BackgroundAdjuvant immune checkpoint inhibitors (ICI) have improved survival in stage III cutaneous melanoma, yet many patients do not benefit. The tumor microenvironment is pivotal for durable responses; defining cellular composition can pinpoint immune components promoting anti-tumor activity and identify biomarkers associated with improved outcomes.MethodRegional lymph nodes (RLNs) were obtained at surgery from 29 patients with stage III melanoma. Eligible patients received adjuvant anti-PD-1 (αPD-1; pembrolizumab or nivolumab). Mass cytometry (CyTOF) was used to determine cellular composition of pre-treatment surgical specimens. NanoString bulk gene expression data from 125 patients receiving surgery without adjuvant therapy were used to evaluate trends observed in the CyTOF dataset.ResultsHigher proportions of CD103+PD-1+CD8+ (TRM) T cells and plasmablast-like CD38hiCD19dim cells were associated with improved prognosis in the CyTOF cohort. In the untreated cohort, a “B cell excluded” subgroup (< 2.5% tumor-infiltrating lymphocyte pathology score) had worse outcome, showing reduced B cell score and lower expression of activation genes including CD38, without change in CD8+ T cell score.ConclusionBaseline infiltration of CD8+ TRM and plasmablast-like CD38hiCD19dim cells in RLN is strongly associated with prolonged distant metastasis-free survival in patients receiving αPD-1, supporting their potential as prognostic biomarkers in stage III melanoma.

The objective of this study was to analyze the diagnostic role of HEG homolog 1 (HEG1) in cancers affecting the serosal cavities. HEG1 protein expression by immunohistochemistry was analyzed in 534 specimens (341 effusions and 193 surgical specimens). Effusions consisted of 151 tubo-ovarian carcinomas, 59 breast carcinomas, 44 mesotheliomas, 37 lung carcinomas, 29 uterine corpus and cervical carcinomas, 17 gastrointestinal carcinomas and 4 genitourinary carcinomas. Surgical specimens consisted of 139 tubo-ovarian carcinomas, 42 mesotheliomas, 7 multicystic mesothelial proliferations and 5 papillary mesothelial tumors. HEG1 expression was found in 43/44 (98%) mesothelioma effusions and 39/42 (93%) surgical mesothelioma specimens, as well as all multicystic and papillary mesothelial tumors. HEG1 was infrequently expressed in breast carcinoma (4/59; 7%), lung carcinoma (2/37; 5%) and cervical/uterine carcinoma effusions (3/29; 10%), but was often detected in tubo-ovarian carcinoma effusions (80/151; 53%) and surgical specimens (99/139, 71%). HEG1 was additionally consistently expressed by reactive mesothelial cells in effusions and in endothelial cells in surgical specimens. HEG1 had sensitivity of 95% for diagnosing malignant mesothelioma in all studied specimens, with a specificity of 38% in the differential diagnosis from tubo-ovarian carcinoma and 93% in the differential diagnosis from non-tubo-ovarian carcinomas. Of 179 HEG1-positive carcinomas, 172 expressed the epithelial marker claudin-4. In conclusion, HEG1 is a highly sensitive marker of both benign and malignant mesothelial cells. It shows high specificity in the differentiation of mesothelioma from lung or breast carcinoma but is of little value in differentiating mesothelioma from tubo-ovarian carcinoma. The potential role of HEG1 as vascular marker merits further research.

