Pan-tumor survey of ROS1 fusions detected by next-generation RNA and whole transcriptome sequencing.

BACKGROUND: Two ROS1 tyrosine kinase inhibitors have been approved for ROS1 fusion positive (ROS1+) non-small cell lung cancer (NSCLC) tumors. We performed a pan-tumor analysis of the incidence of ROS1 fusions to assess if more ROS1+ patients who could benefit from ROS1 TKIs could be identified. METHODS: A retrospective analysis of ROS1 positive solid malignancies identified by targeted RNA sequencing and whole transcriptome sequencing of clinical tumor samples performed at Caris Life Science (Phoenix, AZ). RESULTS: A total of 259 ROS1+ solid malignancies were identified from approximately 175,350 tumors that underwent next-generation sequencing (12% from targeted RNA sequencing [Archer]; 88% from whole transcriptome sequencing). ROS1+ NSCLC constituted 78.8% of the ROS1+ solid malignancies, follow by glioblastoma (GBM) (6.9%), and breast cancer (2.7%). The frequency of ROS1 fusion was approximately 0.47% among NSCLC, 0.29% for GBM, 0.04% of breast cancer. The mean tumor mutation burden for all ROS1+ tumors was 4.8 mutations/megabase. The distribution of PD-L1 (22C3) expression among all ROS1+ malignancies were 0% (18.6%), 1%-49% (29.4%), and ≥ 50% (60.3%) [for NSCLC: 0% (17.8%); 1-49% (27.7%); ≥ 50% (53.9%). The most common genetic co-alterations of ROS1+ NSCLC were TP53 (29.1%), SETD2 (7.3%), ARIAD1A (6.3%), and U2AF1 (5.6%). CONCLUSIONS: ROS1+ NSCLC tumors constituted the majority of ROS1+ solid malignancies with four major fusion partners. Given that > 20% of ROS1+ solid tumors may benefit from ROS1 TKIs treatment, comprehensive genomic profiling should be performed on all solid tumors.

Eosinophilic vacuolated tumor (EVT) of kidney demonstrates sporadic TSC/MTOR mutations: next-generation sequencing multi-institutional study of 19 cases.

A distinct renal tumor has recently been described as "high-grade oncocytic renal tumor" and "sporadic renal cell carcinoma with eosinophilic and vacuolated cytoplasm". The Genitourinary Pathology Society (GUPS) consensus proposed a unifying name "eosinophilic vacuolated tumor" (EVT) for this emerging entity. In this multi-institutional study, we evaluated 19 EVTs, particularly their molecular features and mutation profile, using next-generation sequencing. All cases were sporadic and none of the patients had a tuberous sclerosis complex. There were 8 men and 11 women, with a mean age of 47 years (median 50; range 15-72 years). Average tumor size was 4.3 cm (median 3.8 cm; range 1.5-11.5 cm). All patients with available follow-up data (18/19) were alive and without evidence of disease recurrence or progression during the follow-up, ranging from 12 to 198 months (mean 56.3, median 41.5 months). The tumors were well circumscribed, but lacked a well-formed capsule, had nested to solid growth, focal tubular architecture, and showed ubiquitous, large intracytoplasmic vacuoles, round to oval nuclei, and prominent nucleoli. Immunohistochemically, cathepsin K, CD117, CD10, and antimitochondrial antigen were expressed in all cases. Other positive stains included: PAX8, AE1/AE3 and CK18. CK7 was typically restricted only to rare scattered cells. Vimentin, HMB45, melan-A, and TFE3 were negative in all cases. All tumors showed retained SDHB. All cases (19/19) showed non-overlapping mutations of the mTOR pathway genes: TSC1 (4), TSC2 (7), and MTOR (8); one case with MTOR mutation showed a coexistent RICTOR missense mutation. Low mutational rates were found in all samples (ranged from 0 to 6 mutations/Mbp). Microsatellite instability and copy number variations were not found in any of the 17 analyzable cases. EVT represents an emerging renal entity that shows a characteristic and readily identifiable morphology, consistent immunohistochemical profile, indolent behavior, and mutations in either TSC1, TSC2, or MTOR genes.

