Advancing the PD-L1 CPS test in metastatic TNBC: Insights from pathologists and findings from a nationwide survey.

Pembrolizumab has received approval as a first-line treatment for unresectable/metastatic triple-negative breast cancer (mTNBC) with a PD-L1 combined positive score (CPS) of ≥ 10. However, assessing CPS in mTNBC poses challenges. Firstly, it represents a novel analysis for breast pathologists. Secondly, the heterogeneity of PD-L1 expression in mTNBC further complicates the assessment. Lastly, the lack of standardized assays and staining platforms adds to the complexity. In KEYNOTE trials, PD-L1 expression was evaluated using the IHC 22C3 pharmDx kit as a companion diagnostic test. However, both the 22C3 pharmDx and VENTANA PD-L1 (SP263) assays are validated for CPS assessment. Consequently, assay-platform choice, staining conditions, and scoring methods can significantly impact the testing outcomes. This consensus paper aims to discuss the intricacies of PD-L1 CPS testing in mTNBC and provide practical recommendations for pathologists. Additionally, we present findings from a nationwide Italian survey elucidating the state-of-the-art in PD-L1 CPS testing in mTNBC.

Systematically higher Ki67 scores on core biopsy samples compared to corresponding resection specimen in breast cancer: a multi-operator and multi-institutional study.

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.

ß-Galactosylceramidase Promotes Melanoma Growth via Modulation of Ceramide Metabolism.

Disturbance of sphingolipid metabolism may represent a novel therapeutic target in metastatic melanoma, the most lethal form of skin cancer. ß-Galactosylceramidase (GALC) removes ß-galactose from galactosylceramide and other sphingolipids. In this study, we show that downregulation of galcb, a zebrafish ortholog of human GALC, affects melanoblast and melanocyte differentiation in zebrafish embryos, suggesting a possible role for GALC in melanoma. On this basis, the impact of GALC expression in murine B16-F10 and human A2058 melanoma cells was investigated following its silencing or upregulation. Galc knockdown hampered growth, motility, and invasive capacity of B16-F10 cells and their tumorigenic and metastatic activity when grafted in syngeneic mice or zebrafish embryos. Galc-silenced cells displayed altered sphingolipid metabolism and increased intracellular levels of ceramide, paralleled by a nonredundant upregulation of Smpd3, which encodes for the ceramide-generating enzyme neutral sphingomyelinase 2. Accordingly, GALC downregulation caused SMPD3 upregulation, increased ceramide levels, and inhibited the tumorigenic activity of human melanoma A2058 cells, whereas GALC upregulation exerted opposite effects. In concordance with information from melanoma database mining, RNAscope analysis demonstrated a progressive increase of GALC expression from common nevi to stage IV human melanoma samples that was paralleled by increases in microphthalmia transcription factor and tyrosinase immunoreactivity inversely related to SMPD3 and ceramide levels. Overall, these findings indicate that GALC may play an oncogenic role in melanoma by modulating the levels of intracellular ceramide, thus providing novel opportunities for melanoma therapy. SIGNIFICANCE: Data from zebrafish embryos, murine and human cell melanoma lines, and patient-derived tumor specimens indicate that ß-galactosylceramidase plays an oncogenic role in melanoma and may serve as a therapeutic target.

Correlation of size and focality with prognosis in small breast carcinoma: a single institution case series.

AIM OF THE STUDY: The clinical behavior and prognosis of small multifocal and microinvasive breast cancers are still debated together with the best method of assessing tumor size in multiple invasive carcinomas. This study evaluates the clinico-pathological features of single and multiple breast cancers up to 0.5 cm in order to evaluate the rate of recurrences. MATERIALS AND METHODS: We retrospectively analyzed 170 node-negative patients consecutively treated at European Institute of Oncology from 2001 to 2006. We divided them into Group I (pT1mi) and Group II (pT1a) furtherly divided in subgroups, according to focality and aggregate diameter. For each group we assessed tumor size, (multi)focality, extensive in situ component (EIC), histology, grade, peritumoral vascular invasion (PVI), hormonal receptor status (HR), HER-2 expression, Ki67 expression. RESULTS: We observed that the frequency of local recurrences and distant metastases in group I was higher among those with a single focus; whereas in group II, it was higher in multifocal carcinomas. Then, by comparing the two groups, the prognosis was better in multiple pT1mi than in similarly sized unifocal pT1a. CONCLUSIONS: Microinvasive carcinomas are associated with a good prognosis, even if they seem to have a more aggressive intrinsic biological behavior. Multifocality seems to be correlated with a worse prognosis in case of invasive carcinomas pT1a. In case of microinvasive carcinomas, by contrast, multifocality per se does not seem to affect the recurrence rate.

