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Invasive micropapillary breast carcinoma (IMPC) is a rare breast cancer subtype characterized by small tumor cell clusters with loss of stromal attachment, an inside-out growth appearance, and lymphotropism. IMPC is associated with high incidence of lymphovascular invasion (LVI) and lymph node metastasis. Activated Wnt signaling has been implicated in the metastasis of other aggressive breast tumors, including triple-negative and basal-like carcinomas. In this study, we tested whether activated Wnt signaling could be detected in IMPC. Upon ligand binding, the central mediator of the Wnt pathway, β-catenin, accumulates in the cytosol and translocates to the nucleus where it forms a complex with lymphoid enhancer-binding factor 1 (LEF1) to regulate transcription. We performed immunostaining for β-catenin and LEF1 on a well-annotated cohort of 40 breast tumors and nodal metastases displaying micropapillary histopathology. Strong nuclear accumulation of β-catenin was not observed, however a dim cytosolic and/or nuclear accumulation of β-catenin was sometimes seen in IMPC and this expression pattern was significantly associated with nodal metastasis. β-catenin expression correlated with the upregulation of LEF1 in IMPC. LEF1 expression was detected in 26 of 40 (65%) cases and was specifically enriched at the invasive front of the tumor and in tumor clusters undergoing LVI. Detection of LEF1 expression in the primary tumor was associated with an increased rate of LVI, lymph node metastasis, and disease relapse. LEF1 and β-catenin expression levels were significantly higher in metastases compared with primary tumors. In summary, this study demonstrates an association between the upregulation of β-catenin/LEF1 and the metastasis of IMPC.

Objectives Clinical and demographic features of cystic neutrophilic granulomatous mastitis (CNGM) have not been fully explored due to the rarity of the disease. Herein we studied clinicopathologic characteristics of CNGM in a sizable hospital-based cohort. Methods A case-control study was performed to compare clinicopathologic characteristics between patients with CNGM and granulomatous mastitis other than CNGM and between CNGM with and without Corynebacterium identification. Results Cases of CNGM (n = 31) and non-CNGM (n = 30) were included. Compared with the non-CNGM group, patients with CNGM were statistically significantly younger (median age: 38 vs 43 years), were less likely to be smokers (9% vs 40%), were more likely to have a painful lesion (97% vs 77%) or a larger mass-like lesion (median size: 4.6 vs 1.9 cm), and tended to have a higher Breast Imaging Reporting and Data System score in radiologic studies (score ≥4: 81% vs 53%), positive Corynebacterium identification results (36% vs 0%), and a longer resolving time (12 vs 6 months; all P values for above comparisons Conclusions Our study further demonstrated that CNGM is a unique infectious disease with distinct clinicopathologic features.

COVID-19 has been associated with several adverse pregnancy outcomes, including perinatal loss. Differential effects of COVID-19 in a twin pregnancy may provide unique insights into virus-placental interactions. We present a case of perinatal loss of a female fetus with survival of the male co-twin in a pregnancy complicated by COVID-19 and premature delivery.Viral detection methods recommended by the NICHD task force were used to identify SARS-CoV-2 and its viral receptors in the placentas and fetal tissue (Antoun et al., 2020) [1] RESULTS: Compared with the surviving twin, we found a more severe intervillous necrosis and a relatively low detection of ACE2 membranous expression in the syncytiotrophoblasts of the female twin that succumbed.The interactions of SARS-CoV-2 and ACE2 at the maternal fetal interface within the placenta may play a significant role in perinatal loss, and the effects of fetal sex and gestational age at time of infection need to be explored further.

Context.— Breast carcinoma grade, as determined by the Nottingham Grading System (NGS), is an important criterion for determining prognosis. The NGS is based on 3 parameters: tubule formation (TF), nuclear pleomorphism (NP), and mitotic count (MC). The advent of digital pathology and artificial intelligence (AI) have increased interest in virtual microscopy using digital whole slide imaging (WSI) more broadly. Objective.— To compare concordance in breast carcinoma grading between AI and a multi-institutional group of breast pathologists using digital WSI. Design.— We have developed an automated NGS framework using deep learning. Six pathologists and AI independently reviewed a digitally scanned slide from 137 invasive carcinomas and assigned a grade based on scoring of the TF, NP, and MC. Results.— Interobserver agreement for the pathologists and AI for overall grade was moderate (κ = 0.471). Agreement was good (κ = 0.681), moderate (κ = 0.442), and fair (κ = 0.368) for grades 1, 3, and 2, respectively. Observer pair concordance for AI and individual pathologists ranged from fair to good (κ = 0.313–0.606). Perfect agreement was observed in 25 cases (27.4%). Interobserver agreement for the individual components was best for TF (κ = 0.471 each) followed by NP (κ = 0.342) and was worst for MC (κ = 0.233). There were no observed differences in concordance amongst pathologists alone versus pathologists + AI. Conclusions.— Ours is the first study comparing concordance in breast carcinoma grading between a multi-institutional group of pathologists using virtual microscopy to a newly developed WSI AI methodology. Using explainable methods, AI demonstrated similar concordance to pathologists alone.

Neaodjuvant chemotherapy is used to treat high risk triple-negative breast cancer (TNBC). Residual cancer burden (RCB) is used to predict risk of relapse after neoadjuvant chemotherapy (NAC); however, it cannot predict disease recurrence with certainty. EZH2 is a targetable oncogenic protein overexpressed in TNBC and associated with metastasis and stem cell expansion. We quantified EZH2 protein expression in TNBC before NAC to examine potential utility as a predictive and prognostic biomarker.We retrospectively identified 63 patients with localized TNBC treated with NAC. We quantified EZH2 nuclear expression in pretherapy biopsies using a score which included intensity and percent of positive cells at each intensity. EZH2 expression was evaluated as a continuous variable and dichotomized at a score of 210. Logistic regression analysis was used to determine association between EZH2 expression and RCB, tumor-infiltrating lymphocytes, clinicopathologic features and disease-free survival.There was no significant association between EZH2 score and posttreatment RCB class evaluated as a continuous variable (P=0.831) or dichotomized at 210 (P=0.546). On multivariable logistic regression, adjusted for covariates including RCB, EZH2210 was associated with development of metastasis (odds ratio=14.35, 95% confidence interval: 2.69-76.66; P=0.002). Logistic regression was run with EZH2 scores as a continuous variable and increased EZH2 score was associated with metastasis (odds ratio=1.10, 95% confidence interval: 1.00-1.03; P=0.047).In our study of TNBC treated with NAC, high EZH2 expression in pretherapy core biopsies was significantly associated with metastatic recurrence independent of RCB. The potential value of EZH2 as a biomarker to improve stratification of outcome after NAC should be explored further.

Breast carcinoma grading is an important prognostic feature recently incorporated into the AJCC Cancer Staging Manual. There is increased interest in applying virtual microscopy (VM) using digital whole slide imaging (WSI) more broadly. Little is known regarding concordance in grading using VM and how such variability might affect AJCC prognostic staging (PS). We evaluated interobserver variability amongst a multi-institutional group of breast pathologists using digital WSI and how discrepancies in grading would affect PS. A digitally scanned slide from 143 invasive carcinomas was independently reviewed by 6 pathologists and assigned grades based on established criteria for tubule formation (TF), nuclear pleomorphism (NP), and mitotic count (MC). Statistical analysis was performed. Interobserver agreement for grade was moderate (κ = 0.497). Agreement was fair (κ = 0.375), moderate (κ = 0.491), and good (κ = 0.705) for grades 2, 3, and 1, respectively. Observer pair concordance ranged from fair to good (κ = 0.354-0.684) Perfect agreement was observed in 43 cases (30%). Interobserver agreement for the individual components was best for TF (κ = 0.503) and worst for MC (κ = 0.281). Seventeen of 86 (19.8%) discrepant cases would have resulted in changes in PS and discrepancies most frequently resulted in a PS change from IA to IB (n = 9). For two of these nine cases, Oncotype DX results would have led to a PS of 1A regardless of grade. Using VM, a multi-institutional cohort of pathologists showed moderate concordance for breast cancer grading, similar to studies using light microscopy. Agreement was the best at the extremes of grade and for evaluation of TF. Whether the higher variability noted for MC is a consequence of VM grading warrants further investigation. Discordance in grading infrequently leads to clinically meaningful changes in the prognostic stage.

