Marilyn Leitch

Whole Genome Analysis Informs Breast Cancer Response to Aromatase Inhibition

To correlate the variable clinical features of oestrogen-receptor-positive breast cancer with somatic alterations, we studied pretreatment tumour biopsies accrued from patients in two studies of neoadjuvant aromatase inhibitor therapy by massively parallel sequencing and analysis. Eighteen significantly mutated genes were identified, including five genes (RUNX1, CBFB, MYH9, MLL3 and SF3B1) previously linked to haematopoietic disorders. Mutant MAP3K1 was associated with luminal A status, low-grade histology and low proliferation rates, whereas mutant TP53 was associated with the opposite pattern. Moreover, mutant GATA3 correlated with suppression of proliferation upon aromatase inhibitor treatment. Pathway analysis demonstrated that mutations in MAP2K4, a MAP3K1 substrate, produced similar perturbations as MAP3K1 loss. Distinct phenotypes in oestrogen-receptor-positive breast cancer are associated with specific patterns of somatic mutations that map into cellular pathways linked to tumour biology, but most recurrent mutations are relatively infrequent. Prospective clinical trials based on these findings will require comprehensive genome sequencing.

Circulating Tumor Cells in Patients with Breast Cancer Dormancy

Purpose: The purpose of this study was to test the hypothesis that circulating tumor cells (CTCs) are present in patients many years after mastectomy without evidence of disease and that these CTCs are shed from persisting tumor in patients with breast cancer dormancy. Experimental Design: We searched for CTCs in 36 dormancy candidate patients and 26 age-matched controls using stringent criteria for cytomorphology, immunophenotype, and aneusomy. Results: Thirteen of 36 dormancy candidates, 7 to 22 years after mastectomy and without evidence of clinical disease, had CTCs, usually on more than one occasion. Only 1 of 26 controls had a possible CTC (no aneusomy). The statistical difference of these two distributions was significant (exact P = 0.0043). The CTCs in patients whose primary breast cancer was just removed had a half-life measured in 1 to 2.4 hours. Conclusions: The CTCs that are dying must be replenished every few hours by replicating tumor cells somewhere in the tissues. Hence, there appears to be a balance between tumor replication and cell death for as long as 22 years in dormancy candidates. We conclude that this is one mechanism underlying tumor dormancy.

Randomized Phase II Neoadjuvant Comparison Between Letrozole, Anastrozole, and Exemestane for Postmenopausal Women With Estrogen Receptor–Rich Stage 2 to 3 Breast Cancer: Clinical and Biomarker Outcomes and Predictive Value of the Baseline PAM50-Based Intrinsic Subtype—ACOSOG Z1031

PURPOSE Preoperative aromatase inhibitor (AI) treatment promotes breast-conserving surgery (BCS) for estrogen receptor (ER)-positive breast cancer. To study this treatment option, responses to three AIs were compared in a randomized phase II neoadjuvant trial designed to select agents for phase III investigations. PATIENTS AND METHODS Three hundred seventy-seven postmenopausal women with clinical stage II to III ER-positive (Allred score 6-8) breast cancer were randomly assigned to receive neoadjuvant exemestane, letrozole, or anastrozole. The primary end point was clinical response. Secondary end points included BCS, Ki67 proliferation marker changes, the Preoperative Endocrine Prognostic Index (PEPI), and PAM50-based intrinsic subtype analysis. RESULTS On the basis of clinical response rates, letrozole and anastrozole were selected for further investigation; however, no other differences in surgical outcome, PEPI score, or Ki67 suppression were detected. The BCS rate for mastectomy-only patients at presentation was 51%. PAM50 analysis identified AI-unresponsive nonluminal subtypes (human epidermal growth factor receptor 2 enriched or basal-like) in 3.3% of patients. Clinical response and surgical outcomes were similar in luminal A (LumA) versus luminal B tumors; however, a PEPI of 0 (best prognostic group) was highest in the LumA subset (27.1% v 10.7%; P = .004). CONCLUSION Neoadjuvant AI treatment markedly improved surgical outcomes. Ki67 and PEPI data demonstrated that the three agents tested are biologically equivalent and therefore likely to have similar adjuvant activities. LumA tumors were more likely to have favorable biomarker characteristics after treatment; however, occasional paradoxical increases in Ki67 (12% of tumors with > 5% increase after therapy) suggest treatment-resistant cells, present in some LumA tumors, can be detected by post-treatment profiling.

