Lynn G. Dressler

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.

Molecular classification of head and neck squamous cell carcinomas using patterns of gene expression.

The prognostication of head and neck squamous cell carcinoma (HNSCC) is largely based upon the tumor size and location and the presence of lymph node metastases. Here we show that gene expression patterns from 60 HNSCC samples assayed on cDNA microarrays allowed categorization of these tumors into four distinct subtypes. These subtypes showed statistically significant differences in recurrence-free survival and included a subtype with a possible EGFR-pathway signature, a mesenchymal-enriched subtype, a normal epithelium-like subtype, and a subtype with high levels of antioxidant enzymes. Supervised analyses to predict lymph node metastasis status were approximately 80% accurate when tumor subsite and pathological node status were considered simultaneously. This work represents an important step toward the identification of clinically significant biomarkers for HNSCC.

Adjuvant chemotherapy in older women with early-stage breast cancer.

BACKGROUND Older women with breast cancer are underrepresented in clinical trials, and data on the effects of adjuvant chemotherapy in such patients are scant. We tested for the noninferiority of capecitabine as compared with standard chemotherapy in women with breast cancer who were 65 years of age or older. METHODS We randomly assigned patients with stage I, II, IIIA, or IIIB breast cancer to standard chemotherapy (either cyclophosphamide, methotrexate, and fluorouracil or cyclophosphamide plus doxorubicin) or capecitabine. Endocrine therapy was recommended after chemotherapy in patients with hormone-receptor-positive tumors. A Bayesian statistical design was used with a range in sample size from 600 to 1800 patients. The primary end point was relapse-free survival. RESULTS When the 600th patient was enrolled, the probability that, with longer follow-up, capecitabine therapy was highly likely to be inferior to standard chemotherapy met a prescribed level, and enrollment was discontinued. After an additional year of follow-up, the hazard ratio for disease recurrence or death in the capecitabine group was 2.09 (95% confidence interval, 1.38 to 3.17; P<0.001). Patients who were randomly assigned to capecitabine were twice as likely to have a relapse and almost twice as likely to die as patients who were randomly assigned to standard chemotherapy (P=0.02). At 3 years, the rate of relapse-free survival was 68% in the capecitabine group versus 85% in the standard-chemotherapy group, and the overall survival rate was 86% versus 91%. Two patients in the capecitabine group died of treatment-related complications; as compared with patients receiving capecitabine, twice as many patients receiving standard chemotherapy had moderate-to-severe toxic effects (64% vs. 33%). CONCLUSIONS Standard adjuvant chemotherapy is superior to capecitabine in patients with early-stage breast cancer who are 65 years of age or older. (ClinicalTrials.gov number, NCT00024102.)

Pathologic Complete Response Predicts Recurrence-Free Survival More Effectively by Cancer Subset: Results From the I-SPY 1 TRIAL—CALGB 150007/150012, ACRIN 6657

PURPOSE Neoadjuvant chemotherapy for breast cancer provides critical information about tumor response; how best to leverage this for predicting recurrence-free survival (RFS) is not established. The I-SPY 1 TRIAL (Investigation of Serial Studies to Predict Your Therapeutic Response With Imaging and Molecular Analysis) was a multicenter breast cancer study integrating clinical, imaging, and genomic data to evaluate pathologic response, RFS, and their relationship and predictability based on tumor biomarkers. PATIENTS AND METHODS Eligible patients had tumors ≥ 3 cm and received neoadjuvant chemotherapy. We determined associations between pathologic complete response (pCR; defined as the absence of invasive cancer in breast and nodes) and RFS, overall and within receptor subsets. RESULTS In 221 evaluable patients (median tumor size, 6.0 cm; median age, 49 years; 91% classified as poor risk on the basis of the 70-gene prognosis profile), 41% were hormone receptor (HR) negative, and 31% were human epidermal growth factor receptor 2 (HER2) positive. For 190 patients treated without neoadjuvant trastuzumab, pCR was highest for HR-negative/HER2-positive patients (45%) and lowest for HR-positive/HER2-negative patients (9%). Achieving pCR predicted favorable RFS. For 172 patients treated without trastuzumab, the hazard ratio for RFS of pCR versus no pCR was 0.29 (95% CI, 0.07 to 0.82). pCR was more predictive of RFS by multivariate analysis when subtype was taken into account, and point estimates of hazard ratios within the HR-positive/HER2-negative (hazard ratio, 0.00; 95% CI, 0.00 to 0.93), HR-negative/HER2-negative (hazard ratio, 0.25; 95% CI, 0.04 to 0.97), and HER2-positive (hazard ratio, 0.14; 95% CI, 0.01 to 1.0) subtypes are lower. Ki67 further improved the prediction of pCR within subsets. CONCLUSION In this biologically high-risk group, pCR differs by receptor subset. pCR is more highly predictive of RFS within every established receptor subset than overall, demonstrating that the extent of outcome advantage conferred by pCR is specific to tumor biology.

