Maggie Cheang

Immunohistochemical and Clinical Characterization of the Basal-Like Subtype of Invasive Breast Carcinoma

Purpose: Expression profiling studies classified breast carcinomas into estrogen receptor (ER)+/luminal, normal breast-like, HER2 overexpressing, and basal-like groups, with the latter two associated with poor outcomes. Currently, there exist clinical assays that identify ER+/luminal and HER2-overexpressing tumors, and we sought to develop a clinical assay for breast basal-like tumors. Experimental Design: To identify an immunohistochemical profile for breast basal-like tumors, we collected a series of known basal-like tumors and tested them for protein patterns that are characteristic of this subtype. Next, we examined the significance of these protein patterns using tissue microarrays and evaluated the prognostic significance of these findings. Results: Using a panel of 21 basal-like tumors, which was determined using gene expression profiles, we saw that this subtype was typically immunohistochemically negative for estrogen receptor and HER2 but positive for basal cytokeratins, HER1, and/or c-KIT. Using breast carcinoma tissue microarrays representing 930 patients with 17.4-year mean follow-up, basal cytokeratin expression was associated with low disease-specific survival. HER1 expression was observed in 54% of cases positive for basal cytokeratins (versus 11% of negative cases) and was associated with poor survival independent of nodal status and size. c-KIT expression was more common in basal-like tumors than in other breast cancers but did not influence prognosis. Conclusions: A panel of four antibodies (ER, HER1, HER2, and cytokeratin 5/6) can accurately identify basal-like tumors using standard available clinical tools and shows high specificity. These studies show that many basal-like tumors express HER1, which suggests candidate drugs for evaluation in these patients.

Ki67 Index, HER2 Status, and Prognosis of Patients With Luminal B Breast Cancer

Background Gene expression profiling of breast cancer has identified two biologically distinct estrogen receptor (ER)-positive subtypes of breast cancer: luminal A and luminal B. Luminal B tumors have higher proliferation and poorer prognosis than luminal A tumors. In this study, we developed a clinically practical immunohistochemistry assay to distinguish luminal B from luminal A tumors and investigated its ability to separate tumors according to breast cancer recurrence-free and disease-specific survival. Methods Tumors from a cohort of 357 patients with invasive breast carcinomas were subtyped by gene expression profile. Hormone receptor status, HER2 status, and the Ki67 index (percentage of Ki67-positive cancer nuclei) were determined immunohistochemically. Receiver operating characteristic curves were used to determine the Ki67 cut point to distinguish luminal B from luminal A tumors. The prognostic value of the immunohistochemical assignment for breast cancer recurrence-free and disease-specific survival was investigated with an independent tissue microarray series of 4046 breast cancers by use of Kaplan–Meier curves and multivariable Cox regression. Results Gene expression profiling classified 101 (28%) of the 357 tumors as luminal A and 69 (19%) as luminal B. The best Ki67 index cut point to distinguish luminal B from luminal A tumors was 13.25%. In an independent cohort of 4046 patients with breast cancer, 2847 had hormone receptor–positive tumors. When HER2 immunohistochemistry and the Ki67 index were used to subtype these 2847 tumors, we classified 1530 (59%, 95% confidence interval [CI] = 57% to 61%) as luminal A, 846 (33%, 95% CI = 31% to 34%) as luminal B, and 222 (9%, 95% CI = 7% to 10%) as luminal–HER2 positive. Luminal B and luminal–HER2-positive breast cancers were statistically significantly associated with poor breast cancer recurrence-free and disease-specific survival in all adjuvant systemic treatment categories. Of particular relevance are women who received tamoxifen as their sole adjuvant systemic therapy, among whom the 10-year breast cancer–specific survival was 79% (95% CI = 76% to 83%) for luminal A, 64% (95% CI = 59% to 70%) for luminal B, and 57% (95% CI = 47% to 69%) for luminal–HER2 subtypes. Conclusion Expression of ER, progesterone receptor, and HER2 proteins and the Ki67 index appear to distinguish luminal A from luminal B breast cancer subtypes.

