Prognostic Risk Assessment and Prediction of Radiotherapy Benefit for Women with Ductal Carcinoma In Situ (DCIS) of the Breast, in a Randomized Clinical Trial (SweDCIS).

Prediction of radiotherapy (RT) benefit after breast-conserving surgery (BCS) for DCIS is crucial. The aim was to validate a biosignature, DCISionRT®, in the SweDCIS randomized trial. Women were randomly assigned to RT or not after BCS, between 1987 and 2000. Tumor blocks were collected, and slides were sent to PreludeDxTM for testing. In 504 women with complete data and negative margins, DCISionRT divided 52% women into Elevated (DS > 3) and 48% in Low (DS ≤ 3) Risk groups. In the Elevated Risk group, RT significantly decreased relative 10-year ipsilateral total recurrence (TotBE) and 10-year ipsilateral invasive recurrence (InvBE) rates, HR 0.32 and HR 0.24, with absolute decreases of 15.5% and 9.3%. In the Low Risk group, there were no significant risk differences observed with radiotherapy. Using a cutoff of DS > 3.0, the test was not predictive for RT benefit (p = 0.093); however, above DS > 2.8 RT benefit was greater for InvBE (interaction p = 0.038). Recurrences at 10 years without radiotherapy increased significantly per 5 DS units (TotBE HR:1.5 and InvBE HR:1.5). Continuous DS was prognostic for TotBE risk although categorical DS did not reach significance. Absolute 10-year TotBE and InvBE risks appear sufficiently different to indicate that DCISionRT can aid physicians in selecting individualized adjuvant DCIS treatment strategies. Further analyses are planned in combined cohorts to increase statistical power.

Validation of a Ductal Carcinoma In Situ Biomarker Profile for Risk of Recurrence after Breast-Conserving Surgery with and without Radiotherapy.

PURPOSE: A major challenge in ductal carcinoma in situ (DCIS) treatment is selection of the most appropriate therapeutic approach for individual patients. We conducted an external prospective-retrospective clinical validation of a DCIS biologic risk signature, DCISionRT, in a population-based observational cohort of women diagnosed with DCIS and treated with breast-conserving surgery (BCS). EXPERIMENTAL DESIGN: Participants were 455 health plan members of Kaiser Permanente Northwest diagnosed with DCIS and treated with BCS with or without radiotherapy from 1990 to 2007. The biologic signature combined seven protein tumor markers assessed in formalin-fixed, paraffin-embedded tumor tissue with four clinicopathologic factors to provide a DCISionRT test result, termed decision score (DS). Cox regression and Kaplan-Meier analysis were used to measure the association of the DS, continuous (linear) or categorical (DS ≤ 3 vs. DS > 3), and subsequent total ipsilateral breast events and invasive ipsilateral breast events at least 6 months after initial surgery. RESULTS: In Cox regression, the continuous and categorical DS variables were positively associated with total and invasive breast event risk after adjustment for radiotherapy. In a subset analysis by treatment group, categorical Kaplan-Meier analyses showed at least 2-fold differences in 10-year risk of total breast events between the elevated-risk and low-risk DS categories. CONCLUSIONS: In this first external validation study of the DCISionRT test, the DS was prognostic for the risk of later breast events for women diagnosed with DCIS, following BCS.

A Biological Signature for Breast Ductal Carcinoma In Situ to Predict Radiotherapy Benefit and Assess Recurrence Risk.

PURPOSE: Ductal carcinoma in situ (DCIS) patients and their physicians currently face challenging treatment decisions with limited information about the individual's subsequent breast cancer risk or treatment benefit. The DCISionRT biological signature developed in this study provides recurrence risk and predicts radiotherapy (RT) benefit for DCIS patients following breast-conserving surgery (BCS). EXPERIMENTAL DESIGN: A biological signature that calculates an individualized Decision Score (DS) was developed and cross-validated in 526 DCIS patients treated with BCS ± RT. The relationship was assessed between DS and 10-year risk of invasive breast cancer (IBC) or any ipsilateral breast event (IBE), including IBC or DCIS. RT benefit was evaluated by risk group and as a function of DS. RESULTS: The DS was significantly associated with IBC and IBE risk, HR (per 5 units) of 4.2 and 3.1, respectively. For patients treated without RT, DS identified a Low Group with 10-year IBC risk of 4% (7% IBE) and an Elevated Risk Group with IBC risk of 15% (23% IBE). In analysis of DS and RT by group, the Elevated Risk Group received significant RT benefit, HR of 0.3 for IBC and IBE. In a clinicopathologically low-risk subset, DS reclassified 42% of patients into the Elevated Risk Group. In an interaction analysis of DS and RT, patients with elevated DS had significant RT benefit over baseline. CONCLUSIONS: The DS was prognostic for risk and predicted RT benefit for DCIS patients. DS identified a clinically meaningful low-risk group and a group with elevated 10-year risks that received substantial RT benefit over baseline.

