Serous fluid: Metastatic sarcomas, melanoma, and other non-epithelial neoplasms.

While most tumors metastatic to the serous membranes are of epithelial origin, cytologists should be aware that non-epithelial neoplasms can also cause malignant effusions including sarcomas, melanomas, germ cell tumors, and, more rarely, brain tumors. The differential diagnosis of a malignant effusion is accordingly broad, especially for the small round blue cell tumors that includes not only mesenchymal tumors, but also non-mesenchymal tumors, such as neuroblastoma and Wilms tumor. Diagnosing non-epithelial malignancies in effusion specimens based entirely upon their cytomorphologic features is difficult because these neoplasms often exhibit considerable morphological overlap and their cytomorphology can differ from the original tumor. As malignant cells have a tendency to round up in body fluids these non-epithelial neoplasms can therefore mimic reactive mesothelial cells and metastatic adenocarcinoma. The use of ancillary studies including immunostaining, FISH, and molecular studies is thus often critical to reach a definitive diagnosis. This review article will be incorporated finally as one of the chapters in CMAS (CytoJournal Monograph/Atlas Series) #2. It is modified slightly from the chapter by the initial authors in the first edition of Diagnostic Cytopathology of Serous Fluids.

HER2 testing across practices-Have we come to a consensus on the ideal method of testing? A systematic literature review.

Human epidermal growth factor (HER2) is an oncogene that codes for HER2 protein. The gene is amplified, and protein is overexpressed in 20% of patients and carries a poor prognosis. HER2-positive tumors tend to be more aggressive and highly proliferative. In 2006, trastuzumab, a monoclonal antibody targeting HER2, was approved for use with chemotherapy as an adjuvant treatment for women with HER2-positive breast cancer. The drug has shown improved survival rates for women with HER2-positive breast cancer. As promised for survival in HER2-positive patients continues with new research, and as novel drug approvals emerge, HER2 assessment plays a significant role in adjuvant and in metastatic setting. HER2 assays approved by the US Food and Drug administration include immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), and bright field dual in situ hybridization (DISH). Accuracy of HER2 testing across laboratories has an impact on treatment as false-negative testing can deprive the patients of trastuzumab therapy. The current review will focus on the variability of HER2 testing across laboratories and the potential impact on treatment.

Interobserver Reproducibility Among Gynecologic Pathologists in Diagnosing Heterologous Osteosarcomatous Component in Gynecologic Tract Carcinosarcomas.

Distinguishing hyalinized stroma from osteoid production by a heterologous osteosarcomatous component can be challenging in gynecologic tract carcinosarcomas. As heterologous components in a carcinosarcoma may have prognostic and therapeutic implications, it is important that these are recognized. This study examines interobserver reproducibility among gynecologic pathologists in the diagnosis of osteosarcomatous components, and its correlation with expression of the novel antibody SATB2 (marker of osteoblastic differentiation) in these osteosarcomatous foci. Digital H&E images from 20 gynecologic tract carcinosarcomas were reviewed by 22 gynecologic pathologists with a request to determine the presence or absence of an osteosarcomatous component. The 20 preselected cases included areas of classic heterologous osteosarcoma (malignant cells producing osteoid; n=10) and osteosarcoma mimics (malignant cells with admixed nonosteoid matrix; n=10). Interobserver agreement was evaluated and SATB2 scored on all 20 cases and compared with the original diagnoses. Moderate agreement (Fleiss' κ=0.483) was identified for the 22 raters scoring the 20 cases with a median sensitivity of 7/10 and a median specificity of 9/10 for the diagnosis of osteosarcoma. SATB2 showed 100% sensitivity (10/10) and 60% (6/10) specificity in discriminating classic osteosarcoma from osteosarcoma mimics. Utilizing negative SATB2 as a surrogate marker to exclude osteosarcoma, 73% (16/22) of the reviewers would have downgraded at least 1 case to not contain an osteosarcomatous component (range, 1-6 cases, median 1 case). Gynecologic pathologists demonstrate only a moderate level of agreement in the diagnosis of heterologous osteosarcoma based on morphologic grounds. In such instances, a negative SATB2 staining may assist in increasing accuracy in the diagnosis of an osteosarcomatous component.

