Multi-institutional Assessment of Pathologist Scoring HER2 Immunohistochemistry.

The HercepTest was approved 20+ years ago as the companion diagnostic test for trastuzumab in human epidermal growth factor 2 (HER2) or ERBB2 gene-amplified/overexpressing breast cancers. Subsequent HER2 immunohistochemistry (IHC) assays followed, including the now most common Ventana 4B5 assay. Although this IHC assay has become the clinical standard, its reliability, reproducibility, and accuracy have largely been approved and accepted on the basis of concordance among small numbers of pathologists without validation in a real-world setting. In this study, we evaluated the concordance and interrater reliability of scoring HER2 IHC in 170 breast cancer biopsies by 18 breast cancer-specialized pathologists from 15 institutions. We used the Observers Needed to Evaluate Subjective Tests method to determine the plateau of concordance and the minimum number of pathologists needed to estimate interrater agreement values for large numbers of raters, as seen in the real-world setting. We report substantial discordance within the intermediate categories (<1% agreement for 1+ and 3.6% agreement for 2+) in the 4-category HER2 IHC scoring system. The discordance within the IHC 0 cases is also substantial with an overall percent agreement (OPA) of only 25% and poor interrater reliability metrics (0.49 Fleiss' kappa, 0.55 intraclass correlation coefficient). This discordance can be partially reduced by using a 3-category system (28.8% vs 46.5% OPA for 4-category and 3-category scoring systems, respectively). Observers Needed to Evaluate Subjective Tests plots suggest that the OPA for the task of determining a HER2 IHC score 0 from not 0 plateaus statistically around 59.4% at 10 raters. Conversely, at the task of scoring HER2 IHC as 3+ or not 3+ pathologists' concordance was much higher with an OPA that plateaus at 87.1% with 6 raters. This suggests that legacy HER2 IHC remains valuable for finding the patients in whom the ERBB2 gene is amplified but unacceptably discordant in assigning HER2-low or HER2-negative status for the emerging HER2-low therapies.

Determination of the number of observers needed to evaluate a subjective test and its application in two PD-L1 studies.

In pathological studies, subjective assays, especially companion diagnostic tests, can dramatically affect treatment of cancer. Binary diagnostic test results (ie, positive vs negative) may vary between pathologists or observers who read the tumor slides. Some tests have clearly defined criteria resulting in highly concordant outcomes, even with minimal training. Other tests are more challenging. Observers may achieve poor concordance even with training. While there are many statistically rigorous methods for measuring concordance between observers, we are unaware of a method that can identify how many observers are needed to determine whether a test can reach an acceptable concordance, if at all. Here we introduce a statistical approach to the assessment of test performance when the test is read by multiple observers, as would occur in the real world. By plotting the number of observers against the estimated overall agreement proportion, we can obtain a curve that plateaus to the average observer concordance. Diagnostic tests that are well-defined and easily judged show high concordance and plateau with few interobserver comparisons. More challenging tests do not plateau until many interobserver comparisons are made, and typically reach a lower plateau or even 0. We further propose a statistical test of whether the overall agreement proportion will drop to 0 with a large number of pathologists. The proposed analytical framework can be used to evaluate the difficulty in the interpretation of pathological test criteria and platforms, and to determine how pathology-based subjective tests will perform in the real world. The method could also be used outside of pathology, where concordance of a diagnosis or decision point relies on the subjective application of multiple criteria. We apply this method in two recent PD-L1 studies to test whether the curve of overall agreement proportion will converge to 0 and determine the minimal sufficient number of observers required to estimate the concordance plateau of their reads.

HER-2/neu-positive breast cancer neoadjuvant chemotherapy response after implementation of 2018 ASCO/CAP focused update.