The genetics of interdigitating dendritic cell sarcoma share some changes with Langerhans cell histiocytosis in select cases.

Histiocytic disorders have been noted to have evidence of transdifferentiation; examples of cases with combinations of different lineages have been shown. In our index case, we identified interdigitating dendritic cell (IDC) differentiation in a case of Langerhans cell histiocytosis (LCH). Little is currently known about the genetics of IDC sarcoma (IDCS) because they are exceedingly rare. Using array comparative genomic hybridization (aCGH), we evaluated 4 cases of IDCS and compared them with our index case, as well as genetic abnormalities previously found in LCH. Four cases of paraffin-embedded samples of IDCS and 1 case of LCH with IDC differentiation were evaluated using aCGH. Array CGH results showed no abnormalities in a case of LCH with interdigitating cell differentiation. In 3 of 4 cases of IDCS, genetic abnormalities were identified; 1 case had no identifiable abnormalities. Interdigitating dendritic cell sarcoma case 1 had gains of 3q and 13q; IDCS case 2 had trisomy 12; IDCS case 3 had deletions of 7p, 12p, 16p, 18q, 19q, and 22q; and IDCS case 4 had no detectable abnormalities. Our index case, LCH with IDC differentiation, showed no abnormalities by aCGH. A number of LCH cases do not have detectable genetic abnormalities. In contrast, 3 of 4 cases of IDCS evaluated had identifiable abnormalities by aCGH. Furthermore, 2 of these shared abnormalities, albeit of large genetic regions, with published abnormalities seen in LCH. No recurrent abnormalities were identified in the IDCS cases. However, the possibility of a relationship between IDCS and LCH cannot be entirely excluded by these results.

Molecular cytogenetics as a clinical test for prognostic and predictive biomarkers in newly diagnosed ovarian cancer.

BACKGROUND: There is a clinical need for routinely available genomic biomarker testing in newly diagnosed ovarian cancer. In the current study we performed molecular cytogenetics using a validated array based comparative genomic hybridization (array CGH) assay to screen for the presence of predictive and prognostic biomarkers in archival diagnostic tissue from ovarian cancer patients. We hypothesized that biomarkers of high-risk disease would be detectable in tumor samples from patients with treatment refractory, advanced disease, and would be detected less frequently in tumor samples from patients with more favorable outcomes. In addition, we predicted that the use of a genome-wide copy number analysis (CNA) testing platform would enable us to identify novel potentially targetable chromosomal alterations of therapeutic significance in a percentage of cases. METHODS: Formalin-fixed paraffin-embedded tissue (FFPE) tumor bank specimens were retrieved from the initial surgical resection for 18 ovarian cancer patients. Molecular cytogenetics was performed by array CGH for the detection of somatic chromosomal alterations associated with high-risk disease including amplifications of the CCNE1 and HER2 genes. Genomic risk stratification results were correlated with available clinical data. CGH data from each patient's tumor genome was also surveyed for the presence of potentially targetable aberrations. Relevant therapeutic agents and open studies for investigational drugs were reported for each patient. RESULTS: High-risk genomic alterations were identified in 12/18 (67%) of cases and all patients with high-risk markers had advanced, treatment refractory disease. Three tumors with minimal genomic changes had no high-risk markers and were from patients with Stage I/II disease that had been completely resected and under surveillance for recurrence. Eleven patients (61%) had at least one potentially targetable genomic alteration including CCNE1, HER2, KRAS gene amplifications, and somatic BRCA1 and/or BRCA2 gene deletions. Bi-allelic PTEN gene deletion was detected in one patient's tumor. CONCLUSIONS: Clinical genomic profiling of ovarian tumors by array CGH augments pathologic grade and stage to help stratify newly diagnosed ovarian cancer into high and low-risk disease. This personalized genomic information can also help guide treatment planning and disease monitoring by identifying novel potentially targetable genomic alterations that can be used by clinicians to choose rational directed therapies for patients with chemo-resistant disease.

