Segregation, immunohistochemical, molecular and functional analyses classify a novel missense variant in fumarate hydratase (FH) as pathogenic.

Hereditary leiomyomatosis and renal cell cancer (HLRCC) is an autosomal dominant cancer predisposition syndrome characterized by cutaneous leiomyomas, uterine leiomyomas, and aggressive renal cancer. Germline variants in the fumarate hydratase (FH) gene predispose to HLRCC. Identifying germline pathogenic FH variants enables lifetime renal cancer screening and genetic testing for family members. In this report, we present a FH missense variant (c.1039T>C (p.S347P)), initially classified as a variant of uncertain significance. Clinical assessment, histopathological findings, molecular genetic studies, and enzymatic activity studies support the re-classification of the FH c.1039T>C variant to "pathogenic" based on ACMG/AMP criteria. Further insights into pathological recognition of FH-deficient renal cancer are discussed and should be recognized. This study has shown how (a) detailed multi-disciplinary analyses of a single variant can reclassify rare missense variants in FH and (b) careful pathological review of renal cancers is obligatory when HLRCC is suspected.

Unusual Aspects of Small Cell Carcinoma of the Ovary of Hypercalcaemic Type: Retained SMARCA4 Immunohistochemical Staining and Positive Staining With TLE1.

Small cell carcinoma of the ovary of hypercalcaemic type (SCCOHT) is a rare and aggressive ovarian neoplasm that is most common in the second and third decades. Molecular studies have established inactivating SMARCA4 alterations as the driver of SCCOHT, these being present in over 95% of these neoplasms. SMARCA4 alterations almost always result in loss of immunoreactivity with SMARCA4 (BRG1) antibody, and this is an extremely useful adjunct in the diagnosis of SCCOHT. Herein, we report 7 cases of SCCOHT (2 from the same patient) with retention of nuclear immunoreactivity with SMARCA4, but with SMARCA4 alterations identified on molecular testing. All cases exhibited loss of SMARCA2 (BRM) immunoreactivity. In addition, following the identification of diffuse TLE1 immunoreactivity in one of these cases (which did not exhibit an SS18 gene rearrangement characteristic of synovial sarcoma), we stained a total of 63 cases of SCCOHT (14 on whole tissue sections: 49 on tissue microarray) with this marker and 7 of 14 (50%) and 22 of 49 (45%) were positive on whole sections and tissue microarray, respectively. Most cases were focally positive but occasional cases exhibited diffuse immunoreactivity. Our observations highlight the importance of SMARCA2 immunohistochemical staining and molecular testing in suspected cases of SCCOHT that exhibit retained SMARCA4 immunoreactivity. Th common expression of TLE1 in these neoplasms represents a potential diagnostic pitfall since synovial sarcoma may be considered in the differential, especially in cases with retained SMARCA4 immunohistochemistry.

Is Biannual Surveillance for Pancreatic Cancer Sufficient in Individuals With Genetic Syndromes or Familial Pancreatic Cancer?

BACKGROUND: Individuals with a family history of pancreatic adenocarcinoma (PC) or with a germline mutation in a PC susceptibility gene are at increased risk of developing PC. These high-risk individuals (HRIs) may benefit from PC surveillance. METHODS: A PC surveillance program was developed to evaluate the detection of premalignant lesions and early-stage PCs using biannual imaging and to determine whether locally advanced or metastatic PCs develop despite biannual surveillance. From January 2013 to April 2020, asymptomatic HRIs were enrolled and followed with alternating MRI and endoscopic ultrasound every 6 months. RESULTS: Of 75 HRIs, 43 (57.3%) had a germline mutation in a PC susceptibility gene and 32 (42.7%) had a familial pancreatic cancer (FPC) pedigree. Branch-duct intraductal papillary mucinous neoplasms (BD-IPMNs) were identified in 26 individuals (34.7%), but only 2 developed progressive lesions. One patient with Peutz-Jeghers syndrome (PJS) developed locally advanced PC arising from a BD-IPMN. Whole-genome sequencing of this patient's PC and of a second patient with PJS-associated PC from the same kindred revealed biallelic inactivation of STK11 in a KRAS-independent manner. A review of 3,853 patients from 2 PC registries identified an additional patient with PJS-associated PC. All 3 patients with PJS developed advanced PC consistent with the malignant transformation of an underlying BD-IPMN in <6 months. The other surveillance patient with a progressive lesion had FPC and underwent resection of a mixed-type IPMN that harbored polyclonal KRAS mutations. CONCLUSIONS: PC surveillance identifies a high prevalence of BD-IPMNs in HRIs. Patients with PJS with BD-IPMNs may be at risk for accelerated malignant transformation.

