P53 and TLR4 expression are prognostic markers informing progression free survival of advanced stage high grade serous ovarian cancer.

OBJECTIVE: New prognostic biomarkers, and bio-signatures, are urgently needed to facilitate a precision medicine-based approach to more effectively treat patients with high-grade serous ovarian cancer (HGSC). In this study, we analysed the expression patterns of a series of candidate protein biomarkers. METHODS: The panel of markers which included MyD88, TLR4, MAD2, PR, OR, WT1, p53, p16, CD10 and Ki67 was assessed using immunohistochemistry in a tissue microarray (TMA) cohort of n = 80 patients, composed of stage 3-4 HGSCs. Each marker was analysed for their potential to predict both overall survival (OS) and progression-free survival (PFS). RESULTS: TLR4 and p53 were found to be individually predictive of poorer PFS (Log Rank, p = 0.017, p = 0.030 respectively). Cox regression analysis also identified high p53 and TLR4 expression as prognostic factors for reduced PFS (p53; HR=1.785, CI=1.036-3.074, p = 0.037 and TLR4; HR=2.175, CI=1.112-4.253, p = 0.023). Multivariate forward conditional Cox regression analysis, examining all markers, identified a combined signature composed of p53 and TLR4 as prognostic for reduced PFS (p = 0.023). CONCLUSION: Combined p53 and TLR4 marker assessment may help to aid treatment stratification for patients diagnosed with advanced-stage HGSC.

Molecular Landscape of Mullerian Clear Cell Carcinomas Identifies The Cancer Genome Atlas-like Prognostic Subgroups.

Mullerian clear cell carcinoma (CCC) is often aggressive and chemoresistant. The prognostic significance of molecular subclassification of endometrioid carcinomas is well established. However, less is known about the molecular landscape of CCC. The aim of this study was to better characterize the genetic landscape of a large cohort of CCC and correlate these findings with clinicopathologic features. CCC of the ovary (n = 72), endometrium (n = 24), and peritoneum/abdominal wall (n = 5) were retrospectively identified. Tumors had undergone tumor-only targeted sequencing using a hybrid capture next-generation sequencing panel. Median tumor mutational burden was 6.8 mutations/megabase (range, 1.3-185, 21% were ≥10 mutations/Mb). The most frequently mutated genes were ARID1A (48%), PIK3CA (45%), TP53 (23%), and PTEN (10%). ERBB2 amplification occurred in 4%. When classified according to the Cancer Genome Atlas/the Proactive Molecular Risk Classifier for Endometrial Cancer endometrial carcinoma molecular subgroups, 3 (3%) were POLE ultramutated, 5 (5%) were microsatellite instability-high (MSI-H), 20 (20%) were TP53-mutant subgroup, and 73 (72%) were no specific molecular profile (NSMP). Immunohistochemical expression of estrogen receptor, progesterone receptor, and programmed death-ligand 1 were not associated with the molecular subgroup. POLE and MSI-H tumors were characterized by an excellent prognosis, and the TP53-mutant subgroup had a worse disease-free survival than NSMP. NSMP tumors could be further substratified as high-risk NSMP if they lacked PIK3CA, PIK3R1, and ARID1A mutations, and/or harbored a TERT-promoter mutation. The Cancer Genome Atlas and NSMP-specific stratifications were prognostic for both the entire cohort and the subset of stage I ovarian tumors. On multivariable analysis, stage, lymphovascular invasion, and tumor mutational burden were prognostic for disease-free survival, whereas advanced stage and TP53-mutant subgroup - but not a TP53 mutation in isolation - were negative prognostic factors for overall survival. These data suggest that routine molecular profiling of Mullerian CCC may be warranted for both prognosis and identification of potential targeted treatments, such as immunotherapy and anti-HER2 agents.

NTRK -Rearranged Uterine Sarcomas: Clinicopathologic Features of 15 Cases, Literature Review, and Risk Stratification.