Histopathological findings in 29 lymph node biopsies with increased IgG4 plasma cells.

IgG4-related sclerosing disease encompasses a family of disorders associated with increased numbers of IgG4 plasma cells and mass forming lesions in various tissues. Lymphadenopathy is a common finding, seen in up to 80% of cases. In the largest series of cases to date, we describe histologic, immunohistochemical, special stain and flow cytometric findings in 29 cases of enlarged lymph nodes with increased IgG4 plasma cells. Lymph node biopsies showed all resection specimens; no needle core biopsies of tissue were evaluated. Cases were considered to have increased numbers of IgG4 plasma cells using the histological criteria outlined by Cheuk and Chan (2010): IgG4 plasma cells >50 cells in a high-power field and >40% of IgG-positive plasma cells positive for IgG4. Additionally, increased intrafollicular plasma cells were a common finding. The lymph nodes showed a variety of reactive histological features including follicular hyperplasia, progressive transformation of germinal centers, interfollicular expansions, variable degrees of fibrosis, increased histiocytes and occasionally an appearance similar to that of plasma cell Castleman disease.

Co-occurrence of Langerhans cell histiocytosis and Rosai-Dorfman disease: possible relationship of two histiocytic disorders in rare cases.

Rosai-Dorfman disease and Langerhans cell histiocytosis are both disorders of accessory immune cells. Two cases have been previously reported of concurrent Langerhans cell histiocytosis and Rosai-Dorfman disease. In this report, we characterize the findings and selected molecular studies in nine additional cases. Histology was reviewed. Immunohistochemical stains were performed on all cases in which slides or blocks were available. A combination of CD1a, S-100, CD3, CD20, langerin, CD68, CD163, CD21, CD35 and CD123 immunohistochemical stains were performed. High-resolution array comparative genomic hybridization was performed on six samples from five cases. In these cases, seven were female and two male, with an average age of 25 years (15 months-59 years). A majority of the cases were identified in lymph node. Areas of Langerhans cell histiocytosis had a typical appearance with the existence of bland 'coffee-bean' nuclei, clear cytoplasm and associated eosinophils. The immunophenotype was typical, including expression of CD1a, S100, CD68 and langerin. In areas of Rosai-Dorfman disease, there was emperipolesis seen in all cases. Cells were intermediate-large in size with large round nuclei and ample clear or pale cytoplasm. The lesional cells were positive for S100, CD68, CD163, without expression of langerin or CD1a. Array comparative genomic hybridization showed gains and/or losses in four of the six samples. One case showed no gains or losses and one additional case showed gains and losses in the Langerhans cell histiocytosis, while no abnormalities were discovered in the Rosai-Dorfman disease component. These findings are comparable to those seen in previous studies of Langerhans cell histiocytosis. We report the clinical and pathologic findings of the combination of Langerhans cell histiocytosis and Rosai-Dorfman disease. Furthermore, we suggest on the basis of evidence from our cases that, when simultaneous, the two entities may be pathophysiologically related.

Immunomodulator agent-related lymphoproliferative disorders.

