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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 HE 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.

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.

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.

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.

Malignant mixed mullerian tumors (MMMTs) are an aggressive form of endometrial cancer (EC) with malignant epithelial and stromal components. Although MMMTs comprise less than 5% of ECs, 5-year overall survival (OS) rates for this subtype are extremely low, ranging from 20% to 35%.1–7 In comparison, low-grade endometrioid (EM) ECs have a 5-year OS rate approaching 90%.8 The significant morbidities associated with the MMMT subtype (ie, local and distant recurrences) represent challenges in the management of this invasive malignancy. Between 33% and 55% of MMMT cases have recurrence within 2 years of diagnosis.7,9 Studies that examine the MMMT subtype exclusively report that extent of the tumor, intraperitoneal metastasis, lymph node involvement, age, race, and stage predict recurrence and death.7,10–16 In the 1988 International Federation of Gynecology and Obstetrics (FIGO) staging criteria, MMMTs were grouped as uterine sarcomas owing to the presence of a sarcomatous component and poor survival in comparison to epithelial-derived EC. Consequently, MMMTs were excluded from FIGO staging.17 Pathologically, several theories of MMMT histogenesis exist; the most commonly accepted hypothesis is that most MMMTs are monoclonal, that is, derived from a single stem cell.18 The sarcomatous component of the tumor is derived either from the carcinoma during the progression of the tumor or from divergent differentiation of the 2 components from the same stem cell that occurs early in the development of the tumor.18 Evidence from Silverberg19 and Nordal13 suggests that the malignant epithelial component of MMMTs drives the aggressive behavior of this tumor. Malignant mixed mullerian tumors with a papillary serous (PS) or clear cell (CC) carcinomatous elements had a greater tendency for distant metastasis and poor survival compared to a low-grade EM element. Furthermore, MMMTs tend to metastasize via the lymphatic system, similar to the pattern of spread in epithelial EC subtypes. In contrast, 2 uterine sarcoma subtypes, leiyomyosarcomas and endometrial stromal sarcomas, metastasize primarily through the hematogenous route.20 Malignant mixed mullerian tumors share epidemiologic risk factors with EM-type ECs, including increasing weight, estrogen replacement therapy, and nulliparity.21,22 Based on this evidence, the most recent revision of the FIGO staging guidelines concluded that MMMTs should be staged as epithelial EC.23,24 Three studies have examined survival differences between MMMT and high-grade EM, CC, and PS EC patients.25–27 Similar to MMMTs, these epithelial subtypes are less common and associated with poor outcomes. The purpose of these studies was to discern whether MMMTs should be incorporated into clinical treatment trials with these other high-risk subtypes. Collectively, evidence from these studies suggests that MMMTs have significantly worse OS compared to high-risk epithelial subtypes and should not be included with these subtypes. Although advanced stage is associated with poor prognosis in MMMTs, shorter OS times have been observed in patients with MMMTs confined to the uterus.26,27 In adjusted Cox regression models, MMMTs had worse OS compared to non-EM cases (CC and PS subtypes combined) adjusted for stage27 and worse OS and RFS than high-grade EM cases adjusted for stage and vascular invasion.25 Although valuable, these studies are limited by small sample sizes and lack of control for important prognostic factors in survival outcomes. Our primary goals were to confirm or challenge the findings from previous studies, assess survival endpoints, and examine possible effect modification between histologic subtype and prognostic factors.

Ovarian serous papillary carcinoma, although rarely metastasizing to the breast, is often challenging based on morphology alone, particularly from the micropapillary variant of breast carcinoma. Gross cystic disease fluid protein-15, although a specific marker, can be negative in up to 50% of breast carcinomas. Wilm's tumor gene 1 (WT-1) has been identified as a useful marker to differentiate metastatic ovarian serous papillary carcinoma from primary breast carcinoma; however, it has recently been shown in the micropapillary variant of the primary breast carcinoma making it a less specific marker. PAX 2, a nuclear transcription factor, was recently observed in ovarian serous papillary carcinomas. In this study of 89 breast carcinoma cases, 26 micropapillary carcinoma, and 63 nonmicropapillary carcinoma types were retrieved from our pathology archives, represented on a single tissue microarray (TMA) with a 3-fold redundancy (TMA-1, TMA-2). In addition, whole tissue sections of a variety of benign and neoplastic müllerian tissues were surveyed with the PAX 2 immunostain. All cases were stained with rabbit polyclonal PAX 2 antibody and, in addition, the 5 metastatic ovarian serous carcinoma cases were stained with WT-1 as well for comparison. Only nuclear staining was considered positive. All primary breast carcinomas represented on TMA-1 and TMA-2 were entirely negative for PAX 2 100% (89/89), whereas 100% (5/5) of all metastatic ovarian serous carcinomas showed moderate-to-strong staining. PAX 2 expression was comparable with WT-1 as well in the metastatic ovarian serous carcinoma group. We therefore conclude that PAX 2 is a promising new, sensitive, and specific müllerian immunomarker for ovarian serous carcinomas (primary and metastatic).

The basal-like phenotype (BLP) subtype of breast carcinoma has been identified as 1 of 5 tumor subtypes first revealed by microarray profiling. This phenotype tends to be more aggressive, is more often associated with BRCA1 mutations, and carries a poor prognosis. Few studies have morphologically characterized BLP on resected breast specimens (RS), and no studies have evaluated these diagnostic parameters in core needle biopsies (CNB) of breast. We identified a group of 35 RS that demonstrated BLP by morphology and/or immunophenotype based on the criteria used in the literature. Retrospectively, we reviewed the CNB of these RS for the following morphologic features: growth pattern, nuclear grade, mitotic rate, presence of ductal carcinoma in situ, necrosis, and lymphocytic response. Of these histologic features, solid growth pattern [88.6% (31/35)] with nuclear grade 3 [100% (35/35)], marked lymphocytic infiltrate [74.3% (26/35)], and absence or

Lobular neoplasia (LN) and columnar cell alterations (CCAs) may share similar genetic abnormalities, but there is no appreciable literature that addresses the simultaneous occurrence of these lesions in breast core biopsy (CNB) specimens or resection specimens. Three groups of breast tissue were examined: group 1, 68 CNB specimens targeted for "suspicious" microcalcifications (Breast Imaging Reporting and Data System [BI-RADS] 4) and diagnosed with LN; group 2, 2,516 CNB reports for a 1-year period; and group 3, 400 consecutive breast carcinoma resection specimens analyzed for LN and CCAs within the vicinity of carcinoma. In group 1, LN was associated with CCAs in 54% of cases (37/68). In group 2, LN was found in association with CCA in 1.3% of cases (32/2,516). In group 3, 13.0% of cases of CCAs (52/400) were associated with LN. Our study suggests the association of these two lesions in breast tissue is nonrandom and that they may have a common progenitor pathway of neoplastic development.

