Beiyun Chen

Impact of PTEN Protein Expression on Benefit From Adjuvant Trastuzumab in Early-Stage Human Epidermal Growth Factor Receptor 2–Positive Breast Cancer in the North Central Cancer Treatment Group N9831 Trial

PURPOSE It has been suggested that PTEN, a negative regulator of PI3K/AKT signaling, is involved in tumor sensitivity to trastuzumab. We investigated the association between tumor PTEN protein expression and disease-free survival (DFS) of patients randomly assigned to receive chemotherapy alone (arm A) or chemotherapy with sequential (arm B) or concurrent trastuzumab (arm C) in the phase III early-stage human epidermal growth factor receptor 2 (HER2) -positive trial-North Central Cancer Treatment Group (NCCTG) N9831. PATIENTS AND METHODS The intensity and percentage of invasive cells with cytoplasmic PTEN staining were determined in tissue microarray sections containing three cores per block (n = 1,286) or in whole tissue sections (WS; n = 516) by using standard immunohistochemistry (138G6 monoclonal antibody). Tumors were considered positive for PTEN (PTEN-positive) if any core or WS had any invasive cells with ≥ 1+ staining. Median follow-up was 6.0 years. RESULTS Of 1,802 patients included in this analysis (of 3,505 patients registered to N9831), 1,342 (74%) had PTEN-positive tumors. PTEN positivity was associated with hormone receptor negativity (χ(2) P < .001) and nodal positivity (χ(2) P = .04). PTEN did not have an impact on DFS within the various arms. Comparing DFS of arm C to arm A, patients with PTEN-positive and PTEN-negative tumors had hazard ratios (HRs) of 0.65 (P = .003) and 0.47 (P = .005), respectively (interaction P = .16). For arm B versus arm A, patients with PTEN-positive and PTEN-negative tumors had HRs of 0.70 (P = .009) and 0.85 (P = .44), respectively (interaction P = .47). CONCLUSION In contrast to selected preclinical and limited clinical studies suggesting a decrease in trastuzumab sensitivity in patients with PTEN-negative tumors, our data show benefit of adjuvant trastuzumab for patients with HER2-positive breast cancer, independent of tumor PTEN status.

Predictability of Adjuvant Trastuzumab Benefit in N9831 Patients Using the ASCO/CAP HER2-Positivity Criteria

The 2007 American Society of Clinical Oncology and College of American Pathologists (ASCO/CAP) joint guidelines defined criteria for HER2 positivity of tumors that modified those of the US Food and Drug Administration (FDA), causing some confusion and uncertainty among clinicians. Using data from the HER2-positive breast cancer adjuvant trial N9831, we compared eligibility for patients who met both criteria, and disease-free survival (DFS) was assessed by Cox proportional hazards regression. The number of patients in the N9831 trial retrospectively eligible for trastuzumab therapy was decreased when ASCO/CAP criteria vs FDA criteria were applied to immunohistochemistry and/or fluorescence in situ hybridization results (107 [3.7%] of 2904 patients with immunohistochemistry results, 37 [1.3%] of 2809 patients with fluorescence in situ hybridization results, and 47 [1.7%] of 2809 patients with both results). Improvement in DFS was similar among patients treated with trastuzumab under either set of criteria (concurrent trastuzumab and chemotherapy compared with chemotherapy alone: by ASCO/CAP criteria, hazard ratio of DFS = 0.59, 95% confidence interval = 0.48 to 0.73; by FDA criteria but not ASCO/CAP criteria, hazard ratio = 0.60, 95% confidence interval = 0.12 to 3.13; number needed to treat to prevent one additional DFS event at 5 years: 10 and 11.2 patients, respectively). Following the 2007 ASCO/CAP criteria for HER2 positivity would negate the option of potentially life-saving trastuzumab therapy for a small but meaningful group of patients. We recommend using FDA-approved HER2 criteria for therapeutic decision making.

