Gary Tozbikian

Mesothelin expression in triple negative breast carcinomas correlates significantly with basal-like phenotype, distant metastases and decreased survival.

Mesothelin is a cell surface associated antigen expressed on mesothelial cells and in some malignant neoplasms. Mesothelin-targeted therapies are in phase I/II clinical trials. The clinicopathologic and prognostic significance of mesothelin expression in triple negative breast carcinomas (TNBC) has not been fully assessed. We evaluated the expression of mesothelin and of basal markers in tissue microarrays of 226 TNBC and 88 non-TNBC and assessed the clinicopathologic features of mesothelin-expressing breast carcinomas. Furthermore, we investigated the impact of mesothelin expression on the disease-free and overall survival of patients with TNBC. We found that mesothelin expression is significantly more frequent in TNBC than in non-TNBC (36% vs 16%, respectively; p = 0.0006), and is significantly correlated with immunoreactivity for basal keratins, but not for EGFR. Mesothelin-positive and mesothelin-negative TNBC were not significantly different by patients’ race, tumor size, histologic grade, tumor subtype, lymphovascular invasion and lymph node metastases. Patients with mesothelin-positive TNBC were older than patients with mesothelin-negative TNBC, developed more distant metastases with a shorter interval, and had significantly lower overall and disease-free survival. Based on our results, patients with mesothelin-positive TNBC could benefit from mesothelin-targeted therapies.

Atypical Ductal Hyperplasia Bordering on Ductal Carcinoma In Situ Interobserver Variability and Outcomes in 105 Cases

Background. The clinical implications of the diagnosis of atypical ductal hyperplasia (ADH) and ductal carcinoma in situ (DCIS) are very different. Yet there are “borderline” breast lesions that have characteristics of both ADH and DCIS. We examined interobserver diagnostic variability for such lesions and correlated pathologic features of the lesions with clinical outcomes. Methods. We identified all cases of borderline ADH/DCIS lesions treated at our center from 1997 to 2010. Five specialized breast pathologists blinded to clinical outcomes independently reviewed all available slides from each case and were instructed to classify each as benign, ADH, or DCIS. A majority diagnosis (MajDx) was defined as a diagnosis agreed upon by ≥3 pathologists. Results. A total of 105 women with borderline ADH/DCIS and slides available for review were identified. The MajDx was ADH in 84 (80%), and DCIS in 18 (17%). There were split diagnoses in 3 (3%). MajDx of DCIS correlated significantly with lesion size and nuclear grade. There was diagnostic agreement by all 5 pathologists in 30% of cases, 4 pathologists in 42%, and 3 pathologists in 25%. At a median follow-up of 37 months, 4 (3.8%) patients developed subsequent ipsilateral breast carcinoma (2 invasive, 2 DCIS); all 4 cases had MajDx of ADH. Conclusions. Borderline ADH/DCIS represents an entity for which reproducible categorization as ADH or DCIS cannot be achieved. Furthermore, histologic features of borderline lesions resulting in MajDx of ADH vs. DCIS are not prognostic for risk of subsequent breast carcinoma.

Impact of the 2013 ASCO/CAP HER2 revised guidelines on HER2 results in breast core biopsies with invasive breast carcinoma: a retrospective study.

Human epidermal growth factor receptor 2 (HER2) is overexpressed in up to 15–20% of invasive breast carcinomas and has both prognostic and predictive value [1–5]. In 2013, ASCO/CAP revised the HER2 guideline recommendations and interpretation criteria for both immunohistochemistry (IHC) and fluorescent in situ hybridization (FISH) assays. Most recent studies that have investigated the potential impact of the 2013 ASCO/CAP Guidelines on HER2 rates [6–10] are hampered by weaknesses in study design that either introduce bias or limit the generalizability of their results. In most of these series, HER2 FISH analysis was performed as a reflex test on cases in which HER2 was initially equivocal by IHC. Using this sample population introduces selection bias skewed towards greater numbers of HER2 amplified tumors, and limits the ability to understand how the revised ASCO/CAP Guidelines would impact the majority of patients with breast cancer whose tumors demonstrate low HER2 expression by IHC. The inclusion of multiple specimen types (breast core biopsies, primary breast resections, and metastases) in their analyses introduces selection bias due to overrepresentation of high stage, biologically aggressive, and HER2 positive tumors. Typically, these studies included both in-house and referred cases. Combining multiple specimen types and including cases from multiple sources (in-house and consult cases) causes potential analytical bias related to pre-analytical variables, such as heterogeneity in intra-laboratory tissue preparation, and differences in fixation and cold ischemic times between specimen types. Furthermore, the inclusion of both biopsies and excision specimens limits the ability to examine the efficacy of repeat testing as proffered by the 2013 ASCO/CAP guidelines in the resolution equivocal HER2 status. Prior to the 2013 ASCO/CAP HER2 Guidelines, the incidence of HER2 equivocal breast cancer cases at our institution (1 %) [11] and others (3.6 %) was low [12]. In comparison to the 2007 ASCO/CAP HER2 Guidelines, the 2013 ASCO/ CAP HER2 Guidelines generally lowered the threshold for all diagnostic categories for both HER2 IHC and FISH [13, 14]. As a result, the revised guidelines have the potential to recategorize the HER2 status of some patients, and increase the rate of bothHER2 positive and equivocal results. To assess the impact of the 2013 ASCO/CAP HER2 Guidelines on the HER2 rates in breast carcinoma diagnosed at our center, we conducted a retrospective study of a consecutive series of breast core needle biopsies with a diagnosis of invasive breast cancer performed at our institution comparing HER2 results during two intervals: a pre-revision cohort under the 2007 ASCO/CAP HER2 Guidelines (2007 Guideline Cohort), and a post-revision cohort under the 2013 ASCO/CAP HER2 Guidelines (2013 Guideline Cohort). HER2 IHC and FISH were performed on all biopsies. Changes in HER2 positive, equivocal, and negative rates for HER2 IHC, HER2 FISH, and overall HER2 status (taking into consideration the results * Gary H. Tozbikian Gary.Tozbikian@osumc.edu

