Search

The utility of prognostic and predictive immunohistochemistry biomarkers in the context of cancer is plagued by inconsistent interpretation of results which can lead to poor rates of adoption or inappropriate use of novel therapeutic strategies. To monitor immunohistochemistry assay performance, a new on-slide control motif, Immunohistochemistry Critical Assay Performance Controls (ICAPC) was developed. We hypothesized that the use of these controls by the diagnosing pathologist to interpret BRAFV600E would result in reduced interobserver and intraobserver interpretation errors. A cross-sectional, sequentially obtained sample of surgical pathology cases stained for BRAFV600E was assembled from a single hospital in Vancouver, British Columbia. Half of the cases had normal on-slide controls and the remainder with ICAPC. Results from 6 independent and blinded readers were compared with each other and to the gold-standard pathologic diagnosis with the goal of demonstrating superior interrater agreement with ICAPC relative to standard on-slide controls. Cohen’s κ was used to compute pair-wise reader agreements, whereas Fleiss’ κ was used to compare to the gold standard. The implementation of ICAPC resulted in statistically significant improvements in the interobserver agreement of BRAFmutation status ascertained by BRAFV600E immunohistochemistry. Half of the readers demonstrated significant improvements in agreement with the gold-standard diagnosis with the addition of ICAPC. Across all readers, the mean increase in κ was 0.14 with a 95% confidence interval of 0.01-0.28 (P=0.04). This study demonstrates that the addition of ICAPC serves to significantly reduce interobserver variability in the assessment of BRAFV600E immunohistochemistry. As such, we recommend that this approach should be used as part of a comprehensive quality management strategy in the setting of histopathology.

Introduction

Inhibitors of the programmed cell death 1 (PD-1) signaling axis have recently demonstrated efficacy and are rapidly being incorporated into the treatment of non–small cell lung cancers (NSCLCs). Despite clear benefits to certain patients, the association of these responses with a predictive biomarker remains uncertain. Several different biomarkers have been proposed, with differing results and conclusions. This study compares multiple methods of biomarker testing for treatment of NSCLCs with PD1-axis inhibitors. Tissue microarrays of matched primary and metastatic NSCLCs were used to compare four different PD-1 ligand (PD-L1) IHC techniques, as well as RNA ISH. Additional cases with whole genome and transcriptome data were assessed for molecular correlates of PD-L1 overexpression. Eighty cases were included in the IHC study. Multiple IHC methodologies showed a high rate of agreement (Kappa = 0.67). When calibrated to RNA expression, agreement improved significantly (Kappa = 0.90, p=0.0049). PD-L1 status of primary and metastatic tumors was discordant in 17 (22%) cases. This study suggests that different IHC methodologies for PD-L1 assessment provide slightly different results. There is significant discordance between the PD-L1 status of primary tumors and lymph node metastases. RNA ISH may be a useful adjunct to complement PD-L1 IHC testing.

Deficiencies in DNA mismatch repair have been associated with inferior response to 5-FU in colorectal cancer. Pancreatic ductal adenocarcinoma is similarly treated with pyrimidine analogs, yet the predictive value of mismatch repair status for response to these agents has not been examined in this malignancy. A tissue microarray with associated clinical outcome, comprising 254 resected pancreatic ductal adenocarcinoma patients was stained for four mismatch repair proteins (MLH1, MSH2, MSH6 and PMS2). Mismatch repair deficiency and proficiency was determined by the absence or presence of uniform nuclear staining in tumor cells, respectively. Cases identified as mismatch repair deficient on the tissue microarray were confirmed by immunohistochemistry on whole slide sections. Of the 265 cases, 78 (29%) received adjuvant treatment with a pyrimidine analog and 41 (15%) showed a mismatch repair-deficient immunoprofile. Multivariable disease-specific survival in the mismatch repair-proficient cohort demonstrated that adjuvant chemotherapy, regional lymph-node status, gender, and the presence of tumor budding were significant independent prognostic variables (P≤0.04); however, none of the eight clinico-pathologic covariates examined in the mismatch repair-deficient cohort were of independent prognostic significance. Univariable assessment of disease-specific survival revealed an almost identical survival profile for both treated and untreated patients with a mismatch repair-deficient profile, while treatment in the mismatch repair-proficient cohort conferred a greater than 10-month median disease-specific survival advantage over their untreated counterparts (P=0.0018). In this cohort, adjuvant chemotherapy with a pyrimidine analog conferred no survival advantage to mismatch repair-deficient pancreatic ductal adenocarcinoma patients. Mismatch repair immunoprofiling is a feasible predictive marker in pancreatic ductal adenocarcinoma patients, and further prospective evaluation of this finding is warranted.

