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CTNNB1 mutations convey increased risk of recurrence in low-risk endometrial endometrioid carcinoma (EEC). Results from previous high-intermediate risk (HIR) cohorts are mixed. The aims of this study were to correlate CTNNB1 mutational status with clinical outcomes and to evaluate the relationship between CTNNB1 mutations and the 4 prognostic subgroups defined by The Cancer Genome Atlas in HIR EEC. CTNNB1 mutational status was determined by Sanger sequencing of exon 3 of the CTNNB1 gene. Mismatch repair, POLE, p53, and L1 cell-adhesion molecule (L1CAM) status were also evaluated. Descriptive statistics and survival analyses were performed. Eighty-eight cases of HIR EEC were identified, of which 22 (25%) were CTNNB1 mutant (CTNNB1-mut) and 66 (75%) were wild-type (CTNNB1-WT). Median follow-up was 60 mo. Recurrence occurred in 13/88 (15%) patients. Recurrence rates were not significantly different between patients with CTNNB1-mut and CTNNB1-WT tumors (14% vs. 15%, P=0.86). Recurrence-free survival and overall survival were not significantly different (recurrence-free survival hazard ratio: 0.97, 95% confidence interval: 0.27-3.52, P=0.96; overall survival hazard ratio: 0.23, 95% confidence interval: 0.03-1.71, P=0.15). Mismatch repair deficiency was more prevalent in CTNNB1-WT compared with CTNNB1-mut tumors (46% vs. 14%, P=0.01); prevalence of POLE mutations and aberrant p53 were not significantly different. In contrast to patients with low-risk EEC, no differences in recurrence or survival were found in patients with HIR EEC with CTNNB1-mut compared with CTNNB1-WT tumors.

Pilocytic astrocytoma is the most frequent pediatric glioma. Despite its overall good prognosis, complete surgical resection is sometimes unfeasible, especially for patients with deep-seated tumors. For these patients, the identification of targetable genetic alterations such as NTRK fusions, raised as a new hope for therapy. The presence of gene fusions involving NTRK2 has been rarely reported in pilocytic astrocytoma. The aim of the present study was to investigate the frequency of NTRK2 alterations in a series of Brazilian pilocytic astrocytomas.Sixty-nine pilocytic astrocytomas, previously characterized for BRAF and FGFR1 alterations were evaluated. The analysis of NTRK2 alterations was performed using a dual color break apart fluorescence in situ hybridization (FISH) assay.NTRK2 fusions were successfully evaluated by FISH in 62 of the 69 cases. Neither evidence of NTRK2 gene rearrangements nor NTRK2 copy number alterations were found.NTRK2 alterations are uncommon genetic events in pilocytic astrocytomas, regardless of patients' clinicopathological and molecular features.

Purpose: Determine whether specific CTNNB1 or APC mutations in patients with desmoid tumor were associated with differences in clinical responses to systemic treatments. Experimental Design: We established a multi-institutional dataset of previously treated patients with desmoid tumor across four U.S. sarcoma centers, including demographic and clinicopathologic characteristics, treatment regimens, and clinical and radiographic responses. CTNNB1 or APC mutation status was determined from prior pathology records, or archival tissue was requested and analyzed by Sanger sequencing and/or next-generation sequencing. Evaluable patients with mutation results were analyzed to determine clinical progression-free survival (cPFS), RECIST 1.1 PFS (rPFS), time to next treatment (TTNT), and overall survival (OS). Kaplan–Meier analysis and Cox proportional hazards regression were performed to identify differences in cPFS, rPFS, TTNT, and OS by mutation subtype, desmoid tumor location, and treatment regimen. Results: A total of 259 evaluable patients were analyzed for at least one of the survival outcomes, with 177 patients having mutation data. First- and second-line cPFS, rPFS, and TTNT were not significantly affected by mutation subtype; however, APC-mutant desmoid tumors demonstrated nonstatistically significant inferior outcomes. Extremity/trunk desmoid tumor location and treatment with doxorubicin-based, methotrexate/vinca alkaloids and sorafenib regimens were associated with better clinical outcomes compared with surgery or “other” therapies, including estrogen-receptor blockade and imatinib. OS was significantly worse with APC or CTNNB1 negative/other mutations. Conclusions: Mutation subtype did not affect responses to specific systemic therapies. APC mutations and nonextremity desmoid tumor locations remain prognostic for worse outcomes, and earlier initiation of systemic therapy for these higher-risk desmoid tumors should be prospectively evaluated. See related commentary by Greene and Van Tine, p. 3911