We assessed associations between PD-L1 protein expression, RS, tumor grade, and stromal tumor-infiltrating lymphocyte (TIL) count in early-stage ER + cancers.FFPE tissue blocks of 213 patients with RS in 2012-2017 were identified. PD-L1 immunohistochemistry was performed with SP142 assay, cases with ≥ 1% tumor-infiltrating immune cell positivity in the tumor area were considered PD-L1 + . TIL scores were determined following the international TIL counting guidelines. PD-L1 expression positivity rates were compared across RS ( 11, 11-25, 25) and TIL categories ( 10%, 10-29%, 30%), and tumor grade using Wilcoxon and Chi-square tests. Multivariate analysis was performed using logistic regression.PD-L1 and TIL results were available for 201 and 203 patients. Overall, 53% of cases were PD-L1 +. PD-L1 expression was higher among cases with RS 25, versus RS 11 (p = 0.00019) and RS 11-25 (p = 0.0017). PD-L1 positivity also correlated with TIL score, tumor grade, and tumor size. Among cancers with TIL 30%, 92% were PD-L1 + versus 44% PD-L1 + among TIL 10% (p = 2.8 × 10PD-L1 expression is significantly more frequent and higher in larger tumors (T2, T3), grade 3 cancers, and in cancers with RS 25. PD-L1 expression also correlates with TIL score.

Triple-negative breast cancers (TNBCs) represent approximately 12-17% of all breast cancers and have distinctively aggressive clinical courses. Because routine biomarkers for breast cancer do not apply for TNBCs, it is essential to find novel prognostic markers and potential targets for therapeutic agents. p16 and SOX10 are emerging biomarkers with relatively unexplored expressions in TNBCs. We present an analysis of the expression of p16 and SOX10 in combination with that of androgen receptor (AR) and cytokeratin (CK) 5/6 in TNBCs. In addition, we used tissue microarrays (TMAs) to compare frequencies of p16 and SOX10 between TNBCs and non-TNBCs. Fifty-six TNBC samples with clinical data were stained immunohistochemically with p16, SOX10, AR, and CK5/6. Fifty-four cases (96.4%) were invasive ductal carcinoma, not otherwise specified, and 46 cases (82.1%) were Nottingham histologic grade 3. The majority of TNBC cases were positive for p16 (n = 44; 78.6%) and SOX10 (n = 48; 85.7%). AR was positive in 15 cases (26.8%). CK5/6 was positive in 24 cases (42.9%), which were classified as basal-like breast cancer (BLBC) subtype. The frequencies of p16 and SOX10 expression in BLBC and non-BLBC subtypes did not reveal significant statistical difference in a separate analysis. Using archived TNBC and non-TNBC TMAs, we observed that 56% of TNBC cases were positive for p16 compared with 16% of non-TNBC cases (p-value0.0001). SOX10 was positive in 80% of TNBC cases compared with 35% of non-TNBC cases (p-value0.0001). A significant correlation was observed between p16 and SOX10 coexpression in TNBC cases (n = 56/80, p = 0.02) but not in non-TNBC cases (n = 23/348; p = 0.626). In conclusion, p16 and SOX10 are frequently expressed in TNBC, regardless of CK5/6 expression. Furthermore, p16 and SOX10 are often coexpressed in TNBCs compared with non-TNBCs.

Introduction: Automated image analysis provides an objective, quantitative, and reproducible method of measurement of biomarkers. Image quantification is particularly well suited for the analysis of tissue microarrays which has played a major pivotal role in the rapid assessment of molecular biomarkers. Data acquired from grinding up bulk tissue samples miss spatial information regarding cellular localization; therefore, methods that allow for spatial cell phenotyping at high resolution have proven to be valuable in many biomarker discovery assays. Here, we focus our attention on breast cancer as an example of a tumor type that has benefited from quantitative biomarker studies using tissue microarray format.Areas covered: The history of immunofluorescence and immunohistochemistry and the current status of these techniques, including multiplexing technologies (spectral and non-spectral) and image analysis software will be addressed. Finally, we will turn our attention to studies that have provided proof-of-principle evidence that have been impacted from the use of these techniques.Expert opinion: Assessment of prognostic and predictive biomarkers on tissue sections and TMA using Quantitative immunohistochemistry is an important advancement in the investigation of biologic markers. The challenges in standardization of quantitative technologies for accurate assessment are required for adoption into routine clinical practice.

IMPORTANCE: Trastuzumab deruxtecan (T-DXd) has shown efficacy in patients with breast cancer with ERBB2 immunohistochemistry (IHC) scores of 1+ or 2+ but not 0 as read in central pathology laboratories. The drug is currently being tested in large randomized clinical trials with registration intent for this patient population. OBJECTIVE: To determine the suitability of the current standard ERBB2 IHC assays to select patients with low ERBB2 positivity for treatment with T-DXd. DESIGN AND SETTING: Assessment of data from College of American Pathologists surveys and assessment of analytic data from a Yale University–based study of concordance of 18 pathologists reading 170 breast cancer biopsies. RESULTS: The total survey data set included scores over 2 years from 1391 to 1452 laboratories of 40 ERBB2 cores from each laboratory (20 cores twice a year for a total of 80). College of American Pathologists surveys show that 19% of cases read by the laboratories generate results with less than or equal to 70% concordance for IHC ERBB2 score 0 vs 1+. When 18 pathologists read the scanned slides from a selected set of breast cancer biopsies using a 4-point scale, there was only 26% concordance between 0 and 1+ compared with 58% concordance between 2+ and 3+. CONCLUSIONS AND RELEVANCE: In this study using a current standard ERBB2 IHC assay, the scoring accuracy for ERBB2 IHC in the low range (0 and 1+) was poor. This inaccuracy in the real world could lead to misassignment of many patients for treatment with T-DXd.

Summary 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.

Objectives Recent studies have shown that trichorhinophalangeal syndrome type 1 (TRPS1) is a sensitive and specific marker that shows positive staining in breast carcinoma. We conducted this study to evaluate the role of TRPS1 immunohistochemistry (IHC) in differentiating breast primary vs tumors from other primary sites in malignant pleural effusion cytology specimens (MPECSs). Methods We selected 61 MPECS cases with cell block material available to analyze TRPS1 IHC staining. Of these 61 cases, 38 cases were metastatic carcinoma (MC) from a breast primary. We primarily selected MC cases confirmed as breast origin based on GATA binding protein 3 IHC positivity, except in two of the cases. The remaining 23 MPECS cases were from various primary sites, including urothelial (n = 6), Müllerian (n = 6), lung adenocarcinoma (n = 6), malignant melanoma (MM; n = 3), and squamous cell carcinoma (SqCC; n = 2). Results TRPS1 expression was observed in 35 (92%) of 38 MCs of breast origin. The staining intensity was variable, with 18 (47%) cases showing strong nuclear expression. In comparison, no TRPS1 expression was seen in any cases of urothelial carcinoma, MM, and SqCC. However, four of six Müllerian MC cases demonstrated TRPS1 expression. Conclusions TRPS1 is a new marker that can be used in an IHC panel to investigate breast origin in MPECS.

Invasive apocrine carcinoma is a rare breast cancer that is frequently triple negative. Little is known about the characteristics of its molecular subtypes. We compared the incidence, demographics, and clinicopathologic features of this cancer with non-apocrine carcinomas stratified by molecular subtype.Women with invasive apocrine cancer were retrospectively identified from the Surveillance, Epidemiology, and End Results (SEER) database. Clinicopathologic and demographic features were compared with non-apocrine carcinomas, both overall using data from 2004 to 2017 and stratified by molecular subtypes using data from 2010 to 2017. The life table method was used to determine the 7-year breast cancer-specific survival.Compared with non-apocrine cancers, apocrine cancers presented at a younger age, with larger, higher-grade tumors that were much more likely to be triple negative (50% vs. 11%) or human epidermal growth factor receptor 2 (HER2)-positive (28% vs. 15%) and less likely to be luminal (22% vs. 74%); however, the 7-year survival was the same at 85%. The characteristics varied dramatically by molecular type. Compared with non-apocrine triple-negative, apocrine triple-negative patients were less likely to be African American and were much older, with smaller, lower-grade tumors and much better survival (86% vs. 74%). In contrast, compared with luminal non-apocrine, apocrine luminal patients had larger, higher-grade tumors and worse survival (79% vs. 89%).Invasive apocrine carcinomas have more aggressive features than non-apocrine carcinomas but the breast cancer-specific survival is the same. Half of these apocrine tumors are triple negative but these have more favorable features and much better survival than non-apocrine triple-negative cancers.