INTRODUCTION:FAT1 is mutated in many types of cancer. The objective of this study was to analyze the diagnostic role of FAT1 protein in cancers affecting the serosal cavities. METHODS:FAT1 protein expression by immunohistochemistry was analyzed in 256 effusions (139 peritoneal, 116 pleural, 1 pericardial), consisting of 96 tubo-ovarian carcinomas, 56 breast carcinomas, 44 mesotheliomas, 28 uterine corpus and cervical carcinomas, 15 lung carcinomas, 13 gastrointestinal carcinomas, and 4 cancers of other origin. RESULTS:FAT1 expression was most common in gastrointestinal carcinomas (12/13; 92%), followed by lung (9/15; 60%), breast (31/56; 55%), and uterine cervical (4/8; 50%) carcinomas, with infrequent expression in other tumors, including tubo-ovarian carcinomas (5/96; 5%). Mesotheliomas were uniformly negative. Staining of a limited series of surgical specimens showed comparable results for patient-matched breast carcinomas (29/41; 71%), whereas tubo-ovarian carcinomas were more frequently positive (16/57; 28%), though often focally, compared to effusions. Eight epithelioid mesotheliomas were negative (0/8; 0%). Survival analysis for 42 breast carcinoma patients with effusion for whom clinical data were available showed no association with overall (p = 0.398) or disease-free (p = 0.255) survival. CONCLUSION:FAT1 is commonly expressed in carcinomas of gastrointestinal, lung, or breast origin but is rarely expressed in tubo-ovarian carcinoma effusions and is absent in mesothelioma. Whether this difference is of use in the diagnostic setting remains to be established.

The objective of this study was to analyze the expression and prognostic role of cancer-associated proteins in uterine leiomyosarcoma (uLMS). p53, DAXX, ATRX, HMGA2, IMP3, Stathmin, and phospho-Stathmin (p-Stathmin) protein expression by immunohistochemistry was analyzed in tissue microarrays from 244 uLMS. Expression was assessed for association with clinicopathologic parameters in 173 patients with available data. Tissue microarrays were informative in 230 cases. p53 was aberrant in 44% of tumors. DAXX, ATRX, HMGA2, IMP3, and Stathmin were expressed in 90%, 55%, 40%, 33%, and 97% uLMS, respectively. Cytoplasmic and nuclear p-Stathmin staining was seen in 77% and 68% of tumors, respectively. Stathmin expression was significantly related to higher mitotic count ( P < 0.001), a higher degree of atypia ( P = 0.006), and vascular invasion ( P = 0.016), whereas p-Stathmin expression was significantly related to advanced stage ( P < 0.001), higher mitotic count ( P < 0.001), and vascular invasion ( P = 0.001). In univariate survival analysis for 165 patients with informative tissue microarrays, aberrant p53 ( P = 0.026) and higher IMP3 ( P = 0.024), Stathmin ( P < 0.001), cytoplasmic p-Stathmin ( P < 0.001), and nuclear p-Stathmin ( P < 0.001) expression was associated with poor disease-specific survival. Clinicopathologic parameters significantly related to poor disease-specific survival were older age ( P = 0.006), extrauterine disease at diagnosis (International Federation of Gynecology and Obstetrics (FIGO) stage ≥2; P < 0.001), high mitotic count ( P = 0.02), and grade 2 to 3 atypia ( P = 0.017). In multivariate analysis, age ( P = 0.002), FIGO stage ( P < 0.001), and Stathmin expression ( P < 0.001) were independent prognosticators. Stathmin was the only prognosticator in a multivariate analysis limited to patients with FIGO stage I disease ( P = 0.013). In conclusion, Stathmin expression is strongly associated with poor survival in uLMS and may be a new prognostic marker in this malignancy.

Background: Patients with high-grade serous carcinoma (HGSC) are commonly diagnosed at late disease stages and after primary tumors have disseminated in the peritoneum. The overexpression of tight junction proteins has been associated with poor prognosis in this setting, potentially reflecting the tumors adaptive changes in the disease cascade. Methods: By performing immunohistochemistry in a large single-center cohort of a total of 705 HGSC, we test the hypothesis that the protein expression of PReferentially expressed Antigen of MElanoma (PRAME) contains prognostic, predictive or clinically translatable information. We further examine its co-expression with tight junction proteins. Results: We confirmed the nuclear expression of PRAME in 442 (63 %) of specimens with comparable expression levels in peritoneal and pleural effusions (p = 0.72), and in effusions versus surgical specimens (p = 0.339). In effusions, any degree of expression of PRAME was significantly associated with suboptimal debulking surgery during primary treatment (p = 0.034). In surgical specimens, higher expression of PRAME was significantly linked to more advanced FIGO stage (p = 0.021). PRAME expression was not associated with other clinicopathologic factors as age, CA125 levels, chemoresistance or survival, but correlated with PRAME mRNA levels. Significant correlation was found between expression levels of PRAME and the tight junction protein Occludin (p = 0.002). Conclusion: Taken together, our study confirms PRAME to be expressed in the majority of HGSC effusions and surgical samples. The association of high levels of PRAME expression with incomplete surgical resection status and advanced stage disease may suggest PRAME expression as adaptative mechanism during disease dissemination. This finding warrants confirmation in independent series.