Supplementary Data from Systemic Chemotherapies Retain Antitumor Activity in Desmoid Tumors Independent of Specific Mutations in CTNNB1 or APC: A Multi-institutional Retrospective Study

Purpose:Determine whether specific CTNNB1 or APC mutations in patients with desmoid tumor were associated with differences in clinical responses to systemic treatments.Experimental Design:We established a multi-institutional dataset of previously treated patients with desmoid tumor across four U.S. sarcoma centers, including demographic and clinicopathologic characteristics, treatment regimens, and clinical and radiographic responses. CTNNB1 or APC mutation status was determined from prior pathology records, or archival tissue was requested and analyzed by Sanger sequencing and/or next-generation sequencing. Evaluable patients with mutation results were analyzed to determine clinical progression-free survival (cPFS), RECIST 1.1 PFS (rPFS), time to next treatment (TTNT), and overall survival (OS). Kaplan–Meier analysis and Cox proportional hazards regression were performed to identify differences in cPFS, rPFS, TTNT, and OS by mutation subtype, desmoid tumor location, and treatment regimen.Results:A total of 259 evaluable patients were analyzed for at least one of the survival outcomes, with 177 patients having mutation data. First- and second-line cPFS, rPFS, and TTNT were not significantly affected by mutation subtype; however, APC-mutant desmoid tumors demonstrated nonstatistically significant inferior outcomes. Extremity/trunk desmoid tumor location and treatment with doxorubicin-based, methotrexate/vinca alkaloids and sorafenib regimens were associated with better clinical outcomes compared with surgery or “other” therapies, including estrogen-receptor blockade and imatinib. OS was significantly worse with APC or CTNNB1 negative/other mutations.Conclusions:Mutation subtype did not affect responses to specific systemic therapies. APC mutations and nonextremity desmoid tumor locations remain prognostic for worse outcomes, and earlier initiation of systemic therapy for these higher-risk desmoid tumors should be prospectively evaluated.See related commentary by Greene and Van Tine, p. 3911

Supplementary Figure from Systemic Chemotherapies Retain Antitumor Activity in Desmoid Tumors Independent of Specific Mutations in CTNNB1 or APC: A Multi-institutional Retrospective Study

Introduction ROS1 gene fusions are a well-characterized class of oncogenic driver found in approximately 1% to 2% of NSCLC patients. ROS1-directed therapy in these patients is more efficacious and is associated with fewer side effects compared to chemotherapy and is thus now considered standard-of-care for patients with advanced disease. Consequently, accurate detection of ROS1 rearrangements/fusions in clinical tumor samples is vital. In this study, we compared the performance of three common molecular testing approaches on a cohort of ROS1 rearrangement/fusion-positive patient samples. Methods Twenty-three ROS1 rearrangement/fusion-positive clinical samples were assessed by at least two of the following molecular testing methodologies: break-apart fluorescence in situ hybridization, DNA-based hybrid capture library preparation followed by next-generation sequencing (NGS), and RNA-based anchored multiplex polymerase chain reaction library preparation followed by NGS. Results None of the testing methodologies demonstrated 100% sensitivity in detection of ROS1 rearrangements/fusions. Fluorescence in situ hybridization results were negative in 2 of 20 tested samples, the DNA-based NGS assay was negative in 4 of 18 tested samples, and the RNA-based NGS assay was negative in 3 of 19 tested samples. For all three testing approaches, we identified assay characteristics that likely contributed to false-negative results. Additionally, we report that genomic breakpoints are an unreliable predictor of breakpoints at the transcript level, likely due to alternative splicing. Conclusions ROS1 rearrangement/fusion detection in the clinical setting is complex and all methodologies have inherent limitations of which users must be aware to correctly interpret results.