Human Epidermal Growth Factor Receptor 2 (HER2), a routinely tested breast cancer marker, is associated with worse prognosis yet increased sensitivity to targeted neoadjuvant therapy (NAT) in breast cancer patients. The presence of HER2 in breast carcinoma can be detected with either immunohistochemistry (IHC) or in situ hybridization (ISH). In this study, we examine the relationship between clinicopathological features, HER2 detection method (IHC vs ISH), and prognostic outcomes in NAT-treated HER2-positive breast cancer patients. We included 99 HER2-positive patients from three academic institutions following 2018 HER2 testing updates and conducted a retrospective correlational study. Seventy-one (72%) were HER2-positive by IHC and 28 (28%) were positive following reflexive ISH. Multivariate analysis showed biomarker status to be significantly associated with pathologic complete response (pCR) (p = 0.003), Residual Cancer Burden (RCB) (p = 0.007), and tumor size downstaging (p = 0.002) and HER2 detection method of IHC to be significantly associated with pCR (p = 0.05), RCB (p = 0.004), and nodal downstaging (p= 0.03). In conclusion, HER2 detection method and biomarker subtype allow for further prognostic stratification of HER2-positive patients when 2018 American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) guideline updates are applied.

Characteristics and Long-Term Risk of Breast Angiosarcoma.

BACKGROUND: Angiosarcoma of the breast is rare and aggressive. It can occur as a de novo tumor or secondary to breast cancer treatment. The purpose of this study is to analyze differences between patients with primary and secondary angiosarcoma of the breast and investigate potential risk factors for its development. PATIENTS AND METHODS: The Surveillance, Epidemiology, and End Results program of the National Cancer Institute database was queried to identify patients with angiosarcoma of the breast, trunk, shoulder, and upper arm. The population-based incidence was analyzed. Primary and secondary angiosarcoma cases were identified and compared. Breast cancer characteristics of secondary angiosarcoma patients were compared with all breast cancer patients in the database who did not develop angiosarcoma. RESULTS: Overall, 904 patients were included, and 65.4% were secondary angiosarcomas. These patients had worse survival, were older, more likely to be White, more likely to have regionally advanced disease, and had angiosarcoma tumors of higher pathologic grade. Independent factors associated with development of secondary angiosarcoma among breast cancer patients included White race, older age, invasive tumor, lymph node removal, lumpectomy, radiation treatment, and left-sided tumor. Although the mean time to develop angiosarcoma after breast cancer diagnosis was 8.2 years, the risk continues to increase up to 30 years after breast cancer treatment. CONCLUSION: Angiosarcoma is rare but increasing in incidence. Secondary angiosarcomas are more common and exhibit more aggressive behavior. Several factors for angiosarcoma after breast cancer treatment could be identified, which may help us counsel and identify patients at risk.

Prospective multi-institutional evaluation of pathologist assessment of PD-L1 assays for patient selection in triple negative breast cancer.

The US Food and Drug Administration (FDA) approved the PD-L1 immunohistochemical assay, SP142, as a companion test to determine eligibility for atezolizumab therapy in patients with advanced triple negative breast cancer (TNBC) but data in lung cancer studies suggest the assay suffers from poor reproducibility. We sought to evaluate reproducibility and concordance in PD-L1 scoring across multiple pathologists. Full TNBC sections were stained with SP142 and SP263 assays and interpreted for percentage (%) immune cell (IC) staining by 19 pathologists from 14 academic institutions. Proportion of PD-L1 positive cases (defined as ≥1% IC) was determined for each assay as well as concordance across observers. We utilized a new method we call Observers Needed to Evaluate Subjective Tests (ONEST) to determine the minimum number of evaluators needed to estimate concordance between large numbers of readers, as occurs in the real-world setting. PD-L1 was interpreted as positive with the SP142 assay in an average 58% of cases compared with 78% with SP263 (p < 0.0001). IC positive continuous scores ranged from 1 to 95% (mean = 20%) and 1 to 90% (mean = 10%) for SP263 and SP142, respectively. With SP142, 26 cases (38%) showed complete two category (<1% vs. ≥1%) concordance; with SP263, 38 cases (50%) showed complete agreement. The intraclass correlation coefficient (ICC) for two category scoring of SP263 and SP142 was 0.513 and 0.560. ONEST plots showed decreasing overall percent agreement (OPA) as observer number increased, reaching a low plateau of 0.46 at ten observers for SP263 and 0.41 at eight observers for SP142. IC scoring with both assays showed poor reproducibility across multiple pathologists with ONEST analysis suggesting more than half of pathologists will disagree about IC scores. This could lead to many patients either receiving atezolizumab when they are unlikely to benefit, or not receiving atezolizumab when they may benefit.