C-MYC Alterations and Association With Patient Outcome in Early-Stage HER2-Positive Breast Cancer From the North Central Cancer Treatment Group N9831 Adjuvant Trastuzumab Trial

PURPOSE Findings from the human epidermal growth factor receptor 2 (HER2) -positive National Surgical Adjuvant Breast and Bowel Project (NSABP) B31 trial suggested that MYC/HER2 coamplification (> 5.0 copies/nucleus) was associated with additional benefit from adjuvant trastuzumab in patients with early-stage breast cancer. To further explore this relationship, we investigated associations between MYC amplification and disease-free survival (DFS) in a similar adjuvant trastuzumab HER2-positive breast cancer trial-North Central Cancer Treatment Group (NCCTG) N9831. PATIENTS AND METHODS This analysis included 799 patients randomly assigned to receive chemotherapy alone or with concurrent trastuzumab on N9831. Fluorescence in situ hybridization (FISH) was performed by using a dual-probe mixture for MYC and centromere 8 (MYC:CEP8) on tissue microarrays. MYC amplification was prespecified as MYC:CEP8 ratio > 2.2 or average MYC copies/nucleus > 5.0. Exploratory variables included polysomy 8. RESULTS In comparing DFS (median follow-up, 4.0 years) between treatments, patients with MYC:CEP8 ratio ≤ 2.2 (n = 618; 77%) and > 2.2 (n = 181; 23%) had hazard ratios (HRs) of 0.46 (P < .001) and 0.67 (P = .33), respectively (interaction P = .38). Patients with MYC copies/nucleus ≤ 5.0 (n = 534; 67%) and > 5.0 (n = 265; 33%) had HRs of 0.52 (P = .002) and 0.48 (P = .02), respectively (interaction P = .94). Patients with MYC:CEP8 ratio < 1.3 with normal chromosome 8 copy number (n = 141; 18%) and ≥ 1.3 or < 1.3 with polysomy 8 (n = 658; 82%) had HRs of 0.66 (P = .28) and 0.44 (P < .001), respectively (interaction P = .23). Patients with MYC copies/nucleus < 2.5 (n = 130; 16%) and ≥ 2.5 (n = 669; 84%) had HRs of 1.07 (P = .87) and 0.42 (P < .001), respectively (interaction P = .05). CONCLUSION We did not confirm the B31 association between MYC amplification and additional trastuzumab benefit. Exploratory analyses revealed potential associations between alternative MYC/chromosome 8 copy number alterations and differential benefit of adjuvant trastuzumab.

Testing for HER2 in Breast Cancer: A Continuing Evolution

Human epidermal growth factor receptor 2 (HER2) is an important prognostic and predictive factor in breast cancer. HER2 is overexpressed in approximately 15%–20% of invasive breast carcinomas and is associated with earlier recurrence, shortened disease free survival, and poor prognosis. Trastuzumab (Herceptin) a “humanized” monoclonal antibody targets the extracellular domain of HER2 and is widely used in the management of HER2 positive breast cancers. Accurate assessment of HER2 is thus critical in the management of breast cancer. The aim of this paper is to present a comprehensive review of HER2 with reference to its discovery and biology, clinical significance, prognostic value, targeted therapy, current and new testing modalities, and the interpretation guidelines and pitfalls.

Comparison of Central HER2 Testing With Quantitative Total HER2 Expression and HER2 Homodimer Measurements Using a Novel Proximity-Based Assay

The accuracy and reliability of immunohistochemical analysis and in situ hybridization for the assessment of HER2 status remains a subject of debate. We developed a novel assay (HERmark Breast Cancer Assay, Monogram Biosciences, South San Francisco, CA) that provides precise quantification of total HER2 protein expression (H2T) and HER2 homodimers (H2D) in formalin-fixed, paraffin-embedded tissue specimens. H2T and H2D results of 237 breast cancers were compared with those of immunohistochemical studies and fluorescence in situ hybridization (FISH) centrally performed at the Mayo Clinic, Rochester, MN. H2T described a continuum across a wide dynamic range ( approximately 2.5 log). Excluding the equivocal cases, HERmark showed 98% concordance with immunohistochemical studies for positive and negative assay values. For the 94 immunohistochemically equivocal cases, 67% and 39% concordance values were observed between HERmark and FISH for positive and negative assay values, respectively. Polysomy 17 in the absence of HER2 gene amplification did not result in HER2 overexpression as evaluated quantitatively using the HERmark assay.