During breast cancer staging, histological evaluation of axillary sentinel lymph nodes (SLN) is usually straightforward. However, the exact characterization of a small epithelial deposit in an SLN can be challenging, especially during the frozen section examination. We report the first case of endosalpingiosis involving bilateral axillary lymph nodes. We review published literature on axillary endosalpingiosis and discuss the differential diagnosis of small epithelial deposits in an axillary SLN. Pathologists should consider benign epithelial rests and displaced epithelium as differential diagnoses for the microscopic epithelial nodal deposit, especially during the frozen section examination.

Background Prior studies have shown the presence of immunohistochemical staining for the SARS-CoV-2 spike protein (SP) in endothelial cells and eccrine epithelium of acral perniosis classified as "COVID toes." Yet, other studies have been unable to detect SARS-CoV-2 RNA in skin biopsies of "COVID toes" by reverse-transcriptase polymerase chain reaction testing. Objective In order to address these apparently conflicting findings, we compared detection of SARS-CoV-2 SP, through RNA in situ hybridization (ISH) vs immunohistochemistry (IHC), in skin biopsies of acral perniotic lesions presenting during the COVID-19 pandemic. Results Three of six cases showed positive immunohistochemical labeling of endothelial cells, with one of three cases with sufficient depth also having labeling of eccrine glands, using an anti-SP SARS-CoV-2 antibody. These three cases positive with IHC were negative for SP by RNA ISH. Conclusion While the gold standard for detection of SARS-CoV-2 in tissue sections has yet to be determined, the detection of SARS-CoV-2 SP alone without spike RNA suggests that cleaved SP may be present in cutaneous endothelial cells and eccrine epithelium, providing a potential pathogenetic mechanism of COVID-19 endotheliitis.

Circulating tumor cells (CTC) and more recently, CTC clusters are implicated as a fundamental mechanism by which tumor cells break away from the primary site and travel to distant sites. Enumeration of CTC and CTC clusters represents a new approach to prognosis, prediction, and response to therapy in patients with early and metastatic breast cancer. Several recent studies have shown the predictive importance of monitoring CTCs levels in progression-free and overall survival in breast cancer patients. This review will focus on CTC enumeration and characterization in breast cancers.We will provide a historical perspective and clinical background of CTC detection in peripheral blood. The current methodologies for studying CTCs and newer technologies for CTC detection will be reviewed together with the current state of the art of CTCs as a biomarker in risk stratification and prognostication in breast cancers.Currently, there is an FDA approved CTC assessment method for clinical use. While CTC enumeration, is a marker for prognostication and survival, molecular characterization of CTC, may be more accurate in monitoring response to treatment due to tumor heterogeneity rather than the tumor phenotype at the primary or metastatic sites.

BACKGROUND: The effects of the novel coronavirus disease 2019 (COVID-19) in pregnancy remain relatively unknown. We present a case of second trimester pregnancy with symptomatic COVID-19 complicated by severe preeclampsia and placental abruption. METHODS: We analyzed the placenta for the presence of severe acute respiratory syndrome coronavirus 2 (SARS–CoV-2) through molecular and immunohistochemical assays and by and electron microscopy and measured the maternal antibody response in the blood to this infection. RESULTS: SARS–CoV-2 localized predominantly to syncytiotrophoblast cells at the materno-fetal interface of the placenta. Histological examination of the placenta revealed a dense macrophage infiltrate, but no evidence for the vasculopathy typically associated with preeclampsia. CONCLUSION: This case demonstrates SARS–CoV-2 invasion of the placenta, highlighting the potential for severe morbidity among pregnant women with COVID-19. FUNDING: Beatrice Kleinberg Neuwirth Fund and Fast Grant Emergent Ventures funding from the Mercatus Center at George Mason University. The funding bodies did not have roles in the design of the study or data collection, analysis, and interpretation and played no role in writing the manuscript.

Background Little is known about how the immune microenvironment of breast cancer evolves during disease progression. Patients and methods We compared tumor infiltrating lymphocyte (TIL) count, programmed death-ligand 1 (PD-L1) protein expression by immunohistochemistry and mRNA levels of 730 immune-related genes using Nanostring technology in primary and metastatic cancer samples. Results TIL counts and PD-L1 positivity were significantly lower in metastases. Immune cell metagenes corresponding to CD8, T-helper, T-reg, Cytotoxic T, Dendritic and Mastoid cells, and expression of 13 of 29 immuno-oncology therapeutic targets in clinical development including PD1, PD-L1, and CTLA4 were significantly lower in metastases. There was also coordinated down regulation of chemoattractant ligand/receptor pairs (CCL19/CCR7, CXCL9/CXCR3, IL15/IL15R), interferon regulated genes (STAT1, IRF-1,-4,-7, IFI-27,-35), granzyme/granulysin, MHC class I and immune proteasome (PSMB-8,-9,-10) expression in metastases. Immunotherapy response predictive signatures were also lower. The expression of macrophage markers (CD163, CCL2/CCR2, CSF1/CSFR1, CXCR4/CXCL12), protumorigenic toll-like receptor pathway genes (CD14/TLR-1,-2,-4,-5,-6/MyD88), HLA-E, ecto-nuclease CD73/NT5E and inhibitory complement receptors (CD-59,-55,-46) remained high in metastases and represent potential therapeutic targets. Conclusions Metastatic breast cancers are immunologically more inert than the corresponding primary tumors but some immune-oncology targets and macrophage and angiogenesis signatures show preserved expression and suggest therapeutic combinations for clinical testing.

The SHAVE trial is a prospective trial in which 235 women with stage 0 to 3 disease undergoing partial mastectomy were randomized intraoperatively to either have cavity shave's (CSMs) taken at the time of initial surgery or not. In this study, 9 of the 76 patients (11.8%) with initially negative margins randomized to the “shave” group had occult cancers found in the CSM. Seven of these patients had sufficient primary tumor tissue available for further analysis. These patients were age and stage matched in a 1:2 ratio to other patients in the trial who had no further disease found in their CSM. A tissue microarray was created, stained for E-cadherin, MUC1, and beta-catenin, and evaluated by two independent pathologists (blinded to outcome). There were no significant differences between cases and controls in terms of median invasive tumor size, ductal carcinoma in situ size, volume of initial resection, and volume of CSM. Further, no differences were noted between cases and controls for median (staining intensity x per cent of cells staining) for each marker. Hence, although nearly 12 per cent of breast cancer patients with negative margins will have occult disease, this could not be predicted by primary tumor markers in this study.

KIT Proto-Oncogene Receptor Tyrosine Kinase (KIT) is a transmembrane receptor tyrosine kinase which plays an important role in regulation of cell proliferation, survival and migration. Interestingly, the role of c-KIT in malignant transformation seems to be highly tissue-specific and it can act either as an oncogene or tumor suppressor gene. Here we analyzed the expression of c-KIT in normal breast tissues and tissues from different stages encompassing major steps of breast tumor development. Our study showed, that the c-KIT protein expression is gradually lost during the process of breast tissue transformation. The analysis of previously published datasets revealed that c-KIT expression in breast malignancies was downregulated at mRNA level. Because sequencing studies did not identify any recurrent mutations or copy number alterations, we proposed a potential epigenetic mechanism for the downregulation of c-KIT expression. In-silico analysis of the KIT promoter revealed the presence of CpG islands, therefore we performed bisulfite sequencing of normal breast epithelial tissues as well as breast tumor samples. We found, that KIT promoter is hypermethylated in breast tumors compared to normal breast tissues. Furthermore, treatment of breast cancer cell lines, that lack the expression of c-KIT, with methyltransferase inhibitor 5-Azacytidine (5Aza-2dC) resulted in increased expression of c-KIT mRNA. Collectively, our studies demonstrate that c-KIT expression is epigenetically downregulated during breast epithelium transformation and cancer development via KIT promoter hypermethylation.