Association between low estrogen receptor positive breast cancer and staining performance.

Estrogen receptor (ER) expression in breast carcinomas, determined by immunohistochemistry, indicates statistically significant benefit to endocrine therapy in patients with tumors expressing ER in ≥1% of tumor cells. Rare cases with low ER expression (1-10%) lead to the dilemma of treating these tumors as ER positive or negative. We hypothesize that low ER positive result from poor staining performance and that we may detect this artefact by assessing the average dynamic range of normal ducts adjacent to low ER positive tumors. Using quantitative tools, we compare the dynamic range of normal background ER expression in patients with low (1-10%) ER tumors to dynamic range of ER expression in normal epithelium from control patient populations, to determine if low ER cases are accompanied by decreased dynamic range. Low ER cases were infrequent (1% of invasive breast carcinomas). Twenty-one cases with low ER staining and two control cohorts, including a tissue microarray (TMA) of 10 benign breast sections and a group of 34 control breast carcinomas (reported as ER negative or >10% ER positive) with normal background epithelium, were digitally scanned. QuPath was utilized to quantify ER staining for each cell as the mean optical density of nuclear DAB staining. The dynamic range of ER expression in normal epithelium surrounding low ER tumors was significantly lower (range 2-240, median 16.5) than that of the benign epithelium in the control tumors (range 3-475, median 30.8; p < 0.001) and benign TMA sections (range 38-212, median 114; p < 0.001) suggesting inconsistent stainer performance.

Interobserver variability in breast carcinoma grading results in prognostic stage differences.

The AJCC Cancer Staging Manual 8th edition included tumor grade in the pathologic prognostic stage for breast carcinomas. Due to the known subjectivity of tumor grading, we aimed to assess the degree of interobserver agreement for invasive carcinoma grade among pathologists and determine its effect on pathologic prognostic stage. One hundred consecutive cases of invasive stage II carcinomas were independently graded twice, with an 4-week intervening wash-out period, by 6 breast pathologists utilizing established Nottingham grading criteria. Inter- and intra-observer variability was determined for overall grade and for each of the 3 scoring components. Interobserver variability was good to very good (κ range = 0.582-0.850) with even better intra-observer variability (mean κ = 0.766). Tubule score was the most reproducible element (κ = 0.588). Complete concordance was reached in 54 cases and 58 cases in rounds 1 and 2 respectively. In round 1 this resulted in different pathologic prognostic stage in only 25 of discordant cases, 18 of which were stage IA versus IB. In conclusion, grading agreement between pathologists was good to very good and discordant grades resulted in small changes to pathologic prognostic stage.

Differences in immunohistochemistry utilization by general and breast subspecialty pathologists at a large academic institution.

OBJECTIVES: Immunohistochemistry (IHC) can be a useful adjunct in diagnostic breast pathology, but best practices have not been clearly established. We sought to compare the patterns of p63 utilization between general pathologists (GP) and subspecialized breast pathologists (BP), analyze diagnostic discrepancy rates, and identify types of lesions requiring immunohistochemistry. METHODS: The pathology database was searched over 6-year period to identify breast needle core biopsy cases utilizing p63 and subsequent excision results. RESULTS: P63 was ordered more frequently by BP (2.3% of cores) compared to GP (1.1% of cores, p = 0.0005). The most frequent utilization of p63 by GP for benign lesions (44.0%) followed by invasive carcinomas (36.0%) while BP most frequently ordered p63 on invasive carcinomas (49.5%) and DCIS (26.6%). CONCLUSIONS: While IHC use may be thought to be most helpful to those with less experience or knowledge in breast pathology, these results suggest that utilization is increased with subspecialty training.

Can tumor-associated macrophages in ductal carcinoma in situ on biopsy predict invasive carcinoma on excision?