Impact of American Society of Clinical Oncology/College of American Pathologists guideline recommendations on HER2 interpretation in breast cancer

Accurate assessment of human epidermal growth factor receptor 2 is critical for the management of patients with breast cancer. We set out to study the impact of the 2007 American Society of Clinical Oncology/College of American Pathologists guidelines on the interpretation of human epidermal growth factor receptor 2 IHC results and its correlation with fluorescence in situ hybridization results. Invasive breast carcinomas with IHC HercepTest 3+ were retrieved from the archive of Mayo Clinic Rochester. The human epidermal growth factor receptor 2 slides were rereviewed, and results were recorded as percentage of invasive tumor cells with 3+, 2+, 1+, and 0 staining intensity. Human epidermal growth factor receptor 2 gene amplification by fluorescence in situ hybridization was performed on all tumors with 3+ staining in 70% or less of tumor cells. Of the 141 cases studied, 12 cases showed intense membrane staining in 11% to 30% of the invasive tumor cells and would have been scored as 2+ according to the new American Society of Clinical Oncology/College of American Pathologists guidelines. Of these 12 cases, 6 were positive for human epidermal growth factor receptor 2 gene amplification by fluorescence in situ hybridization (ratio >2.2), 4 cases were negative (HER2/CEP17 ratio of < 1.8), and 2 cases were equivocal (ratio of 1.8-2.2). One human epidermal growth factor receptor 2-positive case showed dramatic intratumoral heterogeneity with high-level amplification (ratio of 12.2) in the IHC 3+ area and no amplification (ratio of 1.0) in the IHC 1+/2+ areas. The 2007 American Society of Clinical Oncology/College of American Pathologists guidelines down-scored 2.8% of tumors from human epidermal growth factor receptor 2-positive (IHC 3+) to human epidermal growth factor receptor 2-negative (IHC 2+ equivocal and fluorescence in situ hybridization negative) in this study. Clinical studies are needed to determine whether the updated guidelines are better at predicting response to anti-human epidermal growth factor receptor 2 therapy.

Amyloidosis of the breast: predominantly AL type and over half have concurrent breast hematologic disorders

Amyloidosis is a disorder characterized by extracellular deposition of proteins in an abnormal fibrillar configuration. Amyloidosis can be localized or systemic and may affect any organ. Breast involvement by amyloidosis has rarely been reported. In this study, we described the characteristics of 40 cases of breast amyloidosis that were reviewed at the Division of Anatomic Pathology at Mayo Clinic from 1995 to 2011. The cohort included 39 women and 1 man with a mean age of 60 years. The type of amyloidosis, determined by immunohistochemistry or mass spectrometry-based proteomics in 26 patients, was immunoglobulin-associated in all cases (AL-kappa type in 15 (58%) cases, AL-lambda in 10 (38%) and mixed heavy and light chains (AH/AL) in 1 (4%) case). Mass spectrometry-based proteomics was able to determine the type of amyloidosis in 95% of cases tested compared with 69% of cases by immunohistochemistry. In addition to amyloidosis, the breast biopsy showed a hematologic disorder in 55% of cases, most commonly MALT lymphoma. One patient had concurrent intraductal carcinoma, but none had invasive carcinoma. Of the 15 patients seen in our institution, 53% had localized amyloidosis and 47% had extramammary amyloid involvement, which was diagnosed before breast amyloidosis in most patients. M-spike was detected in the blood in 62%. After a median follow-up of 33.5 months in 12 patients, 5 died, mostly of complications of lymphoma or leukemia. In conclusion, our findings indicate that breast amyloidosis is of the AL type in the vast majority of patients (usually kappa). It is associated with systemic amyloidosis in close to half of patients and with hematologic malignancy in the breast in over half of patients. Therefore, further work up to rule out hematologic malignancy and/or systemic amyloidosis is recommended. Mass spectrometry-based proteomics is superior to immunohistochemistry for typing of breast amyloidosis.