Deficiencies in DNA mismatch repair have been associated with inferior response to 5-FU in colorectal cancer. Pancreatic ductal adenocarcinoma is similarly treated with pyrimidine analogs, yet the predictive value of mismatch repair status for response to these agents has not been examined in this malignancy. A tissue microarray with associated clinical outcome, comprising 254 resected pancreatic ductal adenocarcinoma patients was stained for four mismatch repair proteins (MLH1, MSH2, MSH6 and PMS2). Mismatch repair deficiency and proficiency was determined by the absence or presence of uniform nuclear staining in tumor cells, respectively. Cases identified as mismatch repair deficient on the tissue microarray were confirmed by immunohistochemistry on whole slide sections. Of the 265 cases, 78 (29%) received adjuvant treatment with a pyrimidine analog and 41 (15%) showed a mismatch repair-deficient immunoprofile. Multivariable disease-specific survival in the mismatch repair-proficient cohort demonstrated that adjuvant chemotherapy, regional lymph-node status, gender, and the presence of tumor budding were significant independent prognostic variables (P≤0.04); however, none of the eight clinico-pathologic covariates examined in the mismatch repair-deficient cohort were of independent prognostic significance. Univariable assessment of disease-specific survival revealed an almost identical survival profile for both treated and untreated patients with a mismatch repair-deficient profile, while treatment in the mismatch repair-proficient cohort conferred a greater than 10-month median disease-specific survival advantage over their untreated counterparts (P=0.0018). In this cohort, adjuvant chemotherapy with a pyrimidine analog conferred no survival advantage to mismatch repair-deficient pancreatic ductal adenocarcinoma patients. Mismatch repair immunoprofiling is a feasible predictive marker in pancreatic ductal adenocarcinoma patients, and further prospective evaluation of this finding is warranted.

The contemporary oncologic pathology report conveys diagnostic, prognostic, predictive, and hereditary predisposition information. Each component may be premised on a morphologic feature or a biomarker. Clinical validity and reproducibility are paramount as is standardization of reporting and clinical response to ensure individualization of patient care. Regarding hereditary predisposition, morphology-based genetic referral systems in some instances have eclipsed genealogy-based systems, for example, cell type in ovarian cancer and BRCA screening. In other instances such as Lynch syndrome, morphology-based schemas supplement clinical schemas and there is an emerging standard of care for reflex biomarker testing. Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) syndrome predisposes patients to uterine and cutaneous leiomyomas (LMs) and renal cell carcinomas (RCCs). Several authors have emphasized the role pathologists may play in identifying this syndrome by recognizing the morphologic characteristics of syndromic uterine LMs and RCCs. Recently immunohistochemical overexpression of S-(2-succinyl) cysteine (2SC) has been demonstrated as a robust biomarker of mutation status in tumors from HLRCC patients. In this blinded control-cohort study we demonstrate that the proposed morphologic criteria used to identify uterine LMs in HLRCC syndrome are largely irreproducible among pathologists and lack sufficient robustness to serve as a trigger to triage cases for 2SC immunohistochemistry or patients for further familypersonal history inquiry. Although refinement of morphologic criteria can be considered, in view of the availability of a clinically robust biomarker, consideration should be given to reflex testing of uterine LMs with an appropriate age cut off or in the setting of a suspicious family history.

Ovarian cancer treatments and outcomes vary substantially, yielding a diverse group of survivors. Few data exist on quality of life (QoL) concerns and the foremost needs of these patients. Our goal was to conduct a pilot study to determine the QoL needs of ovarian cancer survivors to establish priorities for future interventions.

Subclassification of endometrial carcinoma according to histological type shows variable interobserver agreement. The aim of this study was to assess specifically the interobserver agreement of histological type in high-grade endometrial carcinomas, recorded by gynecological pathologists from five academic centers across Canada. In a secondary aim, the agreement of consensus diagnosis with immunohistochemical marker combinations was assessed including six routine (TP53, CDKN2A (p16), ER, PGR, Ki67, and VIM) and six experimental immunohistochemical markers (PTEN, ARID1A, CTNNB1, IGF2BP3, HNF1B, and TFF3). The paired interobserver agreement ranged from κ 0.50 to 0.63 (median 0.58) and the intraobserver agreement from κ 0.49 to 0.67 (median 0.61). Consensus about histological type based on morphological assessment was reached in 72% of high-grade endometrial carcinomas. A seven-marker immunohistochemical panel differentiated FIGO grade 3 endometrioid from serous carcinoma with a 100% concordance rate compared with the consensus diagnosis. More practically, a three-marker panel including TP53, ER, and CDKN2A (p16) can aid in the differential diagnosis of FIGO grade 3 endometrioid from endometrial serous carcinoma. Our study demonstrates that the inter- and intraobserver reproducibility of histological type based on morphology alone are mostly moderate. Ancillary techniques such as immunohistochemical marker panels are likely needed to improve diagnostic reproducibility of histological types within high-grade endometrial carcinomas.

Subclassification of endometrial carcinoma according to histological type shows variable interobserver agreement. The aim of this study was to assess specifically the interobserver agreement of histological type in high-grade endometrial carcinomas, recorded by gynecological pathologists from five academic centers across Canada. In a secondary aim, the agreement of consensus diagnosis with immunohistochemical marker combinations was assessed including six routine (TP53, CDKN2A (p16), ER, PGR, Ki67, and VIM) and six experimental immunohistochemical markers (PTEN, ARID1A, CTNNB1, IGF2BP3, HNF1B, and TFF3). The paired interobserver agreement ranged from κ0.50 to 0.63 (median 0.58) and the intraobserver agreement from κ0.49 to 0.67 (median 0.61). Consensus about histological type based on morphological assessment was reached in 72% of high-grade endometrial carcinomas. A seven-marker immunohistochemical panel differentiated FIGO grade 3 endometrioid from serous carcinoma with a 100% concordance rate compared with the consensus diagnosis. More practically, a three-marker panel including TP53, ER, and CDKN2A (p16) can aid in the differential diagnosis of FIGO grade 3 endometrioid from endometrial serous carcinoma. Our study demonstrates that the inter- and intraobserver reproducibility of histological type based on morphology alone are mostly moderate. Ancillary techniques such as immunohistochemical marker panels are likely needed to improve diagnostic reproducibility of histological types within high-grade endometrial carcinomas.