Basal-Like Breast Cancer Defined by Five Biomarkers Has Superior Prognostic Value than Triple-Negative Phenotype

Purpose: Basal-like breast cancer is associated with high grade, poor prognosis, and younger patient age. Clinically, a triple-negative phenotype definition [estrogen receptor, progesterone receptor, and human epidermal growth factor receptor (HER)-2, all negative] is commonly used to identify such cases. EGFR and cytokeratin 5/6 are readily available positive markers of basal-like breast cancer applicable to standard pathology specimens. This study directly compares the prognostic significance between three- and five-biomarker surrogate panels to define intrinsic breast cancer subtypes, using a large clinically annotated series of breast tumors. Experimental Design: Four thousand forty-six invasive breast cancers were assembled into tissue microarrays. All had staging, pathology, treatment, and outcome information; median follow-up was 12.5 years. Cox regression analyses and likelihood ratio tests compared the prognostic significance for breast cancer death-specific survival (BCSS) of the two immunohistochemical panels. Results: Among 3,744 interpretable cases, 17% were basal using the triple-negative definition (10-year BCSS, 6 7%) and 9% were basal using the five-marker method (10-year BCSS, 62%). Likelihood ratio tests of multivariable Cox models including standard clinical variables show that the five-marker panel is significantly more prognostic than the three-marker panel. The poor prognosis of triple-negative phenotype is conferred almost entirely by those tumors positive for basal markers. Among triple-negative patients treated with adjuvant anthracycline-based chemotherapy, the additional positive basal markers identified a cohort of patients with significantly worse outcome. Conclusions: The expanded surrogate immunopanel of estrogen receptor, progesterone receptor, human HER-2, EGFR, and cytokeratin 5/6 provides a more specific definition of basal-like breast cancer that better predicts breast cancer survival.

Metastatic behavior of breast cancer subtypes.

PURPOSE Prognostic and predictive factors are well established in early-stage breast cancer, but less is known about which metastatic sites will be affected. METHODS Patients with early-stage breast cancer diagnosed between 1986 and 1992 with archival tissue were included. Subtypes were defined as luminal A, luminal B, luminal/human epidermal growth factor receptor 2 (HER2), HER2 enriched, basal-like, and triple negative (TN) nonbasal. Distant sites were classified as brain, liver, lung, bone, distant nodal, pleural/peritoneal, and other. Cumulative incidence curves were estimated for each site according to competing risks methods. Association between the site of relapse and subtype was assessed in multivariate models using logistic regression. RESULTS Median follow-up time among 3,726 eligible patients was 14.8 years. Median durations of survival with distant metastasis were 2.2 (luminal A), 1.6 (luminal B), 1.3 (luminal/HER2), 0.7 (HER2 enriched), and 0.5 years (basal-like; P < .001). Bone was the most common metastatic site in all subtypes except basal-like tumors. In multivariate analysis, compared with luminal A tumors, luminal/HER2 and HER2-enriched tumors were associated with a significantly higher rate of brain, liver, and lung metastases. Basal-like tumors had a higher rate of brain, lung, and distant nodal metastases but a significantly lower rate of liver and bone metastases. TN nonbasal tumors demonstrated a similar pattern but were not associated with fewer liver metastases. CONCLUSION Breast cancer subtypes are associated with distinct patterns of metastatic spread with notable differences in survival after relapse.

Breast Cancer Subtypes and the Risk of Local and Regional Relapse

PURPOSE The risk of local and regional relapse associated with each breast cancer molecular subtype was determined in a large cohort of patients with breast cancer. Subtype assignment was accomplished using a validated six-marker immunohistochemical panel applied to tissue microarrays. PATIENTS AND METHODS Semiquantitative analysis of estrogen receptor (ER), progesterone receptor (PR), Ki-67, human epidermal growth factor receptor 2 (HER2), epidermal growth factor receptor (EGFR), and cytokeratin (CK) 5/6 was performed on tissue microarrays constructed from 2,985 patients with early invasive breast cancer. Patients were classified into the following categories: luminal A, luminal B, luminal-HER2, HER2 enriched, basal-like, or triple-negative phenotype-nonbasal. Multivariable Cox analysis was used to determine the risk of local or regional relapse associated the intrinsic subtypes, adjusting for standard clinicopathologic factors. RESULTS The intrinsic molecular subtype was successfully determined in 2,985 tumors. The median follow-up time was 12 years, and there have been a total of 325 local recurrences and 227 regional lymph node recurrences. Luminal A tumors (ER or PR positive, HER2 negative, Ki-67 < 1%) had the best prognosis and the lowest rate of local or regional relapse. For patients undergoing breast conservation, HER2-enriched and basal subtypes demonstrated an increased risk of regional recurrence, and this was statistically significant on multivariable analysis. After mastectomy, luminal B, luminal-HER2, HER2-enriched, and basal subtypes were all associated with an increased risk of local and regional relapse on multivariable analysis. CONCLUSION Luminal A tumors are associated with a low risk of local or regional recurrence. Molecular subtyping of breast tumors using a six-marker immunohistochemical panel can identify patients at increased risk of local and regional recurrence.