NTRK -rearranged uterine sarcomas are rare spindle cell neoplasms that typically arise in the uterine cervix of young women. Some tumors recur or metastasize, but features which predict behavior have not been identified to date. Distinguishing these tumors from morphologic mimics is significant because patients with advanced stage disease may be treated with TRK inhibitors. Herein, we present 15 cases of NTRK- rearranged uterine sarcomas, the largest series to date. Median patient age was 35 years (range: 16 to 61). The majority arose in the uterine cervix (n=14) and all but 2 were organ-confined at diagnosis. Tumors were composed of an infiltrative, fascicular proliferation of spindle cells and most showed mild-to-moderate cytologic atypia. All were pan-TRK positive by immunohistochemistry (13/13); S100 (11/13) and CD34 (6/10) were usually positive. RNA or DNA sequencing found NTRK1 (10/13) and NTRK3 (3/13) fusions with partners TPR , TPM3 , EML4 , TFG , SPECC1L , C16orf72 , and IRF2BP2 . Unusual morphology was seen in 2 tumors which were originally diagnosed as unclassifiable uterine sarcomas, 1 of which also harbored TP53 mutations. Follow up was available for 9 patients, of whom 3 died of disease. By incorporating outcome data of previously reported tumors, adverse prognostic features were identified, including a mitotic index ≥8 per 10 high-power fields, lymphovascular invasion, necrosis, and NTRK3 fusion. Patients with tumors which lacked any of these 4 features had an excellent prognosis. This study expands the morphologic spectrum of NTRK -rearranged uterine sarcomas and identifies features which can be used for risk stratification.

Low-grade Fibromyxoid Sarcoma of the Vulva and Vagina: Clinical, Pathologic, and Molecular Characterization of 7 Cases and Review of the Literature.

Low-grade fibromyxoid sarcoma (LGFMS) is a malignancy with propensity for late relapse that principally affects deep soft tissues of the extremities and trunk. Its occurrence in the lower female genital tract is rare, and thus it may not be always considered in the differential diagnosis. We describe the salient features of 7 vulvovaginal LGFMS identified in the authors' consultation files. Clinical information was obtained from referring pathologists. Archival slides were reviewed, and immunohistochemistry and fluorescence in situ hybridization were performed in cases with available material. Median age was 40 years (range, 34 to 58 y). Primary sites included vulva (n=6) and vagina (n=1). Tumors were 1.2 to 8.7 cm (median, 5.0 cm) in size and grossly circumscribed with firm to focally gelatinous cut surfaces. Microscopically, 5/7 had infiltrative edges. All tumors showed fibrous and myxoid areas, with lobulated myxoid foci in 5/7, comprising storiform, patternless, or (less often) fascicular arrangement of spindled to stellate cells with bland, slender to ovoid nuclei. In all cases, mitoses were <1/2.4 mm 2 , and necrosis was absent. Capillary "arcades" were seen in 3/7. Margins were positive in 3/6. Immunohistochemistry showed positive epithelial membrane antigen in 4/6 and MUC4 in 5/6. Fluorescence in situ hybridization detected FUS rearrangement in 5/7. Both tumors without FUS rearrangement were also negative for EWSR1 rearrangement. All 5 patients with available follow-up were alive and disease-free 10 to 150 months (median, 57 mo) after diagnosis. However, a review of vulvovaginal/pelvic LGFMS previously reported shows recurrences as late as 45 years after initial diagnosis. Pathologists need to be aware that LGFMS can arise in the vulvovaginal region. Tumor lobulation, capillary arcades, and positive MUC4 are helpful features distinguishing LGFMS from other bland myxoid spindle cell neoplasms in the lower female genital tract. Molecular testing can be useful in challenging cases. Complete excision is feasible for most vulvovaginal LGFMS. Long-term surveillance is required as local and/or distant spread can occur decades after diagnosis.

Correlation of integrated ERG/PTEN assessment with biochemical recurrence in prostate cancer.

INTRODUCTION: Prostate cancer is a heterogeneous disease, with a complex molecular landscape that evolves throughout disease progression. Common alterations in genes such as ERG and PTEN have been attributed to worse prognosis. This study aimed to further examine the clinical relevance of PTEN and ERG expression in a cohort of patients with prostate cancer post radical prostatectomy. METHODS: Tissue microarrays were constructed from 132 patients with prostate cancer from the Irish Prostate Cancer Research Consortium and University Hospital of Orebro, Sweden. Patients were divided into three groups - Group 1: biochemical recurrence, Group 2: no biochemical recurrence and Group 3: immediate progression after surgery. PTEN and ERG immunohistochemical analysis was performed and the association between expression levels and clinical parameters were compared. RESULTS: Pathological stage pT3 tumours were more common at borderline significantly higher levels amongst patients who biochemically recurred when compared to patients who did not recur after radical prostatectomy (p = 0.05). ERG and PTEN expression levels were compared separately and concurrently across all three patient groups. Lack of ERG expression was strongly associated with immediate progression after surgery (p = 0.029). Loss of/low PTEN trended towards an association with immediate progression, however this was not statistically significant (p = 0.066). CONCLUSION: In this study, negative ERG expression was strongly associated with immediate biochemical progression after radical prostatectomy. Moreover, a trend towards a relationship between aberrant PTEN expression and progression was observed. Additional studies with long-term follow up data may provide further clinical insight into the genomic heterogeneity in this population.