Ultrasound Guided Core Biopsy versus Fine Needle Aspiration for Evaluation of Axillary Lymphadenopathy in Patients with Breast Cancer.

Rationale and Objectives. To compare the sensitivities of ultrasound guided core biopsy and fine needle aspiration (FNA) for detection of axillary lymph node metastases in patients with a current diagnosis of ipsilateral breast cancer. Materials and Methods. From December 2008 to December 2010, 105 patients with breast cancer and abnormal appearing lymph nodes in the ipsilateral axilla consented to undergo FNA of an axillary node immediately followed by core biopsy of the same node, both with ultrasound guidance. Experienced pathologists evaluated the aspirate cytology without knowledge of the core histology. Cytology and core biopsy results were compared to sentinel node excision or axillary dissection pathology. Sensitivities were compared using McNemar's test. Results. Of 70 patients with axillary node metastases, FNA was positive in 55/70 (78.6%) and core was positive in 61/70 (87.1%) (P = 0.18). The FNA and core results were discordant in 14/70 (20%) patients. Ten cases were FNA negative/core positive. Four cases were FNA positive/core negative. Conclusion. Core biopsy detected six (8.6%) more cases of metastatic lymphadenopathy than FNA but the difference in sensitivities was not statistically significant. Core biopsy should be considered if the node is clearly imaged and readily accessible. FNA is a good alternative when a smaller needle is desired due to node location or other patient factors. This trial is registered with NCT01920139.

Interobserver agreement among pathologists for semiquantitative hormone receptor scoring in breast carcinoma.

The American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) guidelines recommend reporting of hormone receptor test results in a semiquantitative manner. This study used 74 resected estrogen receptor (ER)-positive invasive breast cancers to determine reproducibility of semiquantitative scoring of hormone receptors using the H-score method. Four pathologists independently scored each slide. Agreement among observers was analyzed via Fleiss κ statistics on ER and progesterone receptor (PR) categorical scores. Intraclass correlation coefficient (ICC) was used to estimate the interobserver agreement for ER and PR H-scores on a continuous scale (0-300). There was 100% agreement for categorical ER results (κ = 1) and 97% agreement (κ = 0.823, P < .001) for categorical PR results. For quantitative H-scores, ICC agreement was 0.85 (95% confidence interval [CI] = 0.79-0.90) for ER and 0.87 (95% CI = 0.82-0.92) for PR. Because the H-score provides a continuous measure of tumor hormone receptor content, we suggest universal adoption of this method.

Is PAX2 a reliable marker in differentiating diffuse malignant mesotheliomas of peritoneum from serous carcinomas of müllerian origin?

Diffuse peritoneal malignant mesotheliomas (DPMM) are often disseminated in the peritoneal cavity as multiple nodules including localized masses in the ovaries that are clinically and histologically similar to serous adenocarcinomas of müllerian origin. It is imperative to differentiate these tumors given their diverse responses to chemotherapy and/or radiotherapy. PAX2 gene was recently demonstrated in benign epithelial cells of the female genital tract and in serous carcinomas (SC) of müllerian origin. The aim of our study was to determine if PAX2 can reliably be used in differentiating DPMM from SC. A total of 59 cases to include 25 cases of DPMM and 34 cases of SC were retrieved. All cases were stained with PAX2, Wilm tumor gene 1, calretinin and the results were compared. Our results demonstrate that PAX2 can be used reliably as a Müllerian marker in formulating an efficient panel to differentiate DPMM and SC.

The effect of 96-hour formalin fixation on the immunohistochemical evaluation of estrogen receptor, progesterone receptor, and HER2 expression in invasive breast carcinoma.