Low-grade serous carcinoma (LGSC) of the ovary has only recently been recognized as a disease entity distinct from the more common high-grade serous carcinoma (HGSC). When confined to the ovary, LGSC is associated with a very favorable prognosis, and chemotherapy is typically not recommended. There is little available information on the prognosis of patients with LGSC with extraovarian spread, from population-based tumor registries where there has been full pathology review. Thirty-two cases of Stage II to IV ovarian LGSC were identified in the Cheryl Brown Ovarian Cancer Outcomes Unit (1984–2000). In 19 cases, blocks were available and immunostaining for p53, p16, Ki-67, WT1, and E-cadherin was performed. Expression of these markers was then compared with a series of >400 cases of HGSC from the outcomes unit. LGSCs presented at Stage II in 1032 cases, Stage III in 2132 cases, and Stage IV in 132 cases. On follow-up, most patients died of disease, with <30 survival at 10 years. Compared with HGSCs, the LGSCs were significantly less likely to express p16 at high levels, or to show abnormal p53expression (P=0.049 and <0.0001, respectively). Ki-67 staining indices were lower in the LGSCs (P<0.0001). There were no significant differences between LGSCs and HGSCs with respect to expression of WT1 and E-cadherin (P=0.27 and 0.62, respectively). This population-based series of LGSC with extraovarian spread at presentation had an unfavorable prognosis, similar to that of HGSC. As previously reported, LGSC shows lower tumor proliferation and fewer p53abnormalities than in HGSC.

Low-grade serous carcinomas and serous borderline tumors, combined herein and referred to as low-grade serous tumors, show distinct molecular alterations and clinical behaviors compared with high-grade serous carcinomas. The discrimination between low-grade serous tumors and high-grade serous carcinomas can be challenging on small tissue samples, such as cell blocks of paracentesis fluid or biopsies from omental disease. The purpose of this study was to test the ability of TP53 and CDKN2A immunohistochemistry to distinguish between high-grade serous carcinomas and low-grade serous tumors on small tissue samples. Tissue microarrays containing 582 high-grade serous carcinomas, 45 low-grade serous carcinomas, and 49 serous borderline tumors, confirmed by contemporary histopathological review, were stained for TP53 and CDKN2A (DO7 and E6H4 antibody clones, respectively). TP53 was scored as completely absent, wild-type pattern or overexpressed (>60%), and CDKN2A was scored as either negative/patchy (<90%) or block expression (>90%). The combination of the two markers, ie, the TP53 wild-type pattern and CDKN2A patchy expression, had sensitivity for low-grade serous tumors of 89%, a specificity of 93%, a positive predictive value of 68%, and a negative predictive value of 98%. These markers can, therefore, be used on small biopsies/cell blocks to refute a diagnosis of low-grade serous tumors. These findings may inform emerging neoadjuvant therapeutic strategies in advanced ovarian cancers and may be crucial for future clinical trials on molecular-based therapies.

Low-grade serous carcinomas and serous borderline tumors, combined herein and referred to as low-grade serous tumors, show distinct molecular alterations and clinical behaviors compared with high-grade serous carcinomas. The discrimination between low-grade serous tumors and high-grade serous carcinomas can be challenging on small tissue samples, such as cell blocks of paracentesis fluid or biopsies from omental disease. The purpose of this study was to test the ability of TP53 and CDKN2A immunohistochemistry to distinguish between high-grade serous carcinomas and low-grade serous tumors on small tissue samples. Tissue microarrays containing 582 high-grade serous carcinomas, 45 low-grade serous carcinomas, and 49 serous borderline tumors, confirmed by contemporary histopathological review, were stained for TP53 and CDKN2A (DO7 and E6H4 antibody clones, respectively). TP53 was scored as completely absent, wild-type pattern or overexpressed (>60%), and CDKN2A was scored as either negative/patchy (<90%) or block expression (>90%). The combination of the two markers, ie, the TP53 wild-type pattern and CDKN2A patchy expression, had sensitivity for low-grade serous tumors of 89%, a specificity of 93%, a positive predictive value of 68%, and a negative predictive value of 98%. These markers can, therefore, be used on small biopsies/cell blocks to refute a diagnosis of low-grade serous tumors. These findings may inform emerging neoadjuvant therapeutic strategies in advanced ovarian cancers and may be crucial for future clinical trials on molecular-based therapies.