HER2 oncoprotein is overexpressed in 15-20% of breast carcinomas and is associated with poor outcome. The 2+ group is considered equivocal, since gene amplification is observed in some but not others. The aim of our study is to ascertain if there is clinical significance to heterogeneity of HER2 immunohistologic expression in breast core-needle biopsies vs surgical resection specimens. A total of 37 invasive breast carcinomas diagnosed on core-needle biopsies and scored 2+ by HER2 immunohistochemical assay were selected from our files. The results were obtained on these selected cases, of which 19 cases were nonamplified and 18 cases were amplified. The follow-up resection specimens were reviewed and two additional tumor blocks were selected in each case for HER2 immunostaining. The 74 tissue blocks were examined for HER2 using antibody clone CB11 on the Benchmark XT and scored as negative (score 0 or 1+), weakly positive (2+) or strongly positive (3+). Results within the amplified group, 56% (11/18) showed significant areas with 3+ score in both blocks, 28% (5/18) remained as 2+, 11% (2/18) showed score 0-1+. In the nonamplified group, 42% (8/19) had score 0-1+, 37% (7/19) remained as 2+, 0% (0/19) had score 3+. Five (5) cases showed heterogeneous staining in both the groups. In the amplified group, 56% of cases showed strong 3+ in both the blocks of which half of these cases had areas of 2+. Fluorescence in situ hybridization was performed on a representative resection specimen block. In the amplified group 72% (13/18) cases were amplified, 22% (4/18) were nonamplified. In the nonamplified group, no amplification is detected in a great majority of cases 89% (17/19). HER2 immunohistochemistry on core-needle biopsies is usually predictive of tumor HER2 status. However, performing fluorescence in situ hybridization on core-needle biopsies almost completely resolves the issue of heterogeneous expression of HER2.

Pleomorphic lobular carcinoma of the breast is a high nuclear grade variant of lobular carcinoma. E-cadherin, a tumor-invasion suppressor gene, codes for a transmembrane protein that functions in intercellular adhesion. The E-cadherin protein internal domain binds with alpha, beta, gamma, and p120 catenins to anchor the E-cadherin complex to the actin cytoskeleton of the cell. The E-cadherin gene is routinely mutated in lobular neoplasia. This study examines the morphomolecular spectrum of the components of the E-cadherin-catenin complex in lobular neoplasia. Fifteen cases of pleomorphic lobular neoplasia, 8 cases of classic lobular neoplasia and 4 ductal carcinomas were studied. Normal breast epithelium and invasive ductal carcinomas all showed intense linear cell membrane immunostaining with antibodies to E-cadherin, alpha, beta, gamma, and P120 catenins. Membrane immunostaining of the catenin antibodies in lobular neoplasia was negative, except for rare cases that displayed beaded or dotlike patterns. Cytoplasmic immunostaining patterns for all lobular lesions included coarse paranuclear granules of beta catenin or diffuse intense cytoplasmic staining for P120 catenin. These immunostaining patterns demonstrate that catenins alpha, beta, gamma, and p120 are routinely dislocated from the cell membrane into the cytoplasm in lobular neoplasia and that the disrupted catenin patterns parallel absence of membrane E-cadherin in all cases. The diffuse cytoplasmic immunostaining of p120 in lobular neoplasia may be useful diagnostically as a positive marker for lobular neoplasia.

Gynandroblastoma is a rare ovarian tumor that is composed of both Sertoli cells and granulosa cells. Only 23 cases have been reported in the literature, and recurrence has never been described. We report the first case of a recurrent gynandroblastoma along with its molecular analysis and immunohistochemical studies. A 49-year-old Gravida 0 woman with a 10-year prior diagnosis of ovarian-mixed stromal tissue tumor (well-differentiated Sertoli cell and granulosa cell tumor) and staging laparotomy, presented now with a retroperitoneal mass and an elevated inhibin level. CT scan was suspicious for recurrence. The patient had no prior adjuvant therapy. The histomorphological features of the recurrent tumor had both Sertoli cell and granulosa cell tumor. The molecular analysis of both primary and recurrent tumor showed minor genetic instability in the 17q12.2 gene locus with no dedifferentiation or progression, which is consistent with a low-grade tumor. The immunohistochemical staining profile showed positivity for CD99, inhibin, calretinin, and vimentin; focal positivity for cytokeratin AE1/AE3 and negative for EMA and melan-A. All the previously mentioned immunostainings support the diagnosis. We report the first case of a recurrent gynandroblastoma 10 years after initial presentation along with its molecular analysis and immunohistochemical studies.

The basal phenotype of breast carcinoma was demonstrated from a study of gene expression profiles, which demonstrated five carcinoma phenotypes with differing immunohistologic profiles and outcomes. The basal phenotype, so-named because of an immunohistologic profile that is similar to myoepithelial cells of the breast, has poor outcomes. While the invasive basal phenotype has been described, there is a paucity of literature regarding the existence or recognition of a precursor lesion. We searched our CoPath database for breast carcinomas in the age group of 37 years or less, and this yielded 98 cases from the years 2001 to April 2006. Pathology reports were screened for those cases that were negative for estrogen and progesterone receptors and HER-2/neu (triple negative). A total of 16 cases (16/98, 16%) fulfilled these criteria. Histology was reviewed and immunostains were performed for Cytokeratins 14, 17, and 5/6, vimentin, EGFR, c-kit, smooth muscle actin and p63. All 16 cases had a high-grade invasive ductal carcinoma, Nottingham score 9/9, with geographic necrosis, good circumscription and lymphoid infiltrates. Of the 16 cases, 13 exhibited at least one area of ductal carcinoma in situ (DCIS). The DCIS types were solid, flat or micropapillary, high nuclear grade, with comedonecrosis and invariably associated with intense lymphoid inflammatory cell infiltration. Of 16 invasive cases, 14 (88%) were positive for CK14, CK17, CK5/6 and EGFR; 94% were vimentin positive, while half or less of cases were positive for smooth muscle actin, c-kit or p63. All of the DCIS components demonstrated the same immunohistologic profile as the invasive component. A DCIS component of solid, flat or micropapillary type exists in the basal phenotype of breast carcinoma, and it demonstrates the same immunophenotype as the invasive carcinoma, typically positive for CK5/6, CK14, CK17, vimentin and EGFR, but negative for ER/PR and HER-2/neu.

