed in 22% of the patients. • HER2-low expression is dynamic and may be enriched in the advanced-stage setting. [ABSTRACT FROM AUTHOR]

* Purpose.--To update key recommendations of the American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) human epidermal growth factor receptor 2 (HER2) testing in breast cancer guideline. Methods.--Based on the signals approach, an Expert Panel reviewed published literature and research survey results on the observed frequency of less common in situ hybridization (ISH) patterns to update the recommendations. Recommendations.--Two recommendations addressed via correspondence in 2015 are included. First, immunohistochemistry (IHC) 2+ is defined as invasive breast cancer with weak to moderate complete membrane staining observed in .10% of tumor cells. Second, if the initial HER2 test result in a core needle biopsy specimen of a primary breast cancer is negative, a new HER2 test may (not "must") be ordered on the excision specimen based on specific clinical criteria. The HER2 testing algorithm for breast cancer is updated to address the recommended workup for less common clinical scenarios (approximately 5% of cases) observed when using a dual-probe ISH assay. These scenarios are described as ISH group 2 (HER2/chromosome enumeration probe 17 [CEP17] ratio ≥2.0; average HER2 copy number <4.0 signals per cell), ISH group 3 (HER2/CEP17 ratio <2.0; average HER2 copy number ≥6.0 signals per cell), and ISH group 4 (HER2/CEP17 ratio,2.0; average HER2 copy number ≥4.0 and <6.0 signals per cell). The diagnostic approach includes more rigorous interpretation criteria for ISH and requires concomitant IHC review for dual-probe ISH groups 2 to 4 to arrive at the most accurate HER2 status designation (positive or negative) based on combined interpretation of the ISH and IHC assays. The Expert Panel recommends that laboratories using single-probe ISH assays include concomitant IHC review as part of the interpretation of all single-probe ISH assay results. [ABSTRACT FROM AUTHOR]

Abstract: The YOMICS™ antibody library (http://www.yomics.com/) presented in this article is a new collection of 1559 murine polyclonal antibodies specific for 1287 distinct human proteins. This antibody library is designed to target marginally characterized membrane-associated and secreted proteins. It was generated against human proteins annotated as transmembrane or secreted in GenBank, EnsEMBL, Vega and Uniprot databases, described in no or very few dedicated PubMed-linked publications. The selected proteins/protein regions were expressed in E. coli, purified and used to raise antibodies in the mouse. The capability of YOMICS™ antibodies to specifically recognize their target proteins either as recombinant form or as expressed in cells and tissues was confirmed through several experimental approaches, including Western blot, confocal microscopy and immunohistochemistry (IHC). Moreover, to show the applicability of the library for biomarker investigation by IHC, five antibodies against proteins either known to be expressed in some cancers or homologous to tumor-associated proteins were tested on tissue microarrays carrying tumor and normal tissues from breast, colon, lung, ovary and prostate. A consistent differential expression in cancer was observed. Our results indicate that the YOMICS™ antibody library is a tool for systematic protein expression profile analysis that nicely complements the already available commercial antibody collections. [Copyright &y& Elsevier]

Background: Tools able to predict pathological complete response (pCR) to preoperative chemotherapy might improve treatment outcome. Patients and methods: Data from 783 patients with invasive ductal carcinoma treated with preoperative chemotherapy and operated at the European Institute of Oncology were used to develop a nomogram using logistic regression model based on both categorical (clinical T and N, HER2/neu, grade and primary therapy) and continuous variables (age, oestrogen receptor (ER), progesterone receptor (PgR), Ki-67 expression and number of chemotherapy courses). The performance of the resulting nomogram was internally evaluated through bootstrapping methods. Finally the model was externally validated on a patient set treated in other institutions and subsequently operated at the EIO. Results: At multivariable analysis the probability of pCR was directly associated with Ki-67 expression (OR for 10% increase in the percentage of positive cells, 1.15, 95% confidence interval (CI), 1.03, 1.29) and number of chemotherapy courses (OR for one cycle increase, 1.31, 95% CI, 1.12, 1.53) and inversely associated with ER and PgR expression (ORs for 10% increase in the percentage of positive cells, 0.86, 95% CI 0.79, 0.93 and 0.82, 95% CI 0.69, 0.99, respectively). The nomogram for pCR based on these variables had good discrimination in training as well in validation set (AUC, 0.78 and 0.77). Conclusion: The use of a nomogram based on the number of preoperative courses, degree of Ki-67 and steroid hormone receptors expression may be useful for predicting the probability of pCR and for the design of the proper therapeutic algorithm in locally advanced breast cancer. [Copyright &y& Elsevier]

Immunoreactivity for fascin, an actin-bundling protein related to cell motility, has been reported in breast, ovary, pancreas, skin, and non-small cell carcinomas, and associated with more advanced disease stage and poorer prognosis. Data on pulmonary neuroendocrine (NE) tumors, however, are lacking. We evaluated the expression of fascin by immunohistochemistry—using two different monoclonal antibodies—in surgical specimens of pulmonary NE tumors of all the diverse histological types from 128 consecutive patients recruited between 1987 and 2001, and investigated its relationship with the presence of lymph node metastases. Overall, fascin immunoreactivity was detected in 5% of 38 typical carcinoids (TC), 35% of 23 atypical carcinoids (AC), 83% of 40 large-cell neuroendocrine carcinomas (LCNEC), and 100% of 27 small-cell lung carcinomas (SCLC) (P<0.001), Normal NE cells or hyperplastic NE tumorlets were consistently unreactive. No statistically significant differences in fascin immunoreactivity were found between the two antibodies. In TC and AC but not high-grade NE tumors, fascin immunoreactivity closely correlated with the occurrence of lymph node metastases, the pN class and the number of involved lymph nodes (P<0.001). It was also significantly associated with an increased proliferative activity (Ki-67 labeling index >5%) (P=0.020), and with either down-regulation or altered subcellular compartmentalization of E-cadherin (P<0.001) and CD99 (P=0.030), two cell adhesion complexes in pulmonary NE tumors. At multivariate analysis, only fascin emerged as an independent predictor of lymph node metastases in this tumor group (HR 30.28; 95% confidence intervals: 1.59–574.49; P=0.023). This study indicates that fascin immunoreactivity may identify subsets of pulmonary carcinoid patients with different metastatic potential to regional lymph nodes. Targeting the fascin pathway could be a novel therapeutic strategy of pulmonary carcinoids. [Copyright &y& Elsevier]