Intra-Tumoral CD8+ T-Cell Infiltration and PD-L1 Positivity in Homologous Recombination Deficient Pancreatic Ductal Adenocarcinoma.

The immune contexture of pancreatic ductal adenocarcinoma (PDAC) is generally immunosuppressive. A role for immune checkpoint inhibitors (ICIs) in PDAC has only been demonstrated for the rare and hypermutated mismatch repair (MMR) deficient (MMR-d) subtype. Homologous recombination repair (HR) deficient (HR-d) PDAC is more prevalent and may encompass up to 20% of PDAC. Its genomic instability may promote a T-cell mediated anti-tumor response with therapeutic sensitivity to ICIs. To investigate the immunogenicity of HR-d PDAC, we used multiplex immunohistochemistry (IHC) to compare the density and spatial distribution of CD8+ cytotoxic T-cells, FOXP3+ regulatory T-cells (Tregs), and CD68+ tumor-associated macrophages (TAMs) in HR-d versus HR/MMR-intact PDAC. We also evaluated the IHC positivity of programmed death-ligand 1 (PD-L1) across the subgroups. 192 tumors were evaluated and classified as HR/MMR-intact (n=166), HR-d (n=25) or MMR-d (n=1) based on germline testing and tumor molecular hallmarks. Intra-tumoral CD8+ T-cell infiltration was higher in HR-d versus HR/MMR-intact PDAC (p<0.0001), while CD8+ T-cell densities in the peri-tumoral and stromal regions were similar in both groups. HR-d PDAC also displayed increased intra-tumoral FOXP3+ Tregs (p=0.049) and had a higher CD8+:FOXP3+ ratio (p=0.023). CD68+ TAM expression was similar in HR-d and HR/MMR-intact PDAC. Finally, 6 of the 25 HR-d cases showed a PD-L1 Combined Positive Score of >=1, whereas none of the HR/MMR-intact cases met this threshold (p<0.00001). These results provide immunohistochemical evidence for intra-tumoral CD8+ T-cell enrichment and PD-L1 positivity in HR-d PDAC, suggesting that HR-d PDAC may be amenable to ICI treatment strategies.

Oncology clinic-based germline genetic testing for exocrine pancreatic cancer enables timely return of results and unveils low uptake of cascade testing.

BACKGROUND: Traditional medical genetics models are unable to meet the growing demand for germline genetic testing (GT) in patients with exocrine pancreatic cancer (PC). This study investigates the impact of an ambulatory oncology clinic-based GT model. METHODS: From 2012 to 2021, patients with PC were prospectively enrolled and considered for GT. Two chronological cohorts were compared: (1) the preuniversal genetic testing (pre-UGT) cohort, which received GT based on clinical criteria or family history; and (2) the post-UGT cohort, where an 86-gene panel was offered to all patients with PC. RESULTS: Of 847 eligible patients, 735 (86.8%) were enrolled (pre-UGT, n=579; post-UGT, n=156). A higher proportion of the post-UGT cohort received prospective GT (97.4% vs 58.5%, p<0.001). The rate of pathogenic germline alterations (PGA) across both cohorts was 9.9%, with 8.0% of PGAs in PC susceptibility genes. The post-UGT cohort had a higher prevalence of overall PGAs (17.2% vs 6.6%, p<0.001) and PGAs in PC susceptibility genes (11.9% vs 6.3%, p<0.001). The median turnaround time from enrolment to GT report was shorter in the post-UGT cohort (13 days vs 42 days, p<0.001). Probands with a PGA disclosed their GT results to 84% of their first-degree relatives (FDRs). However, only 31% of informed FDRs underwent GT, and the number of new cases per index case was 0.52. CONCLUSION: A point-of-care GT model is feasible and expedites access to GT for patients with PC. Strategies to increase the uptake of cascade testing are needed to maximise the clinical impact of an oncology clinic-based GT model.