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.

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.

Lobular carcinoma in situ (LCIS) is considered a risk factor for development of invasive carcinoma (IC). Many variants of LCIS have been described based on pathologic features such as nuclear grade, pleomorphism, and necrosis, but little is known about the biology of these variants. The proposed 3-tier grading system for LCIS has not been validated or endorsed across laboratories. We found significant upstaging of pure pleomorphic LCIS (LCIS with nuclear grade [NG] 3), up to 25% in core needle biopsy (CNB) specimens, in an earlier study. The aim of the current study was to address the importance of pure classical LCIS (NGs 1 and 2) in CNB specimens along with clinicopathologic follow-up. In follow-up resection specimens, IC or ductal carcinoma in situ was seen in 18% (7/39), a high incidence of residual LCIS was seen in 69% (27/39), and other high-risk lesions, such as atypical ductal hyperplasia, were seen in 36% (14/39) of LCIS NG 2 cases. Our study illustrates the importance of grading LCIS; we recommend follow-up excision in LCIS NG 2 cases owing to a high incidence of residual LCIS and the likelihood of identifying other high-risk lesions.

Flat epithelial atypia (FEA) is an emerging entity of uncertain clinical significance, and outcome data are sparse. The aim of this study was to evaluate the clinicopathologic significance of this entity for proper management. All core needle biopsy (CNB) specimens diagnosed as atypical ductal hyperplasia (ADH) from January 2006 to April 2008 were retrieved. H&E-stained slides of 5 levels on each case were reviewed. The differences in upstaging in subsequent excisions in the FEA and ADH group (31/189 [16.4%]) vs the pure FEA group (5/35 [14%]) and pure FEA (5/35 [14%]) vs pure ADH (5/45 [11%]) were not statistically significant. We observed that FEA evolved into ADH at the same site at an average of 3 to 4 levels. Our study concludes that there is an association of FEA with ADH on multiple levels of CNB specimens, and follow-up surgical excision findings for FEA are clinically significant.

Regressive changes (RC) have been described in malignant melanoma, carcinomas of the prostate and cervix. The presence of RC in these neoplasms may signify some degree of host response to tumor and seems to be a sign of poor prognosis for some neoplasms. RC in breast cancer is vaguely defined in the older literature. We have observed periodically similar RC in a subset of high-grade ductal carcinoma in situ (HGDCIS) in breast specimens. The aim of our study is to demonstrate how to recognize RC in the diagnostic setting and an attempt to understand the biologic behavior in this subset of HGDCIS cases. Fifty-nine cases of HG-DCIS (35 cases with RC and 24 cases without RC) were included. We defined RC in our study as demonstrating thick periductal fibrosis, dense lymphocytic infiltrate, and a thin rim of intact neoplastic cells. A short panel of immunomarkers to study this entity included myoepithelial markers. Reduced expression of myoepithelial markers (p63 and smooth muscle heavy chain myosin) were seen more frequently in the HGDCIS group with RC than without RC cases. Invasion as well as metastatic disease was seen in association with HGDCIS with RC nearly 4 times as often. It is also critically important to recognize HGDCIS-RC for diagnostic purposes, as the differential diagnosis of RC includes, benign associations such as papilloma, fibrocystic changes and periductal mastitis. HGDCIS-RC may also be a sign of an aggressive phenotype than other HGDCIS subtypes. Further outcome studies are necessary to determine if it has a clinical impact akin to other tumors with RC.

The distinction between lobular and ductal lesions of the breast is important in several circumstances. Diagnostic reproducibility of lobular versus ductal lesions, based on histology alone, is less than optimal. The proper distinction between atypical lobular hyperplasia, lobular carcinoma in situ and low-grade ductal carcinoma in situ is critical for patient management. Patients who have a core biopsy of invasive lobular carcinoma often have preoperative magnetic resonance imaging to prepare the surgeon for proper margin attainment. E-cadherin, a negative membrane marker for lobular neoplasia, is useful in the distinction of lobular versus ductal neoplasia, but as a negative marker, can be difficult to interpret in particularly challenging cases. In this study, we surveyed primary and metastatic ductal lesions (62) and lobular lesions (64) of the breast to determine if P120 catenin is useful in the diagnostic distinction between lobular and ductal neoplasia. Primary breast ductal and lobular preneoplastic and neoplastic lesions were immunostained with E-cadherin and P120ctn and independently classified as ductal or lobular lesions. In addition, a wide array of carcinomas of different types were surveyed with P120ctn in tissue microarrays to ascertain whether the cytoplasmic P120ctn immunostaining pattern observed in lobular neoplasia was unique. Accurate categorization of ductal versus lobular neoplasia in the breast with P120ctn immunostaining was effective in all cases. Separation of low-grade ductal carcinoma in situ from lobular neoplasia was efficient. Diagnostically, P120ctn was particularly useful in identifying early lesions of lobular neoplasia. Of the other tumors that may morphologically mimic lobular carcinoma, only the diffusely infiltrating variants of rectal and gastric carcinomas showed diffuse cytoplasmic P120ctn immunostaining. Caution should be exercised when examining tumors in metastatic sites with P120ctn, with the incorporation of an appropriate panel of immunostains.

Aim: The aim was to analyze the expression of novel biological transcription markers, forkhead-box A1 (FOXA1), GATA binding protein 3 (GATA-3), and established markers such as Ki-67 (MIB-1) and human epidermal growth factor receptor 2 (HER2) in estrogen receptor (ER(+)) and ER(-) ductal carcinoma in situ (DCIS) patients with/without recurrence. Methods: Two hundred and ninety-one cases of DCIS were retrieved from our pathology database, with complete data available for 219 cases. The follow-up period is from 1988 to 2009. Recurrence is defined in terms of DCIS or invasive carcinoma (IC). No recurrence was seen in 88% (196/219) of cases; 12% (26/219) had a recurrence (IC: 13, DCIS: 13). We are reporting the results of biological marker expression in terms of recurrence and ER status. Results: Our study demonstrates strong expression of GATA-3 in the ER(+) DCIS in recurrence and nonrecurrence groups similar to previously described in IC. A reduced expression of GATA-3 was observed in ER(-) recurrence and nonrecurrence groups. A strong HER2 protein expression, as well as high proliferation index, was seen in recurrence group (DCIS and IC). FOXA1 expression is reduced across the groups though not statistically significant. Conclusion: This is the first study to analyze novel transcription markers FOXA1 and GATA-3 in DCIS. Further work needs to be done on a larger cohort of DCIS cases with recurrence to better understand, which variables are best able to predict recurrence and guide therapy decision strategies. Maintenance of FOXA1 and GATA-3 expression in ER(-) DCIS may offer new promising targets for therapy in future.