We studied the impact of 96 hours of formalin fixation on estrogen receptor (ER), progesterone receptor (PR), and HER2 testing by comparing immunohistochemical results from core biopsy specimens fixed under current American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) guidelines with results for corresponding resection samples fixed for 96 hours. Samples enriched with cases showing weak to moderate receptor expression on core biopsy were included in the study. Cases were scored using ASCO/CAP guidelines. Of the 47 cases, only 1 case (2%) showed a qualitative change in result. However, this change was a positive ER result (H score, 1) on the 96-hour fixed resected sample compared with a negative ER result (H score, 0) for the core biopsy. Minimal changes in semiquantitative H scoring were noted for ER and PR that were likely due to tumor heterogeneity and/or intraobserver variability as the variation occurred in both directions. ER, PR, and HER2 immunohistochemical results should be considered valid for cases fixed up to 96 hours.

Risk of malignancy when microscopic radial scars and microscopic papillomas are found at percutaneous biopsy.

OBJECTIVE: The objective of our study was to assess the incidence of associated malignancy when microscopic radial scars and microscopic intraductal papillomas are encountered at percutaneous biopsy for lesions that otherwise reveal benign histopathology. MATERIALS AND METHODS: A search of the pathology database for the period from December 14, 2006, through December 21, 2009, identified patients with a microscopic radial scar, a microscopic intraductal papilloma, or both at percutaneous biopsy. Patients whose percutaneous biopsy was performed for a lesion that revealed carcinoma or a high-risk pathology result were excluded to avoid confounding bias, as were patients who had only imaging follow-up. Only patients who underwent surgery solely for the study lesion were included. The lesion type that prompted core biopsy, biopsy guidance and device, sample number, and surgical outcomes were recorded. The incidences of benign, high-risk, and malignant pathology findings from surgery were calculated. RESULTS: The search revealed 35 patients (18 microscopic radial scars, 17 microscopic papillomas) who underwent surgery solely for the study lesion. Stereotactic guidance was used for 15 (43%); ultrasound, for 12 (34%); and MRI, for eight (23%). At surgery, 12 patients (34%) had high-risk histopathology results and 23 (66%) had benign results. No study lesions were upgraded to malignancy. CONCLUSION: Our study found no evidence of associated malignancy at surgical excision when microscopic radial scars and microscopic intraductal papillomas were encountered at percutaneous biopsy in patients who otherwise had benign histopathology results; thus, routine imaging follow-up may be performed.

Survival outcomes in endometrial cancer patients are associated with CXCL12 and estrogen receptor expression.

CXCL12 is a chemotactic cytokine that has pro-metastatic functions in several malignancies through interactions with its receptor, CXCR4. CXCL12 is an estrogen-regulated gene, and notably, estrogen is a major risk factor for endometrial cancer (EC) development. As few studies examine concurrent CXCL12, CXCR4, and estrogen receptor (ER) expression in EC patients, we examined this pathway in 199 EC patients with data from the University of Pittsburgh Medical Center Cancer Registry. Immunohistochemistry (IHC) was used to detect CXCR4, CXCL12 and ER protein expression. As CXCR4 expression was positive in all cases, this investigation focused on associations between CXCL12 and ER expression, clinicopathologic factors and survival outcomes using chi-square tests, Kaplan-Meier graphs, and log-rank tests. CXCL12 expression was negative in 63 cases (32%) and positive in 136 cases (68%). Negative CXCL12 expression was borderline significantly associated with metastasis (χ(2) p = 0.07). ER expression was negative in 75 cases (38%) and positive in 124 cases (62%). Positive ER expression was significantly associated with low grade and early stage tumors (χ(2) p < 0.001). CXCL12 and ER were not significantly associated (χ(2) p = 0.11). Positive CXCL12 expression was associated with longer overall survival (OS) (log-rank p = 0.006) and longer recurrence-free survival (RFS) (log-rank p = 0.01) in ER negative patients, but not in ER positive patients. We identified a unique molecular signature associated with better OS and RFS in EC patients. In addition to pathological characteristics of the tumor, expression of CXCL12 and ER may be clinically useful for assigning adjuvant treatment to EC cases.

Prognostic utility of the breast cancer index and comparison to Adjuvant! Online in a clinical case series of early breast cancer.