A Preclinical Trial and Molecularly Annotated Patient Cohort Identify Predictive Biomarkers in Homologous Recombination-deficient Pancreatic Cancer.

PURPOSE: Pancreatic ductal adenocarcinoma (PDAC) arising in patients with a germline BRCA1 or BRCA2 (gBRCA) mutation may be sensitive to platinum and PARP inhibitors (PARPi). However, treatment stratification based on gBRCA mutational status alone is associated with heterogeneous responses. EXPERIMENTAL DESIGN: We performed a seven-arm preclinical trial consisting of 471 mice, representing 12 unique PDAC patient-derived xenografts, of which nine were gBRCA mutated. From 179 patients whose PDAC was whole-genome and transcriptome sequenced, we identified 21 cases with homologous recombination deficiency (HRD), and investigated prognostic biomarkers. RESULTS: We found that biallelic inactivation of BRCA1/BRCA2 is associated with genomic hallmarks of HRD and required for cisplatin and talazoparib (PARPi) sensitivity. However, HRD genomic hallmarks persisted in xenografts despite the emergence of therapy resistance, indicating the presence of a genomic scar. We identified tumor polyploidy and a low Ki67 index as predictors of poor cisplatin and talazoparib response. In patients with HRD PDAC, tumor polyploidy and a basal-like transcriptomic subtype were independent predictors of shorter survival. To facilitate clinical assignment of transcriptomic subtype, we developed a novel pragmatic two-marker assay (GATA6:KRT17). CONCLUSIONS: In summary, we propose a predictive and prognostic model of gBRCA-mutated PDAC on the basis of HRD genomic hallmarks, Ki67 index, tumor ploidy, and transcriptomic subtype.

A region-based gene association study combined with a leave-one-out sensitivity analysis identifies SMG1 as a pancreatic cancer susceptibility gene.

Pancreatic adenocarcinoma (PC) is a lethal malignancy that is familial or associated with genetic syndromes in 10% of cases. Gene-based surveillance strategies for at-risk individuals may improve clinical outcomes. However, familial PC (FPC) is plagued by genetic heterogeneity and the genetic basis for the majority of FPC remains elusive, hampering the development of gene-based surveillance programs. The study was powered to identify genes with a cumulative pathogenic variant prevalence of at least 3%, which includes the most prevalent PC susceptibility gene, BRCA2. Since the majority of known PC susceptibility genes are involved in DNA repair, we focused on genes implicated in these pathways. We performed a region-based association study using the Mixed-Effects Score Test, followed by leave-one-out characterization of PC-associated gene regions and variants to identify the genes and variants driving risk associations. We evaluated 398 cases from two case series and 987 controls without a personal history of cancer. The first case series consisted of 109 patients with either FPC (n = 101) or PC at ≤50 years of age (n = 8). The second case series was composed of 289 unselected PC cases. We validated this discovery strategy by identifying known pathogenic BRCA2 variants, and also identified SMG1, encoding a serine/threonine protein kinase, to be significantly associated with PC following correction for multiple testing (p = 3.22x10-7). The SMG1 association was validated in a second independent series of 532 FPC cases and 753 controls (p<0.0062, OR = 1.88, 95%CI 1.17-3.03). We showed segregation of the c.4249A>G SMG1 variant in 3 affected relatives in a FPC kindred, and we found c.103G>A to be a recurrent SMG1 variant associating with PC in both the discovery and validation series. These results suggest that SMG1 is a novel PC susceptibility gene, and we identified specific SMG1 gene variants associated with PC risk.