Tubulolobular carcinoma is a type of mammary carcinoma that displays an admixture of invasive tubules and lobular-like cells. Previous reports have shown it to share clinical similarities to lobular carcinoma, whereas more recent studies have shown it to be E-cadherin positive. The aim of the current study was to further explore the immunophenotype of tubulolobular carcinoma, and to document its natural behavior. Nineteen cases of tubulolobular carcinoma and 10 cases each of tubular and lobular carcinoma were retrieved for comparison analysis. Immunohistochemistry was performed with antibodies against estrogen receptor, progesterone receptor, HER2/neu, 34betaE12, E-cadherin, and the catenins. Twenty-five percent of patients with tubulolobular carcinoma presented with greater than stage I disease, compared to 0 and 60% of patients with tubular and lobular carcinoma, respectively. Two patients with tubulolobular carcinoma had tumor recurrence, one of whom also developed metastasis. The majority of all carcinomas were estrogen and progesterone receptor positive. E-cadherin displayed membranous staining in all tubular and tubulolobular carcinomas, and was negative in all lobular carcinomas. Half of each carcinoma subtype displayed granular cytoplasmic 34betaE12 immunoreactivity. alpha-Catenin exhibited partial or complete membranous staining in all tubulolobular and tubular carcinomas, and was negative in all lobular carcinomas. beta-Catenin displayed membranous staining in tubulolobular and tubular carcinomas, whereas all lobular carcinomas had coarse cytoplasmic immunoreactivity. p120 and gamma-catenin displayed membranous staining in 100% of tubulolobular and tubular carcinomas and cytoplasmic staining in 100% of lobular carcinomas. Tubulolobular carcinoma of the breast is thus a distinct type of mammary carcinoma that displays both tubular and lobular patterns histologically but displays the membranous E-cadherin/catenin complex characteristic of the ductal immunophenotype. Tubulolobular carcinoma appears to be more aggressive than tubular carcinoma, as 16% of patients had lymph node metastases, although all were alive at a mean follow-up of 40 months.

As an extension of our previous study (Shidham et al., Acta Cytol 2000;44:1015-1022), we evaluated the interference by methods of cytology smear preparation on the immunoreactivity of cytokeratin-7, cytokeratin-20, estrogen receptor, progesterone receptor, chromogranin, synaptophysin, and vimentin. Scrape cytology smears of 34 fresh specimens submitted for intraoperative consultation were studied. They were processed by three different methods--A: wet-fixed in 95% ethanol; B: air-dried saline rehydrated smears fixed in alcoholic formalin; and C: air-dried saline rehydrated smears fixed in 95% ethanol with 5% acetic acid. The intensity scores of immunostaining were estimated semiquantitatively and compared with corresponding formalin-fixed paraffin-embedded tissue sections (FPTS). Except vimentin, all immunomarkers showed higher intensity scores with method A or B than with method C. Vimentin showed the best results with method A. Our results indicate that the immunoreactivity pattern with each immunomarker is affected by the method of cytology smear processing. Most importantly, method C, which is the desired choice for cytomorphological staining, was not suitable for immunostaining.

Context.—The differential diagnosis of gastrointestinal stromal tumors (GIST) and solitary fibrous tumors (SFT) may be a diagnostic challenging because of overlapping clinicopathologic features. Many studies have shown consistent immunoreactivity for CD117 (c-Kit) in GIST. However, only a few studies have evaluated CD117 expression in SFT, and these studies have used an antibody from Santa Cruz Biotechnology. In non-GIST lesions, reactivity with this antibody has been shown to differ from that with a CD117 antibody from Dako Corporation. The immunoreactivity of SFT with the Dako CD117 antibody has not been reported. Conversely, CD99 is a marker for SFT, and its expression in GIST has not been evaluated. Objective.—To study the immunohistochemical profiles of GIST and SFT to evaluate their diagnostic overlap. Design.—We studied the immunoreactivity of 27 unequivocal GIST and 19 unequivocal extra-abdominal SFT for CD117, CD34, CD99, α-smooth muscle actin, vimentin, CD31, S100 protein, and muscle-specific actin. All antibodies, including CD117, were from Dako Corporation. Results.—We found positive immunoreactivity for CD117 in 100% of GIST and none of SFT; for CD34 in 89% of GIST, and 100% of SFT; for CD99 in 89% of GIST and 100% of SFT; for α-smooth muscle actin in 48% of GIST and 31% of SFT; for vimentin in 89% of GIST and 90% of SFT; and for muscle-specific actin in 22% of GIST and none of SFT. None of the GIST or SFT showed immunoreactivity for CD31 and S100 protein. Conclusions.—The major difference between GIST and SFT was strong CD117 immunoexpression in all GIST and an absence of this expression in all SFT. With the exception of muscle-specific actin, the prevalence of immunoreactivity for the markers studied did not differ substantially between these 2 tumors. We conclude that GIST and SFT show distinctly divergent immunoprofiles with respect to CD117 and muscle-specific actin.

Hematoxylin-eosin (H&E)-stained sections may not allow proper evaluation of birefringence properties of the crystals in the lesions of pseudogout, gout, and tumoral calcinosis. This study was undertaken to verify the application of a special stain that could facilitate the evaluation of the birefringence properties of these crystals for definitive diagnosis. We evaluated previously described nonaqueous alcoholic eosin staining (NAES) method based on the principle of using alcoholic eosin without hematoxylin and any other aqueous reagents for staining of formalin-fixed, paraffin-embedded tissue sections. Two observers, in a blinded fashion, evaluated the sections stained with routine H&E and NEAS method without the knowledge about clinical diagnosis. All pseudogout (nine sections from seven cases) and gout (eight sections from five cases) lesions demonstrated birefringence in the sections stained with NAES method. H&E-stained sections showing the respective diagnostic histomorphology failed to demonstrate the birefringent crystals by polarizing microscopy in all the eight sections from gout and in seven of nine sections from pseudogout. Only two H&E-stained sections showed scant calcium pyrophosphate dihydrate (CPPD) crystals in pseudogout. None of the three sections from two cases of tumoral calcinosis showed birefringence with either stain. We conclude that CPPD in pseudogout and monosodium urate in gout may not polarize in the routine H&E-stained sections. However, polarizing microscopy of sections stained with NAES method allowed demonstration of CPPD crystals with positive birefringence in pseudogout, MSU crystals with negative birefringence in gout, and calcium hydroxyapatite crystals without birefringence in tumoral calcinosis. Section stained with NAES method is a significantly useful adjunct to the routine H&E stain for proper evaluation of the crystals under polarizing microscope in these lesions.