Breast cancer biomarkers before and after neoadjuvant chemotherapy: does repeat testing impact therapeutic management?

In patients treated with neoadjuvant chemotherapy (NAC), there is no consensus on retesting biomarkers within the excision specimen. Our aim was to investigate the clinical relevance of biomarker changes post-NAC at a large tertiary medical center. A retrospective search was performed to identify cases from 2012 to 2015 with needle biopsy-confirmed invasive breast carcinoma treated with NAC and subsequent excision containing residual invasive tumor. Biomarkers (estrogen receptor [ER], progesterone receptor [PR], and HER2/neu [HER2]) were performed on all pre-NAC biopsies. One hundred fifty-four NAC-treated cases were identified in which 83 (54%) had repeat testing of at least 1 biomarker on the surgical specimen. Twenty-five (30%) of 83 repeated cases demonstrated changes in pre-NAC biopsy versus post-NAC resection biomarker status. There was no impact of age or grade on biomarker status changes. Tumors that were triple negative at biopsy were more likely to remain triple negative. Clinically relevant changes were identified including the following: (1) ER negative to ER positive, 2 (3%) of 75; (2) PR negative to PR positive with ER negative both pre- and post-NAC, 2 (3%) of 73; and (3) HER2 negative to positive, 1 (1%) of 77. Four of 5 of the changes led to modifications of the adjuvant treatment regimen, including the addition of adjuvant tamoxifen, anastrazole, or trastuzumab. In summary, post-NAC biomarker repeat testing in patients with breast cancer impacts therapeutic management in a small subset of patients and therefore, repeat testing may be considered for patients that are hormone receptor and/or HER2 negative before NAC.

Stabilization of bone marrow infiltration by metastatic breast cancer with continuous doxorubicin

Complete bone marrow infiltration with profound pancytopenia is very uncommon in breast cancer. Bone marrow metastasis can frequently occur following development of metastatic breast cancer. However, bone marrow failure as the herald of this disease is not typically seen. Very limited data exists as to the safest and most efficacious manner to treat patients with profound pancytopenia due to metastatic solid tumor involvement. In this case, the patient’s thrombocytopenia was particularly worrisome, requiring daily platelet transfusions. There was also concern that cytotoxic chemotherapy would exacerbate the patient’s thrombocytopenia and increase bleeding risk. The patient’s dramatic response to chemotherapy with full platelet recovery is also highly unusual. For our patient, continuous doxorubicin successfully “unpacked” the bone marrow despite a low baseline platelet level, and without increasing the need for more frequent platelet transfusion or risk of catastrophic bleeding. Given the rarity of this presentation, it is currently unknown if the majority of similar patients experience near full recovery of hematopoietic function after initiation of appropriate systemic treatment for metastatic disease.

Using the Modified Magee Equation to Identify Patients Unlikely to Benefit From the 21-Gene Recurrence Score Assay (Oncotype DX Assay)

Objectives This study aimed to compare a modified Magee equation with Oncotype DX (Genomic Health, Redwood City, CA) recurrence score (RS) and identify patients who are unlikely to benefit from Oncotype DX. Methods Magee equation RS was calculated in 438 cases and correlated with Oncotype DX RS. Results The Pearson correlation coefficient ( r ) for the Magee equation and Oncotype DX RS was 0.6645 ( P  < .00001), and the overall agreement was 66.4%. All cases (11.6%) with a Magee equation RS greater than 30 or 11 or less had been correctly predicted to have either high Oncotype DX RS or low Oncotype DX RS, respectively. Conclusions The modified Magee equation is able to identify up to 12% patients who are unlikely to benefit from Oncotype DX testing. Using the modified Magee equation RS on these patients would be an alternative to Oncotype DX, leading to cost savings.

