Context-dependent environmental associations with endometrial cancer histotype and genotype.

OBJECTIVE: MLH1 loss due to MLH1 methylation, detected during Lynch syndrome screening, is one of the most common molecular changes in endometrial cancer. It is well established that environmental influences such as nutritional state can impact gene methylation, both in the germline and in a tumor. In colorectal cancer and other cancer types, aging is associated with changes in gene methylation. The objective of this study was to determine if there was an association between aging or body mass index on MLH1 methylation in sporadic endometrial cancer. METHODS: A retrospective review of patients with endometrial cancer was performed. Tumors were screened for Lynch syndrome via immunohistochemistry, with MLH1 methylation analysis performed when there was loss of MLH1 expression. Clinical information was abstracted from the medical record. RESULTS: There were 114 patients with mismatch repair deficient tumors associated with MLH1 methylation, and 349 with mismatch repair proficient tumors. Patients with mismatch repair deficient tumors were older than those whose tumors were proficient. Mismatch repair deficient tumors had a higher incidence of lymphatic/vascular space invasion. When stratified by endometrioid grade, associations with body mass index and age became apparent. Patients with endometrioid grades 1 and 2 tumors and somatic mismatch repair deficiency were significantly older, but body mass index was comparable with that of the mismatch repair intact group. For endometrioid grade 3, patient age did not significantly vary between the somatic mismatch repair deficient group and the mismatch repair intact group. In contrast, body mass index was significantly higher in the patients with grade 3 tumors with somatic mismatch repair deficiency. CONCLUSION: The relationship of MLH1 methylated endometrial cancer with age and body mass index is complex and somewhat dependent on tumor grade. As body mass index is modifiable, it is possible that weight loss induces a 'molecular switch' to alter the histologic characteristics of an endometrial cancer.

Comparative Tumor Microenvironment Analysis of Primary and Recurrent Ovarian Granulosa Cell Tumors.

Adult-type granulosa cell tumors (aGCT) are rare ovarian sex cord tumors with few effective treatments for recurrent disease. The objective of this study was to characterize the tumor microenvironment (TME) of primary and recurrent aGCTs and to identify correlates of disease recurrence. Total RNA sequencing (RNA-seq) was performed on 24 pathologically confirmed, cryopreserved aGCT samples, including 8 primary and 16 recurrent tumors. After read alignment and quality-control filtering, DESeq2 was used to identify differentially expressed genes (DEG) between primary and recurrent tumors. Functional enrichment pathway analysis and gene set enrichment analysis was performed using "clusterProfiler" and "GSVA" R packages. TME composition was investigated through the analysis and integration of multiple published RNA-seq deconvolution algorithms. TME analysis results were externally validated using data from independent previously published RNA-seq datasets. A total of 31 DEGs were identified between primary and recurrent aGCTs. These included genes with known function in hormone signaling such as LHCGR and INSL3 (more abundant in primary tumors) and CYP19A1 (more abundant in recurrent tumors). Gene set enrichment analysis revealed that primarily immune-related and hormone-regulated gene sets expression was increased in recurrent tumors. Integrative TME analysis demonstrated statistically significant depletion of cancer-associated fibroblasts in recurrent tumors. This finding was confirmed in multiple independent datasets. IMPLICATIONS: Recurrent aGCTs exhibit alterations in hormone pathway gene expression as well as decreased infiltration of cancer-associated fibroblasts, suggesting dual roles for hormonal signaling and TME remodeling underpinning disease relapse.

Mismatch Repair and Microsatellite Instability Testing for Immune Checkpoint Inhibitor Therapy: ASCO Endorsement of College of American Pathologists Guideline.

