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AIMS: The LUMINA trial demonstrated a very low local recurrence rate in women ≥55 years with low-risk luminal A breast cancer (defined as grade I-II, T1N0, hormone receptor positive, HER2 negative and Ki67 index ≤13.25%) treated with breast-conserving surgery and endocrine therapy (but no other systemic therapy), supporting the safe omission of radiation in these women. Here we describe the protocol for Ki67 assessment, the companion diagnostic used to guide omission of adjuvant radiotherapy. METHODS: Ki67 immunohistochemistry was performed on full-face sections at one of three regional labs. Pathologists trained in the International Ki67 in Breast Cancer Working Group (IKWG) method demarcated tumour areas on scanned slides and scored 100 nuclei from each of at least five randomly selected 1-mm fields. For cases with high Ki67 heterogeneity, further virtual cores were selected and scored in order to confidently assign a case as luminal A (≤13.25%) or B (>13.25%). Interlaboratory variability was assessed through an annual quality assurance programme during the study period. RESULTS: From the quality assurance programme, the mean Ki67 index across all cases/labs was 13%. The observed intraclass correlation coefficient (ICC) and kappa statistics were ≥0.9 and ≥0.7, respectively, indicating a substantial level of agreement. Median scoring time was 4 min per case. The IKWG-recommended scoring method, performed directly from slides, requiring up to four scored fields, is concordant with the LUMINA scoring method (ICC ≥ 0.9). CONCLUSION: Ki67 is a practical, reproducible, and inexpensive biomarker that can identify low-risk luminal A breast cancers as potential candidates for radiation de-escalation. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov number, NCT01791829.
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Neoplasias de la Mama , Femenino , Humanos , Neoplasias de la Mama/diagnóstico , Antígeno Ki-67 , InmunohistoquímicaRESUMEN
Ki67 has potential clinical importance in breast cancer but has yet to see broad acceptance due to inter-laboratory variability. Here we tested an open source and calibrated automated digital image analysis (DIA) platform to: (i) investigate the comparability of Ki67 measurement across corresponding core biopsy and resection specimen cases, and (ii) assess section to section differences in Ki67 scoring. Two sets of 60 previously stained slides containing 30 core-cut biopsy and 30 corresponding resection specimens from 30 estrogen receptor-positive breast cancer patients were sent to 17 participating labs for automated assessment of average Ki67 expression. The blocks were centrally cut and immunohistochemically (IHC) stained for Ki67 (MIB-1 antibody). The QuPath platform was used to evaluate tumoral Ki67 expression. Calibration of the DIA method was performed as in published studies. A guideline for building an automated Ki67 scoring algorithm was sent to participating labs. Very high correlation and no systematic error (p = 0.08) was found between consecutive Ki67 IHC sections. Ki67 scores were higher for core biopsy slides compared to paired whole sections from resections (p ≤ 0.001; median difference: 5.31%). The systematic discrepancy between core biopsy and corresponding whole sections was likely due to pre-analytical factors (tissue handling, fixation). Therefore, Ki67 IHC should be tested on core biopsy samples to best reflect the biological status of the tumor.
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Neoplasias de la Mama , Biomarcadores de Tumor/análisis , Biopsia , Neoplasias de la Mama/patología , Femenino , Humanos , Procesamiento de Imagen Asistido por Computador/métodos , Inmunohistoquímica , Antígeno Ki-67/análisis , Receptores de EstrógenosRESUMEN
PURPOSE: Alterations to mismatch repair (MMR) pathways are a known cause of cancer, particularly colorectal and endometrial carcinomas. Recently, checkpoint inhibitors have been approved for use in MMR-deficient cancers of any type (Prasad et al. in JAMA Oncol 4:157-158, 2018). Functional studies in breast cancer have shown associations between MMR loss, resistance to aromatase inhibitors and sensitivity to palbociclib (Haricharan et al. in Cancer Discov 7:1168-1183, 2017). Herein, we investigate the clinical meaning of MMR deficiency in breast cancer by immunohistochemical assessment of MSH2, MSH6, MLH1 and PMS2 on a large series of breast cancers linked to detailed biomarker and long-term outcome data. METHODS: Cases were classified as MMR intact when all four markers expressed nuclear reactivity, but MMR-deficient when at least one of the four biomarkers displayed loss of nuclear staining in the presence of positive internal stromal controls on the tissue microarray core. RESULTS: Among the 1635 cases with interpretable staining, we identified 31 (1.9%) as MMR-deficient. In our cohort, MMR deficiency was present across all major breast cancer subtypes, and was associated with high-grade, low-progesterone receptor expression and high tumor-infiltrating lymphocyte counts. MMR deficiency is significantly associated with inferior overall (HR 2.29, 95% CI 1.02-5.17, p = 0.040) and disease-specific survival (HR 2.71, 95% CI 1.00-7.35, p = 0.042) in the 431 estrogen receptor-positive patients who were uniformly treated with tamoxifen as their sole adjuvant systemic therapy. CONCLUSION: Overall, this study supports the concept that breast cancer patients with MMR deficiency as assessed by immunohistochemistry may be good candidates for alternative treatment approaches such as immune checkpoint or CDK4 inhibitors.