Adjuvant immunotherapy has significantly improved survival for patients with stage III cutaneous melanoma, yet a fraction of patients will not benefit from immune checkpoint inhibitors (ICI). The tumor microenvironment plays a pivotal role in generating durable responses to ICI. By analyzing the cellular composition of tumor-associated subsets, key immune components essential for promoting an anti-tumor environment can be pinpointed. This will allow for both patient stratification and identification of biomarkers associated with improved patient outcome. Regional lymph nodes were obtained from patients with stage III melanoma at surgery (n=29). Patients eligible for anti-PD-1 therapy (αPD-1; pembrolizumab or nivolumab) received adjuvant treatment for up to one year. CyTOF was used to determine cellular composition in pre-treatment surgical specimens. Bulk gene expression data generated by NanoString from patients receiving surgery without adjuvant therapy (n=125) was implemented for evaluating trends observed in the CyTOF dataset. Although no significant differences were observed across major hierarchical immune cell types between patients who developed distant metastasis after surgery and those that did not, an increased proportion of CD103 + PD-1 + CD8 + (T RM ) T cells and plasmablast-like CD38 hi CD19 dim cells were associated with improved prognosis in the CyTOF cohort. In the untreated cohort, a subset of patients defined as “Ultra-cold” (< 2.5 % tumor-infiltrating lymphocyte (TIL) scored by a pathologist) had significantly worse outcome than those with higher TIL infiltration. This Ultra-cold TIL group was associated with reduced B cell score, but not CD8 + T cell score, as well as reduced expression of activation genes like CD38 . In this study, CD103 + PD-1 + CD8 + (T RM ) T cells and plasmablast-like CD38 hi CD19 dim cell populations were found to be strongly associated with prolonged distant metastasis-free survival in regional lymph nodes from patients with stage III melanoma treated with αPD-1. This suggests an association between progression and infiltration of these cell types at baseline and highlights the potential of using immune cell subsets as prognostic biomarkers. What is already known on this topic – Patients being diagnosed with stage III melanoma will receive immune checkpoint inhibitors but will often be cured by surgery alone. Selection of which patients might benefit from treatment is still unresolved. Accurate biomarkers would aid in treatment stratification, to avoid overtreatment, and unnecessary toxicities. What this study adds – This study highlights how baseline CD103 + PD-1 + CD8 + (T RM ) T cells and plasmablast-like CD38 hi CD19 dim cell populations in the regional lymph node, is strongly associated with improved outcome in patients receiving anti-PD-1 therapy. How this study might affect research, practice or policy – The strong association between baseline plasmablast-like cell infiltration in RLN and prolonged distant metastasis free- survival, highlights this cell type as a potential treatment stratification criterion to identify patients with good prognosis.