We describe the architectural patterns of advanced ovarianpelvic high-grade serous carcinomas that have been treated with upfront surgery, followed by adjuvant chemotherapy or neoadjuvant chemotherapy, followed by interval debulking to explore the association with the chemotherapeutic response. For 70 cases of advanced (i.e. stage IIIIV) high-grade serous carcinomas (33 platinum resistantintermediate, 37 platinum sensitive; 24 neoadjuvantly treated, 44 primary surgery), all tumor-containing histologic slides were reviewed by 1 of 3 pathologists. Histologic type was confirmed and the following features were assessed: major architectural pattern and the presence of any of 8 predefined minor architectural patterns (papillary, transitional cell carcinoma-like, micropapillary, microcystic, nested papillary, slit-like, glandular, solid). A semiquantitative assessment of psammoma bodies, histiocytic response, necrosis, nuclear atypia, and single-cell invasion was performed. Mitotic count was performed in 10 HPF and 1 HPF was counted for intraepithelial lymphocytes. The morphologic features were tested for an association with previous neoadjuvant chemotherapy and response to chemotherapy (resistantintermediate versus chemotherapy-sensitive cases stratified by neoadjuvant chemotherapy), which was carried out using 2tests for categorical variables and analysis of variance for continuous data. Combinations of features were analyzed using unsupervised clustering (Wald). Although 8 of 18 features were significantly different when samples from neoadjuvantly treated patients were compared with those not previously treated, no individual histomorphologic feature or a combination of features was associated with response to chemotherapy. Further subtyping of high-grade serous carcinomas will likely need ancillary molecular markers that may have a greater potential to identify cases that will not respond to platinum-based chemotherapy.

We characterized BRCA1and BRCA2status (mutation/methylation) in a consecutive series of cases of ovarian carcinoma in order to identify differences in clinicopathological features, molecular characteristics, and outcome between the pelvic high-grade serous cancers with (i) germline or somatic mutations in BRCA1or BRCA2, (ii) methylation of BRCA1, and (iii) normal BRCA1or BRCA2. In all, 131 women were identified prospectively, who were undergoing surgical staging and agreed to germline testing for BRCA1and BRCA2 mutations. Histopathology, germline and somatic BRCA1or BRCA2 mutations, BRCA1methylation, and BRCA1and BRCA2mRNA expression levels distinguished four subgroups. In all, 103 cases were high-grade serous carcinoma and of these 31 (30%) had germline or somatic BRCA1 or BRCA2mutations (20% BRCA1and 10% BRCA2) (group 1), 21 (20%) had methylation of BRCA1(group 2), and in 51 (50%) there was no BRCA loss (group 3). Group 4 consisted of 28 cases of non-high-grade serous, none of which had BRCA loss. BRCA1and BRCA2mRNA expression levels correlated with designated group (P=0.0008). Among high-grade serous carcinomas, there were no differences between groups 1–3 with respect to stage, ascites, CA125 level, platinum sensitivity, cytoreduction rate, neoadjuvant chemotherapy, or survival. Tumors with BRCA1or BRCA2mutations had increased immune infiltrates (CD20 and TIA-1) compared with high-grade serous without mutations (P=0.034, 0.027). TP53expression differed between groups (P<0.0001), with abnormal TP53expression in 49/50 tumors from groups 1 and 2. Wild-type TP53expression was associated with worse outcome in high-grade serous (P<0.001). BRCA loss (mutation/methylation) is a common event in the pelvic high-grade serous (50%). TP53abnormalities and increased immune cell infiltrates are significantly more common in high-grade serous with germline and somatic mutations in BRCA1or BRCA2, compared with tumors lacking BRCA abnormalities.

We characterized BRCA1 and BRCA2 status (mutation/methylation) in a consecutive series of cases of ovarian carcinoma in order to identify differences in clinicopathological features, molecular characteristics, and outcome between the pelvic high-grade serous cancers with (i) germline or somatic mutations in BRCA1 or BRCA2, (ii) methylation of BRCA1, and (iii) normal BRCA1 or BRCA2. In all, 131 women were identified prospectively, who were undergoing surgical staging and agreed to germline testing for BRCA1 and BRCA2 mutations. Histopathology, germline and somatic BRCA1 or BRCA2 mutations, BRCA1 methylation, and BRCA1 and BRCA2 mRNA expression levels distinguished four subgroups. In all, 103 cases were high-grade serous carcinoma and of these 31 (30%) had germline or somatic BRCA1 or BRCA2 mutations (20% BRCA1 and 10% BRCA2) (group 1), 21 (20%) had methylation of BRCA1 (group 2), and in 51 (50%) there was no BRCA loss (group 3). Group 4 consisted of 28 cases of non-high-grade serous, none of which had BRCA loss. BRCA1 and BRCA2 mRNA expression levels correlated with designated group (P=0.0008). Among high-grade serous carcinomas, there were no differences between groups 1–3 with respect to stage, ascites, CA125 level, platinum sensitivity, cytoreduction rate, neoadjuvant chemotherapy, or survival. Tumors with BRCA1 or BRCA2 mutations had increased immune infiltrates (CD20 and TIA-1) compared with high-grade serous without mutations (P=0.034, 0.027). TP53 expression differed between groups (P<0.0001), with abnormal TP53 expression in 49/50 tumors from groups 1 and 2. Wild-type TP53 expression was associated with worse outcome in high-grade serous (P<0.001). BRCA loss (mutation/methylation) is a common event in the pelvic high-grade serous (50%). TP53 abnormalities and increased immune cell infiltrates are significantly more common in high-grade serous with germline and somatic mutations in BRCA1 or BRCA2, compared with tumors lacking BRCA abnormalities.