Cancer and Leukemia Group B Pathology Committee Guidelines for Tissue Microarray Construction Representing Multicenter Prospective Clinical Trial Tissues

Practice-changing evidence requires confirmation, preferably in multi-institutional clinical trials. The collection of tissue within such trials has enabled biomarker studies and evaluation of companion diagnostic tests. Tissue microarrays (TMAs) have become a standard approach in many cooperative oncology groups. A principal goal is to maximize the number of assays with this precious tissue. However, production strategies for these arrays have not been standardized, possibly decreasing the value of the study. In this article, members of the Cancer and Leukemia Group B Pathology Committee relay our experiences as array facility directors and propose guidelines regarding the production of high-quality TMAs for cooperative group studies. We also discuss statistical issues arising from having a proportion of patients available for TMAs and the possibility that patients with TMAs fail to represent the greater study population.

The Use of Molecular Breast Imaging to Assess Response in Women Undergoing Neoadjuvant Therapy for Breast Cancer: A Pilot Study

Purpose of the Report: To report our findings from a prospective pilot study evaluating the accuracy of molecular breast imaging (MBI) in assessing tumor response to neoadjuvant therapy (NT) for breast cancer. Materials and Methods: Twenty patients with newly diagnosed invasive breast cancer who were scheduled to receive NT underwent MBI before beginning and after completing NT before surgery. MBI was performed using a dual-detector cadmium-zinc-telluride gamma camera system mounted on a modified mammography gantry after patients had received an intravenous injection of 20 mCi of 99mTc sestamibi. Tumor extent was measured on MBI, and tumor-to-background (T/B) ratios of radiotracer uptake were determined through region-of-interest analysis. Pathologic measurement of tumor size was used as a standard and compared with post-NT tumor size derived from MBI. Results: Three patients in whom post-NT MBI could not be performed because of scheduling problems were excluded from analysis. Eighteen cancers were diagnosed in 17 patients. A correlation coefficient of r = 0.681 (P = 0.002) was found between MBI and residual tumor size. The average T/B ratio on MBI decreased from a pretreatment value of 3.0 to a posttreatment value of 1.4. The relative decrease in T/B ratio did not appear to be predictive of response. Conclusions: Measurements of tumor size by MBI and T/B ratios are limited in their predictive value regarding the pathologic extent of residual disease in women treated with NT for breast cancer. Alternate tumor-specific radiopharmaceuticals should be evaluated to provide information to improve planning and monitoring of breast cancer treatment.

Image Analysis of HER2 Immunohistochemical Staining Reproducibility and Concordance With Fluorescence In Situ Hybridization of a Laboratory-Validated Scoring Technique

Image analysis of the HER2 immunohistochemical (IHC) stain can help determine which breast cancer patients may benefit from HER2-targeted therapy. We studied the concordance of HER2 IHC and fluorescence in situ hybridization (FISH) as well as reproducibility of surgical pathologist (SP) and cytotechnologist (CT) interpretations using manual and image analysis methodologies on 154 IHC cases. Concordances with FISH were good for IHC negative (0, 1+) cases (range, 97%-100%) and positive (3+) cases (range, 87%-100%). Image analysis had fewer equivocal (2+) results (10.4%) than CT (14.9%) and SP (16.2%) manual methods, with higher concordances to FISH (31%, 26%, and 20% for image analysis, CT manual, and SP manual, respectively). CT manual (κ = 0.747) and image analysis (κ = 0.779) methods had better interobserver reproducibility than SP manual (κ = 0.697). CT image analysis had better intraobserver reproducibility (κ = 0.882) than CT (κ = 0.828) and SP (κ = 0.766) manual methods. HER2 IHC analysis performed by image analysis can produce accurate results with improved reproducibility.