The recent development of inhibitors of key immune response proteins has revolutionized the therapy of autoimmune diseases; these immunomodulator agents include monoclonal antibodies and receptor antagonists. However, as with all therapies, these new agents are not without side effects and complications. In particular, anti-tumor necrosis factor alpha (TNFalpha) agents have been reported to be associated with an increased incidence of lymphoproliferative disorders, infections, and vasculitis. We evaluated the clinicopathological features of 18 cases of immunomodulator agent-related lymphoproliferative disorders (IAR-LPD) from several institutions. These included 6 cases of B-cell lymphoma, 2 cases of T-cell lymphoma, 3 cases of classical Hodgkin lymphoma, and 7 atypical lymphoid proliferations that did not fulfill diagnostic criteria for lymphoma; two of the latter regressed after discontinuation of the immunomodulator agent therapy. All eight lymphoma patients with available information had also received prior chemotherapy (methotrexate or 6-mercaptopurine). EBV was strongly associated with the B-cell and classical Hodgkin lymphomas. This case series illustrates that a broad range of lymphoid proliferations can occur after immunomodulator agent therapy and that these immunomodulator agent-related lymphoproliferative disorders have considerable overlap with other well-defined lymphoproliferative diseases associated with iatrogenic immunosuppression. Further study is warranted to evaluate how these therapies interact with other immunosuppressive agents and the underlying abnormal immune system to enhance the development of lymphomas and atypical lymphoid proliferations.

Genetic heterogeneity in HER2 testing in breast cancer: panel summary and guidelines.

CONTEXT: Intratumoral heterogeneity of HER2 gene amplification has been well documented and represents subclonal diversity within the tumor. The reported incidence of intratumor HER2 amplification genetic heterogeneity ranges in the literature from approximately 5% to 30%. The presence of HER2 genetic heterogeneity may increase subjectivity in HER2 interpretation by the pathologist. OBJECTIVES: To define HER2 genetic heterogeneity and to provide practice guidelines for examining and reporting breast tumors with genetic heterogeneity for improvement of HER2 testing in breast cancer. DESIGN: We convened an expert panel to discuss HER2 gene amplification testing by fluorescence in situ hybridization. Components addressed included a definition of HER2 amplification heterogeneity, practice guidelines for examination of the tissue, and reporting criteria for this analysis. RESULTS: Genetic heterogeneity for amplification of HER2 gene status in invasive breast cancer is defined and guidelines established for assessing and reporting HER2 results in these cases. These guidelines are additive to and expand those published in 2007 by the American Society of Clinical Oncology and the College of American Pathologists. CONCLUSION: Standardized methods for analysis will improve the accuracy and consistency of interpretation of HER2 gene amplification status in breast cancer.

t(11;18)(q21;q21) in extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue in stomach: a study of 48 cases.

Gastric extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue (MZL-MALT) is speculated to be immune mediated and is notable for responding to treatment by Helicobacter pylori eradication. However, the gastric MZL-MALT with t(11;18)(q21;q21) has been shown to be resistant to treatment by H. pylori eradication. We studied the molecular, immunohistochemical, and histological aspects of 48 cases of gastric MZL-MALT and used a reverse transcription real-time PCR assay to assess the presence of a t(11;18)(q21;q21) in formalin-fixed, paraffin-embedded tissue. Florescence in situ hybridization for t(11:18)(q21;q21) was used to confirm the real-time PCR results. Three distinct morphological subtypes were recognized: monocytoid, small lymphocytic, and plasmacytoid. Morphology, immunophenotype, and immunoglobulin heavy chain (IgH) gene rearrangement were correlated with the results of the t(11:18)(q21;q21) assay. Of the 48 analyzed cases, 15 (31%) were positive for t(11;18)(q21;q21) and 33 (69%) were monoclonal for IgH gene rearrangement. Of the 15, 13 (87%) cases with t(11;18)(q21;q21) translocation showed IgH gene rearrangement by PCR. Of the 33 t(11;18)(q21;q21)-negative cases tested, 20 cases (61%) showed IgH gene rearrangement. The 15 t(11;18)(q21;q21) translocation-positive cases had either monocytoid (12 of 15) or small lymphocytic morphology (3 of 15). Aberrant expression of CD43 was observed in 8 of 15 (53%) t(11;18)(q21;q21)-positive cases and 21 of 31 (68%) t(11;18)(q21;q21)-negative cases. Our data show that t(11;18)(q21;q21)-positive MZL-MALTs frequently show monocytoid morphology, less often small lymphocytic morphology, and not purely plasmacytoid morphology. Identification of a t(11;18)(q21;q21) by reverse transcription real-time PCR is highly specific for MZL-MALT and helps in the diagnosis of MZL-MALT. Studying the correlation between this translocation and morphological features may increase our understanding of the role of this translocation in the pathogenesis and the clinical behavior of gastric MZL-MALT.