Androgen receptor (AR) expression is an emerging prognostic marker that has been observed in 25% to 75% of triple-negative breast cancers (TNBCs) that lack estrogen receptor, progesterone receptor and HER2 overexpression. TNBCs are treated with AR-targeted therapies and standardized evaluation of AR expression may help guide patient management. Basal-like TNBCs are a subgroup of TNBCs defined by positive immunoreactivity for CK5/6 or EGFR that carry a worse prognosis. However, it's unclear whether basal-like TNBCs have a different rate of AR positivity or if AR expression is predictive of disease-free survival (DFS) in this patient group. Here, we examined a total of 185 TNBCs for AR and CK5/6 using tissue microarrays (TMAs). Among all samples, 32% were AR positive using a 1% cutoff, and 24% were AR positive using a 10% cutoff. Ninety (49%) TNBCs were CK5/6 positive with lower intensity of AR expression compared to CK5/6-negative cases. There was no significant difference in pathologic stage, tumor size, histologic grade and type, or lymph node stage after stratifying by AR and CK5/6 positivity. AR-positive TNBCs had better overall survival (OS) and DFS compared to AR-negative TNBCs. In addition, increased AR expression carried a dose-dependent effect on DFS. AR expression, nodal status and tumor size were significant predictors of disease-free and overall survival in a multivariable model adjusted for CK5/6 expression, age, histologic grade, and mitotic rate. In conclusion, our data demonstrated that AR positivity is a favorable prognostic factor for triple-negative breast cancers.

Summary GATA3 is a transcription factor used clinically as a marker of breast or urothelial differentiation. A marker is yet needed to distinguish this in the case of the GATA3-positive tumor of unknown origin. We tested classical markers of breast differentiation and hormonal signaling to see which correlated strongest with GATA3 expression in breast cancer and thus which could help correctly identify breast origin in the case of the GATA3-positive tumor of unknown origin. GATA3, estrogen receptor, progesterone receptor, androgen receptor (AR), HER2, GCDFP15, and mammaglobin expression was intercorrelated in a histologically diverse 259-case breast cancer tissue microarray. We show herein a uniquely high level of correlation between GATA3 and AR expression ( r =0.61; 95% confidence interval 0.52-0.68) that was strongest among lobular carcinomas ( r =1; 95% confidence interval 0.73-1) and stronger than any other correlation studied. Separate AR staining of 10 metastatic GATA3+ carcinomas of urothelial origin and 13 metastatic GATA3+ carcinomas of breast origin showed that strong AR staining (>60% of tumor cells) has a sensitivity of 54% and a specificity of 100% for correctly distinguishing GATA3+ carcinoma of mammary origin from urothelial origin in the metastatic setting. Androgen receptor expression is strongly correlated with GATA3 in breast cancer, particularly in tumors with lobular morphology. Strong AR expression (>60% of tumor cells) is an excellent test to rule out urothelial carcinoma in the GATA3+ metastatic setting (specificity 100%) and will effectively identify breast origin in approximately 50% of cases.

BACKGROUND Oncotype Dx is a genetic test that has been incorporated into the 2017 AJCC breast cancer staging system for ER positive, HER2-negative, lymph node-negative patients to predict the risk of recurrence. Recent data suggest that immunohistochemistry (ER, PR, HER2, and Ki-67) and histologic subtype may identify patients that will not benefit from Oncotype Dx testing. METHODS A total of 371 patients underwent Oncotype Dx testing at our institution from 2012 to 2016. Oncotype recurrence score was categorized as low- (ORS = 0-10), intermediate- (11-25), or high risk (26-100). Invasive carcinomas were categorized based on histologic subtype as "favorable" (mucinous, tubular, cribriform, tubulolobular, and lobular) and "unfavorable" (ductal, mixed ductal and lobular, and micropapillary carcinoma). All cases were estrogen receptor positive and HER2-negative. Clinical and histologic predictors of low-risk ORS were assessed in univariate and multivariate logistic regression. RESULTS A total of 371 patients were categorized by ORS as low risk (n = 85, 22.9%), intermediate risk (n = 244, 65.8%), and high risk (n = 42, 11.3%). The histologic subtypes with the highest percentage of high-risk ORS were invasive micropapillary (n = 4/17, 23.5%), pleomorphic lobular (n = 2/10, 20%), and ductal carcinoma (n = 28/235, 11.9%). Low-grade invasive carcinomas with favorable histology rarely had a high-risk ORS (n = 1/97, 1%). In a simple multivariable model, favorable histologic subtype (OR = 2.39, 95% CI: 1.10 to 5.15, P = 0.026), and histologic grade (OR = 1.76, 95% CI: 1.07 to 2.90, P = 0.025) were the only significant predictors of an ORS less than 11 in estrogen receptor positive, HER2-negative, and lymph node-negative patients. CONCLUSION We question the utility of performing Oncotype Dx in subtypes of invasive carcinoma that are associated with excellent prognosis. We propose that immunohistochemistry for ER, PR, and HER2 is sufficient for patients with low-grade invasive carcinomas and can be used as a surrogate for Oncotype Dx.

The aim of the study was to compare costs associated with excision of routine cavity shave margins (CSM) versus standard partial mastectomy (PM) in patients with breast cancer.Excision of CSM reduces re-excision rates by more than 50%. The economic implications of this is, however, unclear.Between October 21, 2011 and November 25, 2013, 235 women undergoing PM for Stage 0-III breast cancer were randomized to undergo either standard PM ("no shave", n = 116) or have additional CSM taken ("shave", n = 119). Costs from both a payer and a hospital perspective were measured for index surgery and breast cancer surgery-related care through subsequent 90 days.The 2 groups were well-matched in terms of baseline characteristics. Those in the "shave" group had a longer operative time at the initial surgery (median 76 vs 66 min, P0.01), but a lower re-excision rate for positive margins (13/119 = 10.9% vs 32/116 = 27.6%, P0.01). Actual direct hospital costs associated with operating room time ($1315 vs. $1137, P = 0.03) and pathology costs ($1195 vs $795, P0.01) were greater for the initial surgery in patients in the "shave" group. Taking into account the index surgery and the subsequent 90 days, there was no significant difference in cost from either the payer ($10,476 vs $11,219, P = 0.40) or hospital perspective ($5090 vs $5116, P = 0.37) between the "shave" and "no shave" groups.Overall costs were not significantly different between the "shave" and "no shave" groups due to significantly fewer reoperative surgeries in the former.

An accurate biomarker for the follow-up of women positive for human papillomavirus type 16 (HPV16) DNA may improve the efficiency of cervical cancer prevention. Previously, we analyzed all 113 HPV16 CpGs in cervical cytology samples and discovered differential methylation at different stages of premalignancy. In the current study, we identified a methylation biomarker consisting of a panel of 12 HPV16 CpG sites in the E5, L2, and L1 open reading frames, and tested whether it fulfilled three necessary conditions of a prospective biomarker. A total of 33 cytology samples from North American and West African women with all grades of cervical intraepithelial neoplasia (CIN) and invasive cervical cancer (ICC) were analyzed by using DNA bisulfite sequencing. The results showed (i) a highly significant trend for increasing HPV16 biomarker methylation with increasing histologic severity (P < 0.0001), (ii) 100% sensitivity for ICC over a wide range of methylation cutoff scores; 80% detection of CIN3 at cutoff scores up to 39% methylation, and (iii) substantially lower detection of CIN2, from 0% to 71%, depending on the cutoff score. Our results support the prognostic potential of the HPV16 methylation biomarker for the triage to colposcopy of women with HPV16-positive screening tests and, eventually, for the management of women with HPV16-positive CIN2. Cancer Prev Res; 7(5); 526–33. ©2014 AACR.