65-year-old woman presented with recurrent right knee pain. She had experienced similar persistent and debilitating symptoms 8 months earlier. Evaluation of arthrocentesis on 2 separate occasions was unremarkable. This was followed 5 months later by arthroscopy. Magnetic resonance imaging of the right knee showed lowsignal-intensity lesions compatible with pigmented villonodular synovitis. Biopsy was performed. Gross examination revealed red to yellow-brown soft tissue fragments, measuring 7 cm in aggregate. Light microscopic examination of formalin-fixed, paraffin-embedded tissue sections stained with hematoxylin-eosin (H&E), revealed hyperplastic synovium lined by a prominent synovial lining with focal edema and fibrosis (Figure 1). The fragments were edematous with focal fibrosis. Immediately beneath the synovial lining were scattered histiocytes and lymphocytes, with some histiocytes containing yellow-brown hemosiderin pigment. This pigment was also

In the era of tailored therapy, the evaluation of systemic treatment of breast cancer has been increasingly dominated by consideration of biological features of the tumor and the host. Established breast cancer prognostic factors — those that determine natural history of breast cancer — include axillary nodal status, tumor size, histological grade, hormone receptor status, HER-2 expression, and presence of lymphovascular invasion. These factors often inform decisions about whether to use adjuvant systemic therapy. In contrast, predictive markers like expression of estrogen receptor (ER) alpha, progesterone receptor, and HER-2 protein are powerful tools to select certain types of therapy. Techniques to examine a myriad of genomic, transcriptional, or proteomic factors simultaneously, so-called ‘omics, currently dominate this field. In this molecular age, it is important to refl ect on the continuing importance of classic histopathology. One such feature is identifi cation of lymphovascular invasion. Its importance in prognosis was suggested at the 2007 St Gallen consensus conference ( 1 ) when extensive lymphovascular invasion was identifi ed as a factor to identify women with moderate risk as opposed to low risk for recurrence. These classic histological studies have been amplifi ed by immunohistochemical analysis. For example, immunohistochemical studies of microvascular density assessed by vascular markers such as CD31 and CD34 antigens have usually shown an association between extent of staining and a greater likelihood of subsequent metastatic disease ( 2 ), shorter relapse-free interval, and reduced overall survival in patients with node-negative, ER-negative breast cancers ( 3 ). The presence of tumor in peritumoral small lymphatic spaces, independent of lymphovascular density, has been shown to play a fundamental role in tumor progression ( 4 ). D2-40 or podoplanin, a novel antibody, selectively stains the endothelium of lymphatic vessels. The utility of this antibody as a specifi c marker for detection of lymphovascular invasion in the routine pathological workup is evolving ( 5 , 6 ). Recent studies ( 7 , 8 ) have demonstrated a higher sensitivity for detection of lymphovascular invasion by D2-40 than by routine histological detection or CD31detected vascular invasion. Controversy continues to exist with regard to the process whereby tumor cells gain access via preexisting lymphatics or via newly formed lymphatics at the invasive front of the tumor; D2-40 – detected lymph channel invasion along with high CD31 microvessel density has been associated with outcome in breast cancer as shown in univariate and multivariable analyses. Several studies have consistently shown that lymphovascular invasion is an adverse prognostic factor for relapse and survival in node-negative patients in combination with other risk factors such as tumor grade and size and receptor status ( 9 ). The need for prospective research to defi ne its individual role has been raised. In this issue of the Journal, Ejlertsen et al. ( 10 ) report a comprehensive analysis of the prognostic value of lymphovascular invasion in tumors from 15 659 women entered into the Danish Breast Cancer Cooperative Group registry from 1996 to 2002. The goal of the study was to assess whether lymphovascular invasion was a single independent prognostic factor in stratifying early breast cancer patients as low risk vs high risk for recurrence. The presence of lymphovascular invasion was noted in only 15% of tumors. A statistically signifi cant difference in 5-year invasive cancer disease – free interval was seen: 79.5% (95% confi dence interval [CI] = 78.7% to 80.2%) for patients without lymphovascular invasion vs 54.5% (95% CI = 52.4% to 56.6%) for patients with lymphovascular invasion. These differences were mirrored in the overall survival rates of 87.3% (95% CI = 86.7% to 87.8%) and 66.0% (95% CI = 64.1% to 67.9%) in patients without and with lymphovascular invasion, respectively, and they persisted in multivariable analysis. Finally, the study gives the unexpected and somewhat disappointing result that lymphovascular invasion was associated with adverse outcome in patients who are at high risk for recurrence by other recognized prognostic factors but not in those who are at low risk by the same criteria. It is therefore apparently not useful as a means to subdivide the low-risk group, the group in which many clinicians and patients would like assistance. This fi nding is at odds with the 2007 St Gallen consensus recommendations, where extensive lymphovascular invasion was felt to be suffi cient to upstage patients from low risk to moderate risk for recurrence. The strengths of this study are several. It is derived from a population-wide database of virtually all women diagnosed with breast cancer in Denmark over a 7-year period, who were treated according to standard algorithms and whose tumors were analyzed in a prespecifi ed way for multiple factors including lymphovascular invasion. It has shown the expected association between lymphovascular invasion and other poor prognostic features such as with positive nodal status, tumor size greater than 2 cm, ductal histology, grade 2 or 3, ER negativity, and use of adjuvant endocrine therapy and/or chemotherapy ( P < .001 for each). This agreement with other work gives credence to the results of this study. Finally, the test is “low tech” and could theoretically be performed in virtually any diagnostic laboratory , although it does require two things — investment of valuable pathologist time and availability of standardized criteria that are easily reproduced across all pathology laboratories. The limitations of the study are also real. Neither the reproducibility of the determination of lymphovascular invasion nor its extent was documented; rather a dichotomous cutoff of present or absent was used. HER-2 testing was not performed, and the study period preceded the routine use of several contemporary adjuvant