Consensus recommendations on estrogen receptor testing in breast cancer by immunohistochemistry.

Estrogen receptor (ER) status in breast cancer is currently the most important predictive biomarker that determines breast cancer prognosis after treatment with endocrine therapy. Although immunohistochemistry has been widely viewed as the gold standard methodology for ER testing in breast cancer, lack of standardized procedures, and lack of regulatory adherence to testing guidelines has resulted in high rates of "false-negative" results worldwide. Standardized testing is only possible after all aspects of ER testing--preanalytical, analytical, and postanalytical, have been closely controlled. A meeting of the "ad-hoc committee" of expert pathologists, technologists, and scientists, representing academic centers, reference laboratories, and various agencies, issued standardization testing recommendations, aimed at optimization of clinical ER testing environment, as a step toward improved standardized testing.

High concordance between immunohistochemistry and fluorescence in situ hybridization testing for HER2 status in breast cancer requires a normalized IHC scoring system.

The American Society of Clinical Oncologists and College of American Pathologists have recently released new guidelines for laboratory testing of HER2 status in breast cancer, which require high levels (95%) of concordance between immunohistochemistry positive (3+) and fluorescence in situ hybridization-amplified cases, and between immunohistochemistry negative (0/1+) and fluorescence in situ hybridization-nonamplified cases; these required levels of concordance are significantly higher than those found in most published studies. We tested the hypothesis that a modification of the HER2 immunohistochemistry scoring system could significantly improve immunohistochemistry and fluorescence in situ hybridization concordance. A total of 6604 breast cancer specimens were evaluated for HER2 status by both immunohistochemistry and fluorescence in situ hybridization using standard methodologies. Results were compared when the standard immunohistochemistry scoring system was replaced by a normalized scoring system in which the HER2 score was derived by subtracting the score on the non-neoplastic breast epithelium from that on the tumor cells. Among the 6604 tumors, using a non-normalized immunohistochemistry scoring system, 267/872 (30.6%) of the immunohistochemistry 3+ cases proved to be fluorescence in situ hybridization nonamplified, whereas using the normalized scoring system only 30/562 (5.3%) of immunohistochemistry 3+ cases proved to be 'false positive'. The concordance rate between immunohistochemistry 3+ and fluorescence in situ hybridization-amplified cases using the normalized scoring method was 94.7%, whereas the concordance using the non-normalized method was only 69.4%. Extremely high concordance between immunohistochemistry and fluorescence in situ hybridization assessment of HER2 status in breast cancer is achievable, but to attain this high level of concordance, modification of the FDA-approved immunohistochemistry scoring system is required.

Estrogen receptor alpha and progesterone receptor expression in ovarian adult granulosa cell tumors and Sertoli-Leydig cell tumors.

The biologic role that estrogen receptor (ER) and progesterone receptor (PR) play in ovarian sex cord-stromal tumors is poorly understood. Furthermore, immunohistochemical data on these hormone receptors in this group of neoplasms are limited and conflicting, with many reports suggesting that expression of ERalpha and/or PR is either infrequent or present at low levels in granulosa and Sertoli cell tumors. Immunohistochemical staining for ERalpha and PR was performed in 69 ovarian sex cord-stromal tumors: 41 adult granulosa cell tumors and 28 Sertoli-Leydig cell tumors. Extent of expression was scored based on the percentage of positive cells: 0, 5% or less; 1+, 6% to 25%; 2+, 26% to 50%; 3+, 51% to 75%; and 4+, 76% to 100%. Estrogen receptor alpha and PR were frequently expressed in adult granulosa cell tumors (66% and 98%, respectively) and Sertoli-Leydig cell tumors (79% and 86%, respectively). Diffuse (3+ or 4+) expression of PR was more common in adult granulosa cell tumors (68% vs. 36%; P = 0.013), whereas diffuse (3+ or 4+) expression of ERalpha was more frequent in Sertoli-Leydig cell tumors (50% vs. 20%; P = 0.010). In cases positive for both markers, adult granulosa cell tumors exhibited a focal (1+ or 2+) ERalpha/diffuse (3+ or 4+) PR coordinate profile more commonly than Sertoli-Leydig cell tumors (52% vs. 18%; P = 0.02), whereas Sertoli-Leydig cell tumors displayed a diffuse (3+ or 4+) ERalpha/focal (1+ or 2+) PR profile more frequently than adult granulosa cell tumors (36% vs. 0%; P = 0.0007). We conclude that expression of hormone receptors (based only on frequency of immunostaining) does not allow for distinction from other tumors in the differential diagnosis that are known to be frequently positive for ERalpha and PR such as endometrioid neoplasms. Most adult granulosa cell tumors and Sertoli-Leydig cell tumors share overlapping patterns of expression of ERalpha and PR with each other, but a subset of cases in each tumor category exhibits unique ERalpha/PR immunoprofiles (eg, focal ERalpha/diffuse PR in adult granulosa cell tumors and diffuse ERalpha/focal PR in Sertoli-Leydig cell tumors). These patterns of expression of ERalpha and PR may aid our understanding of the biologic differences between granulosa and Sertoli cell tumors.