INTRODUCTION: Breast Cancer Index (BCI) combines two independent biomarkers, HOXB13:IL17BR (H:I) and the 5-gene molecular grade index (MGI), that assess estrogen-mediated signalling and tumor grade, respectively. BCI stratifies early-stage estrogen-receptor positive (ER+), lymph-node negative (LN-) breast cancer patients into three risk groups and provides a continuous assessment of individual risk of distant recurrence. Objectives of the current study were to validate BCI in a clinical case series and to compare the prognostic utility of BCI and Adjuvant!Online (AO). METHODS: Tumor samples from 265 ER+LN- tamoxifen-treated patients were identified from a single academic institution's cancer research registry. The BCI assay was performed and scores were assigned based on a pre-determined risk model. Risk was assessed by BCI and AO and correlated to clinical outcomes in the patient cohort. RESULTS: BCI was a significant predictor of outcome in a cohort of 265 ER+LN- patients (median age: 56-y; median follow-up: 10.3-y), treated with adjuvant tamoxifen alone or tamoxifen with chemotherapy (32%). BCI categorized 55%, 21%, and 24% of patients as low, intermediate and high-risk, respectively. The 10-year rates of distant recurrence were 6.6%, 12.1% and 31.9% and of breast cancer-specific mortality were 3.8%, 3.6% and 22.1% in low, intermediate, and high-risk groups, respectively. In a multivariate analysis including clinicopathological factors, BCI was a significant predictor of distant recurrence (HR for 5-unit increase = 5.32 [CI 2.18-13.01; P = 0.0002]) and breast cancer-specific mortality (HR for a 5-unit increase = 9.60 [CI 3.20-28.80; P < 0.0001]). AO was significantly associated with risk of recurrence. In a separate multivariate analysis, both BCI and AO were significantly predictive of outcome. In a time-dependent (10-y) ROC curve accuracy analysis of recurrence risk, the addition of BCI+AO increased predictive accuracy in all patients from 66% (AO only) to 76% (AO+BCI) and in tamoxifen-only treated patients from 65% to 81%. CONCLUSIONS: This study validates the prognostic performance of BCI in ER+LN- patients. In this characteristically low-risk cohort, BCI classified high versus low-risk groups with ~5-fold difference in 10-year risk of distant recurrence and breast cancer-specific death. BCI and AO are independent predictors with BCI having additive utility beyond standard of care parameters that are encompassed in AO.

Limitations of the criteria used to diagnose histologic endometritis in epidemiologic pelvic inflammatory disease research.

While endometrial neutrophils and plasma cells are criteria used to diagnose histologic endometritis in epidemiologic pelvic inflammatory disease (PID) research, plasma cell misidentification and nonspecificity may limit the accuracy of these criteria. Herein, we examined: (1) the identification of endometrial plasma cells with conventional methyl green pyronin-based methodology versus plasma cell-specific (CD138) immunostaining, (2) the prevalence of endometrial plasma cells among women at low risk for PID, and (3) endometrial leukocyte subpopulations among women diagnosed with acute or chronic histologic endometritis by conventional criteria. We observed an absence of CD138+ cells in 25% of endometrial biopsies in which plasma cells had been identified by conventional methodology, while additional immunohistochemical analyses revealed indistinguishable inflammatory infiltrates among women diagnosed with acute or chronic endometritis by conventional criteria. Among women considered at lower risk for PID development, flow cytometric analyses detected plasma cells in 30% of endometrial biopsy specimens, suggesting that these cells, even when accurately identified, only nonspecifically identify upper genital tract inflammatory processes. Combined, our findings underscore the limitations of the criteria used to diagnose histologic endometritis in PID-related research and suggest that satisfactory understanding of PID pathogenesis, treatment, and prevention is hindered by continued use of these criteria.

Carcinomas of Distal Fallopian Tube and Their Association with Tubal Intraepithelial Carcinoma: Do They Share a Common "Precursor" Lesion? Loss of Heterozygosity and Immunohistochemical Analysis Using PAX 2, WT-1, and P53 Markers.