Poroid adnexal skin tumors with YAP1 fusions exhibit similar histopathologic features: A series of six YAP1-rearranged adnexal skin tumors.

BACKGROUND: Adnexal skin tumors are diagnostically challenging with few known molecular signatures. Recently, however, YAP1-MAML2 and YAP1-NUTM1 fusions were identified in poroid adnexal skin tumors. METHODS: Herein, we subjected eight poroid adnexal skin tumors (three poromas and five porocarcinomas) to fusion gene analysis by whole transcriptome sequencing and next-generation DNA sequencing analysis. RESULTS: YAP1 fusions were identified in six cases. YAP1-NUTM1 fusions were identified in two poromas and three porocarcinomas. A single case of porocarcinoma harbored a YAP1-MAML2 fusion. Two cases were negative for gene fusion. All cases that harbored YAP1-NUTM1 fusions showed nuclear protein in testis (NUT) expression by immunohistochemistry, with NUT being negative in the YAP1-MAML2-positive case. In this case series, we provide a detailed histopathologic description of six YAP1-fused poroid skin tumors, which we show harbor reproducible histopathologic features, to include broad, bulbous tumor tongues with admixtures of basaloid, poroid cells punctuated by squamatized cuticles and ductules, with uniform tumor nuclei featuring frequent grooves and pseudonuclear inclusions. CONCLUSIONS: Awareness of the characteristic histopathologic features of YAP1-fused poroid adnexal skin tumor is a step toward a more reproducible classification of adnexal skin tumors as well as a step toward targeted therapy for metastatic and/or unresectable examples of this poroid group of neoplasms.

Novel targetable biomarkers in clear cell carcinoma of the breast uncovered by molecular profiling: A study of nine cases.

We profiled nine pure clear cell carcinomas of the breast using massively parallel DNA and RNA sequencing (NGS), in situ hybridization (ISH), and immunohistochemistry (IHC). All cases were primary mammary clear cell carcinomas that were diagnosed in female patients (mean age: 53.4 years; range: 31-69 years). Based on our findings, we conclude that the majority of clear cell carcinomas are ER/PR positive and consequently amenable to anti-ER treatment modalities. A subset of clear cell carcinomas also harbored alterations in PIK3CA/PTEN/AKT pathway, particularly PTEN, indicating a potential benefit of PI3K/Akt/mTOR inhibitors. The status of I-O biomarkers in clear cell carcinomas indicates a limited therapeutic benefit of immune checkpoint inhibitors (against PD-1/PD-L1).

Molecular characterization of cancers with NTRK gene fusions.

Targeted inhibitors of neurotropic tyrosine kinases are highly effective in selected patients with gene fusions involving NTRK1, NTRK2, or NTRK3. These fusions are consistently detected in rare cancer types (e.g., secretory breast carcinoma and congenital infantile fibrosarcoma), but the occurrence of NTRK fusions in common cancers and their relationship to other therapy biomarkers are largely unexplored. Tissue samples from 11,502 patients were analyzed for 53 gene fusions and sequencing of 592 genes, along with an immunohistochemical evaluation of TrkA/B/C and PD-L1. Thirty-one cases (0.27% of the entire cohort) had NTRK fusions. The most common fusions were ETV6:NTRK3 (n = 10) and TPM3:NTRK1 (n = 6). Gliomas had the highest number of NTRK fusions (14/982, 1.4%), most commonly involving NTRK2 (n = 9). Seventeen non-glioma cases with NTRK fusions included carcinomas of the lungs, thyroid, breast, cervix, colon, nasal cavity, cancer of unknown primary and soft tissue sarcomas. Strong and uniform Trk expression detected with a pan-Trk immunohistochemistry characterized 7/8 NTRK1 fusion cases and 8/9 NTRK2 fusion cases, while NTRK3 fused cases were positive in 6/11 (55%) of cases. 29% of NTRK fusion cases had no other pathogenic genomic alteration. PD-L1 expression was observed in 23% of NTRK fused cases while high tumor DNA microsatellite instability was detected in two cases. We confirm the rarity of NTRK genes fusions outside the brain malignancies. NTRK inhibitors alone or combined with immune checkpoint inhibitors may be a therapeutic option for a substantial proportion of these patients. Strategies for detection of the NTRK fusion-driven cancers may include immunohistochemistry, but gene fusion detection remains the most reliable tool.