Intrinsic Breast Tumor Subtypes, Race, and Long-Term Survival in the Carolina Breast Cancer Study

Purpose: Previous research identified differences in breast cancer–specific mortality across 4 intrinsic tumor subtypes: luminal A, luminal B, basal-like, and human epidermal growth factor receptor 2 positive/estrogen receptor negative (HER2+/ER−). Experimental Design: We used immunohistochemical markers to subtype 1,149 invasive breast cancer patients (518 African American, 631 white) in the Carolina Breast Cancer Study, a population-based study of women diagnosed with breast cancer. Vital status was determined through 2006 using the National Death Index, with median follow-up of 9 years. Results: Cancer subtypes luminal A, luminal B, basal-like, and HER2+/ER− were distributed as 64%, 11%, 11%, and 5% for whites, and 48%, 8%, 22%, and 7% for African Americans, respectively. Breast cancer mortality was higher for participants with HER2+/ER− and basal-like breast cancer compared with luminal A and B. African Americans had higher breast cancer–specific mortality than whites, but the effect of race was statistically significant only among women with luminal A breast cancer. However, when compared with the luminal A subtype within racial categories, mortality for participants with basal-like breast cancer was higher among whites (HR = 2.0, 95% CI: 1.2–3.4) than African Americans (HR = 1.5, 95% CI: 1.0–2.4), with the strongest effect seen in postmenopausal white women (HR = 3.9, 95% CI: 1.5–10.0). Conclusions: Our results confirm the association of basal-like breast cancer with poor prognosis and suggest that basal-like breast cancer is not an inherently more aggressive disease in African American women compared with whites. Additional analyses are needed in populations with known treatment profiles to understand the role of tumor subtypes and race in breast cancer mortality, and in particular our finding that among women with luminal A breast cancer, African Americans have higher mortality than whites. Clin Cancer Res; 16(24); 6100–10. ©2010 AACR.

Mutation of GATA3 in human breast tumors

GATA3 is an essential transcription factor that was first identified as a regulator of immune cell function. In recent microarray analyses of human breast tumors, both normal breast luminal epithelium and estrogen receptor (ESR1)-positive tumors showed high expression of GATA3. We sequenced genomic DNA from 111 breast tumors and three breast-tumor-derived cell lines and identified somatic mutations of GATA3 in five tumors and the MCF-7 cell line. These mutations cluster in the vicinity of the highly conserved second zinc-finger that is required for DNA binding. In addition to these five, we identified using cDNA sequencing a unique mis-splicing variant that caused a frameshift mutation. One of the somatic mutations we identified was identical to a germline GATA3 mutation reported in two kindreds with HDR syndrome/OMIM #146255, which is an autosomal dominant syndrome caused by the haplo-insufficiency of GATA3. The ectopic expression of GATA3 in human 293T cells caused the induction of 73 genes including six cytokeratins, and inhibited cell line doubling times. These data suggest that GATA3 is involved in growth control and the maintenance of the differentiated state in epithelial cells, and that GATA3 variants may contribute to tumorigenesis in ESR1-positive breast tumors.

Prognostic Value of Histologic Grade and Proliferative Activity in Axillary Node–Positive Breast Cancer: Results From the Eastern Cooperative Oncology Group Companion Study, EST 4189

PURPOSE The identification of a subset of patients with axillary lymph node-positive breast cancer with an improved prognosis would be clinically useful. We report the prognostic importance of histologic grading and proliferative activity in a cohort of patients with axillary lymph node-positive breast cancer and compare these parameters with other established prognostic factors. PATIENTS AND METHODS This Eastern Cooperative Oncology Group laboratory companion study (E4189) centered on 560 axillary lymph node-positive patients registered onto one of six eligible clinical protocols. Flow cytometric (ploidy and S-phase fraction [SPF]) and histopathologic analyses (Nottingham Combined Histologic Grade and mitotic index) were performed on paraffin-embedded tissue from 368 patients. RESULTS Disease recurred in 208 patients; in 161 (77%), within the first 5 years. Mitotic index and grade were associated with both ploidy and SPF (P </=.01). Within the first 5 years of follow-up, mitotic index (P =.004), grade (P =.004), ploidy (P =. 006), and SPF (P =.05) were associated with time to recurrence; there was also a significant association with survival. The effect of mitotic index was largely a result of the difference between 0 to 2 mitoses/10 high-power fields (HPF; 5-year recurrence of 31%) and more than 2 mitoses/10 HPF (5-year recurrence of 52%). The 0 to 2 mitoses/10 HPF group was independently associated with improved prognosis at 5 years (P =.002) in regression models that included other standard prognostic factors. CONCLUSION A subset of axillary lymph node-positive patients with improved prognosis may be identified using a lower (< 3 mitoses/10 HPF) mitotic count than is usually performed.