As the role of distal fallopian tube as organ of serous carcinogenesis is emerging, additional literature on the role of tubal intraepithelial carcinoma (TIC) as a precursor lesion in a subset of primary peritoneal serous carcinomas (PPSC is emerging as well. TIC although fallopian tube in origin can be genetically related to ovarian/peritoneal carcinomas. The role of PAX2 in primary fallopian tube carcinomas (PFTC)/PPSC is yet to be defined. The aim of our study was to understand if the biologic properties of tumors arising in the distal fallopian tube that remain as PFTC are different when they seed on to the peritoneal surface (PPSC). A panel of 6 polymorphic microsatellite markers corresponding to p53, PAX2, and WT1 tumor suppressor genes were studied. Invasive carcinomas as well as TIC arising in the distal fallopian tube when remain as PFTC appears to exhibit different LOH patterns in comparison to PPSC. PAX 2 LOH patterns might represent a "hidden PAX 2 signature" analogous to p53 signatures. PAX 2 might be an emerging marker for detection of early serous carcinomas particularly in BRCA + women.

An immunohistochemical panel to differentiate metastatic breast carcinoma to skin from primary sweat gland carcinomas with a review of the literature.

CONTEXT: Approximately 25% of patients with breast cancer develop cutaneous metastases. Sweat gland carcinomas (SGCs) account for about 0.05% of all cutaneous neoplasms. Cutaneous metastases of breast carcinoma (CMBCs) (especially the ductal type) can be difficult to distinguish from SGCs. Treatment and prognoses for these 2 types of tumors differ radically, making accurate histologic diagnosis crucial. Although a few studies attempt to differentiate these entities employing immunohistochemical (IHC) studies (some of which we review here), to date, no panel of IHC stains exists, to our knowledge, to distinguish these entities. OBJECTIVE: To devise a panel of IHC stains to distinguish CMBC from SGC. DESIGN: Twelve cases of ductal CMBCs (11 not otherwise specified type, and 1 basal phenotype), 11 cases of SGCs (5 eccrine carcinomas, 3 porocarcinomas, and 3 microcystic adnexal carcinomas), 2 benign sweat gland neoplasm cases, and 2 primary breast cancer cases were retrieved and analyzed with the following IHC panel: mammaglobin, gross cystic disease fluid protein (GCDFP) 15, p63, basal cytokeratins (CK5, CK14, and CK17), androgen receptor, and PAX5. RESULTS: The p63 was only weakly expressed in 1 of 12 CMBC cases (8.3%), whereas it was strongly expressed in 10 of 11 SGC cases (90.9%) (P < .001). Basal cytokeratins demonstrated a similar immunoprofile in the SGC group, with 10 of 11 cases (90.9%) expressing all 3 markers, and a variable immunoprofile in the CMBC group with 0% (CK14) (P < .001) to 16.7% (2 of 12 cases; CK5 and CK17) (P < .001) expression. Mammaglobin was expressed in 8 of 12 cases (66.7%) of CMBC. CONCLUSIONS: Together, these 5 IHC stains were combined to make a panel that was 100% sensitive and 91% specific in distinguishing between CMBC and SGC.

Rate of detection of high-risk HPV with two assays in women ≥ 30 years of age.

BACKGROUND: High-risk (HR) human papillomavirus (HPV) prevalence rates, as determined by the Cervista(®) HPV HR test, in women aged ≥30 years in a routine screening population have not been studied. OBJECTIVES: The primary objective of this study was to estimate HR HPV prevalence in women negative for intraepithelial lesion or malignancy (NILM) cytology using the CERVISTA HPV HR test. The study also compared HR HPV prevalence rates in women aged ≥30 years and NILM cytology using the CERVISTA HPV HR and Hybrid Capture(®) 2 (hc2) tests. STUDY DESIGN: A multi-center study was conducted to analyze HR HPV prevalence rates using the CERVISTA HPV HR test from residual ThinPrep(®) specimens. HR HPV positive rates were determined for hc2; percent agreement between the CERVISTA HPV HR and the hc2 tests were reported. RESULTS: HR HPV prevalence rates among women with NILM cytology were not statistically different between the CERVISTA HPV HR and hc2 tests (6.92% [98/1417] versus 5.93% [84/1417], respectively; P>0.05). The overall percent agreement between the tests was 95.3% (1351/1417; 95% confidence interval [CI]: 94.1-96.3; κ=0.61, 95% CI: 0.53-0.70). There were no statistically significant differences between tests across age groups or investigational sites. For both tests, there was a statistically significant decrease in HR HPV positive results as age increases (CERVISTA HPV HR, P=0.0009; hc2, P<0.0001). DISCUSSION: There is no statistically significant difference between HR HPV prevalence rates obtained with the CERVISTA HPV HR and hc2 tests in women aged ≥30 years with NILM cytology.