Recent trials have explored surveillance of ductal carcinoma in situ (DCIS) without complete excision, but it is difficult to fully exclude an associated, unsampled invasive focus. Tumor microenvironment, including tumor-associated macrophages, may play a role in the transition from in situ to invasive carcinoma, and the presence of CD163-positive cells with DCIS has been associated with increased risk of progression to invasive carcinoma. We aimed to evaluate the role of DCIS-associated CD163-positive cells on biopsy in predicting associated invasion on excision. Immunohistochemistry for CD163 was performed on 57 total biopsy cases of DCIS of low (n = 13), intermediate (n = 21), and high (n = 23) nuclear grade, 27 (47%) of which showed invasion on the subsequent excision specimen. Positive intratumoral and stromal cells were quantified independently by 2 observers based on the percentage of cells staining. Intratumoral CD163 scores ranged from 0 to 2 (mean, 0.7). Stromal CD163 scores ranged from 0 to 3 (mean, 1.3). Intratumoral and stromal CD163 levels were not significantly associated with the presence of subsequent invasion when evaluated as a whole group (P = .36 and P = .47) or when subdivided into low (P = .36 and P = .17), intermediate (P = .82 and P = .82), or high (P = .09 and P = .68) nuclear grades. There was no correlation between intratumoral CD163 content and DCIS grade (P = .257). A trend for higher stromal CD163 expression was seen with higher-grade DCIS, although not statistically significant (P = .178). In conclusion, CD163 on breast core biopsy does not help select patients who may safely forgo excision of DCIS.

Feasibility of the Less Is More Approach in Treating Low-Risk Ductal Carcinoma In Situ Diagnosed on Core Needle Biopsy: Ten-Year Review of Ductal Carcinoma In Situ Upgraded to Invasion at Surgery.

CONTEXT: - Ductal carcinoma in situ (DCIS) represents 20% of screen-detected breast cancers. The likelihood that certain types of DCIS are slow growing and may never progress to invasion suggests that our current standards of treating DCIS could result in overtreatment. The LORIS (LOw RISk DCIS) and LORD (LOw Risk DCIS) trials address these concerns by randomizing patients with low-risk DCIS to either active surveillance or conventional treatment. OBJECTIVE: - To determine the upgrade rate of DCIS diagnosed on core needle biopsy to invasive carcinoma at surgery and to evaluate the safety of managing low-risk DCIS with surveillance alone, by characterizing the pathologic and clinical features of upgraded cases and applying criteria of the LORD and LORIS trials to these cases. DESIGN: - A 10-year retrospective analysis of DCIS on core needle biopsy with subsequent surgery. RESULTS: - We identified 1271 cases of DCIS on core needle biopsy: 200 (16%) low grade, 649 (51%) intermediate grade, and 422 (33%) high grade. Of the 1271 cases, we found an 8% upgrade rate to invasive carcinoma (n = 105). Nineteen of the 105 upgraded cases (18%) had positive lymph nodes. Low-grade DCIS was least likely to upgrade to invasion, comprising 10% (10 of 105) of upgraded cases. Three of the 105 upgraded cases (3%) met criteria for the LORD trial, and all were low-grade DCIS on core needle biopsy with favorable biology on follow-up. CONCLUSIONS: - There is a clear risk of upgrade to invasion on follow-up excision; however, applying strict criteria of the LORD trial effectively decreases the likelihood of a missed invasive component or missed aggressive pathologic features.

Intraoperative sentinel lymph node evaluation: Optimizing surgical pathology practices in an era of changing clinical management.

Axillary lymph node status is an independent prognostic indicator in breast cancer. Intraoperative identification of metastatic carcinoma in sentinel lymph nodes may allow for concurrent axillary lymph node dissection at the time of primary tumor excision. A retrospective review of patients undergoing primary breast cancer excision with sentinel lymph node sampling was performed. Sensitivity and specificity of imprint cytology (touch prep) with and without the incorporation of gross evaluation was determined using permanent section results as the gold standard. Five hundred sixteen lymph nodes were analyzed by imprint cytology in 213 patients, and 203 lymph nodes were analyzed in 74 patients incorporating gross examination. Sensitivity and specificity for the detection of macrometastases by touch prep alone were 60% and 99% respectively with 4 patients undergoing same-day axillary dissection for only micrometastatic disease. False negative causes included lack of transfer of malignant cells in 8 cases and misinterpretation of tumor cells in 6 cases. Incorporating gross examination in the modified protocol resulted in reduced sensitivity of 38%, but achieved the desired 100% specificity and positive predictive value. Imprint cytology alone did not reliably distinguish between micro- and macrometastatic disease. Gross assessment combined with imprint cytology allows for improved assessment of volume of axillary disease, but is an insensitive technique.

