Genomic Catastrophe (Chromothripsis and Polyploidy) Correlates With Tumor Distribution in Extrauterine High-grade Serous Carcinoma.

Most extrauterine high-grade serous carcinomas (HGSCs) are thought to develop first in the distal fallopian tube. Most models of HGSC assume origin from relatively stable, noninvasive serous tubal intraepithelial carcinomas. However, widespread tumor involvement in the absence of a serous tubal intraepithelial carcinoma could occur after catastrophic genomic events (CGEs; such as chromothripsis or polyploidy). Twenty-six HGSCs assigned to fallopian tube (n = 9, group 1) and/or ovary (n = 9, group 2), and primary peritoneal (n = 8, group 3) were assessed by microarray (Oncoscan). CGEs were identified in 15/26 (57.7%); chromothripsis-like pattern in 13/26 (50.0%) and polyploidy in 6/26 (23.1%). CGE was seen in 4/9 (44.4%), 9/9 (100%), and 2/8 (25%) cases in groups 1. 2, and 3, respectively. Overall, CGEs were seen in 9/9 (100%) cases with grossly evident ovarian parenchymal involvement versus 6/17 (35.3%) without (P = 0.0024). Ovarian size (measured on the long axis) correlated with CGE positivity (P = 0.016). CGEs are significantly more common in HGSCs with ovarian parenchymal involvement compared with those limited to the fallopian tube and/or extraovarian tissues. These associations suggest geographically different tumor growth patterns and support the subdivision of HGSCs according to not only the stage but also tumor distribution. They have implications for clinical and pathologic presentation, trajectory of tumor evolution, and in the case of primary peritoneal HGSCs, potentially unique precursors to tumor transitions that could inform or influence cancer prevention efforts.

Homologous recombination pathway gene variants identified by tumor-only sequencing assays in lung carcinoma patients.

Background: The homologous recombination (HR) repair pathway plays a key role in double-stranded DNA break repair, and germline HR pathway gene variants are associated with increased risk of several cancers, including breast and ovarian cancer. HR deficiency is also a therapeutically targetable phenotype. Methods: Somatic (tumour-only) sequencing was performed on 1,109 cases of lung tumors, and the pathological data were reviewed to filter for lung primary carcinomas. Cases were filtered for variants (disease-associated or of uncertain significance) in 14 HR pathway genes, including BRCA1, BRCA2, and ATM. The clinical, pathological and molecular data were reviewed. Results: Sixty-one HR pathway gene variants in 56 patients with primary lung cancer were identified. Further filtering by variant allele fraction (VAF) of ≥30% identified 17 HR pathway gene variants in 17 patients. ATM gene variants were most the commonly identified (9/17), including two patients with c.7271T>G (p.V2424G), a variant in the germline that is associated with increased familial cancer risk. Four (4/17) patients had a family history of lung cancer, among which three patients had ATM gene variants suspected to be germline in origin. In three other patients with BRCA1/2 or PALB2 gene variants who had undergone germline testing, the variants were confirmed to be germline; lung cancer was the sentinel cancer in two of these patients with a BRCA1 or PALB2 variant. Conclusions: Genomic variants in the HR repair pathway identified in tumor-only sequencing and occurring at higher VAFs (i.e., ≥30%) may suggest a germline origin. Correlating with personal and family history, a subset of these variants is also suggested to be associated with familial cancer risks. Patient age, smoking history and driver mutation status are expected to be a poor screening tool in identifying these patients. Finally, the relative enrichment for ATM variants in our cohort suggests a possible association between ATM mutation and lung cancer risk.

Recurrent WWTR1 S89W mutations and Hippo pathway deregulation in clear cell carcinomas of the cervix.