Optimized generation of high-resolution phantom images using cGAN: Application to quantification of Ki67 breast cancer images

In pathology, Immunohistochemical staining (IHC) of tissue sections is regularly used to diagnose and grade malignant tumors. Typically, IHC stain interpretation is rendered by a trained pathologist using a manual method, which consists of counting each positively- and negatively-stained cell under a microscope. The manual enumeration suffers from poor reproducibility even in the hands of expert pathologists. To facilitate this process, we propose a novel method to create artificial datasets with the known ground truth which allows us to analyze the recall, precision, accuracy, and intra- and inter-observer variability in a systematic manner, enabling us to compare different computer analysis approaches. Our method employs a conditional Generative Adversarial Network that uses a database of Ki67 stained tissues of breast cancer patients to generate synthetic digital slides. Our experiments show that synthetic images are indistinguishable from real images. Six readers (three pathologists and three image analysts) tried to differentiate 15 real from 15 synthetic images and the probability that the average reader would be able to correctly classify an image as synthetic or real more than 50% of the time was only 44.7%.

Stromal PTEN determines mammary epithelial response to radiotherapy

The importance of the tumor–associated stroma in cancer progression is clear. However, it remains uncertain whether early events in the stroma are capable of initiating breast tumorigenesis. Here, we show that in the mammary glands of non-tumor bearing mice, stromal-specific phosphatase and tensin homolog (Pten) deletion invokes radiation-induced genomic instability in neighboring epithelium. In these animals, a single dose of whole-body radiation causes focal mammary lobuloalveolar hyperplasia through paracrine epidermal growth factor receptor (EGFR) activation, and EGFR inhibition abrogates these cellular changes. By analyzing human tissue, we discover that stromal PTEN is lost in a subset of normal breast samples obtained from reduction mammoplasty, and is predictive of recurrence in breast cancer patients. Combined, these data indicate that diagnostic or therapeutic chest radiation may predispose patients with decreased stromal PTEN expression to secondary breast cancer, and that prophylactic EGFR inhibition may reduce this risk.The tumor microenvironment influences tumor progression. Here the authors show that lack of stromal PTEN phosphatase induces DNA repair defects in the neighboring mammary gland epithelial cells via hyperactivation of EGF-receptor signaling, resulting in higher radiation-induced DNA damage and hyperplasia.

HER2 equivocal breast cancer that is positive by alternative probe HER2 FISH are classified as HER2 negative by Oncotype DX

In breast cancer, human epidermal growth factor receptor 2 (HER2) status is determined by immunohistochemistry (IHC) and/or in situ hybridization. Oncotype DX also reports HER2 status by an rt‐PCR‐based assay. Assay concordance between IHC and fluorescent in situ hybridization (FISH) (including alternative probe HER2 FISH) vs Oncotype DX HER2 rt‐PCR has not been described in the post‐2013 American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) HER2 Guideline revision setting. We performed a retrospective review of HER2 equivocal invasive breast carcinoma from 2014 to 2016 with the Oncotype DX HER2 result. Fifteen patients with HER2 equivocal invasive breast cancer had Oncotype DX performed. Of these, 13 underwent alternative probe HER2 FISH yielding 4 negative, 6 equivocal and 3 positive results. All 15 cases were classified as HER2 negative by Oncotype DX rt‐PCR, including the three cases which were positive by alternative probe HER2 FISH, yielding a discordance rate for Oncotype DX rt‐PCR HER2 of 20% (3/15). All three patients with HER2‐positive breast cancer on the basis of alternative probe HER2 FISH received anti‐HER2‐targeted therapy. Treatment decisions based on HER2 status should utilize the IHC/FISH result as Oncotype DX results may incorrectly disqualify some patients from being eligible for anti‐HER2 therapy based on the current 2013 ASCO/CAP HER2 Guidelines.

Relationship between the Ki67 index and its area based approximation in breast cancer

BackgroundThe Ki67 Index has been extensively studied as a prognostic biomarker in breast cancer. However, its clinical adoption is largely hampered by the lack of a standardized method to assess Ki67 that limits inter-laboratory reproducibility. It is important to standardize the computation of the Ki67 Index before it can be effectively used in clincial practice.MethodIn this study, we develop a systematic approach towards standardization of the Ki67 Index. We first create the ground truth consisting of tumor positive and tumor negative nuclei by registering adjacent breast tissue sections stained with Ki67 and H&E. The registration is followed by segmentation of positive and negative nuclei within tumor regions from Ki67 images. The true Ki67 Index is then approximated with a linear model of the area of positive to the total area of tumor nuclei.ResultsWhen tested on 75 images of Ki67 stained breast cancer biopsies, the proposed method resulted in an average root mean square error of 3.34. In comparison, an expert pathologist resulted in an average root mean square error of 9.98 and an existing automated approach produced an average root mean square error of 5.64.ConclusionsWe show that it is possible to approximate the true Ki67 Index accurately without detecting individual nuclei and also statically demonstrate the weaknesses of commonly adopted approaches that use both tumor and non-tumor regions together while compensating for the latter with higher order approximations.