The mismatch repair (MMR) status of tumors is being increasingly recognized as a prognostic, predictive, and possible germline predispositionLynch syndrome (LS) biomarker in colorectal cancer and other cancer types, particularly in endometrial cancer. Current methods (clinical history and tumor morphology) to predict MMR deficiency (dMMR) are suboptimal, and implementation of reflex laboratory testing of appropriate tumors has been recommended, a strategy requiring test standardization and clinical coordination.

Neoadjuvant therapy has an emerging role in the treatment of high-stage ovarian carcinoma. Some ovarian carcinoma subtypes do not respond well to standard chemotherapy, making accurate subtype diagnosis before starting therapy important. This diagnosis is frequently based on omental biopsy specimens. In particular, with very small biopsies, immunostaining for diagnostic biomarkers may be needed. To assess intratumoral heterogeneity of biomarker expression in pelvic high-grade serous carcinoma, we compared the expression of a set of 10 biomarkers between ovarian and omental sites. Tissue microarrays were constructed from 123 high-grade serous carcinomas with paired ovarian and omental tumor samples. These samples were stained with biomarkers that have been used in ovarian carcinoma subtype diagnosis (WT1, TP53p53, MUC16CA125, CDKN2Ap16), and with biomarkers of the tumor microenvironment (CD8, CD163, SPARC, PDGFRB), cell adhesion (CDH1E-Cadherin), and proliferation (Ki67) as well. Expression frequencies in samples from the 2 sites were compared, as was concordance at the 2 sites for individual tumors. The 2 markers of desmoplastic stromal response (PDGFRB, SPARC) were more frequently expressed in the omentum compared with the ovary (P<0.001; McNemar test). The other 8 markers did not show a significant difference in the frequency of expression between sites. Within individual cases, some markers such as Ki67 and CDKN2A showed variability, indicating that these markers are affected by intratumoral heterogeneity. The intratumoral variability for MUC16, TP53, and WT1 was modest. Commonly used diagnostic markers, such as TP53 and WT1, show little variability between ovarian and omental sites, suggesting that they can be successfully used in small biopsy specimens from extraovarian sites. In contrast, markers of host stromal response do vary between sites, suggesting a biologic difference of the microenvironment at different sites that should be taken into account when tissue-based research is carried out.

Sex cord-stromal tumors (SCSTs) of the ovary are relatively uncommon tumors. Diagnosis of SCST rests primarily on the histomorphology of these tumors, and tumors with an atypical or unconventional appearance can pose diagnostic challenges. Previously, we had identified FOXL2 (402C→G) mutation as being characteristic of adult granulosa cell tumors (aGCTs). However, molecular screening for this mutation is not always possible and adds time and cost to the diagnostic process. In this study, we investigated the potential diagnostic use of immunostaining for FOXL2 on formalin-fixed paraffin-embedded tissue sections. Using a commercially available polyclonal antiserum against FOXL2 protein, immunoexpression of FOXL2 was tested in 501 ovarian tumor samples, including 119 SCSTs, using whole tissue sections and tissue microarrays. Staining was correlated with FOXL2 mutation status. In addition, we compared FOXL2 immunoexpression with that of -inhibin and calretinin, the 2 traditional immunomarkers of SCST, in a subset of 89 SCSTs. FOXL2 immunostaining was present in 95 of 119 (80) SCSTs, including >95 of aGCTs, juvenile granulosa cell tumors, fibromas, and sclerosing stromal tumors. Only 50 of Sertoli-Leydig cell tumors (N=40) expressed FOXL2. One of 11 steroid cell tumors and 3 of 3 female adnexal tumors of probable Wolffian origin showed FOXL2 immunoreactivity, whereas all other non-SCSTs tested (N=368) were negative for FOXL2 expression. Thus, the sensitivity and specificity of FOXL2 immunoreactivity for SCST are 80 and 99, respectively. The FOXL2 (402C→G) mutation was confirmed to be both a sensitive and relatively specific indicator of aGCT. Forty-five of 119 SCSTs were mutation positive. These cases were 39 of 42 (93) aGCTs, 3 of 40 Sertoli-Leydig cell tumors, 2 of 5 thecomas, and 1 of 4 (25) SCSTs of unclassified type. SCSTs harboring a FOXL2 mutation consistently immunoexpressed FOXL2 (44 of 45, 98), but FOXL2 immunostaining was also seen in many SCSTs that lacked a mutation (49 of 73, 67). FOXL2 immunostaining showed higher sensitivity for the diagnosis of SCST, compared with -inhibin and calretinin, and FOXL2 staining was typically more intense in positive cases compared with either -inhibin or calretinin. In the SCSTs that were negative for FOXL2 expression, -inhibin andor calretinin immunostaining yielded positive results. In conclusion, FOXL2 is a relatively sensitive and highly specific marker for SCST. FOXL2 staining is present in almost all SCSTs with a FOXL2 mutation, and also in a majority of SCSTs without a mutation. FOXL2, together with -inhibin and calretinin, forms an immunomarker panel that will result in positive staining with 1 or more markers in essentially all cases of SCST.