Objectives: CTNNB1 mutations convey increased risk of recurrence in low risk endometrioid endometrial cancers. However, sub-analyses of high-intermediate risk (HIR) endometrioid endometrial cancers in other cohorts have not demonstrated a clearly increased recurrence risk and have often been equivocal. The objective of this study is to perform a clinicopathologic evaluation of patients with HIR endometrioid endometrial cancer in relation to CTNNB1 mutational status. Methods: A single-institution chart review of patients diagnosed with endometrioid endometrial cancer between 2006 and 2018 was undertaken. Patients were classified as HIR according to GOG249 criteria. Mutational status was determined by Sanger sequencing of exon 3 of the CTNNB1 gene in primary tumor specimens. Immunohistochemical (IHC) analysis of B-catenin was performed to evaluate for the presence of nuclear staining. Factors associated with recurrence were assessed by logistic regression; recurrence-free (RFS) and overall survival (OS) were assessed by Kaplan-Meier survival analysis. Results: 93 cases of HIR endometrioid endometrial cancer were identified, of which 23 (24.7%) were CTNNB1 mutant and 70 (75.3%) were wild type. Median follow-up was 59.7 months. Recurrence occurred in 14/93 (15.1%) patients. Recurrence rates were not significantly different between patients with CTNNB1 mutant and wild type tumors (13.0% vs. 15.9%, p=0.76). In both univariable and multivariable analyses, RFS and OS were not significantly different (RFS HR 0.88, p=0.85, 95% CI 0.24 - 3.17; OS HR 0.20, p=0.12, 95% CI 0.03 - 1.54). Predicted oncogenic mutations comprised 87% of all identified mutations and were present in 100% of recurrent tumors and 85% of non-recurrent tumors (p=0.47). Nuclear B-catenin expression by IHC demonstrated a sensitivity of 87% and a specificity of 83% for CTNNB1 mutations. Conclusions: CTNNB1 mutations have a prevalence of 25% in HIR endometrioid endometrial cancer. In this single institution retrospective study, there were no differences in RFS or OS in patients with CTNNB1 mutant compared to wild-type tumors.

Introduction MNNG HOS Transforming gene (MET) amplification and MET exon 14 (METex14) alterations in lung cancers affect sensitivity to MET proto-oncogene, receptor tyrosine kinase (MET [also known by the alias hepatocyte growth factor receptor]) inhibitors. Fluorescence in situ hybridization (FISH), next-generation sequencing (NGS), and immunohistochemistry (IHC) have been used to evaluate MET dependency. Here, we have determined the association of MET IHC with METex14 mutations and MET amplification. Methods We collected data on a tri-institutional cohort from the Lung Cancer Mutation Consortium. All patients had metastatic lung adenocarcinomas and no prior targeted therapies. MET IHC positivity was defined by an H-score of 200 or higher using SP44 antibody. MET amplification was defined by copy number fold change of 1.8x or more with use of NGS or a MET-to–centromere of chromosome 7 ratio greater than 2.2 with use of FISH. Results We tested tissue from 181 patients for MET IHC, MET amplification, and METex14 mutations. Overall, 71 of 181 patients (39%) were MET IHC–positive, three of 181 (2%) were MET-amplified, and two of 181 (1%) harbored METex14 mutations. Of the MET-amplified cases, two were FISH positive with MET-to–centromere of chromosome 7 ratios of 3.1 and 3.3, one case was NGS positive with a fold change of 4.4x, and one of the three cases was MET IHC–positive. Of the 71 IHC-positive cases, one (1%) was MET-amplified and two (3%) were METex14-mutated. Of the MET IHC–negative cases, two of 110 (2%) were MET-amplified. Conclusions In this study, nearly all MET IHC–positive cases were negative for MET amplification or METex14 mutations. MET IHC can also miss patients with MET amplification. The limited number of MET-amplified cases in this cohort makes it challenging to demonstrate an association between MET IHC and MET amplification. Nevertheless, IHC appears to be an inefficient screen for these genomic changes. MET amplification or METex14 mutations can best be detected by FISH and a multiplex NGS panel.