Ki-67 is a human nuclear protein whose expression is strongly up-regulated in proliferating cells and can be used to determine the growth fraction in clonal cell populations. Although there are some data to suggest that Ki-67 overexpression may be prognostic for endpoints such as survival or postmastectomy recurrence, further elucidation of its prognostic significance is warranted. Specifically after breast conservation therapy (BCT) (defined in this setting as breast-conserving surgery and adjuvant radiation therapy), whether Ki-67 predicts for locoregional recurrence has not been investigated. The purpose of this study was to assess Ki-67 expression in a cohort of early-stage breast cancer patients to determine whether a significant independent association between Ki-67 and locoregional relapse exists.Ki-67 staining was conducted on a tissue microarray of 438 patients previously treated with BCT, and expression was analyzed with clinicopathologic features and outcomes from our database.Ki-67 expression was more prevalent in black patients (37% of black patients vs 17% of white patients, P.01), younger patients (27% of patients aged ≤50 years vs 15% of patients aged50 years, P.01), estrogen receptor (ER)-negative tumors (25% of ER-negative tumors vs 17% of ER-positive tumors, P=.04), human epidermal growth factor receptor 2 (HER2)/neu-positive tumors (35% of HER2-positive tumors vs 18% of HER2-negative tumors, P=.01), and larger tumors (26% of T2 tumors vs 16% of T1 tumors, P=.03). On univariate/multivariate analysis, Ki-67 did not predict for overall survival (74.4% vs 72.6%), cause-specific survival (82.9% vs 82.1%), local relapse-free survival (83.6% vs 88.5%), distant metastasis-free survival (76.1% vs 81.4%), recurrence-free survival (65.5% vs 74.6%), and locoregional recurrence-free survival (81.6% vs 84.7%): P.05 for all.Ki-67 appears to be a surrogate marker for aggressive disease and significantly correlates with known prognostic features such as age, race, hormone receptor status, and HER2 status but independently does not predict for locoregional outcomes after BCT when these other prognostic clinicopathologic features are taken into consideration. The independent associations of Ki-67 with race and age appear to be novel to our study.

Malignant pleural effusion can be a manifestation of many malignancies. Involvement of pleural fluid by metatstatic thyroid carcinoma, though reported, is relatively rare. We present 5 cases of metastatic thyroid carcinoma involving the pleural fluid. The diagnosis of thyroid carcinoma in pleural fluid can be particularly challenging as thyroid transcription factor -1 (TTF-1) which is a marker for carcinoma of thyroid origin is also positive in lung adenocarcinomas (which are more frequently associated with pleural effusions) and thyroglobulin (TG) can often be negative in poorly differentiated/analplastic thyroid carcinomas. In our experience, PAX8 is a particularly useful marker in making the distinction. The diagnosis of metastatic thyroid carcinoma in pleural fluid can be challenging and knowledge of the clinical context and supporting immunohistochemical stains is essential for making the right diagnosis. Diagn. Cytopathol. 2016;44:1085-1089. © 2016 Wiley Periodicals, Inc.

Context.—Our cytology laboratory, like many others, is under pressure to improve quality and provide test results faster while decreasing costs. We sought to address these issues by introducing new technology and lean principles.Objective.—To determine the combined impact of the FocalPoint Guided Screener (GS) Imaging System (BD Diagnostics–TriPath, Burlington, North Carolina) and lean manufacturing principles on the turnaround time (TAT) and productivity of the gynecologic cytology operation.Design.—We established a baseline measure of the TAT for Papanicolaou tests. We then compared that to the performance after implementing the FocalPoint GS Imaging System and lean principles. The latter included value-stream mapping, workflow modification, and a first in–first out policy.Results.—The mean (SD) TAT for Papanicolaou tests before and after the implementation of FocalPoint GS Imaging System and lean principles was 4.38 (1.28) days and 3.20 (1.32) days, respectively. This represented a 27% improvement in the average TAT, which was statistically significant (P < .001). In addition, the productivity of staff improved 17%, as evidenced by the increase in slides screened from 8.85/h to 10.38/h. The false-negative fraction decreased from 1.4% to 0.9%, representing a 36% improvement.Conclusions.—In our laboratory, the implementation of FocalPoint GS Imaging System in conjunction with lean principles resulted in a significant decrease in the average TAT for Papanicolaou tests and a substantial increase in the productivity of cytotechnologists while maintaining the diagnostic quality of gynecologic cytology.

Background: Recently, the Food and Drug Administration approved the use of the location-guided imaging system FocalPoint GS (FPGS), on SurePath Papanicolaou (Pap) tests for primary screening. The objective of the current study was to evaluate the impact of FPGS on the following: distribution of diagnostic categories; rate of high-risk human papillomavirus (HR-HPV)–positive ASC-US cases; and quality control (QC) data before and after FPGS implementation. Methods: A search of the laboratory information system was performed to identify all SurePath Pap tests processed in our laboratory for the first 19 months after FPGS implementation. We also retrieved all SurePath specimens from a 16-month period prior to FPGS implementation to serve as the control. During the period from Janaury 2008 to April 2009, the FocalPoint Slide Profiler was used. Results: Implementation of FPGS resulted in a significantly higher percentage of LSIL and ASC-US interpretations, as well as a significant increase in the detection of candidiasis and bacterial vaginosis. The ASC-to-SIL ratio was 1.4 and 1.9 before and after FPGS implementation, respectively. There was a decrease in the HR-HPV positive rate in ASC-US cases, and a decrease in the estimated false-negative fraction after FPGS implementation. Conclusions: In conclusion, our study seems to demonstrate a favorable performance of FPGS in the routine clinical setting. FPGS may have the potential to be a promising screening tool for gynecologic cytology in a low-risk patient population. Cancer (Cancer Cytopathol) 2012;. © 2011 American Cancer Society.

Purpose Recent misclassification (false negative) incidents have raised awareness concerning limitations of immunohistochemistry (IHC) in assessment of estrogen receptor (ER) in breast cancer. Here we define a new method for standardization of ER measurement and then examine both change in percentage and threshold of intensity (immunoreactivity) to assess sources for test discordance. Methods An assay was developed to quantify ER by using a control tissue microarray (TMA) and a series of cell lines in which ER immunoreactivity was analyzed by quantitative immunoblotting in parallel with the automated quantitative analysis (AQUA) method of quantitative immunofluorescence (QIF). The assay was used to assess the ER protein expression threshold in two independent retrospective cohorts from Yale and was compared with traditional methods. Results Two methods of analysis showed that change in percentage of positive cells from 10% to 1% did not significantly affect the overall number of ER-positive patients. The standardized assay for ER on two Yale TMA cohorts showed that 67.9% and 82.5% of the patients were above the 2-pg/μg immunoreactivity threshold. We found 9.1% and 19.7% of the patients to be QIF-positive/IHC-negative, and 4.0% and 0.4% to be QIF-negative/IHC-positive for a total of 13.1% and 20.1% discrepant cases when compared with pathologists' judgment of threshold. Assessment of survival for both cohorts showed that patients who were QIF-positive/pathologist-negative had outcomes similar to those of patients who had positive results for both assays. Conclusion Assessment of intensity threshold by using a quantitative, standardized assay on two independent cohorts suggests discordance in the 10% to 20% range with current IHC methods, in which patients with discrepant results have prognostic outcomes similar to ER-positive patients with concordant results.

BACKGROUND The role of testing for high-risk human papillomavirus (HR HPV) when triaging women with a cytologic diagnosis of low-grade squamous intraepithelial lesion (LSIL) has not been well established. The objective of the current study was to correlate the status of HR HPV with the incidence of cervical intraepithelial neoplasia 2 and more severe lesions (CIN 2+) on tissue follow-up in women with LSIL. METHODS A total of 1046 women with LSIL and HR HPV testing were identified in the database of a large teaching hospital within a 12-month period. HR HPV testing was performed using the Hybrid Capture 2 assay with 1 relative light unit/cutoff as the cutoff. RESULTS Of the 1046 women with LSIL and concurrent HR HPV testing, 82.3% tested positive for HR HPV, 91.1% of whom were women aged < 30 years and 73% of whom were women aged ≥ 30 years (P < .001). Cytologic and/or histologic follow-up was available in 979 (93.6%) women; 25.5% had negative follow-up, 62.5% were found to have CIN 1 lesions, and 12.0% had CIN 2+ lesions. The sensitivity and negative predictive value of HR HPV status as a marker of CIN 2+ lesions were 98.3% and 98.9%, respectively. The colposcopy rate was 73.3% and 96.9% for women aged ≥ 30 years and women aged < 30 years, respectively (P = .01). CONCLUSIONS Using 1 RLU/CO as the cutoff value, HR HPV testing was found to be highly sensitive for detecting CIN 2+ lesions in women with LSIL. The colposcopy rate was significantly lower in women aged ≥ 30 years compared with women aged < 30 years. Triaging with HR HPV testing may be indicated in women aged ≥ 30 years with LSIL cytology, but not in women aged < 30 years. Cancer (Cancer Cytopathol) 2011;. © 2011 American Cancer Society.