Clear cell carcinoma (CCC) of the cervix (cCCC) is a rare and aggressive type of human papillomavirus (HPV)-negative cervical cancer with limited effective treatment options for recurrent or metastatic disease. Historically, CCCs of the lower genital tract were associated with in utero diethylstilbestrol exposure; however, the genetic landscape of sporadic cCCCs remains unknown. Here we sought to define the molecular underpinning of cCCCs. Using a combination of whole-exome, targeted capture, and RNA-sequencing, we identified pathogenic genetic alterations in the Hippo signaling pathway in 50% (10/20) of cCCCs, including recurrent WWTR1 S89W somatic mutations in 40% (4/10) of the cases harboring mutations in the Hippo pathway. Irrespective of the presence or absence of Hippo pathway genetic alterations, however, all primary cCCCs analyzed in this study (n = 20) harbored features of Hippo pathway deregulation at the transcriptomic and protein levels. In vitro functional analysis revealed that expression of the WWTR1 S89W mutation leads to reduced binding of TAZ to 14-3-3, promoting constitutive nuclear translocation of TAZ and Hippo pathway repression. WWTR1 S89W expression was found to lead to acquisition of oncogenic behavior, including increased proliferation, migration, and colony formation in vitro as well as increased tumorigenesis in vivo, which could be reversed by targeted inhibition of the TAZ/YAP1 complex with verteporfin. Finally, xenografts expressing WWTR1 S89W displayed a shift in tumor phenotype, becoming more infiltrative as well as less differentiated, and were found to be composed of cells with conspicuous cytoplasmic clearing as compared to controls. Our results demonstrate that Hippo pathway alterations are likely drivers of cCCCs and likely contribute to the clear cell phenotype. Therapies targeting this pathway may constitute a new class of treatment for these rare, aggressive tumors. © 2022 The Pathological Society of Great Britain and Ireland.

Genomic catastrophe, the peritoneal cavity and ovarian cancer prevention.

The current theory of carcinogenesis for the deadliest of 'ovarian' cancers-high-grade serous carcinoma (HGSC)-holds that the malignancy develops first in the fallopian tube and spreads to the ovaries, peritoneum, and/or regional lymph nodes. This is based primarily on the observation of early forms of serous neoplasia (serous tubal intraepithelial lesions [STILs], and serous tubal intraepithelial carcinomas [STICS]) in the fimbria of women undergoing risk reduction surgery. However, these lesions are uncommon in the general population, confer a low risk (5%) of HGSC following their removal in at-risk women with germ-line BRCA1/2 mutations, and require 4 or more years to recur as intraperitoneal HGSC. These features suggest that isolated STILs and STICs behave as precursors, with uncertain cancer risk rather than carcinomas. Their evolution to HGSC within, or after, escape from the tube could proceed stepwise with multiple biologic events; however, it is unclear whether tubal or ovarian HGSCs encountered in the setting of advanced disease evolved in the same fashion. The latter scenario could also be explained by a 'catastrophic' model in which STICs suddenly develop with invasive and metastatic potential, overwhelming or obscuring the site of origin. Moreover, a similar model might explain the sudden emergence of HGSC in the peritoneal cavity following escape of precursor cells years before. Long-term follow-up data from opportunistic or prophylactic salpingectomy should shed light on where malignant transformation occurs, as well as the timeline from precursor to metastatic HGSC. © 2022 The Pathological Society of Great Britain and Ireland.

Intra-Tumoral Nerve-Tracing in a Novel Syngeneic Model of High-Grade Serous Ovarian Carcinoma.

Dense tumor innervation is associated with enhanced cancer progression and poor prognosis. We observed innervation in breast, prostate, pancreatic, lung, liver, ovarian, and colon cancers. Defining innervation in high-grade serous ovarian carcinoma (HGSOC) was a focus since sensory innervation was observed whereas the normal tissue contains predominantly sympathetic input. The origin, specific nerve type, and the mechanisms promoting innervation and driving nerve-cancer cell communications in ovarian cancer remain largely unknown. The technique of neuro-tracing enhances the study of tumor innervation by offering a means for identification and mapping of nerve sources that may directly and indirectly affect the tumor microenvironment. Here, we establish a murine model of HGSOC and utilize image-guided microinjections of retrograde neuro-tracer to label tumor-infiltrating peripheral neurons, mapping their source and circuitry. We show that regional sensory neurons innervate HGSOC tumors. Interestingly, the axons within the tumor trace back to local dorsal root ganglia as well as jugular-nodose ganglia. Further manipulations of these tumor projecting neurons may define the neuronal contributions in tumor growth, invasion, metastasis, and responses to therapeutics.

TERT gene rearrangement in chordomas and comparison to other TERT-rearranged solid tumors.