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Neoplasias de la Mama/metabolismo , Proteínas de Unión al ADN/metabolismo , Endonucleasa PMS2 de Reparación del Emparejamiento Incorrecto/metabolismo , Homólogo 1 de la Proteína MutL/metabolismo , Proteína 2 Homóloga a MutS/metabolismo , Neoplasias de la Mama/patología , Colombia Británica , Núcleo Celular/metabolismo , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Clasificación del Tumor , Receptores de Progesterona/metabolismo , Análisis de Supervivencia , Análisis de Matrices TisularesRESUMEN
In the original publication of the article, the funding statement was published incompletely. The corrected funding statement should read as below.
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Ki67 expression has been a valuable prognostic variable in breast cancer, but has not seen broad adoption due to lack of standardization between institutions. Automation could represent a solution. Here we investigate the reproducibility of Ki67 measurement between three image analysis platforms with supervised classifiers performed by the same operator, by multiple operators, and finally we compare their accuracy in prognostic potential. Two breast cancer patient cohorts were used for this study. The standardization was done with the 30 cases of ER+ breast cancer that were used in phase 3 of International Ki67 in Breast Cancer Working Group initiatives where blocks were centrally cut and stained for Ki67. The outcome cohort was from 149 breast cancer cases from the Yale Pathology archives. A tissue microarray was built from representative tissue blocks with median follow-up of 120 months. The Mib-1 antibody (Dako) was used to detect Ki67 (dilution 1:100). HALO (IndicaLab), QuantCenter (3DHistech), and QuPath (open source software) digital image analysis (DIA) platforms were used to evaluate Ki67 expression. Intraclass correlation coefficient (ICC) was used to measure reproducibility. Between-DIA platform reproducibility was excellent (ICC: 0.933, CI: 0.879-0.966). Excellent reproducibility was found between all DIA platforms and the reference standard Ki67 values of Spectrum Webscope (QuPath-Spectrum Webscope ICC: 0.970, CI: 0.936-0.986; HALO-Spectrum Webscope ICC: 0.968, CI: 0.933-0.985; QuantCenter-Spectrum Webscope ICC: 0.964, CI: 0.919-0.983). All platforms showed excellent intra-DIA reproducibility (QuPath ICC: 0.992, CI: 0.986-0.996; HALO ICC: 0.972, CI: 0.924-0.988; QuantCenter ICC: 0.978, CI: 0.932-0.991). Comparing each DIA against outcome, the hazard ratios were similar. The inter-operator reproducibility was particularly high (ICC: 0.962-0.995). Our results showed outstanding reproducibility both within and between-DIA platforms, including one freely available DIA platform (QuPath). We also found the platforms essentially indistinguishable with respect to prediction of breast cancer patient outcome. Results justify multi-institutional DIA studies to assess clinical utility.