Subtyping of breast cancer by immunohistochemistry to investigate a relationship between subtype and short and long term survival: a collaborative analysis of data for 10,159 cases from 12 studies

Paul Pharoah and colleagues evaluate the prognostic significance of immunohistochemical subtype classification in more than 10,000 breast cancer cases with early disease, and examine the influence of a patients survival time on the prediction of future survival.

A Comparison of PAM50 Intrinsic Subtyping with Immunohistochemistry and Clinical Prognostic Factors in Tamoxifen-Treated Estrogen Receptor-Positive Breast Cancer

Purpose: To compare clinical, immunohistochemical (IHC), and gene expression models of prognosis applicable to formalin-fixed, paraffin-embedded blocks in a large series of estrogen receptor (ER)–positive breast cancers from patients uniformly treated with adjuvant tamoxifen. Experimental Design: Quantitative real-time reverse transcription-PCR (qRT-PCR) assays for 50 genes identifying intrinsic breast cancer subtypes were completed on 786 specimens linked to clinical (median follow-up, 11.7 years) and IHC [ER, progesterone receptor (PR), HER2, and Ki67] data. Performance of predefined intrinsic subtype and risk-of-relapse scores was assessed using multivariable Cox models and Kaplan-Meier analysis. Harrells C-index was used to compare fixed models trained in independent data sets, including proliferation signatures. Results: Despite clinical ER positivity, 10% of cases were assigned to nonluminal subtypes. qRT-PCR signatures for proliferation genes gave more prognostic information than clinical assays for hormone receptors or Ki67. In Cox models incorporating standard prognostic variables, hazard ratios for breast cancer disease-specific survival over the first 5 years of follow-up, relative to the most common luminal A subtype, are 1.99 [95% confidence interval (CI), 1.09-3.64] for luminal B, 3.65 (95% CI, 1.64-8.16) for HER2-enriched subtype, and 17.71 (95% CI, 1.71-183.33) for the basal-like subtype. For node-negative disease, PAM50 qRT-PCR–based risk assignment weighted for tumor size and proliferation identifies a group with >95% 10-year survival without chemotherapy. In node-positive disease, PAM50-based prognostic models were also superior. Conclusion: The PAM50 gene expression test for intrinsic biological subtype can be applied to large series of formalin-fixed, paraffin-embedded breast cancers, and gives more prognostic information than clinical factors and IHC using standard cut points. Clin Cancer Res; 16(21); 5222–32. ©2010 AACR.

Breast Cancer Subtypes and Response to Docetaxel in Node-Positive Breast Cancer: Use of an Immunohistochemical Definition in the BCIRG 001 Trial

PURPOSE To investigate the prognostic and predictive significance of subtyping node-positive early breast cancer by immunohistochemistry in a clinical trial of a docetaxel-containing regimen. METHODS Pathologic data from a central laboratory were available for 1,350 patients (91%) from the BCIRG 001 trial of docetaxel, doxorubicin, and cyclophosphamide (TAC) versus fluorouracil, doxorubicin, and cyclophosphamide (FAC) for operable node-positive breast cancer. Patients were classified by tumor characteristics as (1) triple negative (estrogen receptor [ER]-negative, progesterone receptor [PR]-negative, HER2/neu [HER2]-negative), (2) HER2 (HER2-positive, ER-negative, PR-negative), (3) luminal B (ER-positive and/or PR-positive and either HER2-positive and/or Ki67(high)), and (4) luminal A (ER-positive and/or PR-positive and not HER2-positive or Ki67(high)), and assessed for prognostic significance and response to adjuvant chemotherapy. RESULTS Patients were subdivided into triple negative (14.5%), HER2 (8.5%), luminal B (61.1%), and luminal A (15.9%). Three-year disease-free survival (DFS) rates (P values with luminal B as referent) were 67% (P < .0001), 68% (P = .0008), 82% (referent luminal B), and 91% (P = .0027), respectively, with hazard ratios of 2.22, 2.12, and 0.46. Improved 3-year DFS with TAC was found in the luminal B group (P = .025) and a combined ER-positive/HER2-negative group treated with tamoxifen (P = .041), with a marginal trend in the triple negatives (P = .051) and HER2 (P = .068) subtypes. No DFS advantage was seen in the luminal A population. CONCLUSION A simple immunopanel can divide breast cancers into biologic subtypes with strong prognostic effects. TAC significantly complements endocrine therapy in patients with luminal B subtype and, in the absence of targeted therapy, is effective in the triple-negative population.