Chordomas are rare, slow-growing neoplasms thought to arise from the foetal notochord remnant. A limited number of studies that examined the mutational profiles in chordomas identified potential driver mutations, including duplication in the TBXT gene (encoding brachyury), mutations in the PI3K/AKT signaling pathway, and loss of the CDKN2A gene. Most chordomas remain without clear driver mutations, and no fusion genes have been identified thus far. We discovered a novel TERT in-frame fusion involving RPH3AL (exon 5) and TERT (exon 2) in the index chordoma case. We screened a discovery cohort of 18 additional chordoma cases for TERT gene rearrangement by FISH, in which TERT rearrangement was identified in one additional case. In our independent, validation cohort of 36 chordomas, no TERT rearrangement was observed by FISH. Immunohistochemistry optimized for nuclear TERT expression showed at least focal TERT expression in 40/55 (72.7%) chordomas. Selected cases underwent molecular genetic profiling, which showed low tumor mutational burdens (TMBs) without obvious driver oncogenic mutations. We next examined a cohort of 1,913 solid tumor patients for TERT rearrangements, and TERT fusions involving exon 2 were observed in 7/1,913 (0.4%) cases. The seven tumors comprised five glial tumors, and two poorly differentiated carcinomas. In contrast to chordomas, the other TERT-rearranged tumors were notable for higher TMBs, frequent TP53 mutations (6/7) and presence of other driver oncogenic mutations, including a concurrent fusion (TRIM24-MET). In conclusion, TERT gene rearrangements are seen in a small subset (2/55, 3.6%) of chordomas. In contrast to other TERT-rearranged tumors, where the TERT rearrangements are likely passenger events, the possibility that TERT protein overexpression representing a key event in chordoma tumorigenesis is left open.

Interobserver reproducibility of the diagnosis of differentiated exophytic vulvar intraepithelial lesion (DEVIL) and the distinction from its mimics.

AIMS: Most vulvar squamous cell carcinomas are human papillomavirus (HPV)-associated or TP53-mutant. A third category of HPV-independent TP53-wild-type lesions is uncommon and not fully understood. Differentiated exophytic vulvar intraepithelial lesion (DEVIL) has been characterised as a precursor of this latter category. The reproducibility of the diagnosis of DEVIL and its distinction from lesions with overlapping morphology has not been studied. Our aim was to establish the interobserver agreement in the diagnosis of DEVIL and its distinction from neoplastic and reactive conditions of the vulva on haematoxylin and eosin evaluation. METHODS AND RESULTS: A set of 35 slides was evaluated by eight reviewers (two trainees and six practising gynaecological pathologists). The set included DEVIL, condyloma, established vulvar precursors [high-grade squamous intraepithelial lesion (HSIL) and differentiated vulvar intraepithelial neoplasia (dVIN)] with superimposed acanthosis or verruciform growth, lichen simplex chronicus (LSC), and psoriasis. Kappa (κ) values were calculated. Overall, interobserver agreement was moderate (κ = 0.56), improving to substantial (κ = 0.7) when evaluation was performed by practising pathologists. Agreement was strong for the diagnosis of HSIL (κ = 0.88), and substantial for the diagnosis of DEVIL (κ = 0.61), condyloma (κ = 0.79), and LSC (κ = 0.72). Agreement was moderate for the diagnosis of dVIN (κ = 0.59) and psoriasis (κ = 0.53). Perfect agreement (6/6) among practising pathologists was observed in 43% of cases, and majority agreement (5/6 or 4/6) was observed in 48% of cases. CONCLUSIONS: Reproducibility in the diagnosis of verruciform vulvar lesions, including the novel DEVIL, is acceptable overall. Reproducibility is higher for well-known lesions such as HSIL and condyloma than for more challenging diagnoses such as DEVIL, dVIN, and psoriasis. Agreement is higher among practising gynaecological pathologists, suggesting that training and experience improve reproducibility. Our findings support the inclusion of DEVIL as a diagnostic entity in the classification of vulvar squamous lesions.

Endometrial Stem/Progenitor cell (ES/PC) Marker Expression Profile in Adenosarcoma and Endometrial Stromal Sarcoma.