Discovery of cell compartment specific protein-protein interactions using affinity purification combined with tandem mass spectrometry.

Affinity purification combined with tandem mass spectrometry (AP-MS/MS) is a well-established method used to discover interaction partners for a given protein of interest. Because most AP-MS/MS approaches are performed using the soluble fraction of whole cell extracts (WCE), information about the cellular compartments where the interactions occur is lost. More importantly, classical AP-MS/MS often fails to identify interactions that take place in the nonsoluble fraction of the cell, for example, on the chromatin or membranes; consequently, protein complexes that are less soluble are underrepresented. In this paper, we introduce a method called multiple cell compartment AP-MS/MS (MCC-AP-MS/MS), which identifies the interactions of a protein independently in three fractions of the cell: the cytoplasm, the nucleoplasm, and the chromatin. We show that this fractionation improves the sensitivity of the method when compared to the classical affinity purification procedure using soluble WCE while keeping a very high specificity. Using three proteins known to localize in various cell compartments as baits, the CDK9 subunit of transcription elongation factor P-TEFb, the RNA polymerase II (RNAP II)-associated protein 4 (RPAP4), and the largest subunit of RNAP II, POLR2A, we show that MCC-AP-MS/MS reproducibly yields fraction-specific interactions. Finally, we demonstrate that this improvement in sensitivity leads to the discovery of novel interactions of RNAP II carboxyl-terminal domain (CTD) interacting domain (CID) proteins with POLR2A.

Molecular dynamics and mutational analysis of the catalytic and translocation cycle of RNA polymerase.

BACKGROUND: During elongation, multi-subunit RNA polymerases (RNAPs) cycle between phosphodiester bond formation and nucleic acid translocation. In the conformation associated with catalysis, the mobile "trigger loop" of the catalytic subunit closes on the nucleoside triphosphate (NTP) substrate. Closing of the trigger loop is expected to exclude water from the active site, and dehydration may contribute to catalysis and fidelity. In the absence of a NTP substrate in the active site, the trigger loop opens, which may enable translocation. Another notable structural element of the RNAP catalytic center is the "bridge helix" that separates the active site from downstream DNA. The bridge helix may participate in translocation by bending against the RNA/DNA hybrid to induce RNAP forward movement and to vacate the active site for the next NTP loading. The transition between catalytic and translocation conformations of RNAP is not evident from static crystallographic snapshots in which macromolecular motions may be restrained by crystal packing. RESULTS: All atom molecular dynamics simulations of Thermus thermophilus (Tt) RNAP reveal flexible hinges, located within the two helices at the base of the trigger loop, and two glycine hinges clustered near the N-terminal end of the bridge helix. As simulation progresses, these hinges adopt distinct conformations in the closed and open trigger loop structures. A number of residues (described as "switch" residues) trade atomic contacts (ion pairs or hydrogen bonds) in response to changes in hinge orientation. In vivo phenotypes and in vitro activities rendered by mutations in the hinge and switch residues in Saccharomyces cerevisiae (Sc) RNAP II support the importance of conformational changes predicted from simulations in catalysis and translocation. During simulation, the elongation complex with an open trigger loop spontaneously translocates forward relative to the elongation complex with a closed trigger loop. CONCLUSIONS: Switching between catalytic and translocating RNAP forms involves closing and opening of the trigger loop and long-range conformational changes in the atomic contacts of amino acid side chains, some located at a considerable distance from the trigger loop and active site. Trigger loop closing appears to support chemistry and the fidelity of RNA synthesis. Trigger loop opening and limited bridge helix bending appears to promote forward nucleic acid translocation.

Interaction of RNA polymerase II fork loop 2 with downstream non-template DNA regulates transcription elongation.