Endometrial leukocyte subpopulations associated with Chlamydia trachomatis, Neisseria gonorrhoeae, and Trichomonas vaginalis genital tract infection.

OBJECTIVE: The objective of the study was to characterize endometrial inflammation associated with common genital tract pathogens. STUDY DESIGN: The design of the study was the immunohistochemical characterization of the endometrial leukocyte subpopulations from 37 controls and 45 women infected with Chlamydia trachomatis, Neisseria gonorrhoeae, or Trichomonas vaginalis. RESULTS: Compared with uninfected women, endocervical infection with C trachomatis, N gonorrhoeae, or T vaginalis was associated with significant increases in endometrial T cells, B cells, plasma cells, and polymorphonuclear leukocytes. Even more substantial increases in T cell, B cell, and plasma cell numbers were detected among women infected endocervically and endometrially with C trachomatis. CONCLUSION: Because lower genital tract C trachomatis, N gonorrhoeae, or T vaginalis infections were associated with comparable increases in the same endometrial leukocyte subpopulations, our results suggest the underappreciated involvement of T vaginalis in upper genital tract inflammatory processes. The more robust inflammatory infiltrate associated with C trachomatis endometrial ascension may offer insight into host inflammatory responses associated with pelvic inflammatory disease development.

Comparison of survival outcomes between patients with malignant mixed mullerian tumors and high-grade endometrioid, clear cell, and papillary serous endometrial cancers.

INTRODUCTION: Malignant mixed mullerian tumors (MMMTs) are an aggressive subtype of endometrial cancer (EC). Previous studies compare survival between high-grade endometrioid (EM), clear cell (CC), and papillary serous (PS) ECs; yet few studies compare MMMTs to these aggressive subtypes. The goal of this study was to compare recurrence-free survival (RFS), disease-specific survival (DSS), and overall survival (OS) among EC subtypes. METHODS: We conducted a retrospective cohort study of EC cases treated at Magee-Women's Hospital between 1996 and 2008. Kaplan-Meier estimates of RFS, DSS, and OS as well as and log-rank tests were used to compare survival distributions between histologic subtypes. Cox regression was used to estimate hazard ratios for histologic subtypes, adjusted for other significant prognostic factors. Interactions between histologic subtype and prognostic factors were examined to assess effect modification. RESULTS: This cohort included 81 MMMT (15%), 254 high-grade EM (46%), 73 CC (13%), and 147 PS (26%) cases. Compared to high-grade EM (6%) and CC (7%) cases, relatively more MMMT (12%) and PS (12%) cases were nonwhite. Stage differed significantly among the subtypes, with 36%, 34%, 37%, and 51% of MMMT, high-grade EM, CC, and PS cases, respectively, diagnosed at advanced late stage (P<0.001). Kaplan-Meier curves and log-rank tests showed similar RFS, DSS, and OS between MMMT, high-grade EM, CC, and PS cases stratified by stage. In adjusted Cox regression models, RFS and DSS were not significantly different between MMMT and other subtypes. High-grade EM cases had a significantly better OS compared to MMMT cases (HR, 0.63; 95% confidence interval [CI], 0.41-0.98). CONCLUSIONS: This is the first retrospective study to suggest that certain survival outcomes are similar among MMMT, high-grade EM, CC, and PS subtypes. Other large-scale studies are needed to confirm these findings.