BACKGROUND: The uterus is one of the most dynamic organs in the human body, and this dynamic homeostasis is supported by endometrial stem/progenitor cells (ES/PCs), which are heterogeneous in their phenotype and degree of differentiation. ES/PCs are generally localized in the endometrial stroma, the site of origin for adenosarcoma and endometrial stromal sarcoma (ESS). Subsets of ESSs and adenosarcomas harbor SUZ12 or DICER1 gene alterations, two genes with roles in embryonic stem cell biology. However, the possible contribution of ES/PCs to tumorigenesis is unexplored. METHOD: We examined the expression of eleven ES/PC markers, along with three proteins expressed in the mature endometrial stroma (ER, PR and CD10) in 60 uterine tumors (24 low-, 11 high-grade ESS, 25 adenosarcomas). Protein expression profiles were assessed by unsupervised hierarchical clustering. miRNA expression profiles were examined in a subset of adenosarcoma with/without DICER1 mutations, using the NanoString platform. RESULTS: ES/PC markers were variably expressed, and the tumors exhibited limited immunophenotypic resemblance to different ES/PCs. Within the ESSs, the ES/PC marker clustering pattern was prognostic for both overall and disease-free survival. Comparing adenosarcomas and ESSs, most high-grade ESSs clustered with one another, while low-grade ESSs and adenosarcomas tended to cluster with one another. Among the adenosarcomas, the miRNA expression profiles were varied with respect to the DICER1 mutation status, with pathway analysis pointing to dysregulated signal transduction and stem cell biology. CONCLUSIONS: ESSs and adenosarcomas exhibit varying immunophenotypic resemblance to ES/PCs. These expression profiles have prognostic implications and may be genetically driven.

Autophagic Heterogeneity in Gastric Adenocarcinoma.

BACKGROUND AND AIM: Gastric/gastroesophageal junction (GEJ) adenocarcinoma is a heterogeneous disease, with various etiologies and with tumors encompassing a spectrum of histologic and molecular subtypes. "Autophagy" includes two related but distinct homeostatic processes that promote cell survival under adverse conditions, namely macro- and chaperone-mediated autophagy. There is increasing evidence of the roles autophagy may play in tumorigenesis. METHODS: Autophagic pathways were examined in the context of the heterogeneity intrinsic to gastric/GEJ adenocarcinoma, utilizing immunohistochemistry targeting specific proteins within the pathways (p62, LAMP2A, LC3B). We examined whole sections of normal and dysplastic gastric mucosa, as well as a tissue microarray of adenocarcinomas. RESULTS: Dysplastic gastric epithelium was marked by frequent nuclear p62 and aberrant LAMP2A expression compared to normal. Examining the pattern of LC3B/cytoplasmic p62 immuno-reactivity in gastric adenocarcinoma demonstrated a predominant pattern of LC3BHigh/p62High staining (56/86, 65.1%), which has been previously associated with active, but impaired macroautophagy. There were no statistically significant associations seen between LC3B/cytoplasmic p62 staining patterns with tumor grade, histotype, or approximated TCGA molecular subtype. LAMP2A and nuclear p62 and staining patterns were also heterogeneous across the cohort, but with no statistically significant associations seen. The prognostic significance of the three proteins was limited, however high nuclear p62 levels were associated with worse overall survival (log-rank p-value = 0.0396). CONCLUSION: Our data demonstrate the dynamic nature of autophagic proteins in the gastric epithelium, and we expand the biological heterogeneity observed in gastric/GEJ adenocarcinoma to include autophagy.

Mutational spectrum in clinically aggressive low-grade serous carcinoma/serous borderline tumors of the ovary-Clinical significance of BRCA2 gene variants in genomically stable tumors.

OBJECTIVE: The mutational spectra of low-grade serous carcinomas (LGSCs) and serous borderline tumors (SBTs) of the ovary are poorly characterized. We present 17 cases of advanced or recurrent LGSC/SBT patients who underwent molecular profiling. METHODS: Thirteen LGSCs and four SBTs underwent targeted gene panel testing by massively parallel sequencing. Microsatellite stability and tumor mutation burdens (TMBs) were determined based on panel sequencing data. RESULTS: The mean TMB was 5.2 mutations/megabase (range 3-10) in 14 cases. Twelve of twelve (12/12) cases were microsatellite stable. Clear driver mutations were identified in 11 cases, namely KRAS (5/17), BRAF (2/17), NRAS (2/17) and ERBB2 (2/17). Five cases harbored BRCA2 alterations (allele fractions: 44-51%), including two classified as likely benign/benign variants, and three classified as variants of uncertain significance (VUSs), with two variants being confirmed to be germline. The three BRCA2 VUSs were missense variants that were assessed to be of unlikely clinical significance, based on family cancer history and expected impact on protein function. Two patients received PARP inhibitors during their disease course, with neither of the patients demonstrating appreciable response. CONCLUSIONS: The mutational spectra in 17 clinically aggressive SBT/LGSC cases demonstrate genomically stable tumors, frequently driven by the RTK/RAS/MAPK pathway. While BRCA2 variants were identified, our data demonstrate BRCA2 gene variants are at most VUSs and of dubious clinical significance, in contrast to disease-associated BRCA1/2 variants that may be identified in high-grade serous carcinoma. Germline testing and PARP inhibitors are thus expected to provide limited benefit to patients with LGSC/SBTs.