PURPOSE: The College of American Pathologists (CAP) has developed a guideline on testing for mismatch repair (MMR) and microsatellite instability (MSI) for patients considered for immune checkpoint inhibitor therapy. ASCO has a policy and set of procedures for endorsing clinical practice guidelines that have been developed by other professional organizations. METHODS: The CAP guideline was reviewed for developmental rigor by methodologists. An ASCO Endorsement Panel subsequently reviewed the content and the recommendations. RESULTS: The ASCO Endorsement Panel determined that the recommendations from the CAP guideline, published on August 3, 2022, are clear, thorough, and based on the most relevant scientific evidence. ASCO endorses Mismatch Repair and Microsatellite Instability Testing for Immune Checkpoint Inhibitor Therapy: Guideline From the College of American Pathologists in Collaboration With the Association for Molecular Pathology and Fight Colorectal Cancer. RECOMMENDATIONS: Within the guideline, MMR immunohistochemistry (IHC), MSI polymerase chain reaction, and MSI next-generation sequencing are all recommended testing options for colorectal cancer, MMR-IHC and MSI-polymerase chain reaction for gastroesophageal and small bowel cancer, and only MMR-IHC for endometrial cancer. No recommendation in favor of any testing method over another could be made for any other cancer. Tumor mutational burden was not recommended as a surrogate for DNA MMR deficiency. If MMR deficiency consistent with Lynch syndrome is detected, it should be communicated to the treating physician.Additional information is available at www.asco.org/molecular-testing-and-biomarkers-guidelines.

MIG-6 Is Critical for Progesterone Responsiveness in Human Complex Atypical Hyperplasia and Early-Stage Endometrial Cancer.

Women with complex atypical hyperplasia (CAH) or early-stage endometrioid endometrial cancer (EEC) are candidates for fertility preservation. The most common approach is progesterone (P4) therapy and deferral of hysterectomy until after completion of childbearing. However, P4 therapy response rates vary, and molecular mechanisms behind P4 resistance are poorly understood. One potential molecular cause of P4 resistance is a loss or attenuation of PGR expression. Mitogen-inducible gene 6 (MIG-6) is critical for P4 responsiveness. MIG-6 protein expression in the endometrial epithelial and stromal cells from women with CAH and EEC was significantly lower compared to women without CAH or EEC. The P4-responsive women (10/15) exhibited an increase of MIG-6 expression in epithelial and stromal cells compared to P4-resistant women (5/15). In addition, immunohistochemical analysis for PGR results showed that stromal PGR levels are significantly higher in P4-responsive women compared to P4-resistant women, whereas epithelial PGR expression was not different. A reverse correlation of MIG-6 and pAKT levels was observed in early-stage EEC patients. Studies strongly suggest that loss of MIG-6 and PGR and activation of pAKT lead to P4 resistance in CAH and EEC. These results will help to elucidate the molecular mechanism leading to P4 resistance in CAH and EEC.

Mechanisms of mutant ß-catenin in endometrial cancer progression.

Endometrial carcinoma (EC) is the most diagnosed gynecological malignancy in Western countries. Both incidence and mortality rates of EC have steadily risen in recent years. Despite generally favorable prognoses for patients with the endometrioid type of EC, a subset of patients has been identified with decreased progression-free survival. Patients in this group are distinguished from other endometrioid EC patients by the presence of exon 3 hotspot mutations in CTNNB1, the gene encoding for the ß-catenin protein. ß-catenin is an evolutionarily conserved protein with critical functions in both adherens junctions and Wnt-signaling. The exact mechanism by which exon 3 CTNNB1 mutations drive EC progression is not well understood. Further, the potential contribution of mutant ß-catenin to adherens junctions' integrity is not known. Additionally, the magnitude of worsened progression-free survival in patients with CTNNB1 mutations is context dependent, and therefore the importance of this subset of patients can be obscured by improper categorization. This review will examine the history and functions of ß-catenin, how these functions may change and drive EC progression in CTNNB1 mutant patients, and the importance of this patient group in the broader context of the disease.

DNA Damage Repair Classifier Defines Distinct Groups in Hepatocellular Carcinoma.