Background: CTNNB1 mutations convey increased risk of recurrence in low risk endometrioid endometrial cancers. However, sub-analyses of high-intermediate risk (HIR) endometrioid endometrial cancers in other cohorts have not demonstrated a clearly increased recurrence risk and have often been equivocal. The objective of this study is to perform a clinicopathologic evaluation of patients with HIR endometrioid endometrial cancer in relation to CTNNB1 mutational status. Methods: A single-institution chart review of patients diagnosed with endometrioid endometrial cancer between 2006 and 2018 was undertaken. Patients were classified as HIR according to GOG249 criteria. Mutational status was determined by Sanger sequencing of exon 3 of the CTNNB1 gene in primary tumor specimens. Immunohistochemical (IHC) analysis of B-catenin was performed to evaluate for the presence of nuclear staining. Factors associated with recurrence were assessed by logistic regression; recurrence-free (RFS) and overall survival (OS) were assessed by Kaplan-Meier survival analysis. Results: 93 cases of HIR endometrioid endometrial cancer were identified, of which 23 (24.7%) were CTNNB1 mutant and 70 (75.3%) were wild type. Median follow-up was 54.7 months. Recurrence occurred in 14/93 (15.1%) patients. Recurrence rates were not significantly different between patients with CTNNB1 mutant and wild type tumors (13.0% vs. 15.9%, p=0.76). In both univariable and multivariable analyses, RFS and OS were not significantly different (RFS HR 0.88, p=0.85, 95% CI 0.24 – 3.17; OS HR 0.20, p=0.12, 95% CI 0.03 – 1.54). Predicted oncogenic mutations comprised 87% of all identified mutations and were present in 100% of recurrent tumors and 85% of non-recurrent tumors (p=0.47). Nuclear B-catenin expression by IHC demonstrated a sensitivity of 87% and a specificity of 83% for CTNNB1 mutations. Conclusions: CTNNB1 mutations have a prevalence of 25% in HIR endometrioid endometrial cancer. In this single institution retrospective study, there were no differences in RFS or OS in patients with CTNNB1 mutant compared to wild-type tumors. Citation Format: Jennifer G. Haag, Rebecca J. Wolsky, Marisa R. Moroney, Jamie Sheren, Jeanelle Sheeder, Benjamin G. Bitler, Bradley R. Corr. Clinicopathologic evaluation of CTNNB1 gene mutations in high-intermediate risk endometrial cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference: Endometrial Cancer: New Biology Driving Research and Treatment; 2020 Nov 9-10. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(3_Suppl):Abstract nr PO014.

Genomic rearrangements resulting in activating kinase fusions have been increasingly described in a number of cancers including malignant melanoma, but their frequency in specific melanoma subtypes has not been reported. We used break-apart fluorescence in situ hybridization (FISH) to identify genomic rearrangements in tissues from 59 patients with various types of malignant melanoma including acral lentiginous, mucosal, superficial spreading, and nodular. We identified four genomic rearrangements involving the genes BRAF, RET, and ROS1. Of these, three were confirmed by Immunohistochemistry (IHC) or sequencing and one was found to be an ARMC10-BRAF fusion that has not been previously reported in melanoma. These fusions occurred in different subtypes of melanoma but all in tumors lacking known driver mutations. Our data suggest gene fusions are more common than previously thought and should be further explored particularly in melanomas lacking known driver mutations.