The Value of DICER1 Mutation Analysis in "Subtle" Diagnostically Challenging Embryonal Rhabdomyosarcomas of the Uterine Cervix.

Embryonal rhabdomyosarcoma of the uterine cervix is a rare neoplasm which is almost invariably associated with pathogenic somatic or germline DICER1 mutations; patients with germline mutations have DICER1 syndrome. We report 2 subtle cervical embryonal rhabdomyosarcoma, one occurring in a 21-yr-old woman with a known history of DICER1 syndrome and the other in a 19-yr-old woman with no history of DICER1 syndrome or DICER1-associated neoplasms. Both neoplasms focally involved otherwise benign endocervical polyps and were characterized histologically by subtle areas of increased stromal cellularity, nuclear atypia and mitotic activity; there was focal nuclear staining of these areas with the skeletal muscle markers myogenin and myoD1. In both cases, demonstration of a somatic DICER1 RNase IIIb mutation in the tumor was instrumental in establishing the diagnosis. We believe these neoplasms represent the earliest discernible phase of cervical embryonal rhabdomyosarcoma. Pathologists should have a high index of suspicion when atypical stromal elements are present in endocervical polyps and immunohistochemistry together with DICER1 sequencing will assist in diagnosis.

Lgr5 and stem/progenitor gene expression in gastric/gastroesophageal junction carcinoma - significance of potentially retained stemness.

BACKGROUND: Gastric/gastroesophageal junction (GEJ) adenocarcinomas are heterogeneous, comprising four molecularly distinct subtypes, namely EBV-positive, microsatellite instability (MSI), chromosomal instability (CIN) and genomically stable (GS) subtypes, and a part of this heterogeneity may hypothesized to be different cells-of-origin. Stem/progenitor cell hierarchy in the stomach is complex, which include the Lgr5(+) gastric stem cells (GSCs). METHODS: While previous studies have focused on non-nuclear Lgr5 expression, nuclear Lgr5 expression has been reported in a subset of stem cells, and we examined nuclear Lgr5 expression in a local cohort of 95 cases of gastric/GEJ adenocarcinoma. mRNA levels for LGR5 and other stem cell marker genes were examined in the TCGA cohort. RESULTS: We observed nuclear Lgr5 expression in a 18/95 cases. Near mutual exclusivity was seen between nuclear Lgr5 and strong non-nuclear Lgr5. Both strong non-nuclear and nuclear Lgr5 expression tended to be seen more frequently with the intestinal histotype and approximated CIN molecular subtype. With respect to overall survival (OS), nuclear Lgr5 expression appears to be protective, with the worst survival being seen in the cases lacking nuclear Lgr5 and with low non-nuclear Lgr5 expression. When compared to other stem/progenitor cell markers, LGR5 mRNA expression clusters with other GSC marker genes, including VIL1. Higher expression of these GSC marker genes was associated with better OS. CONCLUSIONS: Our results show that Lgr5 expression is dynamic in gastric/GEJ adenocarcinoma and heterogeneous across the several disease attributes. We postulate that this may reflect "retained stemness" in the form of Lgr5High-GSC signature that appears to be associated with better survival.

An Investigation Into the Prognostic Significance of High Proteasome PSB7 Protein Expression in Colorectal Cancer.