DNA repair pathways have been associated with variability in hepatocellular carcinoma (HCC) clinical outcomes, but the mechanism through which DNA repair varies as a function of liver regeneration and other HCC characteristics is poorly understood. We curated a panel of 199 genes representing 15 DNA repair pathways to identify DNA repair expression classes and evaluate their associations with liver features and clinicopathologic variables in The Cancer Genome Atlas (TCGA) HCC study. We identified two groups in HCC, defined by low or high expression across all DNA repair pathways. The low-repair group had lower grade and retained the expression of classical liver markers, whereas the high-repair group had more clinically aggressive features, increased p53 mutant-like gene expression, and high liver regenerative gene expression. These pronounced features overshadowed the variation in the low-repair subset, but when considered separately, the low-repair samples included three subgroups: L1, L2, and L3. L3 had high DNA repair expression with worse progression-free (HR 1.24, 95% CI 0.81-1.91) and overall (HR 1.63, 95% CI 0.98-2.71) survival. High-repair outcomes were also significantly worse compared with the L1 and L2 groups. HCCs vary in DNA repair expression, and a subset of tumors with high regeneration profoundly disrupts liver biology and poor prognosis.

Mismatch Repair and Microsatellite Instability Testing for Immune Checkpoint Inhibitor Therapy: Guideline From the College of American Pathologists in Collaboration With the Association for Molecular Pathology and Fight Colorectal Cancer.

CONTEXT.­: The US Food and Drug Administration (FDA) approved immune checkpoint inhibitor therapy for patients with advanced solid tumors that have DNA mismatch repair defects or high levels of microsatellite instability; however, the FDA provided no guidance on which specific clinical assays should be used to determine mismatch repair status. OBJECTIVE.­: To develop an evidence-based guideline to identify the optimal clinical laboratory test to identify defects in DNA mismatch repair in patients with solid tumor malignancies who are being considered for immune checkpoint inhibitor therapy. DESIGN.­: The College of American Pathologists convened an expert panel to perform a systematic review of the literature and develop recommendations. Using the National Academy of Medicine-endorsed Grading of Recommendations Assessment, Development and Evaluation approach, the recommendations were derived from available evidence, strength of that evidence, open comment feedback, and expert panel consensus. Mismatch repair immunohistochemistry, microsatellite instability derived from both polymerase chain reaction and next-generation sequencing, and tumor mutation burden derived from large panel next-generation sequencing were within scope. RESULTS.­: Six recommendations and 3 good practice statements were developed. More evidence and evidence of higher quality were identified for colorectal cancer and other cancers of the gastrointestinal (GI) tract than for cancers arising outside the GI tract. CONCLUSIONS.­: An optimal assay depends on cancer type. For most cancer types outside of the GI tract and the endometrium, there was insufficient published evidence to recommend a specific clinical assay. Absent published evidence, immunohistochemistry is an acceptable approach readily available in most clinical laboratories.

A phase II study of MK-2206, an AKT inhibitor, in uterine serous carcinoma.

Uterine serous carcinoma (USC) is an uncommon subtype of endometrial cancer with a poor prognosis. USCs have genomic alterations in the PI3K pathway. A prior phase II study of AKT inhibitor MK-2206 (an allosteric AKT inhibitor, primarily affecting AKT1 and AKT2) in endometrial cancers resulted in progression-free survival (PFS) of ≥6 months in five out of seven patients with USC. To further assess the activity of MK-2206 in USC, we designed a phase II, single-stage assessment of MK-2206 in patients with advanced or recurrent high-grade serous endometrial cancer, who had received up to two lines of prior therapy. MK-2206 (135 mg) was administered orally once per week, in continuous 28-day cycles. Fourteen patients received treatment. The most common treatment-related adverse events were diarrhea (36%), acneiform rash (36%), nausea (29%), fatigue (29%), and hyperglycemia (21%); most events were grade 1-2. One confirmed partial response was observed in a patient who was also alive and progression-free at 6 months. One additional patient was alive and progression-free at 6 months. The clinical benefit rate was 14.3% (95% CI: 1.8 to 42.8). Five patients had stable disease (35.7%) and seven had progressive disease (50%); one was unevaluable. Median PFS was 2 months (95% CI: 1.6 to 4.4) and median overall survival was 6.4 months (95% CI: 5.1 to not reached). In summary, MK-2206 had limited activity in USC, although a few patients achieved sustained progression-free intervals in this study and in the previously reported phase II trial of MK-2206. Further investigations are needed to identify features associated with response.