Fork loop 2 is a small semiconservative segment of the larger fork domain in the second largest Rpb2 subunit of RNA polymerase II (Pol II). This flexible loop, juxtaposed at the leading edge of transcription bubble, has been proposed to participate in DNA strand separation, translocation along DNA, and NTP loading to Pol II during elongation. Here we show that the Rpb2 mutant carrying a deletion of the flexible part of the loop is not lethal in yeast. The mutation exhibits no defects in DNA melting and translocation in vitro but confers a moderate decrease of the catalytic activity of the enzyme caused by the impaired sequestration of the NTP substrate in the active center prior to catalysis. In the structural model of the Pol II elongation complex, fork loop 2 directly interacts with an unpaired DNA residue in the non-template DNA strand one nucleotide ahead from the active center (the i+2 position). We showed that elimination of this putative interaction by replacement of the i+2 residue with an abasic site inhibits Pol II activity to the same degree as the deletion of fork loop 2. This replacement has no detectable effect on the activity of the mutant enzyme. We provide direct evidence that interaction of fork loop 2 with the non-template DNA strand facilitates NTP sequestration through interaction with the adjacent segment of the fork domain involved in the active center of Pol II.

Conformational coupling, bridge helix dynamics and active site dehydration in catalysis by RNA polymerase.

Molecular dynamics simulation of Thermus thermophilus (Tt) RNA polymerase (RNAP) in a catalytic conformation demonstrates that the active site dNMP-NTP base pair must be substantially dehydrated to support full active site closing and optimum conditions for phosphodiester bond synthesis. In silico mutant beta R428A RNAP, which was designed based on substitutions at the homologous position (Rpb2 R512) of Saccharomyces cerevisiae (Sc) RNAP II, was used as a reference structure to compare to Tt RNAP in simulations. Long range conformational coupling linking a dynamic segment of the bridge alpha-helix, the extended fork loop, the active site, and the trigger loop-trigger helix is apparent and adversely affected in beta R428A RNAP. Furthermore, bridge helix bending is detected in the catalytic structure, indicating that bridge helix dynamics may regulate phosphodiester bond synthesis as well as translocation. An active site "latch" assembly that includes a key trigger helix residue Tt beta' H1242 and highly conserved active site residues beta E445 and R557 appears to help regulate active site hydration/dehydration. The potential relevance of these observations in understanding RNAP and DNAP induced fit and fidelity is discussed.

Site-directed mutagenesis, purification and assay of Saccharomyces cerevisiae RNA polymerase II.

In order to analyze the structure-function of multi-subunit RNA polymerases (RNAPs), it is necessary to make site-directed mutations in key residues. Because Saccharomyces cerevisiae RNAP II is isolated as a 12 subunit enzyme that has not been amenable to in vitro reconstitution, making site-directed mutations in a particular subunit presents technical issues. In this work, we demonstrate a method to generate and purify site-directed mutants in the second largest (Rpb2) RNAP II subunit from yeast, using a tandem affinity purification tag. Mutants are analyzed for growth defects in vivo and for defects in transcriptional elongation in vitro. We show that Rpb2 R512A/C located just C-terminal to fork loop 2 (Rpb2 500-511) has transcriptional defects that are distinct from surrounding fork loop 2 region mutants. Rpb2 E529A/D replacements are faster and E529Q is slower than wild type RNAP II in elongation. E529 appears to form an ion pair with K987, an essential active site residue. Mutations are also analyzed within the active site region indicating key residues for catalysis and the importance of a Rpb2 R983-E1028 ion pair. Rpb2 R983Q and E1028Q are defective in escape from a transcriptional stall.

Use of site-specific protein-DNA photocrosslinking of purified complexes to analyze the topology of the RNA polymerase II transcription initiation complex.

A method for the photocrosslinking of proteins to DNA in purified complexes is described. It makes use of the juxtaposition of a limited number of photoreactive nucleotides with a limited number of radiolabeled nucleotides at a specific location in a DNA fragment. Protein-DNA complexes are submitted to an electrophoretic mobility shift assay that is then irradiated with UV light in order to crosslink the proteins to DNA. The specific complexes are localized on the gel, purified, and processed for the identification of the crosslinked polypeptides.