Background Adjuvant immunotherapy has significantly improved survival for patients with stage III cutaneous melanoma, yet a fraction of patients will not benefit from immune checkpoint inhibitors (ICI). The tumor microenvironment plays a pivotal role in generating durable responses to ICI. By analyzing the cellular composition of tumor-associated subsets, key immune components essential for promoting an anti-tumor environment can be pinpointed. This will allow for both patient stratification and identification of biomarkers associated with improved patient outcome. Method Regional lymph nodes were obtained from patients with stage III melanoma at surgery (n=29). Patients eligible for anti-PD-1 therapy (αPD-1; pembrolizumab or nivolumab) received adjuvant treatment for up to one year. CyTOF was used to determine cellular composition in pre-treatment surgical specimens. Bulk gene expression data generated by NanoString from patients receiving surgery without adjuvant therapy (n=125) was implemented for evaluating trends observed in the CyTOF dataset. Results Although no significant differences were observed across major hierarchical immune cell types between patients who developed distant metastasis after surgery and those that did not, an increased proportion of CD103+PD-1+CD8+ (TRM) T cells and plasmablast-like CD38hiCD19dim cells were associated with improved prognosis in the CyTOF cohort. In the untreated cohort, a subset of patients defined as “Ultra-cold” (< 2.5 % tumor-infiltrating lymphocyte (TIL) scored by a pathologist) had significantly worse outcome than those with higher TIL infiltration. This Ultra-cold TIL group was associated with reduced B cell score, but not CD8+ T cell score, as well as reduced expression of activation genes like CD38 . Conclusion In this study, CD103+PD-1+CD8+ (TRM) T cells and plasmablast-like CD38hiCD19dim cell populations were found to be strongly associated with prolonged distant metastasis-free survival in regional lymph nodes from patients with stage III melanoma treated with αPD-1. This suggests an association between progression and infiltration of these cell types at baseline and highlights the potential of using immune cell subsets as prognostic biomarkers. What is already known on this topic – Patients being diagnosed with stage III melanoma will receive immune checkpoint inhibitors but will often be cured by surgery alone. Selection of which patients might benefit from treatment is still unresolved. Accurate biomarkers would aid in treatment stratification, to avoid overtreatment, and unnecessary toxicities. What this study adds – This study highlights how baseline CD103+PD-1+CD8+ (TRM) T cells and plasmablast-like CD38hiCD19dim cell populations in the regional lymph node, is strongly associated with improved outcome in patients receiving anti-PD-1 therapy. How this study might affect research, practice or policy – The strong association between baseline plasmablast-like cell infiltration in RLN and prolonged distant metastasis free- survival, highlights this cell type as a potential treatment stratification criterion to identify patients with good prognosis. ### Competing Interest Statement The authors have declared no competing interest. Southern and Eastern Norway Regional Health Authority, https://ror.org/02qx2s478, 2019048, 2016117, 2017100 Norwegian Ministry of Health and Care Services Fondsstiftelsen at Oslo University Hospital

Introduction: Grainyhead-like 2 (GRHL2) regulates epithelial-to-mesenchymal transition (EMT) in cancer. This study analyzed the expression and prognostic role of GRHL2 in high-grade serous carcinoma (HGSC). METHODS:GRHL2 protein expression by immunohistochemistry was analyzed in 411 HGSC (198 effusions, 213 surgical specimens). Expression score was generated by combination of staining extent and intensity and was assessed for association with clinicopathologic parameters and survival. RESULTS:GRHL2 expression was significantly higher in effusions compared to surgical specimens in both analysis of all specimens (p < 0.001) and patient-matched tumors (n = 39 patients; p < 0.001). Expression was additionally higher in post-chemotherapy compared to chemo-naive effusions (p < 0.001). Higher GRHL2 score in effusions was associated with a trend for shorter overall survival (OS; p = 0.088). Higher GRHL2 score in surgical specimens was significantly related to nonoptimal (>0 cm) debulking (p = 0.004), non-complete chemoresponse at diagnosis (p = 0.003), primary chemoresistance (p = 0.045), and shorter OS (p = 0.038) and progression-free survival (PFS; p = 0.024). Both OS and PFS findings remained significant in Cox multivariate analysis (OS: p = 0.048; PFS: p = 0.04). CONCLUSION:GRHL2 is overexpressed in HGSC effusions compared to solid lesions, possibly reflecting altered EMT status. However, high expression in solid lesions is associated with chemoresistance and poor survival, possibly due to mediation of tumor cell migration and invasion. .