With the emerging evidence that the five major ovarian carcinoma subtypes (high-grade serous, clear cell, endometrioid, mucinous, and low-grade serous) are distinct disease entities, management of ovarian carcinoma will become subtype specific in the future. In an effort to improve diagnostic accuracy, we set out to determine if an immunohistochemical panel of molecular markers could reproduce consensus subtype assignment. Immunohistochemical expression of 22 biomarkers were examined on tissue microarrays constructed from 322 archival ovarian carcinoma samples from the British Columbia Cancer Agency archives, for the period between 1984 and 2000, and an independent set of 242 cases of ovarian carcinoma from the Gynaecologic Tissue Bank at Vancouver General Hospital from 2001 to 2008. Nominal logistic regression was used to produce a subtype prediction model for each of these sets of cases. These models were then cross-validated against the other cohort, and then both models were further validated in an independent cohort of 81 ovarian carcinoma samples from five different centers. Starting with data for 22 markers, full model fit, backwards, nominal logistic regression identified the same nine markers (CDKN2A, DKK1, HNF1B, MDM2, PGR, TFF3, TP53, VIM, WT1) as being most predictive of ovarian carcinoma subtype in both the archival and tumor bank cohorts. These models were able to predict subtype in the respective cohort in which they were developed with a high degree of sensitivity and specificity (κ statistics of 0.88±0.02 and 0.86±0.04, respectively). When the models were cross-validated (ie using the model developed in one case series to predict subtype in the other series), the prediction equation's performances were reduced (κ statistics of 0.70±0.04 and 0.61±0.04, respectively) due to differences in frequency of expression of some biomarkers in the two case series. Both models were then validated on the independent series of 81 cases, with very good to excellent ability to predict subtype (κ=0.85±0.06 and 0.78±0.07, respectively). A nine-marker immunohistochemical maker panel can be used to objectively support classification into one of the five major subtypes of ovarian carcinoma.

With the emerging evidence that the five major ovarian carcinoma subtypes (high-grade serous, clear cell, endometrioid, mucinous, and low-grade serous) are distinct disease entities, management of ovarian carcinoma will become subtype specific in the future. In an effort to improve diagnostic accuracy, we set out to determine if an immunohistochemical panel of molecular markers could reproduce consensus subtype assignment. Immunohistochemical expression of 22 biomarkers were examined on tissue microarrays constructed from 322 archival ovarian carcinoma samples from the British Columbia Cancer Agency archives, for the period between 1984 and 2000, and an independent set of 242 cases of ovarian carcinoma from the Gynaecologic Tissue Bank at Vancouver General Hospital from 2001 to 2008. Nominal logistic regression was used to produce a subtype prediction model for each of these sets of cases. These models were then cross-validated against the other cohort, and then both models were further validated in an independent cohort of 81 ovarian carcinoma samples from five different centers. Starting with data for 22 markers, full model fit, backwards, nominal logistic regression identified the same nine markers (CDKN2A, DKK1, HNF1B, MDM2, PGR, TFF3, TP53, VIM, WT1) as being most predictive of ovarian carcinoma subtype in both the archival and tumor bank cohorts. These models were able to predict subtype in the respective cohort in which they were developed with a high degree of sensitivity and specificity (κstatistics of 0.88±0.02 and 0.86±0.04, respectively). When the models were cross-validated (ie using the model developed in one case series to predict subtype in the other series), the prediction equation's performances were reduced (κstatistics of 0.70±0.04 and 0.61±0.04, respectively) due to differences in frequency of expression of some biomarkers in the two case series. Both models were then validated on the independent series of 81 cases, with very good to excellent ability to predict subtype (κ=0.85±0.06 and 0.78±0.07, respectively). A nine-marker immunohistochemical maker panel can be used to objectively support classification into one of the five major subtypes of ovarian carcinoma.

Total laparoscopic hysterectomy (LH) is a minimally invasive technique, which results in comparable morbidity and better cosmesis compared with total abdominal hysterectomy. The literature is discrepant as to whether it is associated with a higher incidence of positive peritoneal cytology compared with total abdominal hysterectomy and recently, associated artifacts, including vascular pseudoinvasion (VPI), have been described. A retrospective histopathologic review of 266 hysterectomy specimens from 2 centers was performed. The observers, blinded to the surgical technique, assessed for the presence of artifactual changes including disruption of the endometrial lining, nuclear crush artifact, VPI, endomyometrial cleft artifact with or without epithelial displacement, inflammatory debris within vessels, serosal carryover, and intratubal contaminants. In addition, the rates of positive peritoneal washings over a 5-year period, and the use of immunohistochemistry (IHC) to aid in cell typing over a 3-year period, were compared between hysterectomies in which a uterine manipulator (UM) device had and had not (nonmanipulated hysterectomies) been used. The hysterectomies were performed for malignant (n=160) and benign (n=102) uterine disease or for ovarian or cervical disease (n=4), and included total abdominal (n=108), vaginal (n=17), laparoscopy-assisted vaginal (n=24), laparoscopy converted to laparotomy (n=10), nonrobotic laparoscopic (n=51), and robot-assisted laparoscopic (n=56) hysterectomies. One hundred and two (38) of these hysterectomies involved the use of a UM. Artifactual changes of disruption of the endometrial lining, endomyometrial clefts, intratubal contaminants, nuclear crush artifact, intravascular inflammatory debris, and VPI were significantly more common with LH and with the use of a UM, independent of whether the endometrial pathology was benign or malignant. IHC to aid in endometrial cancer subtyping was more likely to be used in manipulated hysterectomies (P=0.0166). Furthermore, peritoneal washings were significantly more likely to be positive in hysterectomies in which a UM had been used (P=0.0061). Histologic artifacts are significantly more common in LH and specifically in hysterectomies in which a UM is used. Such artifacts impair the pathologists' interpretation of cell type requiring an increased use of IHC, and displaced epithelial fragments present within vessels or artifactual clefts may result in the misinterpretation of prognostic and staging parameters. Furthermore, there is a significantly higher rate of positive peritoneal cytology in cases that are subjected to uterine manipulation, suggesting dissemination of malignant cells into the abdominal cavity. The clinical significance of this finding needs to be determined.