Clinical and analytical validation of Ki-67 in 9069 patients from IBCSG VIII + IX, BIG1-98 and GeparTrio trial: systematic modulation of interobserver variance in a comprehensive in silico ring trial.

PURPOSE: Ki-67 has been clinically validated for risk assessment in breast cancer, but the analytical validation and cutpoint-definition remain a challenge. Intraclass correlation coefficients (ICCs) are a statistical parameter for Ki-67 interobserver performance. However, the maximum degree of variance among pathologists allowed for meaningful biomarker results has not been defined. METHODS: Different amounts of variance were added to central pathology Ki-67 data (n = 9069) from three cohorts (IBCSGVIII + IX, BIG1-98, GeparTrio) by simulation of 4500 evaluations for each cohort, which were grouped by ICCs, ranging from excellent (ICC = 0.9) to poor concordance (ICC = 0.1). Endpoints were disease-free survival (DFS) and pathological complete response (pCR, GeparTrio). RESULTS: Ki-67 was a significant continuous prognostic marker for DFS over a wide range of cutpoints between 8% and 30% in all three cohorts. In our modelling approach, Ki-67 was a stable prognostic marker despite increased interpathologist variance. Even for a poor ICC of 0.5, one or more significant Ki-67 cutoffs were detected in 86.8% (GeparTrio), 92.4% (IBCSGVIII + IX) and 100% of analyses (BIG1-98). Similarly, in GeparTrio, even with an extremely low ICC of 0.2, 99.6% of analyses were significant for pCR. CONCLUSIONS: Our study shows that Ki-67 is a continuous marker which is extremely robust to pathologist variation. Even if only 50% of variance is attributable to true Ki-67-based proliferation (ICC = 0.5), this information is sufficient to obtain statistically significant differences in clinical cohorts. This stable performance explains the observation that many Ki-67 studies achieve significant results despite relevant interobserver variance and points to a high clinical validity of this biomarker. For clinical decisions based on analysis of individual patient data, ongoing efforts to further reduce interobserver variability, including ring trials and standardized guidelines as well as image analysis approaches, should be continued.

Analytical validation of a standardised scoring protocol for Ki67 immunohistochemistry on breast cancer excision whole sections: an international multicentre collaboration.

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.

Vascular density and inflammatory infiltrate in primary oral squamous cell carcinoma and after allogeneic hematopoietic stem cell transplantation.

Hematopoietic stem cell transplantation (HSCT) recipients have been reported to have an increased risk of chronic graft versus host disease (cGVHD) and hematological and solid cancers. Oral manifestations are the first signs of cGVHD observed in the majority of patients, and oropharyngeal cancer is the most frequent secondary malignancy occurred after HSCT. In this study, we have evaluated the inflammatory infiltrate cell content and correlated with the vascular density in patients affected by primary oral squamous cell carcinoma (OSCC) from previous healthy controls and OSCC after cGVHD. Results have demonstrated that patients with OSCC after GVHD show a more consistent inflammatory infiltrate as compared with the OSCC ones. In detail, the inflammatory background composed of CD3-positive T cells, tryptase-positive mast cells, CD31-positive endothelial cells, and CD68-positive macrophages may be more pronounced in the setting of GVHD + OSCC than in the control group. By contrast, CD20-positive B cells and CD1a-positive dendritic cells were more abundant in the latter population. Finally, a positive correlation was found as between vascular density and inflammatory cell infiltration in both GVHD + OSCC and OSCC groups. Overall, these results confirm the role played by immune cells in enhancing tumor progression and angiogenesis and suggest a potential therapeutic strategy involving inhibition of recruitment of immune cells to the tumor microenvironment and blockade of pro-tumoral effects and pro-angiogenic functions.

Analytical validation of a standardized scoring protocol for Ki67: phase 3 of an international multicenter collaboration.

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.

Outcome and Medial Presentation of Breast Cancer: European Institute of Oncology Experience.

BACKGROUND: No analyses have investigated the prognostic role of medial presentation in breast cancer patients on disease-free survival (DFS) and overall survival according to immunohistochemically-defined subtypes. PATIENTS AND METHODS: We collected information from the institutional clinical database on consecutive breast cancer patients who underwent conservative surgery at the European Institute of Oncology, Milan, Italy, between 1994 and 2008. We compared the outcomes of patients with medial breast cancer with those of patients with nonmedial tumors observed at the institution during the same period. RESULTS: Among 7369 evaluable patients, 2254 (24%) had their primary tumors in medial quadrants and 7015 (76%) in other areas. Five-year DFS was 84.7% and 86.6% (P = .008) in patients with medial and nonmedial disease, respectively. In multivariate analysis, medial location was correlated with greater risk of recurrence (hazard ratio [HR], 1.23; 95% confidence interval [CI], 1.11-1.35; P < .0001) and death (HR, 1.27; 95% CI, 1.09-1.49; P = .0028). CONCLUSION: Medial presentation is an adverse prognostic factor for breast cancer patients.