OBJECTIVE:To analyse the diagnostic role of trefoil factor-1 and -3 (TFF1, TFF3), forkhead box protein A1 (FOXA1), carbonic anhydrase XII (CA XII) and trichorhinophalangeal syndrome type 1 (TRPS1) in serous effusions. The prognostic role of these markers in breast carcinoma was additionally studied. METHODS:Protein expression by immunohistochemistry was analysed in 247 effusions, consisting of 60 breast carcinomas, 54 tubo-ovarian carcinomas, 47 mesotheliomas, 44 lung carcinomas, 20 uterine corpus and cervical carcinomas, 17 gastrointestinal carcinomas and 5 cancers of other origin. RESULTS:TFF1, TFF3, FOXA1, CA XII and TRPS1 expression was found in 67%, 70%, 88%, 82% and 83% of breast carcinomas, respectively. Expression of all markers was seen in some carcinomas of other origin, most commonly in GI metastases, but was least frequent for TRPS1. CA XII expression was additionally seen in mesotheliomas and reactive mesothelial cells. All 5 markers were significantly overexpressed in breast compared to tubo-ovarian carcinoma (all p < 0.001) and lung carcinoma (all p < 0.001 except for FOXA1, p = 0.023). TFF1 (p = 0.003), TFF3 (p = 0.008) and FOXA1 (p = 0.017) expression was significantly higher in receptor-positive compared to receptor-negative primary breast carcinomas. In survival analysis for 44 breast carcinoma patients with clinical data, TFF1 expression was associated with a trend for longer overall (p = 0.096) and disease-free (p = 0.06) survival. CONCLUSION:TFF1, TFF3, FOXA1, CA XII and TRPS1 are sensitive breast carcinoma markers, with FOXA1 performing best in terms of sensitivity and TRPS1 being the most specific. Whether the expression of these markers in breast carcinoma effusions is informative of survival merits further research.

The objective of the present study was to characterize the molecular features of endometrial carcinomas with ambiguous histology. Eighteen carcinomas that could not be conclusively typed based on morphology and immunohistochemistry underwent analysis of mismatch repair (MMR) status, microsatellite status, and whole-exome sequencing. None of the tumors had pathogenic POLE mutation. Twelve tumors (67%) were microsatellite stable, and 6 (33%) had microsatellite instability. Fourteen tumors (78%) harbored TP53 mutations, and 2 (11%) had mutations in MMR genes. Eleven carcinomas (61%) were classified as copy number high and 7 (39%) as MSI-hypermutated, the latter including 3 tumors with TP53 mutation who concomitantly had MSI or mutation in a MMR gene. Other mutations that were found in > 1 tumor affected MUC16 (7 tumors), PIK3CA (6 tumors), PPP2R1A (6 tumors), ARID1A (5 tumors), PTEN (5 tumors), FAT1 (4 tumors), FAT4 (3 tumors), BRCA2 (2 tumors), ERBB2 (2 tumors), FBXW7 (2 tumors), MET (2 tumors), MTOR (2 tumors), JAK1 (2 tumors), and CSMD3 (2 tumors). At the last follow-up (median = 68.6 months), 8 patients had no evidence of disease, 1 patient was alive with disease, 8 patients were dead of disease, and 1 patient died of other cause. In conclusion, based on this series, the molecular landscape of endometrial carcinomas with ambiguous histology is dominated by TP53 mutations and the absence of POLE mutations, with heterogeneous molecular profile with respect to other genes. A high proportion of these tumors is clinically aggressive.

Carcinosarcoma (CS) is an uncommon and clinically aggressive malignancy. The objective of the present study was to characterize the molecular features of CS at various anatomic locations, including serous effusions. Specimens (n = 32) consisted of 25 biopsies/surgical resection specimens and 7 serous effusions (6 peritoneal, 1 pleural) from 25 patients. Fresh-frozen cell pellets and surgical specimens underwent targeted next-generation sequencing covering 50 unique genes. A total of 31 mutations were found in 25 of the 32 tumors studied, of which 1 had 3 mutations, 4 had 2 different mutations, and 20 had a single mutation. The most common mutations were in TP53 (n = 25 in 24 tumors; 1 tumor with 2 different mutations), with less common mutations found in RB1 (n = 2), MET (n = 1), KRAS (n = 1), PTEN (n = 1), and KIT (n = 1). Patient-matched specimens harbored the same TP53 mutation. Tumors with no detected mutations were more common in serous effusion specimens (3/7; 43%) compared with surgical specimens (4/25; 16%). In conclusion, the molecular landscape of CS is dominated by TP53 mutations, reinforcing the observation that the majority of these tumors develop from high-grade serous carcinoma. Whether CS cells in serous effusions differ from their counterparts in solid lesions remains uncertain.