Using unbiased proteomics, we had previously discovered that the catalytic proteasome subunit ß type 7 (PSB7) protein is frequently overexpressed in colorectal adenocarcinomas. In this paper, we validate this finding and derive a prognostic significance for PSB7 by examining an expanded, well-annotated clinical cohort of 318 colorectal cancer patients. We found PSB7 protein levels to be similarly increased in both advanced stage primary disease and metastatic lesions. We then examined the prognostic value of PSB7 protein expression. Elevated PSB7 protein as well as PSMB7 mRNA levels showed associations with lower overall survival, particularly in female patients. The prognostic value of elevated PSB7 protein levels was highest for female patients who were older (>60 years of age at diagnosis) or who had received adjuvant chemotherapy. While high PSB7 did not retain its prognostic significance on multivariate analysis, we discuss the potential significance of PSB7 as a biomarker, considering its differential prognostic strength in different colorectal cancer patient groups and given its role as a subunit of the immunoproteasome for antigen presentation.

Bayesian approach to interpreting somatic cancer sequencing data: a case in point.

Cancer lineage/tissue-of-origin assignment in cancers of unknown primary remains a challenge even when aided by massively parallel sequencing. The stakes are high for patients as many contemporary therapeutic strategies are disease-specific, and the biological differences can influence the patients' responses. Herein, we provide an example of how Bayesian analysis can be used to merge data from clinical history, histology, immunohistochemistry (IHC) and cancer DNA sequencing to assist in tissue-of-origin assignment. Iterative Bayesian analysis is performed through a set of simple calculations to calculate the OR between the differential diagnoses. We illustrate a clinical case, where the distinction between a primary lung versus metastatic bladder cancer was aided meaningfully by iterative Bayesian analyses, incorporating IHC and sequencing data.

PD-L1 lineage-specific quantification in malignant pleural effusions of lung adenocarcinoma by flow cytometry.

OBJECTIVES: Pathologists encounter several challenges with programmed death ligand-1 (PD-L1) immunohistochemistry (IHC) tests in malignant effusions, including lineage specification (distinction between carcinoma vs. immune and mesothelial cells), background staining, sample fixation issues and inter-observer variability. We explored flow cytometric (FC) quantification of PD-L1 expression in malignant pleural effusions of lung adenocarcinoma patients as an alternative, automated, and objective quantification method compared to PD-L1 IHC. MATERIALS AND METHODS: We examined 23 malignant pleural effusions of TTF-1-positive adenocarcinoma were subjected to FC with a panel of antibodies against CD45, CD3, CD200, EpCAM, D2-40 (podoplanin), and PD-L1 (clone MIH1). The PD-L1 gate was established using fluorescence-minus-one (FMO) isotype controls. Lineage-specific PD-L1 surface expression was quantified and the FC tumor proportion score (TPS) was assessed. PD-L1 IHC was performed on cell block sections using Dako PD-L1 IHC 22C3 pharmDx assay and assessed by two cytopathologists blinded to the FC PD-L1 TPS. RESULTS: FC analysis allowed for the distinction between carcinoma cells (CD45-/EpCAM+/D2-40-), leukocytes (CD45+/EpCAM-/D2-40-) and mesothelial cells (CD45-/EpCAM-/D2-40+). FC PD-L1 TPS ranged from 0% to 77 %, while the 22C3 IHC PD-L1 TPS ranged from 0% to 97 %. The FC and IHC TPS values correlated positively (R = 0.8). Best concordance was observed when FC was performed and cell blocks were generated in parallel (R = 0.99). FC also allowed for simultaneous PD-L1 quantification in mesothelial and T-cells. PD-L1 expression on mesothelial cells ranged from 0% to 90.9 %, which also correlated positively with IHC TPS (R = 0.54). PD-L1 expression on T-cells was limited (0.1-2.9 %). CONCLUSION: FC permits rapid, objective and lineage-specific PD-L1 surface expression quantification with limited specimen manipulation. The FC and IHC concordance was impacted by different antibody clones being used, but the positive correlation suggests potential clinical utility, especially in malignant effusion specimens.

A combined FAK, c-MET, and MST1R three-protein panel risk-stratifies colorectal cancer patients.