Objective: To develop a workflow for collecting specimens for proteomic analysis from the peritoneum, fallopian tubes (FT), and ovarian surface epithelium (OSE) at the time of risk-reducing surgery in women at increased risk for developing malignancies due to a germline BRCA mutation. Methods: A pilot set of 6 subjects with BRCA2 mutations were consented for an IRB-approved protocol for surgical specimen collection. During laparoscopic removal of the FT and ovaries, peritoneal washings, OSE brushings, and FT lavage specimens were collected without interfering with standard pathological analysis of the tissues. After immunodepletion, samples were loaded in a stacking gel and digested in-gel with trypsin. Digests were analyzed in duplicate by nanoflow reversed-phase liquid chromatography coupled online to a linear ion trap mass spectrometer. Tandem mass spectra were searched against the UniProt human protein database from the European Bioinformatics Institute using SEQUEST. Differences in protein abundance between samples were derived by spectral counting. Immunohistochemical (IHC) staining for p53 and loss of heterozygosity (LOH) testing for p53 at the D17S-1844, D17S-516, and D17S-786 loci were performed on all paraffin-embedded FT specimens. Results: A total of 265, 328, and 241 different proteins were identified by 2 or more unique peptides each from the any of the OSE, FT and peritoneal samples, respectively. The number of proteins identified on both the right and left side in individual subjects ranged from 16-33% for OSE and 31-36% for FT. 37 common proteins involved in cell signaling, cell growth, cellular assembly, acute phase response, and DNA replication and repair were found in at least 1 specimen from all 6 subjects. 23 of the common proteins are involved in cancer pathways. All surgical pathology was negative both for malignancy and possible precursor lesions such as tubal intraepithelial carcinoma. MUC16 (CA-125) was elevated by proteomic analysis in the right FT of 1 subject; the left FT lavage in this subject could not be collected due to tubal scarring. Although IHC for p53 was normal in both of these FT, LOH for p53 was present in both. IHC was also negative for all remaining FT specimens. LOH was absent in both FT in 3 subjects, present in 1 FT but not the other in 2 subjects, and present bilaterally in 1 subject mentioned above. Conclusions: This workflow allows analysis of the proteomes of peritoneal, FT, and OSE specimens collected at the time of laparoscopic risk-reducing surgery, a technique which has not been described previously. It presents a unique opportunity for closer examination of the tissues at risk for malignant transformation in women with BRCA mutations. Harvesting proteins from the environment where ovarian and related cancers arise will inform current attempts to use the “p53 signature” to evaluate the FT as the potential source of many pelvic serous tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4876. doi:10.1158/1538-7445.AM2011-4876

Background: Estrogen receptor (ER) plays a key role in the development and treatment outcome in breast cancer patients. The two broad groups of invasive carcinoma (IC), namely ER(+) and ER(-), are yet to be well understood in DCIS. Absence of ER was shown to be one significant predictor of IC recurrence in DCIS patients. Recently functional interaction between FOXA1 transcription factor and ER has shown to play a critical role in suppressing ER-dependent breast cancer cell growth and tumorigenesis in vivo. This suggests that FOXA1 may be novel marker that can inhibit tumor proliferation and development of IC. Similarly the GATA-3 transcription factor, highly expressed in the mammary luminal epithelial cells, has emerged as a strong predictor of tumor differentiation, ER status, and clinical outcome. The specific aim of this study is to analyze the expression of novel biological transcription markers FOXA1, GATA-3, and established markers MIB-1 (Ki-67) and HER2/neu in ER(+) and ER(-) DCIS patients.Materials and Methods: Cases of DCIS (N=225) diagnosed on core needle biopsy were retrieved from our Pathology database, with average follow-up from 1995-2009. Cases with recurrence (N=21) and without (N=204) were evaluated, with current data of 153 cases thus far. Recurrence was defined in terms of DCIS or IC. FOXA1, GATA3, ER, PR were evaluated with a cumulative “H score” based on proportionality (PS) and intensity scores (IS) [0-negative, 1-150-low, 151-250-intermediate, 250-300-high]. A 10% or more “nuclear” staining of the tumor cells was considered “positive.” MIB-1 was given a nuclear proliferation index (1-10%-low, 11-25%-intermediate, 26-50%-high, >51%-very high); HER2, a membrane stain, was interpreted as per routine guidelines for IC (0/1+ negative, 2+ weakly positive, 3+ strongly positive]. Pathologic as well as clinical variables were also evaluated.Statistical analysis: Fisher's exact test for testing the dependence between clinical outcome (non-recurrence, recurrent-IDC and recurrent-DCIS) for each biomarker and calculation of p-value.Results: Currently, data on 153 cases is presented. Entire results will be available at time of meeting. No recurrence was seen in 92% (141/153) cases, while 8% (12/153) had recurrence (7 cases with IC, 5 cases with recurrent DCIS). We are reporting the initial results of biological marker expression in terms of recurrence. A high recurrence is seen with expression of low GATA-3 (p=0.78), high/intermediate MIB-1 (p=0.9), and low/absent PR (p=0.07). FOXA1 and GATA-3 expression is maintained in 94% of ER(-) cases.Conclusions: Our study is on-going in comparing the initial biomarker data to other important clinical variables and response to therapy, for clinical predictors of outcome in a novel prediction model. Our initial observation is that a strong expression of FOXA1, GATA-3, low MIB-1 index, and absent Her2 expression are characteristically seen in the ER(+) DCIS group, similar to previously described in IC. However, nearly all of the ER(-) cases expressed FOXA1 and GATA-3, which needs be evaluated further, as these transcription factors may offer new promising targets for therapy. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2115.

Introduction: Basal phenotype breast carcinoma has been described as an aggressive variant with comparatively poor prognosis; however, increasing experience suggests that only a subset show increased mortality. We performed microarray analysis of DNA from 11 basal phenotype carcinomas (from 10 patients) and paired normal breast tissue with copy number and loss of heterozygosity analysis with hierarchical clustering to see if characteristic genetic changes were present that correlated to lymph node metastasis at presentation.Materials and Methods: Electronic search of the medical records from 2006-2008 of our institution by an honest broker for all cases of basal phenotype carcinoma (based on medullary-like histology and immunohistochemistry positive for at least two of CK14, CK17, CK5/6 and EGFR) with known lymph node status at presentation and archived snap frozen fresh and tumor samples returned 10 cases (one with bilateral basal phenotype carcinoma), which were de-identified. 5 of these cases had lymph node metastasis at presentation. DNA isolation by column extraction was performed on 0.25 g of paired tumor and normal samples (confirmed by pathologist review before extraction). An Affymetrix 5.0 mapping microarray was performed on isolated DNA per manufacturer's protocols, and analyzed with Partek Genomics Suite software using copy number/LOH workflows using hidden Markov model and genomic segmentation in separate analyses with unsupervised hierarchical clustering.Results: Copy number and LOH analysis using hidden Markov model and genomic segmentation methods gave equivalent hierarchical clustering for our data set. Adjusting the obtained cluster analysis for known lymph node metastasis showed that changes in chromosomes 9, 7, and 5 that differentially clustered 4 of 5 cases which presented with lymph node metastasis (and 2 closely related initially lymph node negative cases).Discussion: Our results suggest that changes on chromosomes 9, 7 and 5 may correlate with the most important prognostic indicator in breast cancer, lymph node status, in basal phenotype carcinomas. Globally suboptimal amplification prevented accurate quantitation of copy number changes or precise loss of heterozygosity localization on these chromosomes. Additional studies to confirm these results using separate molecular techniques are underway, as well as correlation with 5 year survival when the cohort reaches that milestone. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6133.