The objective of this study was to analyze the expression and prognostic role of methylthioadenosine phosphorylase (MTAP) in mesothelioma. MTAP protein expression by immunohistochemistry was analyzed in 113 mesotheliomas (60 pleural and 53 peritoneal), consisting of 36 effusions and 77 surgical specimens. MTAP expression was fully lost in 38 tumors and partially lost in 8 tumors. Loss of expression was significantly more common in effusions compared with biopsies/surgical resection specimens (20/36 vs. 26/77; P=0.017), and in pleural compared with peritoneal mesotheliomas (35/60 vs. 11/53; P<0.001). MTAP performed less robustly than BAP1 in comparative analysis of 57 tumors previously analyzed for expression of the latter protein (46 vs. 25 cases with loss of expression). In survival analysis for 69 patients with partial clinical data, male gender was significantly associated with shorter overall survival (OS; P=0.042), whereas loss of MTAP was associated with a trend for shorter OS (P=0.058), with no prognostic role for patient age (P=0.379) or anatomic site (P=0.381). The association between loss of MTAP and poor OS became significant when survival analysis was limited to patients with pleural mesothelioma (P=0.018). In conclusion, loss of MTAP expression is more frequent in pleural compared with peritoneal mesothelioma and has limited diagnostic relevance at the latter anatomic site. More frequent loss in effusion specimens suggests a role for this marker in effusion cytology. MTAP loss in pleural mesothelioma is associated with poor survival.

The objective of this study was to analyze the expression and prognostic role of the tight junction protein occludin in high-grade serous carcinoma (HGSC). Occludin protein expression by immunohistochemistry was analyzed in 602 HGSC (417 effusions, 185 surgical specimens). Expression in mesothelioma (n = 87; 45 effusions, 42 surgical specimens) was studied for comparative purposes. Occludin protein expression was found in 587/602 (98%) HGSC vs. 40/87 (46%) mesotheliomas and was predominantly limited to < 5% of cells in the latter (p < 0.001). Occludin was additionally overexpressed in HGSC effusions compared to surgical specimens (p < 0.001) and was overexpressed in post-chemotherapy effusions compared to chemo-naive effusions tapped at diagnosis (p = 0.015). Occludin expression in HGSC surgical specimens was associated with poor chemoresponse (p < 0.001) and primary resistance (p = 0.001). Expression in effusions and surgical specimens was unrelated to survival (p > 0.05). In conclusion, occludin expression is higher in HGSC compared to mesothelioma, and this protein is overexpressed in HGSC effusions, possibly reflecting changes in adhesion related to anchorage-independent growth in this microenvironment. Overexpression in post-chemotherapy compared to chemo-naïve effusions suggest a role in disease progression. Occludin expression in surgical specimens may be related to chemoresistance.

The objective of this study was to identify clinicopathologic parameters associated with disease outcome in FIGO stage I vulvar squamous cell carcinoma (vSqCC). The cohort consisted of 126 patients diagnosed with vSqCC in the period 2006–2016 who underwent primary vulvar surgery and evaluation of groin lymph node status. Tumors were reviewed by an experienced gynecologic pathologist. p16 and p53 protein expression by immunohistochemistry and HPV status were analyzed in 116 tumors. Clinicopathologic parameters, protein expression and HPV status were analyzed for association with progression-free and overall survival (PFS, OS). p16 expression and aberrant p53 were found in 49 (42%) and 61 (53%) tumors, respectively. Sixty-six tumors were HPV-associated (57%). Relapse was diagnosed in 35/126 (28%) of patients, and 23 (18%) died of disease. Tumor diameter > 4 cm ( p = 0.013), lymphovascular space invasion (LVSI; p < 0.001), the presence of lichen sclerosus ( p = 0.019), p16 expression ( p = 0.007), p53 expression ( p = 0.012), HPV status ( p = 0.021), lymph node metastasis ( p < 0.001) and post-operative radiotherapy ( p < 0.001) were significantly related to OS in univariate analysis. Tumor diameter > 4 cm ( p = 0.038), LVSI ( p = 0.003), the presence of lichen sclerosus ( p = 0.004), p16 expression ( p = 0.004), HPV status ( p = 0.039), lymph node metastasis ( p < 0.001) and post-operative treatment ( p < 0.001), were significantly related to PFS in univariate analysis. Age, BMI and surgical resection involvement were not significantly associated with OS or PFS. In multivariate Cox analysis, LVSI and p16 expression were independent prognosticators of OS ( p < 0.001 and p = 0.02, respectively) and PFS ( p = 0.018, p = 0.037). In conclusion, LVSI and p16 expression are independent prognostic factors in stage I vSqCC.