Ovarian mucinous tumors associated with mature cystic teratomas: morphologic and immunohistochemical analysis identifies a subset of potential teratomatous origin that shares features of lower gastrointestinal tract mucinous tumors more commonly encountered as secondary tumors in the ovary.

Most primary ovarian mucinous tumors are of surface epithelial-stromal origin and exhibit diffuse expression of cytokeratin 7 (CK7) combined with variable expression of cytokeratin 20 (CK20); this immunoprofile distinguishes them from most lower gastrointestinal tract tumors secondarily involving the ovaries. The uncommon ovarian mucinous tumors of germ cell (teratomatous) origin have not been extensively evaluated to determine the utility of these markers and other markers of intestinal differentiation for distinguishing these tumors from metastatic gastrointestinal tract mucinous tumors. Immunohistochemical expression of CK7, CK20, CDX2, and villin was assessed in 44 ovarian mucinous tumors associated with a mature cystic teratoma. All cases lacked evidence of a nonovarian primary mucinous tumor. All mucinous tumors were unilateral; 6 cases had bilateral teratomas. All tumors displayed gastrointestinal-type mucinous differentiation, with epithelium that was commonly goblet cell-rich or hypermucinous; 21 were associated with pseudomyxoma ovarii and 3 of these had pseudomyxoma peritonei. Tumor architecture ranged from purely cystadenomatous (n=24), to proliferative (n=13), to carcinomatous (n=6); some tumors had admixtures of these patterns. One tumor had a goblet cell carcinoidlike pattern with pseudomyxoma ovarii. Three carcinomas had a signet ring cell component. Cystadenomatous tumors without pseudomyxoma ovarii (n=15) exhibited all possible CK7/CK20 coordinate expression profiles with nearly equal frequency. All proliferative tumors without pseudomyxoma ovarii (n=8) expressed CK7, most often in combination with CK20 expression. All cystadenomatous and proliferative tumors with pseudomyxoma ovarii (n=9 and n=5) were CK7-/CK20+. All carcinomatous tumors had pseudomyxoma ovarii; 3 were CK7-/CK20+, 2 were CK7+/CK20+, and 1 was CK7+/CK20-. The presence of pseudomyxoma ovarii was significantly associated with a CK7-/CK20+ profile (86% with pseudomyxoma ovarii vs. 13% without, P<0.0001), CDX2 positivity (79% vs. 0%, P<0.0001), and villin positivity (57% vs. 5%, P=0.0009). A subset of mucinous tumors associated with mature cystic teratomas exhibiting morphologic and immunohistochemical features of lower intestinal tract-type mucinous tumors may be teratomatous in origin. In practice, the more common diagnosis of secondary involvement by a lower intestinal tract mucinous tumor should be addressed in the pathology report and in subsequent clinical evaluation; interpretation as a true primary ovarian mucinous tumor of teratomatous origin can be considered as an alternative diagnosis when evaluation and follow-up fail to identify a nonovarian source of the mucinous tumor. Those tumors having CK7 expression with or without CK20 expression may be derived from upper gastrointestinal tract-type or sinonasal-type teratomatous elements but could be independent tumors of surface epithelial-stromal origin.

p16 expression in primary ovarian mucinous and endometrioid tumors and metastatic adenocarcinomas in the ovary: utility for identification of metastatic HPV-related endocervical adenocarcinomas.