NUTM1-rearranged neoplasia: a multi-institution experience yields novel fusion partners and expands the histologic spectrum.

Poorly differentiated neoplasms lacking characteristic histopathologic features represent a significant challenge to the pathologist for diagnostic classification. Classically, NUT carcinoma (previously NUT midline carcinoma) is poorly differentiated but typically exhibits variable degrees of squamous differentiation. Diagnosis is genetically defined by NUTM1 rearrangement, usually with BRD4 as the fusion partner. In this multi-institutional next-generation sequencing and fluorescence in situ hybridization study, 26 new NUTM1-rearranged neoplasms are reported, including 20 NUT carcinomas, 4 sarcomas, and 2 tumors of an uncertain lineage. NUTM1 fusion partners were available in 24 of 26 cases. BRD4 was the fusion partner in 18/24 (75%) cases, NSD3 in 2/24 cases (8.3%), and BRD3 in 1/24 (4.2%) cases. Two novel fusion partners were identified: MGA in two sarcomas (myxoid spindle cell sarcoma and undifferentiated sarcoma) (2/24 cases 8.3%) and MXD4 in a round cell sarcoma in the cecum (1/24 cases 4.2%). Eleven cases tested for NUT immunoexpression were all positive, including the MGA and MXD4-rearranged tumors. Our results confirm that NUTM1 gene rearrangements are found outside the classic clinicopathological setting of NUT carcinoma. In addition, as novel fusion partners like MGA and MXD4 may not be susceptible to targeted therapy with bromodomain inhibitors, detecting the NUTM1 rearrangement may not be enough, and identifying the specific fusion partner may become necessary. Studies to elucidate the mechanism of tumorigenesis of novel fusion partners are needed.

TAF1 Variants Are Associated with Dysmorphic Features, Intellectual Disability, and Neurological Manifestations.

We describe an X-linked genetic syndrome associated with mutations in TAF1 and manifesting with global developmental delay, intellectual disability (ID), characteristic facial dysmorphology, generalized hypotonia, and variable neurologic features, all in male individuals. Simultaneous studies using diverse strategies led to the identification of nine families with overlapping clinical presentations and affected by de novo or maternally inherited single-nucleotide changes. Two additional families harboring large duplications involving TAF1 were also found to share phenotypic overlap with the probands harboring single-nucleotide changes, but they also demonstrated a severe neurodegeneration phenotype. Functional analysis with RNA-seq for one of the families suggested that the phenotype is associated with downregulation of a set of genes notably enriched with genes regulated by E-box proteins. In addition, knockdown and mutant studies of this gene in zebrafish have shown a quantifiable, albeit small, effect on a neuronal phenotype. Our results suggest that mutations in TAF1 play a critical role in the development of this X-linked ID syndrome.

Biochemical and cellular analysis of Ogden syndrome reveals downstream Nt-acetylation defects.

The X-linked lethal Ogden syndrome was the first reported human genetic disorder associated with a mutation in an N-terminal acetyltransferase (NAT) gene. The affected males harbor an Ser37Pro (S37P) mutation in the gene encoding Naa10, the catalytic subunit of NatA, the major human NAT involved in the co-translational acetylation of proteins. Structural models and molecular dynamics simulations of the human NatA and its S37P mutant highlight differences in regions involved in catalysis and at the interface between Naa10 and the auxiliary subunit hNaa15. Biochemical data further demonstrate a reduced catalytic capacity and an impaired interaction between hNaa10 S37P and Naa15 as well as Naa50 (NatE), another interactor of the NatA complex. N-Terminal acetylome analyses revealed a decreased acetylation of a subset of NatA and NatE substrates in Ogden syndrome cells, supporting the genetic findings and our hypothesis regarding reduced Nt-acetylation of a subset of NatA/NatE-type substrates as one etiology for Ogden syndrome. Furthermore, Ogden syndrome fibroblasts display abnormal cell migration and proliferation capacity, possibly linked to a perturbed retinoblastoma pathway. N-Terminal acetylation clearly plays a role in Ogden syndrome, thus revealing the in vivo importance of N-terminal acetylation in human physiology and disease.