Clinicopathologic and Immunohistochemical Correlates of CTNNB1 Mutated Endometrial Endometrioid Carcinoma.

Endometrial endometrioid carcinomas (EECs) with exon 3 CTNNB1 mutations characterize a more aggressive subset of tumors in patients with low-grade low-stage disease. Thus, prospectively identifying these cases may be clinically relevant. The aim of this study was to examine the feasibility of ß-catenin and Cyclin D1 immunohistochemistry to identify EECs harboring CTNNB1 mutations and to evaluate the clinicopathologic features of EECs with exon 3 CTNNB1 mutations. Thirty-nine CTNNB1 mutated EECs and 40 CTNNB1 wild-type EECs were identified from a cohort of previously sequenced endometrial carcinomas using a targeted next-generation sequencing panel. Immunohistochemistry for ß-catenin and Cyclin D1 was performed on all cases. Immunohistochemistry results were correlated with CTNNB1 mutation status and clinicopathologic parameters. Patients with CTNNB1 mutated EECs were younger than those with CTNNB1 wild-type (56.2 vs. 61.5 y; P=0.033). Nuclear ß-catenin expression correlated with exon 3 CTNNB1 mutation (P<0.0001) with a sensitivity of 91% and a specificity of 89%. Cyclin D1 expression correlated with CTNNB1 exon 3 mutation with relatively high specificity (90%) but low sensitivity (29%). Recurrence rate in patients with stage IA disease at diagnosis was significantly higher in patients whose tumors were CTNNB1 mutated compared with CTNNB1 wild-type (30% vs. 0%; P=0.025) and included distant metastases; all recurrent tumors in this group harbored exon 3 mutations and were histologically low grade (5 grade 1, 2 grade 2). Nuclear ß-catenin expression appears to be an acceptable proxy for CTNNB1 mutation.

The extended spectrum of RAS-MAPK pathway mutations in colorectal cancer.

Current clinical guidelines recommend mutation analysis for select codons in KRAS and NRAS exons 2, 3, and 4 and BRAF V600E to guide therapy selection and prognostic stratification in advanced colorectal cancer. This study evaluates the impact of extended molecular testing on the detection of RAS-MAPK pathway mutations. Panel next-generation sequencing results of colorectal cancer specimens from 5795 individuals from the American Association for Cancer Research Project Genomics Evidence Neoplasia Information Exchange (AACR Project GENIE) were included. Mutations in RAS-MAPK pathway genes were analyzed and functionally annotated. Colorectal cancers had recurrent pathogenic pathway activating mutations in KRAS (44%), NRAS (4%), HRAS (<1%), BRAF (10%), MAP2K1 (1%), RAF1 (<1%), and PTPN11 (<1%). The proportion of colorectal cancers with pathogenic RAS pathway mutations was 37% when only KRAS codon 12 and 13 mutations were considered, 46% when also including select KRAS and NRAS exons 2, 3, and 4 mutations, 53% when including BRAF V600E mutations, and 56% when including all pathogenic mutations. Panel next-generation sequencing testing identifies additional RAS-MAPK pathway driver mutations beyond current guideline recommendations. These mutations have potential implications in treatment selection for patients with advanced colorectal cancer.

Defining thyroid spherules: A benign cytomorphologic feature that mimics microfollicles.