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Neoplasias de la Mama/metabolismo , Procesamiento de Imagen Asistido por Computador , Antígeno Ki-67/análisis , Adulto , Anciano , Anciano de 80 o más Años , Estudios de Cohortes , Humanos , Antígeno Ki-67/metabolismo , Persona de Mediana Edad , Variaciones Dependientes del Observador , Reproducibilidad de los ResultadosRESUMEN
The nuclear proliferation biomarker Ki67 has potential prognostic, predictive, and monitoring roles in breast cancer. Unacceptable between-laboratory variability has limited its clinical value. The International Ki67 in Breast Cancer Working Group investigated whether Ki67 immunohistochemistry can be analytically validated and standardized across laboratories using automated machine-based scoring. Sets of pre-stained core-cut biopsy sections of 30 breast tumors were circulated to 14 laboratories for scanning and automated assessment of the average and maximum percentage of tumor cells positive for Ki67. Seven unique scanners and 10 software platforms were involved in this study. Pre-specified analyses included evaluation of reproducibility between all laboratories (primary) as well as among those using scanners from a single vendor (secondary). The primary reproducibility metric was intraclass correlation coefficient between laboratories, with success considered to be intraclass correlation coefficient >0.80. Intraclass correlation coefficient for automated average scores across 16 operators was 0.83 (95% credible interval: 0.73-0.91) and intraclass correlation coefficient for maximum scores across 10 operators was 0.63 (95% credible interval: 0.44-0.80). For the laboratories using scanners from a single vendor (8 score sets), intraclass correlation coefficient for average automated scores was 0.89 (95% credible interval: 0.81-0.96), which was similar to the intraclass correlation coefficient of 0.87 (95% credible interval: 0.81-0.93) achieved using these same slides in a prior visual-reading reproducibility study. Automated machine assessment of average Ki67 has the potential to achieve between-laboratory reproducibility similar to that for a rigorously standardized pathologist-based visual assessment of Ki67. The observed intraclass correlation coefficient was worse for maximum compared to average scoring methods, suggesting that maximum score methods may be suboptimal for consistent measurement of proliferation. Automated average scoring methods show promise for assessment of Ki67 scoring, but requires further standardization and subsequent clinical validation.
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Biomarcadores de Tumor/análisis , Neoplasias de la Mama/patología , Procesamiento de Imagen Asistido por Computador/normas , Inmunohistoquímica/normas , Antígeno Ki-67/análisis , Femenino , Humanos , Inmunohistoquímica/métodos , Reproducibilidad de los ResultadosRESUMEN
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.
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Biomarcadores de Tumor/análisis , Neoplasias de la Mama , Inmunohistoquímica/normas , Antígeno Ki-67/análisis , Patología Clínica/normas , Femenino , Humanos , Variaciones Dependientes del Observador , Reproducibilidad de los ResultadosRESUMEN
AIMS: Vulvar squamous cell carcinoma (VSCC) can be subdivided by human papillomavirus (HPV) status into two clinicopathological entities. Studies on the prognostic significance of HPV in VSCC are discordant. METHODS AND RESULTS: We performed a retrospective analysis of overall survival (OS), disease-specific survival (DSS) and progression-free survival (PFS) in 217 patients with VSCC. Cases were extracted from an era of more aggressive en-bloc radical dissections (1985-95) and more localized radical surgery through separate vulvar and groin excisions (1996-2005). p16 immunohistochemistry was used as a surrogate for HPV status. HPV status could be determined in 197 tumours, 118 HPV-independent and 79 HPV-associated tumours. Patients with HPV-associated tumours were younger (mean 58.8 versus 71.6 years for HPV-independent tumours, P < 0.0001) and more likely to have prior abnormal cervical cytology (41.1 versus 5.6% for HPV-independent tumours, P < 0.0001). In univariable analysis, patients with HPV-associated tumours had superior PFS [hazard ratio (HR): 0.37, 95% confidence interval (CI): 0.18-0.70], DSS (HR: 0.19, 95% CI: 0.08-0.41) and OS (HR: 0.35, 95% CI: 0.21-0.59). This was driven by worse outcomes (PFS, DSS and OS) for patients with HPV-independent tumours compared with HPV-associated tumours who underwent surgery after 1995. After adjusting for age and stage in multivariable analysis, patients with HPV-associated tumours showed superior PFS (HR: 0.25, 95% CI: 0.07-0.77) and DSS (HR: 0.21, 95% CI: 0.04-0.78). CONCLUSIONS: VSCC can be stratified into two prognostically different diseases based on p16 immunostaining. HPV status was associated only with prognosis in the cohort that underwent surgery after 1995, suggesting that more conservative surgery may have led to worse outcomes for patients with HPV-independent tumours.
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Carcinoma de Células Escamosas/patología , Carcinoma de Células Escamosas/virología , Neoplasias de la Vulva/patología , Neoplasias de la Vulva/virología , Adulto , Anciano , Anciano de 80 o más Años , Carcinoma de Células Escamosas/mortalidad , Estudios de Cohortes , Supervivencia sin Enfermedad , Femenino , Humanos , Estimación de Kaplan-Meier , Persona de Mediana Edad , Infecciones por Papillomavirus/complicaciones , Pronóstico , Modelos de Riesgos Proporcionales , Estudios Retrospectivos , Neoplasias de la Vulva/mortalidadRESUMEN
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.