Objective. To analyze the expression and prognostic role of L1CAM in tubo-ovarian high-grade serous carci-noma (HGSC).Methods. L1CAM protein expression by immunohistochemistry was analyzed in 644 HGSC (413 effusions, 231 surgical specimens). Expression was analyzed for association with clinicopathologic parameters and survival.Results. L1CAM protein expression was found in 401/413 (97%) effusions and 209/231 (90%) surgical speci-mens, with significantly higher staining extent in effusions (p < 0.001). L1CAM protein expression in effusions was unrelated to clinicopathologic parameters (p > 0.05). In surgical specimens, higher L1CAM expression was significantly related to primary (intrinsic) chemoresistance (p = 0.017). High (>25%) L1CAM expression in HGSC effusions (p = 0.02), older patient age (p = 0.013), FIGO stage IV disease (p < 0.001) and larger residual disease volume (p = 0.001) were significantly associated with shorter overall survival (OS) in univariate analysis. In Cox multivariate analysis, only FIGO stage (p = 0.001) and residual disease volume (p = 0.003) were indepen-dent prognosticators of OS. L1CAM expression in effusions was unrelated to progression-free survival (PFS). There was no association between L1CAM expression in surgical specimens and survival.Conclusion. L1CAM is overexpressed in HGSC effusions compared to surgical specimens. Its overexpression in effusions is significantly associated with shorter OS, but not independently of established prognostic factors such as FIGO stage and residual disease volume.& COPY; 2023 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY license (http:// creativecommons.org/licenses/by/4.0/).

The objective of this study was to analyze the expression and prognostic role of the tight junction protein claudin-10 in high-grade serous carcinoma (HGSC). Claudin-10 protein expression by immunohistochemistry was analyzed in 588 HGSC (414 effusions, 174 surgical specimens). Expression in mesotheliomas (n = 97; 47 effusions, 50 surgical specimens) was studied for comparative purposes. CLDN10 mRNA expression by quantitative RT-PCR (qRT-PCR) was analyzed in 40 HGSC effusions. Claudin-10 protein expression was found in 360/588 (61%) HGSC vs. 19/97 (20%) mesotheliomas (p < 0.001), and was higher in HGSC surgical specimens compared to effusions (p < 0.001). qRT-PCR confirmed the presence of CLDN10 mRNA in HGSC effusions. High (> 25%) claudin-10 expression in HGSC effusions was significantly associated with shorter overall survival (OS; p = 0.036) and progression-free survival (PFS; p = 0.045) in univariate analysis, and was an independent prognosticator of OS in multivariate analysis (p = 0.045). In conclusion, claudin-10 protein expression is higher in HGSC compared to mesothelioma, although the diagnostic power of this marker appear to be lesser than other claudin family members. Claudin-10 expression in HGSC effusions is marker of more aggressive disease.