The RNF38 gene encodes a RING finger protein of unknown function. Here we demonstrate that RNF38 is a functional ubiquitin protein ligase (E3). We show that RNF38 isoform 1 is localized to the nucleus by a bipartite nuclear localization sequence (NLS). We confirm that RNF38 is a binding partner of p53 and demonstrate that RNF38 can ubiquitinate p53 in vitro and in vivo. Finally, we show that overexpression of RNF38 in HEK293T cells results in relocalization of p53 to discrete foci associated with PML nuclear bodies. These results suggest RNF38 is an E3 ubiquitin ligase that may play a role in regulating p53.

Mesenchymal-epithelial transition factor gene (MET) gene copy number gain may be a predictive biomarker for mesenchymal-epithelial transition factor (MET) inhibition in lung cancer, but the most appropriate method and criteria for defining MET positivity are uncertain.MET copy number was assessed by fluorescence in situ hybridization in lung adenocarcinoma. Positivity criteria included mean MET per cell values greater than 5 (low [≥5 to6], intermediate [≥6 to7], and high [≥7]) and mean MET-to-chromosome 7 centromere ratios (MET/CEP7) of at least 1.8 (low [≥1.8 to ≤2.2], intermediate [2.2 to5], and high [≥5]). Associated clinical and molecular characteristics were captured.Of 686 cases, 99 (14%) had a mean MET per cell value of 5 or greater, 52 of 1164 (4.5%) had a MET/CEP7 ratio of 1.8 or higher. Other oncogenic drivers (in EGFR, KRAS, anaplastic lymphoma receptor tyrosine kinase gene [ALK], erb-b2 receptor tyrosine kinase 2 gene [ERBB2], BRAF, NRAS, ROS1, or ret proto-oncogene [RET]) were detectable in 56% of the group with a mean MET per cell value of 5 or higher and 47% of the group with a MET/CEP7 ratio of 1.8 or higher, suggesting that many MET-positive cases are not truly MET addicted. The rates of concomitant drivers in the groups of patients in the low, indeterminate, and high categories of mean MET per cell were 32 of 52 (62%), 12 of 19 (63%), and 11 of 27 (41%) (p = 0.2), and the rates of concomitant drivers in the low, intermediate, and high categories of MET/CEP7 ratios were 15 of 29 (52%), 9 of 18 (50%), and 0 of 4 (0%), respectively (p = 0.04). A MET/CEP7 ratio of 1.8 or higher in the absence of other oncogenes was associated with a higher rate of adrenal metastases (p = 0.03) but not with never-smoking status.A fluorescence in situ hybridization MET/CEP7 ratio of 5 or higher defined a "MET-positive" group with no oncogenic overlap. As this method and criteria are also associated with the highest response rate to MET inhibition, it represents the clearest definition of a MET copy number gain-addicted state. However, a MET-associated phenotype may also exist across cases with a MET/CEP7 of 1.8 or higher when no other oncogene overlap occurs.

The bacteriophage P1 Cre/loxP site-specific recombination system is a useful tool in a number of genetic engineering processes. The Cre recombinase has been shown to act on DNA sequences that vary considerably from that of its bacteriophage recognition sequence, loxP. However, little is known about the sequence requirements for functional lox-like sequences. In this study, we have implemented a randomized library approach to identify the sequence characteristics of functional lox site domains. We created a randomized spacer library and a randomized arm library, and then tested them for recombination in vivo and in vitro. Results from the spacer library show that, while there is great plasticity, identity between spacer pairs is the most important factor influencing function, especially in in vitro reactions. The presence of one completely randomized arm in a functional loxP recombination reaction revealed that only three wild-type loxP arms are necessary for successful recombination in Cre-expressing bacteria, and that there are nucleotide preferences at the first three and last three positions of the randomized arm for the most efficiently recombined sequences. Finally, we found that in vitro Cre recombination reactions are much more stringent for evaluating which sequences can support efficient recombination compared to the 294-CRE system.