Introduction: Pleomorphic lobular carcinoma in situ (PLCIS) is a rare distinct variant of lobular carcinoma in situ (LCIS) which is morphologically difficult to distinguish from high-grade ductal carcinoma in situ (DCIS). We performed microarray DNA analysis to characterize the genetic changes of this entity in order to determine if such changes were unique to PLCIS.Materials and Methods: 5 Pleomorphic (4 in situ and 1 invasive) and 4 normal non-paired breast tissues were analyzed for copy number changes using the Affymetrix 250K Sty array. Copy Number (CN) changes were detected by the Partek (Partek Inc, St Louis, MI) software. Data processing steps included GC content correction and segmentation to determine regions of amplifications and deletions.Results: Each sample demonstrated a unique pattern of CN changes with amplifications being more frequent than deletions, and between 25 and 66 changes per sample. Chromosomes 1, 11, 14, 16 and 8 exhibit the most frequent changes. Large deletions in chromosome 16q were seen in 4 out of 5 samples and amplification of regions of 1q in 4 out of 5 samples. Focal common amplifications, defined as less than 100kb in length, CN>5 and present in at least 3 samples, were seen in a number of regions including 8q23.1 containing the NUDCD1 gene (5/5 samples), 14q23.3 containing the GPHN (3/5 samples) gene, 12p12.2 containing the gene SLCO1B3 (3/5 samples), and 4p16.1.Discussion: Our results indicate that changes on chromosomes 1, 11, 14, 16 and 8 are frequently observed in pleomorphic lobular carcinoma (PLC) as well as PLCIS. Deletions in chromosome 16q and amplification of regions of 1q seen in our cases are associated with loss of e-cadherin and β-catenin expression and are the characteristic genetic abnormalities seen in classic lobular carcinoma. In addition, the characteristic 8q23.1, 14q23.3 and 12p12.2 reported in high grade DCIS were seen in our cases as well. PLCIS exhibits overlapping features with both classical lobular neoplasa and high grade DCIS. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5004.

e11523 Background: DCIS is a heterogeneous pre-invasive carcinoma with a spectrum of clinical behavior. Patients with ER+ IC have better outcomes compared to ER- patients. FOXA1 and GATA 3 family of transcription factors have been shown to be associated with hormone receptors (ER and PR) and other variables of good prognosis with better overall and relapse-free survival rate. The specific aim of this study is to analyze the expression of these novel biological markers: FOXA1, GATA-3, with recognized markers: MIB-1(Ki-67) and HER2 /neu in DCIS patients with/without associated IC. Methods: Sixty-nine (69) cases of DCIS [(fifty two (52) cases in ER+; seventeen (17) in ER-] were retrieved from our Pathology database. The expressions of the biological markers are analyzed by using a panel of immunohistochemical stains. FOXA1, GATA 3, ER, PR are nuclear stains, a cumulative “H score” is derived based on proportionality (PS) and intensity scores (IS). A proliferation index (PI) is calculated for MIB-1 (Ki67) nuclear stain (low 50%). Her2/neu is scored as per guidelines for HercepTest (0, or 1+ =negative, 2+ =weakly positive, 3+ =strongly positive). Results: DCIS is categorized into low grade (LG) (nuclear grade 1 and 2), high grade (HG) (grade 3). In the HGDCIS (n=48), four (4) cases had IC after a mean of 7.75 years; three cases of recurrent DCIS after a mean 6 years. No recurrent IC or DCIS is seen in the LGDCIS (n=21) group. The results are shown in the Table . Conclusions: (1) Decreased expression of GATA 3 is observed in HGDCIS ER- group may be a contributor to higher recurrence observed in this group (14%) versus (0%) in ER+ group. (2) A strong expression of FOXA, GATA3, low Ki-67 index, absent Her 2 expression are characteristically seen in our ER+ DCIS group, as previously described in IC. 3. Comparing the response to therapy and outcome in the ER+ and ER- groups is on going. [Table: see text] No significant financial relationships to disclose.

e16547 Background: Endometrial papillary serous carcinoma (EPS) can be difficult to differentiate histologically from high grade endometrial endometrioid carcinoma (EE). Both of these tumors often present with solid growth pattern with few papillary features. Recurrences have been found to be more frequent in EPS than high grade EE. These differences were found despite the same preoperative and postoperative radiotherapy and chemotherapy, indicating more aggressive tumor biology. To date, there are no well defined immunophenotypic or molecular methods to differentiate these two tumors or their clinical behavior. Methods: Eleven cases of EE and twenty-three cases of EPS were retrieved from our departmental archives and stained using Pax-2, WT-1 and p16 antibodies. For all three antibodies, a strong nuclear staining is considered positive. The intensity and proportion of cells positive are noted. Seven cases from each group were further examined for their staging and clinical outcome in terms of recurrence, metastasis and treatment. Results: WT-1 is negative in 100% of EE cases. p16 is positive in 95% of EPS cases, and only 55% of EE cases. In addition, out of the seven EPS patients whose clinical information was obtained, the three EPS patients who had strong Pax-2 staining are still alive more than two years status post surgical intervention, while four other EPS patients are deceased. The three patients with the strongly staining PAX-2 EPS tumors all had varying tumor characteristics, recurrence and adjuvant therapies. This finding is now being evaluated in the additional cases. Conclusions: WT-1, Pax-2 and p16 are useful to differentiate high grade EE and EPS cases. Strong Pax-2 staining in three cases of EPS with better outcome is an interesting finding that deserves further investigation. No significant financial relationships to disclose.