Mutation tracking in circulating tumor DNA predicts relapse in early breast cancer.

Noninvasive mutation tracking in plasma can detect circulating tumor DNA arising from residual micrometastatic disease and thus identify patients at high risk of recurrence. Risk of recurrence Predicting whether a cancer patient will relapse remains a formidable challenge in modern medicine. Fortunately, circulating tumor DNA (ctDNA) present in the blood may give clues on residual disease—cancer cells left behind to seed new tumors even after treatment. Garcia-Murillas et al. developed a personalized ctDNA assay based on digital polymerase chain reaction to track mutations over time in patients with early-stage breast cancer who had received apparently curative treatments, surgery, and chemotherapy. Mutation tracking in serial samples accurately predicted metastatic relapse—in several instances, months before clinical relapse (median of ~8 months). Such unprecedented early prediction could allow for intervention before the reappearance of cancer in high-risk patients. In addition, the authors were able to shed light on the genetic events driving such metastases, by massively parallel sequencing of the ctDNA, which could inform new drug-based therapies on the basis of the patients’ individual mutations. The identification of early-stage breast cancer patients at high risk of relapse would allow tailoring of adjuvant therapy approaches. We assessed whether analysis of circulating tumor DNA (ctDNA) in plasma can be used to monitor for minimal residual disease (MRD) in breast cancer. In a prospective cohort of 55 early breast cancer patients receiving neoadjuvant chemotherapy, detection of ctDNA in plasma after completion of apparently curative treatment—either at a single postsurgical time point or with serial follow-up plasma samples—predicted metastatic relapse with high accuracy [hazard ratio, 25.1 (confidence interval, 4.08 to 130.5; log-rank P < 0.0001) or 12.0 (confidence interval, 3.36 to 43.07; log-rank P < 0.0001), respectively]. Mutation tracking in serial samples increased sensitivity for the prediction of relapse, with a median lead time of 7.9 months over clinical relapse. We further demonstrated that targeted capture sequencing analysis of ctDNA could define the genetic events of MRD, and that MRD sequencing predicted the genetic events of the subsequent metastatic relapse more accurately than sequencing of the primary cancer. Mutation tracking can therefore identify early breast cancer patients at high risk of relapse. Subsequent adjuvant therapeutic interventions could be tailored to the genetic events present in the MRD, a therapeutic approach that could in part combat the challenge posed by intratumor genetic heterogeneity.

Prognostic Significance of Progesterone Receptor–Positive Tumor Cells Within Immunohistochemically Defined Luminal A Breast Cancer

PURPOSE Current immunohistochemical (IHC)-based definitions of luminal A and B breast cancers are imperfect when compared with multigene expression-based assays. In this study, we sought to improve the IHC subtyping by examining the pathologic and gene expression characteristics of genomically defined luminal A and B subtypes. PATIENTS AND METHODS Gene expression and pathologic features were collected from primary tumors across five independent cohorts: British Columbia Cancer Agency (BCCA) tamoxifen-treated only, Grupo Español de Investigación en Cáncer de Mama 9906 trial, BCCA no systemic treatment cohort, PAM50 microarray training data set, and a combined publicly available microarray data set. Optimal cutoffs of percentage of progesterone receptor (PR) -positive tumor cells to predict survival were derived and independently tested. Multivariable Cox models were used to test the prognostic significance. RESULTS Clinicopathologic comparisons among luminal A and B subtypes consistently identified higher rates of PR positivity, human epidermal growth factor receptor 2 (HER2) negativity, and histologic grade 1 in luminal A tumors. Quantitative PR gene and protein expression were also found to be significantly higher in luminal A tumors. An empiric cutoff of more than 20% of PR-positive tumor cells was statistically chosen and proved significant for predicting survival differences within IHC-defined luminal A tumors independently of endocrine therapy administration. Finally, no additional prognostic value within hormonal receptor (HR) -positive/HER2-negative disease was observed with the use of the IHC4 score when intrinsic IHC-based subtypes were used that included the more than 20% PR-positive tumor cells and vice versa. CONCLUSION Semiquantitative IHC expression of PR adds prognostic value within the current IHC-based luminal A definition by improving the identification of good outcome breast cancers. The new proposed IHC-based definition of luminal A tumors is HR positive/HER2 negative/Ki-67 less than 14%, and PR more than 20%.