Up to 20% of patients with pilocytic astrocytoma (PA) experience a poor outcome. BRAF alterations and Fibroblast growth factor receptor 1 (FGFR1) point mutations are key molecular alterations in Pas, but their clinical implications are not established. We aimed to determine the frequency and prognostic role of these alterations in a cohort of 69 patients with PAs. We assessed KIAA1549:BRAF fusion by fluorescence in situ hybridization and BRAF (exon 15) mutations by capillary sequencing. In addition, FGFR1 expression was analyzed using immunohistochemistry, and this was compared with gene amplification and hotspot mutations (exons 12 and 14) assessed by fluorescence in situ hybridization and capillary sequencing. KIAA1549:BRAF fusion was identified in almost 60% of cases. Two tumors harbored mutated BRAF. Despite high FGFR1 expression overall, no cases had FGFR1 amplifications. Three cases harbored a FGFR1 p.K656E point mutation. No correlation was observed between BRAF and FGFR1 alterations. The cases were predominantly pediatric (87%), and no statistical differences were observed in molecular alterations-related patient ages. In summary, we confirmed the high frequency of KIAA1549:BRAF fusion in PAs and its association with a better outcome. Oncogenic mutations of FGFR1, although rare, occurred in a subset of patients with worse outcome. These molecular alterations may constitute alternative targets for novel clinical approaches, when radical surgical resection is unachievable., This study was partially supported by CNPq/Universal (475358/2011-2), and FAPESP (2012/19590-0) grants to RMR and to the NIH- P30CA046934 (CCSG Molecular Pathology/Cytogenetics) to MVG and DLA

10067 Background: Pilocytic astrocytoma (PA) often arises in young patients and may be related to type 1 neurofibromatosis (NF1). Although the 5-year survival is high, up to 10% of cases progress poorly. Alterations of BRAF mainly through gene fusions are key genetic events in this tumor type, and are reported to influence patient outcome. Our aim was to assess BRAF molecular changes in PAs for both p.V600E mutation and KIAA1549:BRAF (K:B) fusion and correlate with clinicopathological features in retrospective studies. Methods: Sanger sequencing was used to evaluate BRAF V600E mutation. K:B fusion was evaluated by a customized dual-target, dual-color fluorescence in situ hybridization (FISH) probe set in samples in tissue microarray format. The designed K:BFISH probe was validated in Agilent 8x60K aCGH and RT-PCR assays in 5 cases. Results: A total of 75 samples were evaluated from 69 patients (1.2 M/F), mean age of 11.6 years and long-term follow-up (7-196 months). Cerebellum was the main location (53.6%...

The Cre/lox site-specific recombination system has become a useful tool in a variety of genetic engineering processes. Cre/lox has been used to carry out sequence-specific genomic deletions, insertions, recombinase mediated cassette exchange (RMCE) and to facilitate the building of large, complex DNA constructs. The system essentially consists of the Cre recombinase from bacteriophage P1 and its recombination target site, the 34 base pair loxP sequence. It has been shown that Cre can act on sites who's sequence varies considerably form that of loxP. Use of this characteristic to target endogenous genomic lox-like sequences for site-specific integrations has been explored with limited success. In order to better target endogenous lox-like sites, we are attempting to define the nucleotide sequences that the recombinase can act on. We have created a series of partially randomized lox libraries and tested their recombination proficiency both in vivo and in vitro. The first library consists of sites with loxP arms separated by a randomized 8 base pair spacer. Individual sites from this library were tested for recombination against themselves in an E. coli resistance marker deletion assay and in an in vitro resistance marker deletion assay using purified maltose binding protein tagged Cre. The second library is made up of lox sites with one 13 base pair randomized arm and one wild-type loxP arm and spacer. By pairing the library sites in tandem with a fully wild-type loxP site, functionality was measured in a |[beta]|-galactosidase gain of function assay in E. coli. Using a completely randomized approach to the study of loxP functional domains (arms and spacer) will help define knowledge of the sequence requirements for functional sites and expand the library of useful lox-like sequences.