High-grade endometrial carcinomas are a heterogeneous group of tumors and include grade 3 endometrioid (EC-3), serous (SC), and clear cell carcinomas (CCC). There are conflicting data about the prognosis of these subtypes of high-grade endometrial carcinoma; this may be a result of lack of reproducibility in classifying tumor cell type. The purpose of this study was to examine differences in immunophenotype and outcome in a series of high-grade endometrial carcinomas, focusing on the comparison of EC-3 versus SC. We selected 180 endometrial carcinomas of SC, EC, or CCC type. No mixed carcinomas were included in the study. We chose the following immunohistochemical markers, estrogen receptor (ER), insulin-like growth factor 2 mRNA-binding protein 3 (IMP3), p16, p53, progesterone receptor (PR), and phosphatase and tensin homolog (PTEN) as being significantly differentially expressed in endometrial carcinoma subtypes. The tumors were stratified into 4 groups on the basis of their cell type and grade EC grade 1 or 2, EC-3, SC, and CCC. Univariate survival analysis revealed significant differences in outcome between the 4 groups (P<0.0001), with significantly longer disease-specific survival for grade 1 or 2 EC versus EC-3 (P0.0001), and EC-3 versus SC (P0.0003). p16, PTEN, and IMP3 expression was observed more frequently in SC compared with EC-3 (P<0.0001, P0.021, and P0.031, respectively). These 3 markers showed the highest sensitivity and specificity in distinguishing between EC-3 and SC, with receiver operating characteristics area under the curve of 0.85, 0.69, and 0.71, respectively. ER and p53 approached but did not reach significance for differential expression in EC-3 versus SC (P0.055 and P0.068, respectively). A combination of p16 and PTEN predicts EC-3 versus SC with a sensitivity of 90.0 and specificity of 96.8. p16 and PTEN can aid in distinguishing between EC-3 and SC of the endometrium, and are superior to ER, PR, and p53 for this purpose. EC-3 carcinomas have a significantly better prognosis than SC carcinomas of the endometrium.

Although there are recognized differences in the type of ovarian carcinomas between those tumors diagnosed at low versus high stage, there is a lack of data on stage distribution of ovarian carcinomas diagnosed according to the current histopathologic criteria from large population-based cohorts. We reviewed full slide sets of 1009 cases of 2555 patients diagnosed with ovarian carcinoma that were referred to the British Columbia Cancer Agency over a 16-year period (1984 to 2000). On the basis of the reviewed cases we extrapolated the distribution of tumor type in low-stage (III) and high-stage (IIIIV) tumors. We then compared the frequencies with those seen in a large hospital practice. The overall frequency of tumor types was as follows high-grade serous—68.1, clear-cell—12.2, endometrioid—11.3, mucinous—3.4, low-grade serous—3.4, rare types—1.6. High-grade serous carcinomas accounted for 35.5 of stage III tumors and 87.7 of stage IIIIV tumors. In contrast, clear-cell (26.2 vs. 4.5), endometrioid (26.6 vs. 2.5), and mucinous (7.5 vs. 1.2) carcinomas were relatively more common among the low-stage versus high-stage tumors. This distribution was found to be very similar in 410 consecutive cases from the Washington Hospital Center. The distribution of ovarian carcinoma types differs significantly in patients with low-stage versus high-stage ovarian carcinoma when contemporary diagnostic criteria are used, with consistent results seen in 2 independent case series. These findings reflect important biological differences in the behavior of the major tumor types, with important clinical implications.