Distinction of primary ovarian epithelial tumors from metastatic adenocarcinomas is challenging for tumors exhibiting mucinous, endometrioid, or mixed endometrioid/mucinous differentiation. Metastatic carcinomas with these types of differentiation can be derived from several sites, including the gastrointestinal tract and the uterus. Most endocervical adenocarcinomas exhibit mucinous and/or endometrioid differentiation; they infrequently metastasize to the ovaries but may simulate primary ovarian tumors [both atypical proliferative (borderline) and carcinoma]. Most are high-risk human papillomavirus (HPV)-related and demonstrate diffuse p16 over-expression due to complex molecular mechanisms by which high-risk HPV transforming proteins interact with cell cycle regulatory proteins. The performance of this expression pattern for identifying metastatic endocervical adenocarcinomas in the ovaries among primary ovarian tumors and other metastatic adenocarcinomas having mucinous and/or endometrioid/endometrioidlike differentiation has not been evaluated. Immunohistochemical expression of p16 was assessed in 195 tumors, including 98 primary ovarian tumors (51 mucinous, 47 endometrioid, and 4 mixed mucinous-endometrioid tumors), 93 metastatic adenocarcinomas of known primary sites (colorectum: 34, endocervix: 19, pancreaticobiliary tract: 17, appendix: 7, stomach: 5), 11 metastatic adenocarcinomas of unknown origin (7 established as noncervical), and 4 adenocarcinomas of uncertain (primary ovarian vs. metastatic) origin. The HPV status of the endocervical adenocarcinomas was determined by in situ hybridization and polymerase chain reaction (when in situ hybridization was negative). Expression was assessed based on the percentage of moderately to strongly positive cells, estimated to the nearest 10%. Mean and median expression values for HPV-positive endocervical adenocarcinomas (99%, 100%; range 90% to 100%) were substantially higher than those for primary ovarian mucinous (5%, 0%; range 0% to 70%) and endometrioid (20%, 10%; range 0% to 100%) tumors, HPV-unrelated endocervical adenocarcinomas (0%, 0%; range 0% to 60%), metastatic adenocarcinomas of unknown origin (11%, 0%; range 0% to 30%), and adenocarcinomas of uncertain (primary ovarian vs. metastatic) origin (40%, 35%; range 0% to 90%); only the 15 HPV-positive endocervical adenocarcinomas and 6 other tumors had values of 80% or greater. Diffuse (>75% positive tumor cells) moderate to strong p16 expression is a sensitive (100%) and specific (97%) marker for identifying HPV-related endocervical adenocarcinomas metastatic to the ovary among the primary ovarian tumors and metastatic adenocarcinomas from other sites that are in the differential diagnosis of ovarian tumors having mucinous and/or endometrioid/endometrioidlike differentiation. p16 is useful as part of a panel of immunohistochemical markers for distinguishing primary ovarian tumors from metastases and, when diffusely positive, can suggest the cervix as a potential primary site for metastatic adenocarcinomas of unknown origin.

The specificity of interphase FISH translocation probes in formalin fixed paraffin embedded tissue sections is readily assessed using automated staining and scoring of tissue microarrays constructed from murine xenografts.

Implementation of interphase fluorescence in situ hybridization (FISH) assays in the clinical laboratory requires validation against established methods. Validation tools in common use include exchange of consecutive sections with another institution that has already established the FISH assay, comparison with conventional banded metaphase cytogenetics, confirmation of specificity using probed normal metaphases, consecutive paraffin sections of a validation set tested by a reference laboratory, and specificity assessment against well characterized cell lines. We have investigated the feasibility of using tissue microarrays (TMA) constructed from murine xenografts as a preliminary specificity-screening tool for validation of interphase FISH assays. Cell lines currently in use for FISH controls are used to generate xenografts in SCID mice which are fixed in formalin and paraffin embedded. A TMA is constructed using duplicate donor cores from the xenograft blocks. Xenografts used represent a wide range of translocations used routinely for formalin fixed paraffin embedded sections evaluated by FISH. Probe cocktails (Abbott-Vysis), for several non-random translocations associated with hematologic neoplasms and soft tissue sarcomas have been used in this manner. On-line deparaffinization, cell conditioning, and prehybridization steps are automated using a staining workstation (Ventana Discovery XT); hybridization and stringency washes are performed manually offline. FISH-probed TMAs are tracked using a Metasystems image scanner and analyzed using classifiers specifically developed for each molecular abnormality. FISH results for each xenograft in the TMA correspond exactly to the genotype previously established for the parent cell line from which the xenograft was prepared. Moderate complexity tissue microarrays constructed from murine xenografts are excellent validation tools for initial assessment of interphase FISH probe specificity.