To compare the Affirm VPIII molecular test (Becton Dickinson, Burlington, NC) with morphologic identification used in routine Papanicolaou (Pap) test screening in the detection and identification of Candida species, Trichomonas vaginalis, and Gardnerella vaginalis, we identified 431 cases with a concomitant Pap test and Affirm VPIII assay performed from the archives of a large academic institution. The study population consisted of women ranging in age from 17 to 79 years (mean and median ages, 33 and 31 years, respectively). With a routine Pap test, 60 patients (13.9%) were found to have bacterial vaginosis, 60 (13.9%) candidiasis, and 3 (0.7%) Trichomonas infection. With the Affirm VPIII assay, 183 (42.5%) patients tested positive for G vaginalis, 70 (16.2%) positive for Candida species, and 10 (2.3%) positive for T vaginalis. The differences were statistically significant. The results demonstrate that our patient population had a high incidence of bacterial vaginosis/Candida vaginitis; however, the Affirm VPIII was a more sensitive diagnostic test for the detection and identification of all 3 organisms compared with the Pap test.

Purpose Amplified in breast cancer (AIB1 or SRC-3) is an estrogen receptor coregulatory protein that together with other co-activators like transcription intermediary factor 2 (TIF2) and nuclear receptor co-repressor (NCoR), is implicated in estrogen signaling pathway and estrogen regulated tumor progression. We investigated the prognostic significance of AIB1, TIF2 & NCoR protein expression breast tissue microarray (TMA), and studied the relationship of coregulatory proteins to prognostic biomarkers like estrogen (ER), progesterone (PR) & HER2/neu and between coregulatory proteins. Methods: AIBI, TIF2 & NCoR were studied by fluorescent immunohistochemical staining of a TMA with 670 breast cancer specimens, using AQUA software. Result: Using Cox univariate survival analyses, high AIB1 expression was associated with poor patient outcome (P = 0.002), while no association was noted for TIF2 (P = 0.376) & NCoR (P = 0.12). When subclassified by nodal or ER status, AIB1 was not prognostic in the node positive and ER positive subsets. However, in the ER negative and node negative subsets, high AIB1 expression was associated with poor patient outcome (P = 0.02 and P = 0.007 respectively). AIB1 retained its independent association with survival by multivariate analyses (P = 0.028). There was significant positive correlation between AIB1 and ER and PR status and with other cofators (TIF2 and NCoR) but not with HER2/neu status. Conclusion: High AIB1 expression was predictive of worse overall survival in our study, suggesting that AIB1 may be critical in breast carcinogenesis.

Purpose The variability in scoring of immunohistochemistry, whether a result of true heterogeneity or artifacts in preparation, has led to decreased reliability in companion diagnostics and the recommendation for new standards (eg, the American Society of Clinical Oncology/College of American Pathologists [ASCO-CAP] guidelines). The basis of this problem is the amount of tissue required to be representative of an entire tumor. Because protein expression on tissue microarrays (TMAs) can be rigorously measured and one 0.6-mm spot is equivalent to two to three high-power fields, we used TMAs to assess levels of heterogeneity and to determine optimal representation as a function of outcome. Patients and Methods We analyzed estrogen receptor (ER), progesterone receptor, and human epidermal growth factor receptor 2 (HER-2) expression in two cohorts (n = 676 and n = 152) on a series of four to five separate TMA cores and assessed heterogeneity by linear regression analysis. Minimum, average, and maximum scores were generated for each set, which were then assessed for prognostic and predictive value. Results Each marker shows some heterogeneity, but average r values between 0.7 and 0.8 are seen between TMA spots. Analysis for prognostic value shows that the highest maximum score (of five spots) is the most prognostic for ER, whereas a high HER-2 minimum score is most prognostic for poor outcome and most predictive of response to trastuzumab. Conclusion These results suggest that the representivity required for each biomarker may be a function of its role in tumorigenesis. Furthermore, these results provide scientific basis for the ASCO-CAP guidelines for assessment of HER-2 expression but perhaps suggest that the 30% figure is still too conservative.

BackgroundRoutine resection of cavity shave margins (additional tissue circumferentially around the cavity left by partial mastectomy) may reduce the rates of positive margins (margins positive for tumor) and reexcision among patients undergoing partial mastectomy for breast cancer. MethodsIn this randomized, controlled trial, we assigned, in a 1:1 ratio, 235 patients with breast cancer of stage 0 to III who were undergoing partial mastectomy, with or without resection of selective margins, to have further cavity shave margins resected (shave group) or not to have further cavity shave margins resected (no-shave group). Randomization occurred intraoperatively after surgeons had completed standard partial mastectomy. Positive margins were defined as tumor touching the edge of the specimen that was removed in the case of invasive cancer and tumor that was within 1 mm of the edge of the specimen removed in the case of ductal carcinoma in situ. The rate of positive margins was the primary outcome measure; seco...

BACKGROUND Measures of vascular endothelial growth factor (VEGF) expression in pancreatic cancer typically have been qualitative or semiquantitative. The objective of this study was to use a series of algorithms called AQUA that quantitatively assesses protein expression on tissue microarrays (TMAs) to compare in situ expression of VEGF and its primary receptors, VEGF receptor 1 (FLT-1) and VEGF receptor 1 (FLK-1), on a pancreatic cancer TMA. METHODS TMAs were constructed by arraying 1.5-mm cores from 76 samples of pancreatic adenocarcinoma (1996-2002) that were obtained from the archives of the Yale Department of Pathology. The staining for AQUA was similar to standard immunohistochemistry and involved antigen retrieval and the application of primary antibodies, but with epifluorescence detection. Slides were counterstained with 4′,6-diamidino-2-phenylindole for nuclear visualization and cytokeratin for membrane visualization. The primary antibodies used were VEGF, FLT-1, FLK-1, and cytokeratin. RESULTS Disease stage was highly prognostic for outcome, as expected. Total amounts of VEGF and its receptors were assessed within the tumor mask and were divided into quartiles. Kaplan–Meier survival curves showed that VEGF and FLK-1 were not associated clearly with outcome. However, the expression of FLT-1 was correlated significantly, and the patients who had tumors with the lowest expression FLT-1 levels had the worst survival (P = .0038). In multivariate analysis, FLT-1 expression was an independent prognostic factor for overall survival (P = .0044). CONCLUSIONS VEGF and its 2 principal receptors were expressed to varying degrees in tumors of the pancreas. A significant association was found between low expression of FLT-1 and both poor prognosis and advanced stage, suggesting that tumor expression of this VEGF receptor is a marker of less aggressive disease. Cancer 2006. © 2006 American Cancer Society.

Purpose: The tumor suppressor adhesion molecule E-cadherin is believed to have an anti-invasive role in breast cancer. Lymph node involvement is the best prognostic marker known, yet there is variability in outcome among node-positive patients. We investigated the relationship between E-cadherin expression in primary invasive ductal tumors and corresponding nodal metastases, and determined the prognostic value of E-cadherin expression in node-positive breast cancer. Experimental Design: Membrane E-cadherin expression was studied by immunohistochemical staining of tissue microarrays with fluorescent-labeled antibodies. An objective method of automated quantitative analysis (AQUA) was used. AQUA uses cytokeratin to define pixels as breast cancer (tumor mask) within the array spot, and measures E-cadherin expression using a Cy5-conjugated antibody within the mask. Results: We employed a tissue microarray containing 207 primary and matched nodal metastases suitable for AQUA analysis. There was no significant difference in mean staining intensity between the primary and nodal specimens (P = 0.8). A scattergram was generated which identified a subset of patients (25%) with high E-cadherin expression in nodal metastases, and this top quartile had improved survival (P = 0.028). On univariate analysis, increased E-cadherin expression in nodal metastases was strongly associated with improved survival (P = 0.007), whereas expression in primary tumors was not (P = 0.13). On multivariate analysis, nodal E-cadherin expression retained its independent association with survival, as did tumor size and HER2/neu status. Conclusions: Strong E-cadherin expression in lymph node metastases was highly predictive of improved survival. This suggests that expression of adhesion molecules at metastatic sites portends less aggressive tumor behavior.