Protein Acyltransferase DHHC3 Regulates Breast Tumor Growth, Oxidative Stress, and Senescence.

DHHC-type protein acyltransferases may regulate the localization, stability, and/or activity of their substrates. In this study, we show that the protein palmitoyltransferase DHHC3 is upregulated in malignant and metastatic human breast cancer. Elevated expression of DHHC3 correlated with diminished patient survival in breast cancer and six other human cancer types. ZDHHC3 ablation in human MDA-MB-231 mammary tumor cell xenografts reduced the sizes of both the primary tumor and metastatic lung colonies. Gene array data and fluorescence dye assays documented increased oxidative stress and senescence in ZDHHC3-ablated cells. ZDHHC3-ablated tumors also showed enhanced recruitment of innate immune cells (antitumor macrophages, natural killer cells) associated with clearance of senescent tumors. These antitumor effects were reversed upon reconstitution with wild-type, but not enzyme-active site-deficient DHHC3. Concomitant ablation of the upregulated oxidative stress protein TXNIP substantially negated the effects of ZDHHC3 depletion on oxidative stress and senescence. Diminished DHHC3-dependent palmitoylation of ERGIC3 protein likely played a key role in TXNIP upregulation. In conclusion, DHHC3-mediated protein palmitoylation supports breast tumor growth by modulating cellular oxidative stress and senescence. Cancer Res; 77(24); 6880-90. ©2017 AACR.

Is tumor cellularity in primary invasive breast carcinoma of prognostic significance?

Many methods of analysis to predict survival of invasive mammary carcinoma in the post-neoadjuvant setting utilize tumor cellularity alone or in combination with other tumor features. The goal of this study was to evaluate the prognostic value of tumor cellularity in primary non-treated carcinoma. We used 366 cases of invasive breast carcinoma to determine invasive tumor cellularity (%) by reviewing a representative excisional tumor section and correlated this with breast cancer recurrence (BCR) and overall mortality (OM). Mean patient age was 58 years (range, 21-91) and median follow-up was 87 months (range, 0.7-165). Of the cases, 25% were Nottingham grades I, 41% grade II, and 32% grade III. The Nottingham Prognostic Index (NPI) ranged from 2.06 to 6.8 (mean 3.93). Estrogen receptor was positive in 66% and negative in 25% of cases. Cellularity ranged from 2 to 99% (mean 47.6%). The OM hazard ratio increased by 1.73 for every unit increase in NPI (P < 0.00005; 95% confidence interval 1.45-2.05) The BCR hazard ratio increased by 2.011 for every unit increase in NPI BCR (P < 0.00005; 95% confidence interval 1.62-2.50). Cellularity, unadjusted for other covariates, was not significantly associated with either OM or BCR. When adjusted for NPI, cellularity still showed no significant relation to OM or BCR. The same analysis performed on estrogen receptor-positive and estrogen receptor-negative subgroups continued to show no relation between cellularity and OM or BCR. In conclusion, despite its utility in the neoadjuvant setting, we were unable to show that cellularity is predictive of survival in primary breast carcinomas.

Reporting the greatest linear extent of ductal carcinoma in situ on needle core biopsy.