Final 10-year results of the Breast International Group 2-98 phase III trial and the role of Ki67 in predicting benefit of adjuvant docetaxel in patients with oestrogen receptor positive breast cancer.

AIM: Breast International Group (BIG) 2-98 is a randomised phase III trial that tested the effect of adding docetaxel, either in sequence to or in combination with anthracycline-based adjuvant chemotherapy, in women with node-positive breast cancer (BC). Here, we present the 10-year final trial safety and efficacy analyses. We also report an exploratory analysis on the predictive value of Ki67 for docetaxel efficacy, in the BIG 2-98 and using a pooled analysis of three other randomised trials. PATIENTS AND METHODS: 2887 patients were randomly assigned in a 2×2 trial design to one of four treatments. The primary objective was to evaluate the overall efficacy of docetaxel on disease free survival (DFS). Secondary objectives included comparisons of sequential docetaxel versus sequential control arm, safety and overall survival (OS). Ki67 expression was centrally evaluated by immunohistochemistry. RESULTS: After a median follow-up of 10.1years, the addition of docetaxel did not significantly improve DFS or OS (hazard ratio (HR)=0.91, 95% confidence interval (CI)=0.81-1.04; P=0.16 and HR=0.88, 95% CI=0.76-1.03; P=0.11, respectively). Sequential docetaxel did not improve DFS compared to the sequential control arm (HR=0.86, 95% CI=0.72-1.03; P=0.10). In oestrogen receptor (ER)-positive tumours with Ki67⩾14%, the addition of docetaxel resulted in 5.4% improvement in 10-year OS (P=0.03, test for interaction=0.1). In a multivariate model, there was a trend for improved DFS and OS in ER-positive patients with high Ki67 and treated with docetaxel (HR=0.79, 95% CI=0.63-1.01; P=0.05 and HR=0.76, 95% CI=0.57-1.01; P=0.06, respectively). A pooled analysis of four randomised trials showed a benefit of taxanes in highly proliferative ER-positive disease but not in low proliferating tumours (interaction test P=0.01). CONCLUSION: The DFS benefit previously demonstrated with sequential docetaxel is no longer observed at 10years. However, an exploratory analysis suggested a benefit of docetaxel in patients with highly proliferative ER-positive BC.

An international study to increase concordance in Ki67 scoring.

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.

Proposed new clinicopathological surrogate definitions of luminal A and luminal B (HER2-negative) intrinsic breast cancer subtypes.

INTRODUCTION: The St Gallen International Expert Consensus on the Primary Therapy of Early Breast Cancer 2013 recognized substantial progress in the pathological characterization of breast cancer subtypes. A useful surrogate definition was developed to distinguish luminal A-like breast cancer from luminal B-like disease based on a combination of estrogen receptor (ER), progesterone receptor (PgR) and Ki-67 status, without a requirement for molecular diagnostics. Differences depend upon the choice of the threshold value for Ki-67 and the requirement for substantial PgR positivity. We aimed to verify the suitability of the new surrogate definitions of luminal subtypes in terms of distant disease control in a large series of patients. METHODS: We studied 9,415 women with a median follow-up of 8.1 years who (1) had ER-positive, human epidermal growth factor receptor 2 (HER2)-negative early breast cancer and (2) had undergone surgery at the European Institute of Oncology between 1994 and 2006. We evaluated distant disease-free survival of patients with "low" (<14%), "intermediate" (14% to 19%) or "high" (≥20%) Ki-67 positivity stratified by PgR expression (negative or low versus high). We calculated the cumulative incidence of distant events, considered competing events and performed multivariable analysis adjusted for pathologic tumor stage, pathologic node stage, tumor grade, peritumoral vascular invasion and menopausal status. RESULTS: Lack of substantial PgR positivity was associated with poorer outcomes only for patients with an intermediate Ki-67 level (P<0.001). The 4,890 patients (51.9%) with low Ki-67 level (any PgR expression level) or with intermediate Ki-67 level but substantial PgR positivity had comparably good outcomes and thus may represent a most advantageous grouping of those with luminal A-like disease. CONCLUSIONS: The updated pathological definition of intrinsic molecular subtypes may maximize the number of patients classified as having the luminal A-like intrinsic subtype of breast cancer and for whom the use of cytotoxic drugs could mostly be avoided.