uPAR and HER-2 gene status in individual breast cancer cells from blood and tissues

Overexpression of urokinase plasminogen activator system or HER-2 (erbB-2) in breast cancer is associated with a poor prognosis. HER-2 overexpression is caused by HER-2 gene amplification. The anti-HER-2 antibody trastuzumab significantly improves clinical outcome for HER2-positive breast cancer. Drugs that target the uPA system are in early clinical trials. The aims of this study were to determine whether urokinase plasminogen activator receptor (uPAR) gene amplification occurs and whether analysis of individual tumor cells (TCs) in the blood or tissue can add information to conventional pathological analysis that could help in diagnosis and treatment. Analysis of individual TCs indicates that uPAR amplification occurs in a significant portion of primary breast cancers and also circulating tumor cells (CTCs) from patients with advanced disease. There was complete concordance between touch preps (TPs) and conventional pathological examination of HER-2 and uPAR gene status in primary tumors. There was also excellent concordance of HER-2 gene status between primary tumors and CTCs provided that acquisition of HER-2 gene amplification in CTCs was taken into account. Unexpectedly, gene amplification of HER-2 and uPAR occurred most frequently in the same TC and patient, suggesting a biological bias and potential advantage for coamplification. Expression of HER-2 and uPAR in primary tumors predicted gene status in 100 and 92% of patients, respectively.

Sentinel lymph node biopsy, an alternative to elective axillary dissection for breast cancer

BACKGROUND Axillary metastases remain an important prognostic indicator in breast cancer. Axillary lymphadenectomy (ALND) carries significant morbidity and is unnecessary in most patients with early breast cancer; thus, sentinel lymph node (SLN) biopsy has been advocated for axillary staging. We studied the SLN identification rate and its accuracy in predicting axillary metastases. METHODS One hundred nineteen women with breast carcinoma underwent SLN and ALND. Lymphoscintigraphy was performed using Technetium99 sulfur colloid supplemented by Isosulfan blue dye. Hematoxylin/eosin-stained lymph node sections were examined by light microscopy. RESULTS The SLN identification rate was 81%. One SLN was negative (1%) in a patient with axillary disease. SLN histology correctly predicted the absence of axillary disease in 98.6%. Sensitivity, specificity, and positive and negative predictive values were 96%, 100%, 100%, and 99%, respectively. CONCLUSIONS Sentinel lymph node biopsy accurately predicts total axillary status and is valuable in the surgical staging of breast cancer.

Disparities and Trends in Sentinel Lymph Node Biopsy Among Early-Stage Breast Cancer Patients (1998–2005)

BACKGROUND Sentinel lymph node biopsy (SLNB), an acceptable alternative to axillary lymph node dissection for staging patients with breast cancer, was introduced to clinical practice in the late 1990s. We assessed demographic, clinical, and facility-related factors associated with SLNB in women with early-stage breast cancer and evaluated trends in these factors over time. METHODS Data on early-stage breast cancers (T1a, T1b, T1c, and T2N0) diagnosed between January 1, 1998, and December 31, 2005, were extracted from the National Cancer Database, a hospital-based registry. Patient demographics, tumor stage, type of lymph node surgery, type of breast cancer surgery, health insurance, treatment facility type, and area-level education and income variables were collected. Multivariable logistic regression analyses were performed to assess predictive factors associated with SLNB, temporal differences in factors associated with SLNB, and differences in rates of SLNB by facility type, race/ethnicity, and type of health insurance over time. RESULTS The total analytic study population included 490,899 women. The use of SLNB increased from 26.8% in 1998 to 65.5% in 2005. Factors associated with lower likelihood of SLNB over the study period included being older (odds ratio [OR] = 0.80, 95% confidence interval [CI] = 0.78 to 0.92 for those aged 72 or older compared with those aged 51 or younger), being of racial/ethnic minority (OR = 0.76, 95% CI = 0.74 to 0.78 for African Americans compared with whites), having no health insurance (OR = 0.77, 95% CI = 0.73 to 0.80 for uninsured compared with having private insurance), having certain government insurance plans (for Medicaid, OR = 0.81, 95% CI = 0.78 to 0.84, and for Medicare at age <65 years, OR = 0.83, 95% CI = 0.80 to 0.87, both compared with private insurance), residing in zip codes with lower proportion of high school graduates (OR = 0.88, 95% CI = 0.86 to 0.89) or with lower median income (OR = 0.79, 95% CI = 0.77 to 0.81), and receiving treatment in facility types other than a teaching or research hospital (for community hospital, OR = 0.84, 95% CI = 0.82 to 0.86; for community cancer center, OR = 0.86, 95% CI = 0.84 to 0.87). The associations with insurance status and sociodemographic characteristics were more pronounced in 2005 than in 1998. For example, the adjusted annual rates of SLNB in 1998 were 0.29 in whites, 0.26 in African Americans, and 0.35 in Hispanics; in 2005 the respective rates were 0.70, 0.64, and 0.67. CONCLUSIONS Although use of SLNB increased from 1998 to 2005, disparities persisted in receipt of SLNB that are based on nonclinical factors, including sociodemographic characteristics and insurance status.