BACKGROUND: Distinctive rounded structures termed spherules have been observed in thyroid fine-needle aspiration (FNA) samples. Although they are often small and interpreted as microfollicles, spherules are notable for the even spacing of the follicular cells along the perimeter of the structure. Because they have an orderly architectural arrangement, it has been hypothesized that spherules are benign and do not carry the same risk of malignancy that traditional microfollicles do. The aim of this study was to identify the clinical significance and histopathologic correlate of spherules. METHODS: Thyroid FNAs (n = 637) with an interpretation of atypia of undetermined significance (AUS) were reviewed for the presence of spherules, which were defined as small follicles with a rounded, smooth, sharply defined outer contour, evenly spaced nuclei, and a 3-dimensional appearance. Cases were included if spherules accounted for >50% of follicular cell arrangements. Clinical, histopathologic, molecular, and sonographic follow-up data were obtained. RESULTS: Twenty-five spherule cases were identified, and they represented 3.9% of all AUS cases reviewed. All 24 cases with follow-up had a benign outcome. Eleven were tested with the Afirma gene expression classifier; 8 were benign according to the gene expression classifier, and 3 were suspicious but histologically benign after surgical resection. Four additional cases were histologically benign after surgery. Five patients had repeat benign cytology. Four patients had repeat stable ultrasound findings. CONCLUSIONS: Thyroid nodules diagnosed as AUS, if composed predominantly of spherules, are associated with a benign outcome. Recognizing this distinctive cytomorphologic finding may help to reduce the number of FNAs interpreted as AUS and save patients from unnecessary additional testing and surgery.

Targeted next-generation sequencing in the detection of mismatch repair deficiency in endometrial cancers.

Mismatch repair deficiency represents a biomarker of immunotherapy response and a phenotypic feature of Lynch syndrome-associated endometrial cancers. Using a targeted next-generation sequencing assay, we identified molecular features of mismatch repair deficiency, specifically insertion and deletion mutations in mononucleotide repeats, and established thresholds for the number of such mutations to classify endometrial cancers as mismatch repair deficient, proficient, or indeterminate. Sequencing classification was compared to the loss of MLH1, MSH2, MSH6, or PMS2 expression by immunohistochemistry. A total of 259 endometrial cancers were classified by sequencing as mismatch repair deficient (n = 48, 19%), proficient (n = 199, 77%), or indeterminate (n = 12, 5%). Sequencing findings were concordant with loss of expression of at least one mismatch repair protein in 47 of 48 (98%) cases classified as deficient and retained expression of all four proteins in 190 of 199 (95%) cases classified as proficient. Of the 12 cases classified as indeterminate, 7 (58%) demonstrated mismatch repair protein loss. Overall, targeted next-generation sequencing exhibited a high rate of concordance with immunohistochemistry for mismatch repair deficiency; however, sequencing was indeterminate in a few cases and demonstrated a false negative rate of 5%. Although we recommend implementation of a mismatch repair deficiency algorithm for laboratories performing next-generation sequencing cancer panels, immunohistochemistry remains a cost-effective screening method for mismatch repair deficiency in endometrial cancer.

A Combined Morphologic and Molecular Approach to Retrospectively Identify KRAS-Mutated Mesonephric-like Adenocarcinomas of the Endometrium.

An unusual subset of endometrial carcinoma, the mesonephric-like adenocarcinomas, are morphologically and molecularly similar to mesonephric carcinoma, characterized by KRAS mutation and lack of microsatellite instability. They also have a unique immunohistochemical profile and are usually positive for GATA-3, CD10, TTF-1, and negative for ER and PR. This study implemented a combined morphologic and molecular approach to retrospectively identify mesonephric-like carcinomas of the endometrium. KRAS-mutated microsatellite stable (MSS) endometrial carcinomas were identified from a database of 570 endometrial carcinomas that had undergone massively parallel sequencing. MSS tumors with canonical KRAS mutations that lacked features diagnostic of endometrioid carcinoma (including squamous or mucinous differentiation), were re-reviewed for morphologic features of mesonephric-like adenocarcinomas. Ninty-eight of 570 endometrial carcinomas (17%) harbored canonical KRAS mutations. Of the KRAS-mutated cases, 80 (82%) were MSS and 18 (18%) had microsatellite instability. Of the KRAS-mutated MSS cases with morphology review, 39/61 (64%) had squamous and/or mucinous differentiation while 22 (36%) lacked these histotype-defining features. Eight of these 22 had PTEN mutations and lacked morphologic features of mesonephric-like adenocarcinoma, leaving 14 cases with a possible mesonephric-like adenocarcinoma-like molecular profile that underwent detailed morphologic re-review. Ten of 14 had morphology typical of serous (3), carcinosarcoma (4), or endometrioid (3) carcinoma. In 4 cases, there was striking morphologic, immunophenotypic, and molecular resemblance to mesonephric carcinoma, leading to re-classification as mesonephric-like adenocarcinoma. Two of the 4 cases presented at an advanced stage, and a third case later developed distant metastases. On the basis of this retrospective study, KRAS-mutated mesonephric-like adenocarcinoma represents ∼1% of all endometrial carcinomas. Future prospective recognition of this unusual variant of endometrial carcinoma may be important given its possible aggressive nature.