An international Ki67 reproducibility study.

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.

Axillary dissection versus no axillary dissection in patients with sentinel-node micrometastases (IBCSG 23-01): a phase 3 randomised controlled trial.

BACKGROUND: For patients with breast cancer and metastases in the sentinel nodes, axillary dissection has been standard treatment. However, for patients with limited sentinel-node involvement, axillary dissection might be overtreatment. We designed IBCSG trial 23-01 to determine whether no axillary dissection was non-inferior to axillary dissection in patients with one or more micrometastatic (≤2 mm) sentinel nodes and tumour of maximum 5 cm. METHODS: In this multicentre, randomised, non-inferiority, phase 3 trial, patients were eligible if they had clinically non-palpable axillary lymph node(s) and a primary tumour of 5 cm or less and who, after sentinel-node biopsy, had one or more micrometastatic (≤2 mm) sentinel lymph nodes with no extracapsular extension. Patients were randomly assigned (in a 1:1 ratio) to either undergo axillary dissection or not to undergo axillary dissection. Randomisation was stratified by centre and menopausal status. Treatment assignment was not masked. The primary endpoint was disease-free survival. Non-inferiority was defined as a hazard ratio (HR) of less than 1·25 for no axillary dissection versus axillary dissection. The analysis was by intention to treat. Per protocol, disease and survival information continues to be collected yearly. This trial is registered with ClinicalTrials.gov, NCT00072293. FINDINGS: Between April 1, 2001, and Feb 28, 2010, 465 patients were randomly assigned to axillary dissection and 469 to no axillary dissection. After the exclusion of three patients, 464 patients were in the axillary dissection group and 467 patients were in the no axillary dissection group. After a median follow-up of 5·0 (IQR 3·6-7·3) years, we recorded 69 disease-free survival events in the axillary dissection group and 55 events in the no axillary dissection group. Breast-cancer-related events were recorded in 48 patients in the axillary dissection group and 47 in the no axillary dissection group (ten local recurrences in the axillary dissection group and eight in the no axillary dissection group; three and nine contralateral breast cancers; one and five [corrected] regional recurrences; and 34 and 25 distant relapses). Other non-breast cancer events were recorded in 21 patients in the axillary dissection group and eight in the no axillary dissection group (20 and six second non-breast malignancies; and one and two deaths not due to a cancer event). 5-year disease-free survival was 87·8% (95% CI 84·4-91·2) in the group without axillary dissection and 84·4% (80·7-88·1) in the group with axillary dissection (log-rank p=0·16; HR for no axillary dissection vs axillary dissection was 0·78, 95% CI 0·55-1·11, non-inferiority p=0·0042). Patients with reported long-term surgical events (grade 3-4) included one sensory neuropathy (grade 3), three lymphoedema (two grade 3 and one grade 4), and three motor neuropathy (grade 3), all in the group that underwent axillary dissection, and one grade 3 motor neuropathy in the group without axillary dissection. One serious adverse event was reported, a postoperative infection in the axilla in the group with axillary dissection. INTERPRETATION: Axillary dissection could be avoided in patients with early breast cancer and limited sentinel-node involvement, thus eliminating complications of axillary surgery with no adverse effect on survival. FUNDING: None.

Heterogeneity of triple-negative breast cancer: histologic subtyping to inform the outcome.

BACKGROUND: This study assesses outcome in terms of disease-free survival (DFS) and overall survival (OS) of special types of triple-negative breast cancer (TNBC). PATIENTS AND METHODS: We identified 8801 women with first primary nonmetastatic breast cancer operated on at the European Institute of Oncology between 1997 and 2005. Of these patients, 781 consecutive patients with immunohistochemically defined TNBC were selected for the analyses. We explored patterns of recurrence by histologic type. Median follow-up was 5.7 years (range 0-13 years). RESULTS: The 5-year DFS was 77% for TNBC, 68% for human epidermal growth factor receptor 2 (HER2)-positive breast cancer, and 84% and 95% for luminal B and luminal A breast cancer, respectively. From 781 TNBC subtypes, 693 cases (89%) were classified as ductal not otherwise specified (NOS) (invasive ductal carcinoma [IDC]), 29 were classified as apocrine (3.7%), 18 (2.3%) were classified as lobular, 10 (1.2%) were classified as adenoid cystic, and 10 (1.2%) were classified as metaplastic. Five-year DFS and OS were 77% and 84% for patients with ductal carcinoma, 56% and 89% for patients with metaplastic carcinoma, and both 5-year DFS and OS were 100% for patients with adenoid cystic and medullary carcinomas, respectively. CONCLUSION: Distinct prognostic implications may derive from the specific histotype of TNBC. The identification of these special types has a significant clinical utility and should be considered in therapeutic algorithms.