Adjuvant treatment in early-stage endometrial cancer: context-dependent impact of somatic CTNNB1 mutation on recurrence-free survival.

OBJECTIVE: The primary objective of this study was to determine whether women whose tumors harbor a somatic CTNNB1 mutation have longer recurrence-free survival if they receive traditional adjuvant therapy strategies compared with those who do not. METHODS: A retrospective, stage I endometrial cancer cohort from MD Anderson Cancer Center was assessed. Clinical and pathological characteristics and type of adjuvant therapy (cuff brachytherapy, pelvic radiation, chemotherapy) were obtained by review of medical records. CTNNB1 exon 3 sequencing was performed. Summary statistics were calculated, and recurrence-free survival was measured using the Kaplan-Meier product-limit estimator. RESULTS: The analysis included 253 patients, 245 with information regarding adjuvant therapy. Most patients had tumors of endometrioid histology (210/253, 83%) with superficial myometrial invasion (197/250, 79%) and no lymphatic/vascular space invasion (168/247, 68%). Tumor CTNNB1 mutations were present in 45 (18%) patients. Patients receiving adjuvant therapy were more likely to have higher-grade tumors, non-endometrioid histology, deep myometrial invasion, and lymphatic/vascular invasion. For patients with low-risk features not receiving adjuvant therapy, the presence of CTNNB1 mutation did not significantly impact recurrence-free survival (11.3 years wild-type vs 8.1 years mutant, p=0.65). The cohort was then limited to intermediate-risk tumors, defined as endometrioid histology of any grade with deep myometrial invasion and/or lymphatic/vascular space invasion. When recurrence-free survival was stratified by CTNNB1 mutation status and adjuvant therapy, patients with CTNNB1 mutations and no adjuvant therapy had the shortest recurrence-free survival at 1.6 years, followed by patients with CTNNB1 mutations who received adjuvant therapy (4.0 years), and wild-type CTNNB1 with and without adjuvant therapy (8.5 and 7.2 years, respectively) (comparison for all four groups, p=0.01). CONCLUSION: In patients with intermediate-risk endometrioid endometrial cancers, the use of adjuvant therapy was associated with an improvement in recurrence-free survival for patients with tumor mutations in CTNNB1.

Differences in the microbial profiles of early stage endometrial cancers between Black and White women.

OBJECTIVE: Black women suffer a higher mortality from endometrial cancer (EC) than White women. Potential biological causes for this disparity include a higher prevalence of obesity and more lethal histologic/molecular subtypes. We hypothesize that another biological factor driving this racial disparity could be the EC microbiome. METHODS: Banked tumor specimens of postmenopausal, Black and White women undergoing hysterectomy for early stage endometrioid EC were identified. The microbiota of the tumors were characterized by bacterial 16S rRNA sequencing. The microbial component of endometrioid ECs in The Cancer Genome Atlas (TCGA) database were assessed for comparison. RESULTS: 95 early stage ECs were evaluated: 23 Black (24%) and 72 White (76%). Microbial diversity was increased (p < 0.001), and Firmicutes, Cyanobacteria and OD1 phyla abundance was higher in tumors from Black versus White women (p < 0.001). Genus level abundance of Dietzia and Geobacillus were found to be lower in tumors of obese Black versus obese White women (p < 0.001). Analysis of early stage ECs in TCGA found that microbial diversity was higher in ECs from Black versus White women (p < 0.05). When comparing ECs from obese Black versus obese White women, 5 bacteria distributions were distinct, with higher abundance of Lactobacillus acidophilus in ECs from Black women being the most striking difference. Similarly in TCGA, Dietzia and Geobacillus were more common in ECs from White women compared to Black. CONCLUSION: Increased microbial diversity and the distinct microbial profiles between ECs of obese Black versus obese White women suggests that intra-tumoral bacteria may contribute to EC disparities and pathogenesis.