The objective of the present study was to perform a quantitative analysis of cancer stem cell (CSC) marker expression in ovarian carcinoma effusions. The clinical role of SSEA1 in metastatic high-grade serous carcinoma (HGSC) was additionally analyzed. CD133, Nanog, SOX2, Oct3/4, SSEA1, and SSEA4 protein expressions were quantitatively analyzed using flow cytometry (FCM) in 24 effusions. SSEA1 expression by immunohistochemistry was analyzed in 384 HGSC effusions. Highly variable expression of CSC markers by FCM was observed, ranging from 0 to 78% of Ber-EP4-positive cells in the case of CD133, with the largest number of negative specimens seen for SSEA4. SSEA1 expression by immunohistochemistry was found in HGSC cells in 336/384 (89%) effusions, most commonly focally (< 5% of cells). SSEA1 was overexpressed in post-chemotherapy disease recurrence specimens compared with chemo-naïve HGSC effusions tapped at diagnosis (p = 0.029). In univariate survival analysis, higher SSEA1 expression was significantly associated with poor overall survival (p = 0.047) and progression-free survival (p = 0.018), though it failed to retain its prognostic role in Cox multivariate survival analysis in which it was analyzed with clinical parameters (p = 0.059 and p = 0.111 for overall and progression-free survival, respectively). In conclusion, CSC markers are variably expressed in ovarian carcinoma effusions. SSEA1 expression is associated with disease progression and poor survival in metastatic HGSC. Silencing this molecule may have therapeutic relevance in this cancer.

OBJECTIVE:To analyse the expression and clinical role of the phosphatase PTPN1 (PTP1B) in serous effusions.METHODS:PTPN1 mRNA expression by quantitative RT-PCR was analysed in 83 high-grade serous carcinoma (HGSC) and 15 malignant mesothelioma (MM) effusions. PTP1B and phospho-PTP1B (pPTP1B) protein expression by immunohistochemistry was analysed in 62 HGSC and 44 MM effusions.RESULTS:PTPN1 mRNA (P = .048), PTP1B protein (P = .047) and pPTP1B protein (P < .001) were overexpressed in HGSC compared to MM effusions. PTPN1 mRNA was additionally overexpressed in post-chemotherapy HGSC effusions compared to chemo-naïve effusions (P = .005). However, pPTP1B protein expression was higher in effusions from patients with FIGO stage III compared to stage IV (P = .006), and higher expressions of both PTPN1 mRNA (P = .041) and PTP1B protein (P = .035) in HGSC effusions were associated with better (complete) chemotherapy response at diagnosis. PTPN1 RNA and protein expression was unrelated to survival in HGSC, whereas a trend for shorter overall survival (P = .06) was found for MM patients whose tumours expressed pPTP1B protein.CONCLUSION:PTPN1 is overexpressed in HGSC compared to MM effusions, and may be a marker of better chemotherapy response in the former. Whether PTPN1 activation is informative of adverse outcome in MM merits further investigation.

OBJECTIVE:To analyse the expression and clinical role of the actin-associated molecule palladin in serous effusions.METHODS:PALLD mRNA expression was analysed by quantitative reverse transcription polymerase chain reaction in 83 high-grade serous carcinoma (HGSC) effusions. Fifteen malignant mesothelioma (MM) effusions and 18 surgical HGSC specimens from the ovary were studied for comparative purposes. Palladin protein expression by immunohistochemistry was analysed in another series consisting of 261 HGSC effusions.RESULTS:PALLD mRNA was significantly overexpressed in HGSC compared to MM effusions (P < .001). Palladin expression by immunohistochemistry was found in HGSC cells in 106/261 (41%) effusions, most commonly focally (<5% of cells). PALLD expression was additionally higher in ovarian HGSC specimens compared to HGSC effusions (P < .001). However, immunohistochemistry showed only stromal expression of this protein in surgical specimens. PALLD mRNA expression in HGSC effusions was unrelated to clinicopathological parameters, chemotherapy response or survival. Palladin protein expression was higher in post-chemotherapy, mainly disease recurrence, specimens compared to chemo-naïve effusions tapped at diagnosis (P = .018), although it was unrelated to other clinicopathological parameters or survival.CONCLUSION:PALLD mRNA is overexpressed in HGSC compared to MM effusions, and its protein product is overexpressed in post-chemotherapy compared to pre-chemotherapy HGSC effusions, suggesting upregulation along tumour progression. The presence of this molecule in HGSC effusions, at the mRNA or the protein level, is unrelated to disease outcome.