A risk score based on histopathological features predicts higher risk of distant recurrence in premenopausal patients with lymph node-negative endocrine-responsive breast cancer.

PURPOSE: To develop a Risk Score (RS) to predict distant recurrence among premenopausal women with node-negative endocrine-responsive early breast cancer. METHODS: The Cox model was used to develop the RS using clinical and histopathological features from 378 women participating in the IBCSG Trial VIII who received endocrine therapy alone or following chemotherapy. The performance of the resulting model was validated on a cohort of 1005 patients from a single institution who received endocrine therapy alone. RESULTS: In a multivariable analysis, the risk of distant recurrence was associated with tumor size, ER, Ki-67 and peritumoral vascular invasion. In the validation cohort, patients with high RS were at greater risk of distant recurrence compared to patients with low RS (HR, 17.41; 95% CI, 5.72-52.95). CONCLUSION: In premenopausal women with node-negative endocrine-responsive early breast cancer, the RS identifies patients at higher risk of distant recurrence.

How reliable is Ki-67 immunohistochemistry in grade 2 breast carcinomas? A QA study of the Swiss Working Group of Breast- and Gynecopathologists.

UNLABELLED: 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 self-selected 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 re-examined 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.

Traditional molecular markers and response to adjuvant endocrine or trastuzumab-based therapies.

PURPOSE OF REVIEW: The accurate assessment of traditional molecular markers is essential to inform the choice of the adjuvant systemic treatments for patients with breast cancer. Extensive research efforts have been made to explore whether it is also possible to predict the actual response to the different therapeutic options based on the expression of these markers. RECENT FINDINGS: Endocrine responsiveness of breast cancer has been eventually defined according to the expression of estrogen receptors in at least 1% of invasive tumor cells. The quantitative evaluation of estrogen receptors, progesterone receptors (PgR) and Ki-67 labeling index may help in selecting patients with estrogen receptor-positive and HER2-negative tumors who can be spared or may benefit from the addition of chemotherapy to endocrine therapy. Guideline recommendations for an optimal testing of estrogen receptors and PgR have been issued to assist pathologists in the accurate assessment of these markers. Progress has also been made in the identification of candidate patients to HER2-targeted therapies and in the prediction of response to trastuzumab. SUMMARY: Traditional molecular markers play a major role in the selection of candidate patients to systemic interventions, but they are of limited value in predicting their actual response to the different treatments, especially when the markers are evaluated individually.

Classical cyclophosphamide, methotrexate, and fluorouracil chemotherapy is more effective in triple-negative, node-negative breast cancer: results from two randomized trials of adjuvant chemoendocrine therapy for node-negative breast cancer.

PURPOSE: Retrospective studies suggest that primary breast cancers lacking estrogen receptor (ER) and progesterone receptor (PR) and not overexpressing human epidermal growth factor receptor 2 (HER2; triple-negative tumors) are particularly sensitive to DNA-damaging chemotherapy with alkylating agents. PATIENTS AND METHODS: Patients enrolled in International Breast Cancer Study Group Trials VIII and IX with node-negative, operable breast cancer and centrally assessed ER, PR, and HER2 were included (n = 2,257). The trials compared three or six courses of adjuvant classical cyclophosphamide, methotrexate, and fluorouracil (CMF) with or without endocrine therapy versus endocrine therapy alone. We explored patterns of recurrence by treatment according to three immunohistochemically defined tumor subtypes: triple negative, HER2 positive and endocrine receptor absent, and endocrine receptor present. RESULTS: Patients with triple-negative tumors (303 patients; 13%) were significantly more likely to have tumors > 2 cm and grade 3 compared with those in the HER2-positive, endocrine receptor-absent, and endocrine receptor-present subtypes. No clear chemotherapy benefit was observed in endocrine receptor-present disease (hazard ratio [HR], 0.90; 95% CI, 0.74 to 1.11). A statistically significantly greater benefit for chemotherapy versus no chemotherapy was observed in triple-negative breast cancer (HR, 0.46; 95% CI, 0.29 to 0.73; interaction P = .009 v endocrine receptor-present disease). The magnitude of the chemotherapy effect was lower in HER2-positive endocrine receptor-absent disease (HR, 0.58; 95% CI, 0.29 to 1.17; interaction P = .24 v endocrine receptor-present disease). CONCLUSION: The magnitude of benefit of CMF chemotherapy is largest in patients with triple-negative, node-negative breast cancer.