Diagnostic Immunohistochemistry: what can go wrong?

Although immunohistochemistry (IHC) has become an integral component of the surgical pathology laboratory and practice, it remains a subspecialty in search of a definition. The rapid expansion of this discipline is continually contributing to the struggle of interested pathologists and technologists to identify the proper working conditions of antibody reagents and the right choice of detection systems and instrumentations. Training programs have not, for the most part, successfully incorporated IHC in their curricula. Thus, pitfalls in diagnostic IHC, in our opinion, are becoming more frequent than they used to. In this review, we highlight and attempt to provide solutions for the most common pitfalls in diagnostic IHC as it relates to the routine practice of surgical pathology.

Immunohistochemical expression of CDX2 in primary ovarian mucinous tumors and metastatic mucinous carcinomas involving the ovary: comparison with CK20 and correlation with coordinate expression of CK7.

Recent studies have demonstrated conflicting results regarding the value of CDX2 for distinguishing primary ovarian mucinous tumors from metastatic mucinous carcinomas in the ovary. Utility of coordinate expression of cytokeratins 7 and 20 is restricted to distinction of ovarian mucinous tumors from lower gastrointestinal tract metastases and data comparing coordinate expression of all three markers is limited. Immunohistochemical studies were performed to compare expression of CDX2 and cytokeratin 20, both markers of intestinal differentiation, in conjunction with coordinate expression of cytokeratin 7, in 90 mucinous tumors involving the ovary: 42 primary ovarian mucinous tumors (31 atypical proliferative (borderline) mucinous tumors (gastrointestinal type), 11 mucinous carcinomas) and 48 metastatic mucinous carcinomas of upper (pancreaticobiliary tract: 14; stomach: five) and lower (colon and rectum: 25; appendix: four) gastrointestinal tract origin. Primary ovarian tumors expressed CDX2 (40%) less frequently than cytokeratin 20 (83%) (P<0.0001). CDX2 expression in primary ovarian tumors (40%) was lower than CDX2 expression in metastatic carcinomas of both upper (74%; P=0.016) and lower gastrointestinal tract origin (90%; P<0.0001). Cytokeratin 20 expression was similar in primary ovarian tumors (83%) and metastases of upper (89%; P=0.071) and lower gastrointestinal tract origin (93%; P=0.29). Thus, as a single marker CDX2 offers some advantage over cytokeratin 20 because it is less frequently positive in primary ovarian tumors. In the almost universally cytokeratin 7-positive primary ovarian tumors and metastases of upper gastrointestinal tract origin, CDX2 coordinate expression was less common in primary ovarian tumors (36%) than in metastases of upper gastrointestinal tract origin (63%) (P=0.022) whereas cytokeratin 20 coordinate expression was identical in both tumor types (79%). In the almost universally cytokeratin 7-negative metastases of lower gastrointestinal tract origin, coordinate expression of CDX2 (83%) and cytokeratin 20 (86%) were equivalent (P=1.00). CDX2 was comparable to cytokeratin 20 in distinguishing metastases of lower gastrointestinal tract origin (usually cytokeratin 7-negative and CDX2/cytokeratin 20 positive) from primary ovarian tumors and metastases of upper gastrointestinal tract origin (usually cytokeratin 7-positive and CDX2/cytokeratin 20 variable). CDX2 provided some advantage over cytokeratin 20 for distinguishing primary ovarian mucinous tumors from metastases of upper but not lower gastrointestinal tract origin; however, the advantage in the former was limited due to the occurrence of shared coordinate expression profiles in both tumor types. Cytokeratin 7 provides the predominant discriminatory value among these markers yet is limited to distinction of primary ovarian tumors from metastases of lower gastrointestinal tract origin.