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Biomarcadores de Tumor/análisis , Neoplasias de la Mama/patología , Inmunohistoquímica/normas , Antígeno Ki-67/análisis , Análisis de Matrices Tisulares/normas , Femenino , HumanosRESUMEN
PURPOSE: Endometrioid ovarian carcinoma (ENOC) is the second most-common type of ovarian carcinoma, comprising 10%-20% of cases. Recently, the study of ENOC has benefitted from comparisons to endometrial carcinomas including defining ENOC with four prognostic molecular subtypes. Each subtype suggests differential mechanisms of progression, although tumor-initiating events remain elusive. There is evidence that the ovarian microenvironment may be critical to early lesion establishment and progression. However, while immune infiltrates have been well studied in high-grade serous ovarian carcinoma, studies in ENOC are limited. EXPERIMENTAL DESIGN: We report on 210 ENOC, with clinical follow-up and molecular subtype annotation. Using multiplex IHC and immunofluorescence, we examine the prevalence of T-cell lineage, B-cell lineage, macrophages, and populations with programmed cell death protein 1 or programmed death-ligand 1 across subtypes of ENOC. RESULTS: Immune cell infiltrates in tumor epithelium and stroma showed higher densities in ENOC subtypes with known high mutation burden (POLEmut and MMRd). While molecular subtypes were prognostically significant, immune infiltrates were not (overall survival P > 0.2). Analysis by molecular subtype revealed that immune cell density was prognostically significant in only the no specific molecular profile (NSMP) subtype, where immune infiltrates lacking B cells (TILB minus) had inferior outcome (disease-specific survival: HR, 4.0; 95% confidence interval, 1.1-14.7; P < 0.05). Similar to endometrial carcinomas, molecular subtype stratification was generally superior to immune response in predicting outcomes. CONCLUSIONS: Subtype stratification is critical for better understanding of ENOC, in particular the distribution and prognostic significance of immune cell infiltrates. The role of B cells in the immune response within NSMP tumors warrants further study.
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Carcinoma Endometrioide , Neoplasias Endometriales , Neoplasias Ováricas , Femenino , Humanos , Pronóstico , Biomarcadores de Tumor/genética , Carcinoma Endometrioide/patología , Neoplasias Ováricas/patología , Neoplasias Endometriales/genética , Neoplasias Endometriales/patología , Carcinoma Epitelial de Ovario , Microambiente TumoralRESUMEN
We analysed the reproducibility of Ki67 labelling index (LI) between two scorers using the International Ki67 Working Group (IKWG) global methods on an Android application (APP), correlated the APP and eyeball estimate (EBE) with digital image analysis (DIA) scores and determined the prognostic significance of Ki67LI. Global weighted (GW) and global unweighted (GUW) Ki67 app scores of hormone receptor-positive and HER2 (human epidermal growth factor receptor 2)-negative breast cancer patients were obtained. Reproducibility of Ki67LI between 2 scorers and correlation of APP and EBE scores with DIA scores were performed. The prognostic significance of APP scores and its correlation with other clinico-pathologic variables were evaluated. The intra-class correlation coefficient (ICC) between 2 scorers showed excellent reliability with both GW and GUW methods. ICC between DIA and APP scores was significantly greater than DIA versus EBE. The three categories of APP scores based on median value and cut points of 10%, 18% and 38% were significantly associated with poor DFS. On multivariate analysis, significant association between Ki67LI, tumour size, nodal involvement and DFS was noted. Our study shows that the visual Ki67 scoring app is effective in bringing consistency to KI67LI and APP scores showed significant correlation with DFS.
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Neoplasias de la Mama/diagnóstico , Procesamiento de Imagen Asistido por Computador/métodos , Antígeno Ki-67/metabolismo , Aplicaciones Móviles , Receptores de Estrógenos/metabolismo , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Ojo , Femenino , Humanos , Inmunohistoquímica , Antígeno Ki-67/genética , Pronóstico , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Receptores de Estrógenos/genéticaRESUMEN
Precise biomarkers are needed to guide better diagnostics and therapeutics for basal-like breast cancer, for which DNA-dependent protein kinase catalytic subunit (DNA-PKcs) has been recently reported by the Clinical Proteomic Tumor Analysis Consortium as the most specific biomarker. We evaluated DNA-PKcs expression in clinically-annotated breast cancer tissue microarrays and correlated results with immune biomarkers (training set: n = 300; validation set: n = 2401). Following a pre-specified study design per REMARK criteria, we found that high expression of DNA-PKcs was significantly associated with stromal and CD8 + tumor infiltrating lymphocytes. Within the basal-like subtype, tumors with low DNA-PKcs and high tumor-infiltrating lymphocytes displayed the most favourable survival. DNA-PKcs expression by immunohistochemistry identified estrogen receptor-positive cases with a basal-like gene expression subtype. Non-silent mutations in PRKDC were significantly associated with poor outcomes. Integrating DNA-PKcs expression with validated immune biomarkers could guide patient selection for DNA-PKcs targeting strategies, DNA-damaging agents, and their combination with an immune-checkpoint blockade.