Ki67 proliferation index as a tool for chemotherapy decisions during and after neoadjuvant aromatase inhibitor treatment of breast cancer: Results from the American college of surgeons oncology group Z1031 trial (alliance)

Purpose To determine the pathologic complete response (pCR) rate in estrogen receptor (ER) –positive primary breast cancer triaged to chemotherapy when the protein encoded by the MKI67 gene (Ki67) level was > 10% after 2 to 4 weeks of neoadjuvant aromatase inhibitor (AI) therapy. A second objective was to examine risk of relapse using the Ki67-based Preoperative Endocrine Prognostic Index (PEPI). Methods The American College of Surgeons Oncology Group (ACOSOG) Z1031A trial enrolled postmenopausal women with stage II or III ER-positive (Allred score, 6 to 8) breast cancer whose treatment was randomly assigned to neoadjuvant AI therapy with anastrozole, exemestane, or letrozole. For the trial ACOSOG Z1031B, the protocol was amended to include a tumor Ki67 determination after 2 to 4 weeks of AI. If the Ki67 was > 10%, patients were switched to neoadjuvant chemotherapy. A pCR rate of > 20% was the predefined efficacy threshold. In patients who completed neoadjuvant AI, stratified Cox modeling was used to assess whether time to recurrence differed by PEPI = 0 score (T1 or T2, N0, Ki67 < 2.7%, ER Allred > 2) versus PEPI > 0 disease. Results Only two of the 35 patients in ACOSOG Z1031B who were switched to neoadjuvant chemotherapy experienced a pCR (5.7%; 95% CI, 0.7% to 19.1%). After 5.5 years of median follow-up, four (3.7%) of the 109 patients with a PEPI = 0 score relapsed versus 49 (14.4%) of 341 of patients with PEPI > 0 (recurrence hazard ratio [PEPI = 0 v PEPI > 0], 0.27; P = .014; 95% CI, 0.092 to 0.764). Conclusion Chemotherapy efficacy was lower than expected in ER-positive tumors exhibiting AI-resistant proliferation. The optimal therapy for these patients should be further investigated. For patients with PEPI = 0 disease, the relapse risk over 5 years was only 3.6% without chemotherapy, supporting the study of adjuvant endocrine monotherapy in this group. These Ki67 and PEPI triage approaches are being definitively studied in the ALTERNATE trial (Alternate Approaches for Clinical Stage II or III Estrogen Receptor Positive Breast Cancer Neoadjuvant Treatment in Postmenopausal Women: A Phase III Study; clinical trial information: NCT01953588).

Abstract PD07-01: Z1031B Neoadjuvant Aromatase Inhibitor Trial: A Phase 2 study of Triage to Chemotherapy Based on 2 to 4 week Ki67 level > 10%.