Ductal carcinoma in situ (DCIS) of the breast is staged as pTis regardless of size; however, extent of DCIS correlates with local recurrence rates and likelihood of close or positive margins. As a result, DCIS extent influences patient management and is an important element in the College of American Pathologists tumor summary checklist for excision specimens. There are no recommendations regarding routine reporting of DCIS extent on needle core biopsy material, and to our knowledge, no systematic studies have evaluated the impact of reporting this in biopsy material. Consecutive cases of DCIS performed or reviewed at our institution were identified by pathology report search over a 7-year period. The greatest linear extent of DCIS on core biopsy was compared with the estimated extent in the excision. Of 241 total cases, there were 157 (65%) cases in which the DCIS extent on biopsy was smaller, 13 (5%) cases in which the sizes were equal, and 70 (29%) cases in which the biopsy size was greater, including 30 (12%) with no residual tumor on excision. Mean extent was greater on excision than on core biopsy (16.0 versus 5.7 mm; P < .0001); however, the opposite was seen when only small tumors (≤ 10 mm final size) were considered (4.5 versus. 3.6 mm; P = .0161). There was strong linear correlation (r = 0.9761; P < .0001) between the size change (excision size minus biopsy size) and final pathologic size. For accurate tumor summary checklist completion, DCIS extent should be reported for needle biopsy material, particularly in the setting of small tumors.

The clinical use of a P63/cytokeratin7/18/cytokeratin5/14 antibody cocktail in diagnostic breast pathology.

An antibody cocktail directed against p63, cytokeratin (CK)5/14, and CK7/18 is reported to be useful in distinguishing noninvasive from invasive breast lesions and for the characterization of intraductal epithelial proliferations. However, limited studies evaluate its use in clinical practice. A retrospective review of breast material at a university medical center identified cases that were immunostained with the above antibody cocktail. Additional p63 immunostaining alone was performed to further determine the utility of the antibody cocktail in the evaluation of invasion. Of 50 breast cases identified, the antibody cocktail was used to confirm or exclude invasion in 44 (88%). Twenty-two (50%) of these had easily identifiable p63/CK5/14-positive myoepithelial cells, whereas the remainder lacked such staining, confirming the diagnosis of invasive carcinoma. In 27 cases with available diagnostic material for additional p63 immunostaining, the cocktail better highlighted myoepithelial cells by staining nuclei and cytoplasm. Easier identification of invasion was also facilitated by CK7/18 expression in invasive foci, especially those composed of single cells. Ten cases were immunostained to help determine the nature of an intraductal proliferation. The cocktail demonstrated a mosaic staining pattern of both CK7/18- and CK5/14-positive epithelial cells in 3 (30%) cases consistent with usual hyperplasia; homogenous CK7/18 expression in the remaining cases supported the diagnosis of atypical ductal hyperplasia or carcinoma in situ. In summary, the p63/CK7/18/CK5/14 cocktail stain appears to be a useful tool in diagnostic breast pathology, in the evaluation of possible invasion, particularly in the setting of minute foci of invasion as well as in epithelial proliferations.

MRI-guided breast needle core biopsies: pathologic features of newly diagnosed malignancies.

Magnetic resonance imaging (MRI) of the breast is used for select groups of patients. MRI-guided breast core needle biopsies performed over a 3-year period were retrospectively reviewed to determine the incidence and types of cancers found and to correlate the cancers with the MRI findings and the indication for the study. Patients were stratified based on indication for MRI examination including, evaluation of disease extent in patients with current ipsilateral carcinoma, surveillance for recurrence of prior ipsilateral carcinoma, as a problem-solving method and for screening high-risk patients. The high-risk screening group included those with family history (with or without germline mutations), prior chest wall radiation, and contralateral breast carcinoma (current or prior). Four-hundred and forty-five biopsies were performed on 386 patients. The majority of biopsies (79%) were benign. Biopsies demonstrating ductal carcinoma in situ (DCIS) and invasive carcinoma were more likely to present as nonmass-like and mass-forming enhancements respectively, but with only 52% specificity. The highest rate of malignancy (44%) was seen in the least frequently biopsied patient group (n = 25), those with prior ipsilateral carcinoma. Conversely, the most frequently biopsied group (n = 283), the high-risk screening group, demonstrated the lowest malignancy rate (16%). Within this group, most malignant cases were invasive carcinomas (n = 27), 67% of which were small (≤1 cm), well or moderately differentiated with a good prognostic receptor profile (estrogen receptor positive, human epidermal growth factor receptor 2 negative), and lacked nodal macrometastases. The remaining malignant cases in the high-risk screening group were DCIS with or without microinvasion (n = 18), 78% of which demonstrated high nuclear grade. Overall, enhancement pattern did not correlate with the likelihood of or type of malignancy. The most common types of carcinomas identified by screening were small estrogen receptor positive invasive tumors and high grade DCIS.