Purpose: Functional inactivation of p16 is an early and frequent event in head and neck squamous cell cancers. In this study, we sought to determine whether p16 expression is of prognostic importance in oropharyngeal squamous cell carcinoma. Experimental Design: p16 protein expression was evaluated by immunohistochemistry in a tissue microarray composed of 123 oropharyngeal squamous cell cancers with a mean patient follow-up time of 33 months. Results: p16 overexpression was associated with more advanced Tumor-Node-Metastasis stage and higher histologic grade. Despite this association with unfavorable features, p16 overexpression was associated with decreased 5-year local recurrence rates (11 versus 53%) and increased 5-year disease-free survival (62 versus 19%) and overall survival (60 versus 21%). In multivariate analysis, p16 expression status remained an independent prognostic factor for local recurrence, disease-free survival, and overall survival. Conclusions: In patients with oropharyngeal squamous cell carcinoma, overexpression of p16 as determined by immunohistochemistry is associated with significantly improved prognosis and lower local recurrence rates.

Context.—The new Bethesda System 2001 (TBS 2001) minimized the subclassification of atypical squamous cells of undetermined significance (ASCUS). Objective.—The primary goal of this study was to determine the impact of the new subclassification on the accuracy of Papanicolaou (Pap) test diagnosis by examining the prevalence of human papillomavirus (HPV) DNA in different ASCUS subcategories, as defined by the new TBS 2001 versus the original TBS 1991. The second goal was to identify specific morphologic features of atypical squamous cells that are more frequently associated with HPV detection. Design.—Consecutive cases of ThinPrep Pap tests were retrospectively reviewed by a panel of pathologists to obtain consensus diagnoses. The study group consisted of ASCUS cases; the positive control group consisted of low- and high-grade squamous intraepithelial lesions (LSILs and HSILs, respectively); and the negative control group consisted of cases “negative for intraepithelial lesion or malignancy.” All ASCUS cases were subclassified according to TBS 1991 into the following categories: favor reactive (ASCUS-R), favor LSIL (ASCUS-L), favor HSIL (ASCUS-H), and not otherwise specified (ASCUS-NOS). In a separate review, ASCUS cases were subclassified according to TBS 2001 into the following categories: atypical squamous cells of undetermined significance (ASC-US) and atypical squamous cells, cannot exclude high-grade squamous intraepithelial lesion (ASC-H). Furthermore, morphologic ASCUS subtypes were recorded (atypical mature, immature, parakeratotic, and atrophic cells); in addition, individual morphologic features of atypical cells were recorded. Broad- spectrum HPV DNA amplification and genotyping was performed using short PCR fragment (SPF 10) polymerase chain reaction/Line Probe assays. Results.—In cases classified according to TBS 1991, HPV was detected in 32% of negative, 49% of ASCUS, and 93% of LSIL/HSIL cases. On the second review, using the diagnostic categories of TBS 2001, which eliminated the ASCUS-;R category, the number of ASCUS cases decreased by 45%. The prevalence of HPV DNA in ASCUS cases downgraded to the negative category was 38%, which was not significantly different from HPV prevalence in negative cases as diagnosed under TBS 1991. Furthermore, HPV was detected in 56% of ASC-US and 71% of ASC-H cases. The prevalence of HPV in different morphologic subtypes of ASCUS was not significantly different, and none of the 8 individual morphologic features of atypical cells were more frequently associated with HPV detection. Conclusion.—Elimination of the ASCUS-R category in TBS 2001 resulted in a significant decrease in the number of ASCUS diagnoses. Downgraded cases had a relatively low prevalence of HPV DNA. It is expected that TBS 2001 will increase specificity of the Pap test without compromising its sensitivity.

Sentinel lymph node (SLN) biopsy has emerged during the last few years as a viable option for staging the axilla in the treatment of breast carcinoma. This procedure can potentially identify patients who would be helped by full axillary lymph node dissection (the SLN-positive cases), and those who would not (the SLN-negative cases). Review of the literature confirms the promise of SLN; however, the possible problems in the pathological handling of SLN, including the microscopic misinterpretation of benign structures and "spurious" immunohistochemical staining, need wider recognition.

Studies examining the effects of increased workload on the performance of individual cytotechnologists are limited. Using FocalPoint GS, the performance of 3 cytotechnologists was evaluated. The study consisted of 3 phases. In phase I, cytotechnologists were asked to screen at their usual pace. In phase II, cytotechnologists were asked to screen as fast as possible without feeling that the quality of their work was diminished. In phase III, cytotechnologists were asked to screen at least 15% more than their daily workload from phase II. Productivity was increased by decreasing the percentage of cases that underwent full manual review (from 38% to 19%) and by decreasing the time spent on each slide (from 5.5 min to 3.7 min). Overall, the total abnormal rate decreased by 31.9% from phase I to phase III of the study. In addition, the false-negative fraction increased significantly, from 1% to 6.9%. Our results indicated a negative association between increased cytotechnologist daily workload with FocalPoint GS and CT screening performance. Workloads were increased by decreasing the time spent reviewing 10 fields of view and the percentage of cases that underwent full manual review.

This study compared the performance of Chlamydia trachomatis testing using 2 methods: the BD ProbeTec Chlamydia trachomatis Q(x) Amplified DNA Assay (CTQ) on the BD Viper System with XTR technology (CTQ assay) and the Hybrid Capture (HC) 2 assay. A total of 1,054 Surepath and ThinPrep specimens were tested for C trachomatis nucleic acids using the CTQ assay and the HC2 assay. For positive and discrepant C trachomatis test results, confirmatory test for C trachomatis was performed using a reverse transcriptase-polymerase chain reaction. Of 1,054 liquid-based gynecologic cytology samples tested for C trachomatis using both assays, 1,041 tested negative on both. In 6 (0.57%) samples, findings were discordant. The CTQ assay and the HC2 assay had sensitivity rates of 100% and 66.7%, respectively, with comparable specificity (99.9%). The positive predictive values were 92.3% and 88.9% with the CTQ and HC2 assays, respectively. In this study, the CTQ assay was found to be more sensitive than the HC2 assay in detecting chlamydial infection; the CTQ assay also demonstrated a higher positive predictive value.

The diagnosis of pancreatic endocrine neoplasm (PEN) is often straightforward; however, cytomorphologic variants may impose a diagnostic challenge, particularly in small biopsies such as fine-needle aspiration (FNA). Here we describe prominent fine cytoplasmic vacuoles in three cases of PENs which were initially evaluated by endoscopic ultrasound-guided FNA. The clinicopathologic features and diagnostic pitfalls of this cytomorphologic variant were discussed. Awareness of this rare cytomorphologic feature with application of immunocytochemical studies is crucial for rendering an accurate diagnosis and avoiding diagnostic pitfalls.

Littoral cell angiomas are uncommon primary vascular neoplasms that arise from the sinusoidal lining or littoral cells of the splenic red pulp, and hence are unique to the spleen. We report a case of littoral cell angioma in 34-year-old woman, which was diagnosed by endoscopic ultrasound-guided fine needle aspiration biopsy (EUS-FNAB). The cytologic features of littoral cell angiomas have been described only in three previous case reports, one of which was a bench-top aspirate. In our case, we have utilized the fine-needle aspiration samples obtained by a linear endoscopic ultrasound examination for establishing the diagnosis. The characteristic cytologic features identified on the smears along with immunohistochemical analysis performed on the compact cellblock prepared from the aspirate aided in the confirmation of the diagnosis. We suggest that EUS-FNAB is a safe and reliable method in the diagnosis of vascular lesions of the spleen.