Background: Neoadjuvant aromatase inhibitor (AI) therapy would become a more acceptable alternative to chemotherapy if there was a way to identify AI-resistant cases early for triage to alternate systemic treatment. We therefore conducted a Phase 2 trial of post-menopausal patients with clinical stage 2 or 3 ER+ (Allred Score 6 to 8) breast cancer who were re-biopsied 2 to 4 weeks after initiating neoadjuvant AI therapy. If the tumor Ki67 level was > 10% (AI resistant) the patient was triaged to either chemotherapy or immediate surgery and if Results: 51 patients (21%) had 2 to 4 week Ki67>10%, of these 36 received chemotherapy per protocol (27) anthracycline (A) and taxane (T)-based, 7 T-based, 1 A-only based and 1 other (non-NCCN) regimen but only 2 patients had a pCR (5.5%). 194 patients (79%) had a Ki67 Conclusion. A Ki67 value >10% at 2 to 4 weeks strongly enriches for high-risk molecular subtypes (mainly LumB) however standard neoadjuvant chemotherapy did not meet pre-assigned criteria for adequate Phase 2 activity in this group. Novel neoadjuvant approaches for AI resistant-disease defined by high on treatment Ki67 are therefore a high priority. Management of patients with excellent response to neoadjuvant endocrine therapy (PEPI-0) without adjuvant chemotherapy is feasible. The ALTERNATE trial will enroll over 2000 patients to establish a new standard of care for neoadjuvant treatment of ER+ HER2− disease based on these principles. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr PD07-01.

A randomised trial of nursing interventions supporting recovery of the postmastectomy patient

AIMS AND OBJECTIVES This ABC to recovery study evaluated the combined and separate components of preoperative education and the effectiveness of wearing the Papilla Gown. BACKGROUND Surgical removal of the breast may lead to activity limitation, self-image issues, discomfort and later complication of lymphoedema. Design.  This study used experimental and longitudinal design. METHODS One hundred and forty-five women undergoing mastectomies for stages two and three breast cancer were randomised into four groups: education and Papilla Gown, education only, gown only and control. The outcomes of activity (A), body image (B), comfort (C), knowledge and lymphoedema were assessed at baseline and/or 1 week and 6 months using three measures. All 145 participants completed the study questionnaires at first two measures, and forty-six of these participants completed the questionnaires at 6 months postoperatively. The setting for the study included two clinics and hospitals. To examine statistical significance at each time point after surgery, 2-way anovas were performed on ABC, knowledge and tape measurement to see whether there were any statistically significant differences between the four groups. All reported p-values are two sided. All statistical analyses were performed using sas 9.2 for Windows. RESULTS The mean age of the sample was 55 years. The study revealed that women who received the combined intervention demonstrated greater activity. Women who wore the gown only had a greater comfort level and decreased lymphoedema. Women that received preoperative education experienced increased knowledge. CONCLUSIONS Outcomes suggest that the combined intervention (ABCs to recovery) can improve recovery following mastectomy. Relevance to clinical practice. The results will be used to further modify the intervention and to increase awareness of nurse practitioners and other healthcare professionals of the specific needs of postmastectomy patients.

Identification of Breast Cancer DNA Methylation Markers Optimized for Fine-Needle Aspiration Samples

Background: Random periareolar fine-needle aspiration (RP-FNA) is increasingly used in trials of breast cancer prevention for biomarker assessments. DNA methylation markers may have value as surrogate endpoint biomarkers, but this requires identification of biologically relevant markers suitable for paucicellular, lymphocyte-contaminated clinical samples. Methods: Unbiased whole-genome 5-aza-2′-deoxycytidine (5AZA)–induced gene expression assays, followed by several phases of qualitative and quantitative methylation-specific PCR (MSP) testing, were used to identify novel breast cancer DNA methylation markers optimized for clinical FNA samples. Results: The initial 5AZA experiment identified 453 genes whose expression was potentially regulated by promoter region methylation. Informatics filters excluded 273 genes unlikely to yield useful DNA methylation markers. MSP assays were designed for 271 of the remaining genes and, ultimately, 33 genes were identified that were differentially methylated in clinical breast cancer samples, as compared with benign RP-FNA samples, and never methylated in lymphocytes. A subset of these markers was validated by quantitative multiplex MSP in extended clinical sample sets. Using a novel permutation method for analysis of quantitative methylation data, PSAT1, GNE, CPNE8, and CXCL14 were found to correlate strongly with specific clinical and pathologic features of breast cancer. In general, our approach identified markers methylated in a smaller subpopulation of tumor cells than those identified in published methylation array studies. Conclusions: Clinically relevant DNA methylation markers were identified using a 5AZA-induced gene expression approach. Impact: These breast cancer-relevant, FNA-optimized DNA methylation markers may have value as surrogate endpoint biomarkers in RP-FNA studies. Cancer Epidemiol Biomarkers Prev; 22(12); 2212–21. ©2013 AACR.