Molecular subtypes of breast cancer in relation to paclitaxel response and outcomes in women with metastatic disease: Results from CALGB 9342

IntroductionThe response to paclitaxel varies widely in metastatic breast cancer. We analyzed data from CALGB 9342, which tested three doses of paclitaxel in women with advanced disease, to determine whether response and outcomes differed according to HER2, hormone receptor, and p53 status.MethodsAmong 474 women randomly assigned to paclitaxel at a dose of 175, 210, or 250 mg/m2, adequate primary tumor tissue was available from 175. Immunohistochemistry with two antibodies and fluorescence in situ hybridization were performed to evaluate HER2 status; p53 status was determined by immunohistochemistry and sequencing. Hormone receptor status was obtained from pathology reports.ResultsObjective response rate was not associated with HER2 or p53 status. There was a trend toward a shorter median time to treatment failure among women with HER2-positive tumors (2.3 versus 4.2 months; P = 0.067). HER2 status was not related to overall survival (OS). Hormone receptor expression was not associated with differences in response but was associated with longer OS (P = 0.003). In contrast, women with p53 over-expression had significantly shorter OS than those without p53 over-expression (11.5 versus 14.4 months; P = 0.002). In addition, triple negative tumors were more frequent in African-American than in Caucasian patients, and were associated with a significant reduction in OS (8.7 versus 12.9 months; P = 0.008).ConclusionNone of the biomarkers was predictive of treatment response in women with metastatic breast cancer; however, survival differed according to hormone receptor and p53 status. Triple negative tumors were more frequent in African-American patients and were associated with a shorter survival.

High focal adhesion kinase expression in invasive breast carcinomas is associated with an aggressive phenotype

Focal adhesion kinase (FAK) is a protein tyrosine kinase expressed in invasive breast cancer that regulates antiapoptotic signaling. We have examined FAK expression by immunohistochemistry using anti-FAK 4.47 in breast tumor samples from a large population-based, case–control study of women participating in the University of North Carolina Breast Specialized Programs of Research Excellence (SPORE), Carolina Breast Cancer Study. In this population, 629 formalin-fixed, paraffin-embedded tissue sections were stained for FAK and scored as high (3+ or 4+ intensity and ≥90% positive cells) or otherwise. High FAK expression was associated with poor prognostic indicators including high mitotic index (>10 mitoses per 10 consecutive high-power fields), nuclear grade 3, architectural grade 3, estrogen and progesterone receptor negative, and HER-2/neu overexpressed using CB11 antibody. The association of high FAK expression with HER-2/neu overexpression lends further support that HER-2/neu and FAK collaborate to promote tumorigenesis. The presence of strong FAK expression in many high grade, estrogen- and progesterone-negative breast carcinomas indicates that FAK may be an attractive target for therapeutic intervention.

Comparison of HER2 status by fluorescence in situ hybridization and immunohistochemistry to predict benefit from dose escalation of adjuvant doxorubicin-based therapy in node-positive breast cancer patients

PURPOSE HER2 is a clinically important tumor marker in breast cancer; however, there is controversy regarding which method reliably measures HER2 status. We compared three HER2 laboratory methods: immunohistochemistry (IHC), fluorescence in situ hybridization (FISH) and polymerase chain reaction (PCR), to predict disease-free survival (DFS) and overall survival (OS) after adjuvant doxorubicin-based therapy in node-positive breast cancer patients. METHODS This is a Cancer and Leukemia Group B (CALGB) study, using 524 tumor blocks collected from breast cancer patients registered to clinical trial CALGB 8541. IHC employed CB11 and AO-11-854 monoclonal antibodies; FISH used PathVysion HER2 DNA Probe kit; PCR utilized differential PCR (D-PCR) methodology. RESULTS Cases HER2 positive by IHC, FISH and D-PCR were 24%, 17%, and 18%, respectively. FISH and IHC were clearly related (kappa = 64.8%). All three methods demonstrated a similar relationship for DFS and OS. By any method, for patients with HER2-negative tumors, there was little or no effect of dose of adjuvant doxorubicin-based therapy. For patients with HER2-positive tumors, all three methods predicted a benefit from dose-intense (high-dose) compared with low- or moderate-dose adjuvant doxorubicin-based therapy. CONCLUSION FISH is a reliable method to predict clinical outcome following adjuvant doxorubicin-based therapy for stage II breast cancer patients. There is a moderate level of concordance among the three methods (IHC, FISH, PCR). None of the methods is clearly superior. Although IHC-positive/FISH-positive tumors yielded the greatest interaction with dose of therapy in predicting outcome, no combination of assays tested was statistically superior.