Using VAAST to identify an X-linked disorder resulting in lethality in male infants due to N-terminal acetyltransferase deficiency.

We have identified two families with a previously undescribed lethal X-linked disorder of infancy; the disorder comprises a distinct combination of an aged appearance, craniofacial anomalies, hypotonia, global developmental delays, cryptorchidism, and cardiac arrhythmias. Using X chromosome exon sequencing and a recently developed probabilistic algorithm aimed at discovering disease-causing variants, we identified in one family a c.109T>C (p.Ser37Pro) variant in NAA10, a gene encoding the catalytic subunit of the major human N-terminal acetyltransferase (NAT). A parallel effort on a second unrelated family converged on the same variant. The absence of this variant in controls, the amino acid conservation of this region of the protein, the predicted disruptive change, and the co-occurrence in two unrelated families with the same rare disorder suggest that this is the pathogenic mutation. We confirmed this by demonstrating a significantly impaired biochemical activity of the mutant hNaa10p, and from this we conclude that a reduction in acetylation by hNaa10p causes this disease. Here we provide evidence of a human genetic disorder resulting from direct impairment of N-terminal acetylation, one of the most common protein modifications in humans.

The frequency of previously undetectable deletions involving 3' Exons of the PMS2 gene.

Lynch syndrome is characterized by mutations in one of four mismatch repair genes, MLH1, MSH2, MSH6, or PMS2. Clinical mutation analysis of these genes includes sequencing of exonic regions and deletion/duplication analysis. However, detection of deletions and duplications in PMS2 has previously been confined to Exons 1-11 due to gene conversion between PMS2 and the pseudogene PMS2CL in the remaining 3' exons (Exons 12-15). We have recently described an MLPA-based method that permits detection of deletions of PMS2 Exons 12-15; however, the frequency of such deletions has not yet been determined. To address this question, we tested for 3' deletions in 58 samples that were reported to be negative for PMS2 mutations using previously available methods. All samples were from individuals whose tumors exhibited loss of PMS2 immunohistochemical staining without concomitant loss of MLH1 immunostaining. We identified seven samples in this cohort with deletions in the 3' region of PMS2, including three previously reported samples with deletions of Exons 13-15 (two samples) and Exons 14-15. Also detected were deletions of Exons 12-15, Exon 13, and Exon 14 (two samples). Breakpoint analysis of the intragenic deletions suggests they occurred through Alu-mediated recombination. Our results indicate that ∼12% of samples suspected of harboring a PMS2 mutation based on immunohistochemical staining, for which mutations have not yet been identified, would benefit from testing using the new methodology.

Exploring Co-occurring POLE Exonuclease and Non-exonuclease Domain Mutations and Their Impact on Tumor Mutagenicity.

POLE driver mutations in the exonuclease domain (ExoD driver) are prevalent in several cancers, including colorectal cancer and endometrial cancer, leading to dramatically ultra-high tumor mutation burden (TMB). To understand whether POLE mutations that are not classified as drivers (POLE Variant) contribute to mutagenesis, we assessed TMB in 447 POLE-mutated colorectal cancers, endometrial cancers, and ovarian cancers classified as TMB-high ≥10 mutations/Mb (mut/Mb) or TMB-low <10 mut/Mb. TMB was significantly highest in tumors with "POLE ExoD driver plus POLE Variant" (colorectal cancer and endometrial cancer, P < 0.001; ovarian cancer, P < 0.05). TMB increased with additional POLE variants (P < 0.001), but plateaued at 2, suggesting an association between the presence of these variants and TMB. Integrated analysis of AlphaFold2 POLE models and quantitative stability estimates predicted the impact of multiple POLE variants on POLE functionality. The prevalence of immunogenic neoepitopes was notably higher in the "POLE ExoD driver plus POLE Variant" tumors. Overall, this study reveals a novel correlation between POLE variants in POLE ExoD-driven tumors, and ultra-high TMB. Currently, only select pathogenic ExoD mutations with a reliable association with ultra-high TMB inform clinical practice. Thus, these findings are hypothesis-generating, require functional validation, and could potentially inform tumor classification, treatment responses, and clinical outcomes. SIGNIFICANCE: Somatic POLE ExoD driver mutations cause proofreading deficiency that induces high TMB. This study suggests a novel modifier role for POLE variants in POLE ExoD-driven tumors, associated with ultra-high TMB. These data, in addition to future functional studies, may inform tumor classification, therapeutic response, and patient outcomes.