Pacinian hyperplasia presenting with Raynaud's phenomenon.

Pacinian corpuscle pathology is a rare clinical entity and an uncommonly reported cause of digital pain. While many prior reports implicate hand trauma, we describe a case of Pacinian hyperplasia found in a patient with Raynaud's phenomenon and propose a potential mechanism of disease.

Advances in the clinicopathological and molecular classification of cutaneous mesenchymal neoplasms.

In recent years, there have been several important refinements in the classification of cutaneous mesenchymal neoplasms, including the description of new tumour types, along with the identification of novel and recurrent molecular genetic findings. In addition to providing new insights into tumour biology, many of these advances have had significant clinical consequences with regard to diagnostics, management, and prognostication. Newly described entities include pseudomyogenic haemangioendothelioma, haemosiderotic fibrolipomatous tumour, and fibroblastic connective tissue naevus, which are reviewed in the context of the principal differential diagnoses and significant clinical implications. Genetic characterization of several soft tissue tumour types that occur in the skin has resulted in the identification of diagnostically useful markers: ALK gene rearrangement with corresponding ALK protein expression by immunohistochemistry in epithelioid fibrous histiocytoma; the WWTR1-CAMTA1 fusion gene with CAMTA1 protein expression in epithelioid haemangioendothelioma; MYC amplification and overexpression in radiation-associated angiosarcoma; and EWSR1 gene rearrangement in cutaneous myoepithelial tumours. Finally, the classification of intradermal smooth muscle tumours and unclassified/pleomorphic dermal sarcoma has been refined, resulting in both improved classification and improved prognostication. Many of the tumour types listed above are encountered not only by specialist dermatopathologists, but also by practising general surgical pathologists, and this review should therefore provide a widely applicable update on the histological and molecular classification of cutaneous mesenchymal neoplasms, along with the appropriate use of ancillary diagnostic tests, in particular immunohistochemistry, in the evaluation of such lesions and their histological mimics.

Pulmonary Nodular Lymphoid Hyperplasia presenting cavitating pulmonary nodules.

Pulmonary Nodular Lymphoid Hyperplasia (PNLH) is a rare benign pulmonary disorder characterized by a localized, reactive polyclonal lymphoproliferation. Although the radiological features of this disease have not been clearly defined, they usually consist of a solitary non-cavitatory pulmonary nodule. In this report, we describe two cases of histologically confirmed PNLH presenting as multiple bilateral cavitatory lesions on CT Thorax.

Capsule endoscopy: High negative predictive value in the long term despite a low diagnostic yield in patients with suspected Crohn's disease.

INTRODUCTION: The role of small bowel capsule endoscopy (SBCE) in Crohn's disease (CD) has expanded with greater understanding of the technology. The ability of SBCE to differentiate CD from other causes of inflammation has been questioned. Longitudinal studies are required to assess the long-term impact and significance of SBCE findings in suspected CD. This study aimed to determine the long-term clinical accuracy of SBCE in patients referred with suspected CD. METHODS: A retrospective review was carried out on SBCE procedures performed for suspected CD since 2010. Only patients with at least 6 months of documented follow up were included. A chart review was undertaken to record SBCE findings/correlate with subsequent diagnosis and outcome. RESULTS: In all, 95 patients with sufficient follow up were identified. The mean follow up was 13 months (range 8-24). In total, 72 (76%) SBCEs were negative and 23 (24%) positive for CD. Of the 72 negative tests, two patients (3%) were later diagnosed with CD. The negative predictive value is 96%. There was a strong positive correlation between results of WCE and subsequent clinical diagnosis. CONCLUSIONS: SBCE appears capable of safely out-ruling CD, with only 3% of negative SBCE investigations being diagnosed with CD after 15 months.