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Colony-stimulating factor-1 receptor (CSF-1R) signaling promotes an immune suppressive microenvironment enriched in M2 macrophages. Given that CSF-1R inhibitors are under investigation in clinical trials, including in breast cancer, CSF-1R expression and association with immune biomarkers could identify patients who derive greater benefit from combination with immunotherapies. TIMER2.0 and bc-GenExMiner v4.7 were used to assess the correlation of CSF1R mRNA with immune infiltrates and prognosis. Following a prespecified training-validation approach, an optimized immunohistochemistry assay was applied to assess CSF-1R on carcinoma cells and macrophages on breast cancer tissue microarray series representing 2384 patients, coupled to comprehensive clinicopathological, biomarker, and outcome data. Significant positive correlations were observed between CSF1R mRNA and immune infiltrates. High carcinoma CSF-1R correlated with grade 3 tumors >2 cm, hormone receptor negativity, high Ki67, immune checkpoint biomarkers, and macrophages expressing CSF-1R and CD163. High carcinoma CSF-1R was significantly associated with poor survival in univariate and multivariate analyses. Adverse prognostic associations were retained in ER+ cases regardless of the presence of CD8+ T cells. CSF-1R+ macrophages were not prognostic. High carcinoma CSF-1R is associated with aggressive breast cancer biology and poor prognosis, particularly in ER+ cases, and identifies patients in whom biomarker-directed CSF-1R therapies may yield superior therapeutic responses.
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PURPOSE: Granulocyte colony-stimulating factor (G-CSF) and hypoxia modulate the tumour immune microenvironment. In model systems, hypoxia-induced carbonic anhydrase IX (CAIX) has been associated with G-CSF and immune responses, including M2 polarization of macrophages. We investigated whether these associations exist in human breast cancer specimens, their relation to breast cancer subtypes, and clinical outcome. METHODS: Using validated protocols and prespecified scoring methodology, G-CSF expression on carcinoma cells and CD163 expression on tumour-associated macrophages were assayed by immunohistochemistry and applied to a tissue microarray series of 2960 primary excision specimens linked to clinicopathologic, biomarker, and outcome data. RESULTS: G-CSFhigh expression showed a significant positive association with ER negativity, HER2 positivity, presence of CD163+ M2 macrophages, and CAIX expression. In univariate analysis, G-CSFhigh phenotype was associated with improved survival in non-luminal cases, although the CAIX+ subset had a significantly adverse prognosis. A significant positive association was observed between immune checkpoint biomarkers on tumour-infiltrating lymphocytes and both G-CSF- and CAIX-expressing carcinoma cells. Immune checkpoint biomarkers correlated significantly with favourable prognosis in G-CSFhigh/non-luminal cases independent of standard clinicopathological features. CONCLUSIONS: The prognostic associations linking G-CSF to immune biomarkers and CAIX strongly support their immunomodulatory roles in the tumour microenvironment.
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Ki67 immunohistochemistry (IHC), commonly used as a proliferation marker in breast cancer, has limited value for treatment decisions due to questionable analytical validity. The International Ki67 in Breast Cancer Working Group (IKWG) consensus meeting, held in October 2019, assessed the current evidence for Ki67 IHC analytical validity and clinical utility in breast cancer, including the series of scoring studies the IKWG conducted on centrally stained tissues. Consensus observations and recommendations are: 1) as for estrogen receptor and HER2 testing, preanalytical handling considerations are critical; 2) a standardized visual scoring method has been established and is recommended for adoption; 3) participation in and evaluation of quality assurance and quality control programs is recommended to maintain analytical validity; and 4) the IKWG accepted that Ki67 IHC as a prognostic marker in breast cancer has clinical validity but concluded that clinical utility is evident only for prognosis estimation in anatomically favorable estrogen receptor-positive and HER2-negative patients to identify those who do not need adjuvant chemotherapy. In this T1-2, N0-1 patient group, the IKWG consensus is that Ki67 5% or less, or 30% or more, can be used to estimate prognosis. In conclusion, analytical validity of Ki67 IHC can be reached with careful attention to preanalytical issues and calibrated standardized visual scoring. Currently, clinical utility of Ki67 IHC in breast cancer care remains limited to prognosis assessment in stage I or II breast cancer. Further development of automated scoring might help to overcome some current limitations.