Inactivating Mutations of the IK Gene Weaken Ku80/Ku70-Mediated DNA Repair and Sensitize Endometrial Cancer to Chemotherapy.

IK is a mitotic factor that promotes cell cycle progression. Our previous investigation of 271 endometrial cancer (EC) samples from the Cancer Genome Atlas (TCGA) dataset showed IK somatic mutations were enriched in a cluster of patients with high-grade and high-stage cancers, and this group had longer survival. This study provides insight into how IK somatic mutations contribute to EC pathophysiology. We analyzed the somatic mutational landscape of IK gene in 547 EC patients using expanded TCGA dataset. Co-immunoprecipitation and mass spectrometry were used to identify protein interactions. In vitro and in vivo experiments were used to evaluate IK's role in EC. The patients with IK-inactivating mutations had longer survival during 10-year follow-up. Frameshift and stop-gain were common mutations and were associated with decreased IK expression. IK knockdown led to enrichment of G2/M phase cells, inactivation of DNA repair signaling mediated by heterodimerization of Ku80 and Ku70, and sensitization of EC cells to cisplatin treatment. IK/Ku80 mutations were accompanied by higher mutation rates and associated with significantly better overall survival. Inactivating mutations of IK gene and loss of IK protein expression were associated with weakened Ku80/Ku70-mediated DNA repair, increased mutation burden, and better response to chemotherapy in patients with EC.

Clinical and Functional Characterization of Atypical KRAS/NRAS Mutations in Metastatic Colorectal Cancer.

PURPOSE: Mutations in KRAS/NRAS (RAS) predict lack of anti-EGFR efficacy in metastatic colorectal cancer (mCRC). However, it is unclear if all RAS mutations have similar impact, and atypical mutations beyond those in standard guidelines exist. EXPERIMENTAL DESIGN: We reviewed 7 tissue and 1 cell-free DNA cohorts of 9,485 patients to characterize atypical RAS variants. Using an in vitro cell-based assay (functional annotation for cancer treatment), Ba/F3 transformation, and in vivo xenograft models of transduced isogenic clones, we assessed signaling changes across mutations. RESULTS: KRAS exon 2, extended RAS, and atypical RAS mutations were noted in 37.8%, 9.5%, and 1.2% of patients, respectively. Among atypical variants, KRAS L19F, Q22K, and D33E occurred at prevalence ≥0.1%, whereas no NRAS codon 117/146 and only one NRAS codon 59 mutation was noted. Atypical RAS mutations had worse overall survival than RAS/BRAF wild-type mCRC (HR, 2.90; 95% confidence interval, 1.24-6.80; P = 0.014). We functionally characterized 114 variants with the FACT assay. All KRAS exon 2 and extended RAS mutations appeared activating. Of 57 atypical RAS variants characterized, 18 (31.6%) had signaling below wild-type, 23 (40.4%) had signaling between wild-type and activating control, and 16 (28.1%) were hyperactive beyond the activating control. Ba/F3 transformation (17/18 variants) and xenograft model (7/8 variants) validation was highly concordant with FACT results, and activating atypical variants were those that occurred at highest prevalence in clinical cohorts. CONCLUSIONS: We provide best available evidence to guide treatment when atypical RAS variants are identified. KRAS L19F, Q22K, D33E, and T50I are more prevalent than many guideline-included RAS variants and functionally relevant.