Abstract #3006 Background: Flat epithelial atypia (FEA) is a newly emerging entity of uncertain clinical significance. Defined by the World Health Organization (WHO) in 2003, FEA differs from columnar cell hyperplasia by the presence of nuclear atypia and from atypical ductal hyperplasia (ADH) by the absence of complex architecture. As the term FEA is relatively new, the clinical relevance and the outcome data are sparse. The aim of this study is to evaluate the clinical and pathologic significance of this entity in order to construct the proper management of these lesions. Design: All core-needle biopsies (CNB) diagnosed as ADH, which includes pure FEA at our institution, from January 2006-April 2008 were retrieved from our pathology files. Hematoxylin and eosin (H&E) slides of five levels on each case were reviewed. Statistical analysis was performed with calculation of p-value. Results: Total number of CNB performed from 2006-2008 was 8054. 99% (8051/8054) were stereotactic guided and 1% (3/8054) were ultrasound/MRI guided biopsies. All the CNB were performed for microcalcifications. The mean age of the patients is 54 years (range 29-83). Incidence of ADH was 4% (338/8054). We have reviewed 203 cases so far and the study is ongoing. 32 cases were discarded due to either presence of DCIS or IC in the CNB. Upon review, 9% (18/203) cases were classified as pure FEA and 91% (185/203) cases as FEA+ADH. The upstaging to a significant lesion in pure FEA group is 14% in comparison to 12% in FEA+ADH group (p=0.9471), is not statistically significant. Lobular neoplasia is seen in association with pure FEA group in 33% (6/18) and 11% (21/185) in FEA+ADH group. In 6% (11/185) of cases in FEA+ADH group, we observed FEA evolved into ADH at the same site at an average of 3-4 levels. Conclusions: 1.The incidence of FEA in our CNB targeted for calcifications is 9% 2. The upstaging in the follow-up resections in FEA in comparison to ADH+FEA shows no statistical difference (14% versus 12%). 3. Since FEA and ADH commonly occur together on the same slide, it is prudent to examine deeper tissue levels when pure FEA is encountered on CNB. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3006.

Objective Since the introduction of the liquid-based ThinPrep testing in 1996, most cytology laboratories across the country have adopted the liquid-based cytology (LBC) for Pap test screening. Subsequent to wide-spread adoption of the ThinPrep Pap test, the ThinPrep Imaging System (TIS) Cytyc Corp, Marlborough, MA was introduced to improve the accuracy and efficiency of screening interpretation. We report our initial experience with the TIS at Magee Women's Hospital. We introduced the TIS in December 2004. Methods The imager assisted Pap test results over the first 12 months (December 2004 to December 2005) of implementation were reviewed and analyzed. Our implementation protocol included each cytotechnologist manually prescreening 200 negative slides to gain experience with the imager slides and serve as a quality check for the TIS. We re-screened 3400 slides (200 slides each for 17 cytotechnologists) manually which were initially determined to be negative using the TIS. 104,457 Pap tests were imaged on the TIS. 95,899 manually screened Pap tests, 12 months prior to the introduction of the TIS (December 2003–November 2004) are taken as the historic control group for our study. Results The mean ASC-US rate employing the automated imager was 8.70% [9088/104,457]. The mean LSIL detection rate was 4.22% [4409/104,457]. The imager did not miss any detectible high-grade lesions during these months, with a HSIL (+) detection rate of 0.68% in comparison to 0.60% by manual screening confirmed by follow-up biopsies. The difference is statistically significant with a p value of 0.022. The definition of false negative rate for purposes of this study is calculated as the number of false negative cases identified out of number of negatives re-screened. The TIS false negative rate was estimated at 0.012% [4/3400]. Conclusion The overall performance of the TIS in our lab appears to be highly satisfactory in terms of improving sensitivity in screening cervical precursor lesions. The increased accuracy of detection of HSIL indicates a positive impact of the TIS in our laboratory.

As extra-cranial metastasis of glioblastoma multiforme (GBM) is rare, it may create a diagnostic dilemma especially during interpretation of fine needle aspiration biopsy (FNAB) cytology.

Abstract #2065 Background: Bone is the most common site of breast cancer metastasis. Efforts have been made to understand the complex molecular mechanisms underlying this. It involves invasion, proliferation at metastatic sites, and activity of cytokines (IL1,6,8,11, TNF, and TGFß), chemokines, their receptors (stromal-derived-factor1 and CXC chemokine[CXCR4, also called fusin, involved in HIV tropism]), and matrix metalloproteinases (MMP). Microarray analyses of primary tumors with different metastatic sites have distinct gene expression patterns indicating tumor heterogeneity. Both lung and bone metastasis gene signatures include CXCR4, and MMP1, whereas bone metastasis gene signature also includes IL11. PTHrP, activated by TGFß signaling, also mediate metastasis. A subpopulation of breast cancer cells, expressing CD44 with stem cell properties, was found to promote breast tumor invasion and metastases. Trefoil factor1 (TFF1), a proangiogenic factor, involved in invasion of kidney and colonic cancer cells, has a high correlation with bone metastasis, is expressed under estrogen control, and found in a subset of ER+ breast cancers. New prognostic markers are needed to identify patients at high risk of bone metastases to individualize therapy. This study's aim was, for the first time, to evaluate the expression of selected biomarkers associated with tumor relapse to bone, by immunohistochemistry (IHC) on tissue microarrays (TMA). Materials and Methods: 16 cases of metastastic breast cancer to bone (MBC-B) were retrieved from MWH tumor registry and a TMA with 3-fold redundancy was created (TMA1), consisting of cores with a diameter of 0.6 mm. To study the expression of these markers in primary breast cancers (PBC), a second TMA (TMA2) was created for 63 breast cancers. IHC was performed for selected markers (TFF1,CXCR4, MMP1, PTHrP, CD44, FGFR3, and IL11) on both TMAs. Clinical and other pathologic parameters were available. All patients had received standard chemotherapy/hormonal treatment. Results: In the MBC-B group, 14 cases were infiltrating ductal and 2 cases were infiltrating lobular (average tumor size 2.6cm;range 0.4-8.0cm). 50% (8/16) had axillary lymph node metastases. 94% (15/16) were ER and PR positive and 19% (3/16) were HER2 positive. 44% (7/16) were rapid bone metastasis progressors (1y). Currently, no statistical difference in the expression of markers in MBC-B vs PBC was seen (100% for CXCR4, MMP1, PTHrP, FGFR3, IL11). However, a high expression of ER, CD44, and TFF1 is seen in MBC-B. Ongoing studies are evaluating whether these markers are independent prognostic markers for bone metastases and tumor specific survival. Discussion: We found a high level of expression of CXCR4, MMP1, PTHrP, FGFR3, and IL11 which signifies their importance in both PBC and MBC-B. Overall, expression of ER, CD44, and TFF1 potentially may serve as prognostic markers in identifying, at an early stage, patients at risk of bone metastases. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2065.