Primary ovarian mucinous carcinomas of the intestinal type are uncommon and earlier reports have included cases diagnosed according to older, less stringent, criteria (which would now be considered borderline tumors) and variable numbers of cases of metastatic adenocarcinoma. This study was conducted to identify all cases of primary mucinous carcinoma of the ovary in a population-based registry, diagnosed according to WHO 2003 criteria, and to characterize their histologic features, immunohistochemical expression profile, and outcome. Thirty-one cases of primary ovarian mucinous carcinoma were included in this study. Immunostaining for 33 markers was performed. Mean age of the patients was 55.4±13.5 years. Thirty tumors were stage I or II at presentation. Twenty-six of 31 (83.9) tumors had expansile stromal invasion, 4 of 31 (12.9) showed destructive invasion, and 1 of 31 (3.2) had anaplastic carcinoma in a mural nodule. All cases with destructive invasion showed grade 3 nuclear atypia whereas only 3 of 26 (11.5) cases with expansile invasion had grade 3 nuclear atypia (P0.0003). At follow-up, 6 of 26 patients (23.1) with tumors showing expansile invasion experienced a recurrence, compared with 1 of 4 patients (25) with destructive invasion and the single patient (100) with anaplastic carcinoma. There was CK7 positivity in 26 of 31 cases (86.7), and CK20 and Cdx-2 were each positive in 33.3 of cases. D2-40, calretinin, mesothelin, CA-125, Pax-8, TTF, and WT1 were completely negative in all cases. NHERF1 staining was present in 19 of 26 cases (73) and its expression was associated with poor prognosis (P0.05). Our findings support current diagnostic criteria for primary ovarian mucinous carcinoma, that is, the presence of expansile invasion, in the absence of destructive invasion, warrants a diagnosis of carcinoma. A large majority of mucinous carcinomas show only an expansile pattern of invasion and are confined to the pelvis at diagnosis.

Clear cell carcinoma is an uncommon subtype of ovarian carcinoma, accounting for 10% of cases. Clear cell carcinoma typically presents with stage I or II disease, and in this setting prognostic markers could aid in management decisions, in particular the decision to treat with adjuvant chemotherapy. We tested whether expression of insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3, also known as IMP3) can serve as a new biomarker to predict outcome for patients with clear cell carcinoma and other subtypes of ovarian carcinoma. The expression of IGF2BP3 was evaluated by immunohistochemistry in 475 ovarian carcinomas of different subtypes and correlated with disease-specific survival. IGF2BP3 antibody specificity was validated by correlation of IGF2BP3 protein with mRNA expression level in a series of 35 ovarian carcinomas (r=0.849, P<0.0001). IGF2BP3 protein expression was an independent marker of reduced disease-specific survival (risk ratio 2.9, 95% confidence interval 1.4–5.8) in the clear cell subtype (N=128), but not in high-grade serous (N=198) or endometrioid (N=121) carcinomas. The prognostic significance of IGF2BP3 expression for reduced disease-specific survival (risk ratio 2.6, 95% confidence interval 1.3–5.0) was confirmed in an independent series of cases (N=150) from three different centers in North America. We conclude that IGF2BP3 is the first biomarker of prognostic significance in ovarian clear cell carcinoma that has been validated in an independent case series.

The purpose of this study was to identify perfusion defects of the lung using computed tomography (CT). A balloon catheter was placed in a lobar pulmonary artery of six anesthetized, ventilated, juvenile pigs to simulate occlusive segmental embolus. Contrast medium was injected via a central venous catheter at rates of 1.5, 3, 4.5, and 9 ml/s in each pig. A 40-second single-level cine CT was acquired distal to the inflated balloon during suspended inspiration. Three computer-manipulated images (time to maximal enhancement, change in maximal attenuation, maximal contrast minus precontrast subtraction) were generated using custom software and compared with the unmodified maximal enhancement and precontrast images. Two independent observers identified perfusion defects and scored the level of confidence (5-point scale) on all five images. Regions of interest were drawn in perfused and nonperfused lung and time-attenuation curves were generated. Perfusion defects were accurately (99.8 ± 0.3) and confidently (4.5 ± 0.6) detected and there was excellent interobserver agreement (Kappa 0.99 ± 0.02) on all computer-manipulated images. There was a significant increase in confidence (p< 0.05) between contrast medium injection rates of 1.5 and 9 ml/s. A linear relationship exists (r20.88) between injection rate and change in maximal attenuation. In conclusion, perfusion defects of the lung are seen using computer-manipulated CT images.

Pancreatic neuroendocrine neoplasms (PNENs) are biologically and clinically heterogeneous. Here, we use a multi-omics approach to uncover the molecular factors underlying this heterogeneity. Transcriptomic analysis of 84 PNEN specimens, drawn from two cohorts, is substantiated with proteomic profiling and identifies four subgroups: Proliferative, PDX1-high, Alpha cell-like and Stromal/Mesenchymal. The Proliferative subgroup, consisting of both well- and poorly differentiated specimens, is associated with inferior overall survival probability. The PDX1-high and Alpha cell-like subgroups partially resemble previously described subtypes, and we further uncover distinctive metabolism-related features in the Alpha cell-like subgroup. The Stromal/Mesenchymal subgroup exhibits molecular characteristics of YAP1/WWTR1(TAZ) activation suggestive of Hippo signaling pathway involvement in PNENs. Whole-exome sequencing reveals subgroup-enriched mutational differences, supported by activity inference analysis, and identifies hypermorphic proto-oncogene variants in 14.3% of sequenced PNENs. Our study reveals differences in cellular signaling axes that provide potential directions for PNEN patient stratification and treatment strategies.

Tumor grade has been the most accepted prognostic indicator of disease-specific survival among women with stage I and II ovarian carcinomas. Many investigators have included stage IB, grade 1 at diagnosis as low-risk disease and have suggested that women in this category should not receive adjuvant therapy. However, grading assignment appears to be unreliable because of problems with reproducibility and lack of consideration of biological differences between the different tumor types. Some investigators believe that classification based on tumor typing using new histopathological criteria was more reproducible than grade assignment and would more accurately reflect biological differences.