Clinical and Financial Implications of Second-Opinion Surgical Pathology Review.

OBJECTIVES: Second-opinion pathology review identifies clinically significant diagnostic discrepancies for some patients. Discrepancy rates and laboratory-specific costs in a single health care system for patients referred from regional affiliates to a comprehensive cancer center ("main campus") have not been reported. METHODS: Main campus second-opinion pathology cases for 740 patients from eight affiliated hospitals during 2016 to 2018 were reviewed. Chart review was performed to identify changes in care due to pathology review. To assess costs of pathology interpretation, reimbursement rates for consultation Current Procedural Terminology billing codes were compared with codes that would have been used had the cases originated at the main campus. RESULTS: Diagnostic discrepancies were identified in 104 (14.1%) patients, 30 (4.1%) of which resulted in a change in care. In aggregate, reimbursement for affiliate cases was 65.6% of the reimbursement for the same cases had they originated at the main campus. High-volume organ systems with low relative consultation reimbursement included gynecologic, breast, and thoracic. CONCLUSIONS: Preventable diagnostic errors are reduced by pathology review for patients referred within a single health care system. Although the resulting changes in care potentially lead to overall cost savings, the financial value of referral pathology review could be improved.

Long-term Patterns of Excess Mortality among Endometrial Cancer Survivors.

BACKGROUND: We investigated excess mortality after endometrial cancer using conditional relative survival estimates and standardized mortality ratios (SMR). METHODS: Women diagnosed with endometrial cancer during 2000-2017 (N = 183,153) were identified in the Surveillance Epidemiology and End Results database. SMRs were calculated as observed deaths among endometrial cancer survivors over expected deaths among demographically similar women in the general U.S. POPULATION: Five-year relative survival was estimated at diagnosis and each additional year survived up to 12 years post-diagnosis, conditional on survival up to that year. RESULTS: For the full cohort, 5-year relative survival was 87.7%, 96.2%, and 97.1% at 1, 5, and 10 years post-diagnosis, respectively. Conditional 5-year relative survival first exceeded 95%, reflecting minimal excess mortality compared with the general population, at 4 years post-diagnosis overall. However, in subgroup analyses, conditional relative survival remained lower for Black women (vs. White) and for those with regional/distant stage disease (vs. localized) throughout the study period. The overall SMR for all-cause mortality decreased from 5.90 [95% confidence interval (CI), 5.81-5.99] in the first year after diagnosis to 1.16 (95% CI, 1.13-1.19) at 10+ years; SMRs were consistently higher for non-White women and for those with higher stage or grade disease. CONCLUSIONS: Overall, endometrial cancer survivors had only a small survival deficit beyond 4 years post-diagnosis. However, excess mortality was greater in magnitude and persisted longer into survivorship for Black women and for those with more advanced disease. IMPACT: Strategies to mitigate disparities in mortality after endometrial cancer will be needed as the number of survivors continues to increase.

Clinical significance of homologous recombination deficiency score testing in endometrial Cancer.

BACKGROUND: Homologous recombination deficiency (HRD) score is related to chemotherapy response in some cancers, but its role in endometrial cancer in not known. We determined frequency and clinical significance of alterations in the HR pathway in endometrial cancer. METHODS: 253 endometrioid endometrial adenocarcinoma (EEA) samples from two independent cohorts (discovery and replication) were tested for HRD score using the Myriad HRD assay, microsatellite instability (MSI) and tumor mutation burden (TMB) using a next generation sequencing assay. HRD scores were also generated on endometrial cancer cell lines and in vivo response to olaparib was assessed. RESULTS: ROC curves were employed to determine optimal cutoffs of HRD in relation to survival impact in endometrial cancer and a cutoff of HRD ≥ 4 was suggested for DFS using the discovery cohort. Patients from two independent cohorts with HRD score ≥ 4 trended toward worse survival as compared to those with HRD score < 4. Both cohorts were further separated into four groups according to molecular subtypes (TMB positive; MSI positive; HRD positive; all others). When grouped by molecular subtype, there was a significant difference between groups using an HRD ≥4 cutoff in the initial (p = 0.0024) and replication (p = 0.042) cohorts. The Hec1a model (HRD score = 19) was highly sensitive to olaparib in in vitro and in vivo experiments. CONCLUSIONS: High HRD score was associated with worse DFS in our patient cohort. These findings suggest that HRD score may have clinical utility in patients with advanced or recurrent endometrial cancer.