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Neoplasias de la Mama , Biomarcadores de Tumor , Neoplasias de la Mama/tratamiento farmacológico , Femenino , Humanos , Inmunohistoquímica , Antígeno Ki-67 , Pronóstico , Receptor ErbB-2 , Receptores de EstrógenosRESUMEN
PURPOSE: The two most common molecular subtypes of endometrial cancers, mismatch repair deficient (MMRd) and p53 wild-type (p53wt) comprise the majority of endometrial cancers and have intermediate prognoses where additional risk stratification biomarkers are needed. Isoform switching of FGFR2 from FGFR2b to FGFR2c (normally expressed in mesenchymal cells), has been reported in other solid carcinomas. The objective of this study was to investigate the role of FGFR2c in risk stratification of endometrial cancer. EXPERIMENTAL DESIGN: We have developed and optimized a BaseScope RNA ISH assay to detect FGFR2c. FGFR2c expression was determined in a preliminary screening cohort of 78 endometrial cancers and a clinically and molecularly annotated Vancouver cohort (n = 465). Cox regression model analyses were performed to assess the prognostic value of FGFR2c. RESULTS: Univariate and multivariate analyses revealed FGFR2c expression was significantly associated with shorter disease-specific survival (DSS) and progression-free survival (PFS) in endometrioid endometrial cancer (EEC, n = 302). Notably, FGFR2c expression was significantly associated with shorter PFS and DSS in patients with grade 3 EECs (P < 0.003 and P < 0.002) and the European Society Medical Oncology (ESMO) high-risk group (P < 0.0001 and P < 0.002), respectively. Moreover, within the MMRd subtype, FGFR2c expression was significantly associated with shorter PFS (P < 0.048) and DSS (P < 0.001). CONCLUSIONS: FGFR2c expression appears an independent prognostic biomarker in patients with EEC and further discerns the outcomes within grade 3 tumors, ESMO high-risk groups, as well as within the MMRd and p53wt subtypes. FGFR2c inclusion into future molecular subtyping can further refine risk stratification of EEC.
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Biomarcadores de Tumor/metabolismo , Neoplasias Endometriales/mortalidad , Endometrio/patología , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/metabolismo , Anciano , Biomarcadores de Tumor/genética , Reparación de la Incompatibilidad de ADN , Neoplasias Endometriales/diagnóstico , Neoplasias Endometriales/genética , Neoplasias Endometriales/cirugía , Endometrio/cirugía , Femenino , Estudios de Seguimiento , Regulación Neoplásica de la Expresión Génica , Humanos , Histerectomía , Persona de Mediana Edad , Estadificación de Neoplasias , Pronóstico , Supervivencia sin Progresión , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/genética , Medición de RiesgoRESUMEN
Pathological analysis of the nuclear proliferation biomarker Ki67 has multiple potential roles in breast and other cancers. However, clinical utility of the immunohistochemical (IHC) assay for Ki67 immunohistochemistry has been hampered by unacceptable between-laboratory analytical variability. The International Ki67 Working Group has conducted a series of studies aiming to decrease this variability and improve the evaluation of Ki67. This study tries to assess whether acceptable performance can be achieved on prestained core-cut biopsies using a standardized scoring method. Sections from 30 primary ER+ breast cancer core biopsies were centrally stained for Ki67 and circulated among 22 laboratories in 11 countries. Each laboratory scored Ki67 using three methods: (1) global (4 fields of 100 cells each); (2) weighted global (same as global but weighted by estimated percentages of total area); and (3) hot-spot (single field of 500 cells). The intraclass correlation coefficient (ICC), a measure of interlaboratory agreement, for the unweighted global method (0.87; 95% credible interval (CI): 0.81-0.93) met the prespecified success criterion for scoring reproducibility, whereas that for the weighted global (0.87; 95% CI: 0.7999-0.93) and hot-spot methods (0.84; 95% CI: 0.77-0.92) marginally failed to do so. The unweighted global assessment of Ki67 IHC analysis on core biopsies met the prespecified criterion of success for scoring reproducibility. A few cases still showed large scoring discrepancies. Establishment of external quality assessment schemes is likely to improve the agreement between laboratories further. Additional evaluations are needed to assess staining variability and clinical validity in appropriate cohorts of samples.
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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.