The predictive ability of a CK5/p63/CK8/18 antibody cocktail in stratifying breast papillary lesions on needle biopsy: an algorithmic approach works best.

OBJECTIVES: Immunohistochemical markers have been shown to assist in the stratification of breast papillary lesions. We evaluated the ability of different cytokeratin (CK) and p63 expression profiles on needle biopsy specimens to predict excision diagnoses. METHODS: A CK5/p63/CK8/18 antibody cocktail was applied to 58 needle biopsy specimens (32 papillomas, 7 atypical papillomas, 19 papillary carcinomas on excision). RESULTS: p63 expression was greater in papillomas than in atypical papillomas (P = .044) and papillary carcinomas (P< .0001). Papillary carcinomas and atypical papillomas showed greater CK8/18 expression (and conversely less CK5 expression) than did papillomas (P < .0001). Negative or focal p63 expression was 96% sensitive for diagnosing any atypical lesion (atypical papilloma or papillary carcinoma) on excision, whereas CK8/18 predominant expression (≥80% cells) was 100% sensitive. In contrast, the sensitivity of the original diagnosis was only 81%. The greatest accuracy for the diagnosis of atypical papillary lesions (95%) was achieved when both p63 and cytokeratins were used in combination in an algorithmic fashion. This method also correctly identified all cases that had papillary carcinoma (100% sensitivity) on excision. CONCLUSIONS: Although a single stain or combination cannot independently stratify papillary lesions, a CK5/p63/CK8/18 antibody cocktail is a useful adjunct to morphology for evaluating breast papillary lesions in needle biopsy specimens.

Interobserver concordance in implementing the 2010 ASCO/CAP recommendations for reporting ER in breast carcinomas: a demonstration of the difficulties of consistently reporting low levels of ER expression by manual quantification.

OBJECTIVES: Endocrine therapy reduces recurrence risk by 30% to 50% in estrogen receptor (ER)-positive breast cancer. The ER-positive threshold recommended by the American Society of Clinical Oncology/College of American Pathologists is 1% based on studies using the ER-6F11 antibody. ER-SP1 antibody has a higher sensitivity and is more widely used. METHODS: We report interobserver concordance manually measuring ER in 264 breast cancers using ER-SP1 and 1D5 and 2 scoring methods (H-score and Allred score). RESULTS: With both antibodies, 3% to 4% of cases have a low level of ER expression (1%-10%), more than previously reported (<1%). We find a high level of paired observer concordance with both antibodies and scoring methods (κ = 0.892-0.943) with no significant difference with method of scoring. Despite excellent concordance, positive/negative discordance was almost 5% among 3 observers using either antibody, an underappreciated clinically significant rate. CONCLUSIONS: Discordance overwhelmingly reflected differing opinions recording the proportion of tumor cells positive with low levels of expression (<10% staining; 12/13 cases).

Evaluation of dual immunohistochemistry and chromogenic in situ hybridization for HER2 on a single section.

The evaluation of HER2 status in invasive breast carcinoma can be performed by multiple methods. We assessed the feasibility of performing 2 of these, chromogenic in situ hybridization (CISH) and immunohistochemical staining, on single tissue sections of breast carcinoma. During assay development, sequential performance of immunohistochemical staining after CISH resulted in weaker HER2 expression than that obtained when immunohistochemical staining was performed alone; this was ameliorated by increased antibody incubation time. Performance of both techniques in a combined/hybrid protocol resulted in HER2 protein expression and gene signals identical to those produced by the individual techniques performed alone. Prospective validation of these dual staining protocols in 31 cases of breast carcinoma resulted in 100% concordance with results of CISH when performed alone, but was still associated with a reduced immunohistochemical signal in some cases. Although further testing is needed, we conclude that performance of both immunohistochemical staining and CISH on a single section is possible and could allow for direct "cell-by-cell" comparison of HER2 signals and potentially offer a more economical and real-time method for ongoing validation of HER2 testing.