Loss of CD73 shifts transforming growth factor-ß1 (TGF-ß1) from tumor suppressor to promoter in endometrial cancer.

In many tumors, CD73 (NT5E), a rate-limiting enzyme in adenosine biosynthesis, is upregulated by TGF-ß and drives tumor progression. Conversely, CD73 is downregulated in endometrial carcinomas (EC) despite a TGF-ß-rich environment. Through gene expression analyses of normal endometrium samples of the uterine cancer TCGA data set and genetic and pharmacological studies, we discovered CD73 loss shifts TGF-ß1 from tumor suppressor to promoter in EC. TGF-ß1 upregulated CD73 and epithelial integrity in vivo in the normal endometrium and in vitro in early stage EC cells. With loss of CD73, TGF-ß1-mediated epithelial integrity was abrogated. EC cells developed TGF-ß1-mediated stress fibers and macromolecule permeability, migration, and invasion increased. In human tumors, CD73 is downregulated in deeply invasive stage I EC. Consistent with shifting TGF-ß1 activity, CD73 loss increased TGF-ß1-mediated canonical signaling and upregulated cyclin D1 (CCND1) and downregulated p21 expression. This shift was clinically relevant, as CD73Low/CCND1High expression associated with poor tumor differentiation, increased myometrial and lymphatic/vascular space invasion, and patient death. Further loss of CD73 in CD73Low expressing advanced stage EC cells increased TGF-ß-mediated stress fibers, signaling, and invasiveness, whereby adenosine A1 receptor agonist, CPA, dampened TGF-ß-mediated invasion. These data identify CD73 loss as essential for shifting TGF-ß activity in EC.

Factors Impacting Clinically Relevant RNA Fusion Assays Using Next-Generation Sequencing.

CONTEXT.­: RNA-based next-generation sequencing (NGS) assays are being used with increasing frequency for comprehensive molecular profiling of solid tumors. OBJECTIVE.­: To evaluate factors that might impact clinical assay performance. DESIGN.­: A 4-month retrospective review of cases analyzed by a targeted RNA-based NGS assay to detect fusions was performed. RNA extraction was performed from formalin-fixed, paraffin-embedded tissue sections and/or cytology smears of 767 cases, including 493 in-house and 274 outside referral cases. The types of samples included 422 core needle biopsy specimens (55%), 268 resection specimens (35%), and 77 cytology samples (10%). RESULTS.­: Successful NGS fusion testing was achieved in 697 specimens (90.9%) and correlated positively with RNA yield (P < .001) and negatively with specimen necrosis (P = .002), decalcification (P < .001), and paraffin block age of more than 2 years (P = .001). Of the 697 cases that were successfully sequenced, 50 (7.2%) had clinically relevant fusions. The testing success rates and fusion detection rates were similar between core needle biopsy and cytology samples. In contrast, RNA fusion testing was often less successful using resection specimens (P = .007). Testing success was independent of the tumor percentage in the specimen, given that at least 20% tumor cellularity was present. CONCLUSIONS.­: The success of RNA-based NGS testing is multifactorial and is influenced by RNA quality and quantity. Identification of preanalytical factors affecting RNA quality and yield can improve NGS testing success rates.