Focal adhesion kinase (FAK) is a key tyrosine kinase downstream of c-MET (or hepatocyte growth factor receptor, HGFR) and MST1R (macrophage-stimulating protein receptor or recepteur d'origine Nantais, RON) membrane receptors. The pathway plays an important role in cancer survival and invasion. In this study, we examined the protein expression of FAK, c-MET, and MST1R levels in a well-annotated cohort of 330 colorectal cancer patients. We found FAK to be overexpressed in colorectal adenocarcinomas (p = 0.0002), and FAK levels correlated positively with phospho-FAK levels (R2 = 0.81). In comparison, MST1R levels were not significantly different, and c-MET levels were slightly higher in the normal samples. We then developed a combined 3-protein panel of FAK, c-MET, and MST1R expression signatures that can robustly risk-stratify colorectal cancer across all stages into three clusters that differ in progression-free survival. The colorectal cancer subgroup with high FAK, low c-MET, and low MST1R protein levels showed the worst progression-free survival with particularly early progression of disease (p = 0.0053). Combined FAK, c-MET, and MST1R were independently prognostic for progression-free survival in stage II colorectal cancers in a multivariate model. The 3-protein panel provides a potentially clinically attractive method for risk-stratification and adjuvant therapy guidance, especially in stage II disease.

Stem/progenitor cell marker expression in clear cell renal cell carcinoma: a potential relationship with the immune microenvironment to be explored.

BACKGROUND: Clear cell renal cell carcinoma (ccRCC) is a markedly heterogeneous disease in many aspects, including the tumour microenvironment. Our previous study showed the importance of the tumour microenvironment in ccRCC xeno-transplant success rates. In order to better understand the potential relationship between TICs and the immune microenvironment, we employed a multi-modal approach, examining RNA and protein expression (flow cytometry, immunohistochemistry). METHODS: We first examined the gene expression pattern of 18 stem/progenitor marker genes in the cancer genome atlas (TCGA) ccRCC cohort. Flow cytometry was next employed to examine lineage-specific expression levels of stem/progenitor markers and immune population makeup in six, disaggregated, primary ccRCC specimens. Immunohistochemistry was performed on a commercial ccRCC tissue microarray (TMA). RESULTS: The 18 genes differed with respect to their correlation patterns with one another and to their prognostic significance. By flow cytometry, correlating expression frequency of 12 stem/progenitor markers and CD10 resulted in two clusters-one with CD10 (marker of proximal tubular differentiation), and second cluster containing mostly mesenchymal stem cell (MSC) markers, including CD146. In turn, these clusters differed with respect to their correlation with different CD45+ lineage markers and their expression of immune checkpoint pathway proteins. To confirm these findings, four stem/progenitor marker expression patterns were compared with CD4, CD8 and CD20 in a ccRCC TMA which showed a number of similar trends with respect to frequency of the different tumour-infiltrating leukocytes. CONCLUSION: Taken together, we observed heterogeneous but patterned expression levels of different stem/progenitor markers. Our results suggest a non-random relationship between their expression patterns with the immune microenvironment populations in ccRCC.

An Isothermal, Multiplex Amplification Assay for Detection and Genotyping of Human Papillomaviruses in Formalin-Fixed, Paraffin-Embedded Tissues.

Rapid and accurate identification of human papillomavirus (HPV) is important for both clinical management and population screening. Analytic validation of Atila AmpFire Multiplex HPV assays on formalin-fixed, paraffin-embedded (FFPE) cervix/vulva and oropharynx diagnostic tissue samples was performed. The AmpFire assay incorporates a novel isothermal multiplex amplification coupled with real-time fluorescent detection to detect and genotype 15 high-risk (HR) HPV genotypes. Limits of detection determined by plasmids cloned with HPV genotype-specific sequences were 2 copies/reaction for HPV16, HPV18, and some HR HPV genotypes, and 20 copies/reaction for the remaining HR HPV genotypes. The performance of the AmpFire assays in clinical samples was evaluated using 214 FFPE specimens. The AmpFire assay failed in one clinical specimen for an invalid rate of 0.5%. The AmpFire assay detected HPV in clinical samples with positive percent agreements of 100.0% for HPV16, 100.0% for HPV18, and 94.7% for non-16/18 HR HPV, and 100% negative percent agreements for HPV16, HPV18, and non-16/18 HR HPV. Qualitative detection agreement was obtained in the reproducibility study. In summary, the Atila AmpFire HPV assay demonstrated excellent analytic sensitivity and specificity for detection and genotyping of 15 HR HPV genotypes. Assay parameters of simple specimen processing, small sample size requirement, rapid turnaround time, and being near instrument-free render it well suited for HPV detection and genotyping in FFPE specimens.