Assessment of key breast cancer tissue biomarkers is often done using nonquantitative methods. We hypothesized that use of continuous analysis of expression with the AQUA method of automated quantitative analysis will provide prognostic information beyond that attainable with conventional methods. A tissue microarray was made from 2123 of 3122 patients accrued to SWOG 9313, in which sequential doxorubicin (A) and cyclophosphamide (C) was compared with combination AC and in which all patients except premenopausal estrogen receptor (ER)-negative patients received tamoxifen. Multiplexed assays of 1) HER2 and estrogen receptor and 2) progesterone receptor (PgR) and p53 were performed on the two slides using the immunofluorescence-based AQUA method of automated quantitative analysis. Both ER and PgR showed unimodal distributions and significantly predicted disease-free survival when tested as continuous variables and adjusted for node status, tumor size, treatment, and menopausal status (P = 0.005 and P < 0.001, respectively). HER2, measured as a continuous variable, showed a biphasic effect on disease-free survival. Both high and low expressers of HER2 have worse outcomes (when low levels are equivalent to that seen in normal breast ducts). In patients who were uniformly treated with AC chemotherapy and tamoxifen (when indicated), both ER and PgR, assessed as continuous variables, were highly prognostic, whereas p53 expression was not. This assay method may provide a new companion diagnostic approach for targeted therapies.

Human papillomavirus (HPV) gene expression is dramatically altered during cervical carcinogenesis. Because dysregulated genes frequently show abnormal patterns of DNA methylation, we hypothesized that comprehensive mapping of the HPV methylomes in cervical samples at different stages of progression would reveal patterns of clinical significance. To test this hypothesis, thirteen HPV16-positive samples were obtained from women undergoing routine cervical cancer screening. Complete methylation data were obtained for 98.7% of the HPV16 CpGs in all samples by bisulfite-sequencing. Most HPV16 CpGs were unmethylated or methylated in only one sample. The other CpGs were methylated at levels ranging from 11% to 100% of the HPV16 copies per sample. The results showed three major patterns and two variants of one pattern. The patterns showed minimal or no methylation (A), low level methylation in the E1 and E6 genes (B), and high level methylation at many CpGs in the E5/L2/L1 region (C). Generally, pattern A was associated with negative cytology, pattern B with low-grade lesions, and pattern C with high-grade lesions. The severity of the cervical lesions was then ranked by the HPV16 DNA methylation patterns and, independently, by the pathologic diagnoses. Statistical analysis of the two rating methods showed highly significant agreement. In conclusion, analysis of the HPV16 DNA methylomes in clinical samples of cervical cells led to the identification of distinct methylation patterns which, after validation in larger studies, could have potential utility as biomarkers of neoplastic cervical progression.

Evidence suggests that breast cancer hormone receptor status varies by etiologic factors, but studies have been inconsistent. In a population-based case-control study in Poland that included 2,386 cases and 2,502 controls, we assessed ER-alpha and PR status of tumors based on clinical records according to etiologic exposure data collected via interview. For 842 cancers, we evaluated ER-alpha, ER-beta, PR and HER2 levels by semiquantitative microscopic scoring of immunostained tissue microarrays and a quantitative immunofluorescence method, automated quantitative analysis (AQUAtrade mark). We related marker levels in tumors to etiologic factors, using standard regression models and novel statistical methods, permitting adjustment for both correlated tumor features and exposures. Results obtained with different assays were generally consistent. Receptor levels varied most significantly with body mass index (BMI), a factor that was inversely related to risk among premenopausal women and directly related to risk among postmenopausal women with larger tumors. After adjustment for correlated markers, exposures and pathologic characteristics, PR and HER2 AQUA levels were inversely related to BMI among premenopausal women (p-trend = 0.01, both comparisons), whereas among postmenopausal women, PR levels were associated directly with BMI (p-trend = 0.002). Among postmenopausal women, analyses demonstrated that BMI was related to an interaction of PR and HER2: odds ratio (OR) = 0.86 (95% CI = 0.69-1.07) for low PR and HER2 expression vs. OR = 1.78 (95% CI = 1.25-2.55) for high expression (p-heterogeneity = 0.001). PR and HER2 levels in breast cancer vary by BMI, suggesting a heterogeneous etiology for tumors related to these markers.

Purpose: A multitude of breast cancer mRNA profiling studies has stratified breast cancer and defined gene sets that correlate with outcome. However, the number of genes used to predict patient outcome or define tumor subtypes by RNA expression studies is variable, nonoverlapping, and generally requires specialized technologies that are beyond those used in the routine pathology laboratory. It would be ideal if the familiarity and streamlined nature of immunohistochemistry could be combined with the rigorously quantitative and highly specific properties of nucleic acid–based analysis to predict patient outcome.Experimental Design: We have used AQUA-based objective quantitative analysis of tissue microarrays toward the goal of discovery of a minimal number of markers with maximal prognostic or predictive value that can be applied to the conventional formalin-fixed, paraffin-embedded tissue section.Results: The minimal discovered multiplexed set of tissue biomarkers was GATA3, NAT1, and estrogen receptor. Genetic algorithms were then applied after division of our cohort into a training set of 223 breast cancer patients to discover a prospectively applicable solution that can define a subset of patients with 5-year survival of 96%. This algorithm was then validated on an internal validation set (n = 223, 5-year survival = 95.8%) and further validated on an independent cohort from Sweden, which showed 5-year survival of 92.7% (n = 149).Conclusions: With further validation, this test has both the familiarity and specificity for widespread use in management of breast cancer. More generally, this work illustrates the potential for multiplexed biomarker discovery on the tissue microarray platform.

Purpose We sought to determine the prevalence of biologically relevant human papillomavirus (HPV) in oropharyngeal squamous cell carcinoma (OSCC). Retinoblastoma (Rb) downregulation by HPV E7 results in p16 upregulation. We hypothesized that p16 overexpression in OSCC defines HPV-induced tumors with favorable prognosis. Methods Using real-time polymerase chain reaction for HPV16, we determined HPV16 viral load in a cohort of 79 OSCCs annotated with long-term patient follow-up. A tissue microarray including these cases was also analyzed for p53, p16, and Rb utilizing in situ quantitative protein expression analysis. Seventy-seven tumors were classified into a three-class model on the basis of p16 expression and HPV-DNA presence: class I, HPV–, p16 low; class II, HPV+, p16 low; and class III, HPV+, p16 high. Results Sixty-one percent of OSCCs were HPV16+; HPV status alone was of no prognostic value for local recurrence and was barely significant for survival times. Overall survival was improved in class III (79%) compared with the other two classes (20% and 18%; P = .0095). Disease-free survival for the same class was 75% versus 15% and 13% (P = .0025). The 5-year local recurrence was 14% in class III versus 45% and 74% (P = .03). Only patients in class III had significantly lower p53 and Rb expression (P = .017 and .001, respectively). Multivariable survival analysis confirmed the prognostic value of the three-class model. Conclusion Using this system for classification, we define the molecular profile of HPV+ OSCC with favorable prognosis, namely HPV+/p16 high (class III). This study defines a novel classification scheme that may have value for patient stratification for clinical trials testing HPV-targeted therapies.

Purpose: This study was designed to provide a comprehensive assessment on the role of β1,6-branched oligosaccharides in the metastasis and outcome of breast carcinoma. Generation of these structures on N-glycans is initiated by β1,6-N-acetylglucosaminyltransferase V and used by both myeloid cells and cancer cells in systemic migration. Experimental Design: Tissue microarrays of >700 tumors (>400 patients; 30-year follow-up data) were stained through lectin histochemistry with leukocytic phytohemagglutinin (LPHA), a selective marker for β1,6-branched oligosaccharides. Node-negative and node-positive primary tumors and patient-matched lymph node metastases were scored by blinded observers. Results: Metastases stained at significantly greater intensities than did the patient-matched primary tumors (P < 0.0001), demonstrating for the first time that the abundance of β1,6-branched oligosaccharides was directly associated with breast carcinoma nodal metastasis. Multivariate analyses revealed that β1,6-branched oligosaccharides in primary tumors were a predictor of poor outcome, most notably in node-negative tumors, where an LPHA staining score of 3+ gave a risk factor of 3.3, independent of tumor size, nuclear grade, or patient age (P = 0.007). Conclusions: The data firmly establish a role for β1,6-N-acetylglucosaminyltransferase V activity and β1,6-branched oligosaccharides in breast carcinoma metastasis, and reemphasize the involvement, although poorly understood, of aberrant glycosylation in tumor progression.