Constitutional mismatch repair deficiency presenting in childhood as three simultaneous malignancies.

A 13-year-old child presented with three simultaneous malignancies: glioblastoma multiforme, Burkitt lymphoma, and colonic adenocarcinoma. She was treated for her diseases without success and died 8 months after presentation. Genetic analysis revealed a homozygous mutation in the PMS2 gene, consistent with constitutional mismatch repair deficiency. Her siblings and parents were screened: three of four siblings and both parents were heterozygous for this mutation; the fourth sibling did not have the mutation.

Pan-tumor survey of RET fusions as detected by next-generation RNA sequencing identified RET fusion positive colorectal carcinoma as a unique molecular subset.

BACKGROUND: RET fusions are driver alterations in cancer and are most commonly found in non-small cell lung cancer and well-differentiated thyroid cancer. However, RET fusion have been reported in other solid tumors. MATERIAL AND METHODS: A retrospective analysis of RET+ solid malignancies identified by targeted RNA sequencing and whole transcriptome sequencing of clinical tumor samples performed at Caris Life Science (Phoenix, AZ). RESULTS: As of March 22, 2022, a total of 378 RET+ solid malignancies were identified in 15 different tumor types and carcinoma of unknown primary (CUP) that underwent next-generation RNA sequencing. RET+ NSCLC and RET+ thyroid cancer constituted 66.9% and 11.1% of the RET+ solid malignancies, respectively. RET+ colorectal adenocarcinoma and RET+ breast adenocarcinoma constituted 10.1% and 2.6%, respectively. The estimated frequency of RET fusions within specific tumor types were NSCLC 0.7%, thyroid cancer 3.1%, colorectal cancer 0.2% and breast cancer 0.1%. KIF5B (46.8%) was the most common fusion partner followed by CCDC6 (28.3%) and NCOA4 (13.8%) in RET+ solid tumors. KIF5B-RET was the dominant fusion variant in RET+ NSCLC, NCOA4-RET was the dominant variant in RET+ colorectal carcinoma, and CCDC6-RET was the dominant variant in thyroid cancer. The most common single gene alterations in RET+ tumors were TP53 (34.8%), RASA1 (14.3%) and ARIAD1A (11.6%). RET+ CRC had a high median TMB of 20.0 and were commonly MSI-H. CONCLUSIONS: RET fusions were identified in multiple tumor types. With a higher median TMB and commonly MSI-H, RET fusion positive CRC may be a unique molecular subset of CRC.

Sickle cell disease resulting from uniparental disomy in a child who inherited sickle cell trait.

Sickle cell disease (SCD) is a classic example of a disorder with recessive Mendelian inheritance, in which each parent contributes one mutant allele to an affected offspring. However, there are exceptions to that rule. We describe here the first reported case of conversion of inherited sickle cell trait to SCD by uniparental disomy (UPD) resulting in mosaicism for SS and AS erythrocytes. A 14-year-old boy presented with splenomegaly and hemolysis. Although his father has sickle cell trait, his mother has no abnormal hemoglobin (Hb). DNA sequencing, performed to rule out Hb S/ß-thalassemia, detected homozygous Hb SS. Further studies revealed mosaic UPD of the ß-globin locus, more SS erythroid progenitors than AS, but a reverse ratio of erythrocytes resulting from the survival advantage of AS erythrocytes. This report exemplifies non-Mendelian genetics wherein a patient who inherited sickle cell trait has mild SCD resulting from postzygotic mitotic recombination leading to UPD.

Avoidance of pseudogene interference in the detection of 3' deletions in PMS2.

Lynch syndrome is characterized by mutations in the mismatch repair genes MLH1, MSH2, MSH6, and PMS2. In PMS2, detection of mutations is confounded by numerous pseudogenes. Detection of 3' deletions is particularly complicated by the pseudogene PMS2CL, which has strong similarity to PMS2 exons 9 and 11-15, due to extensive gene conversion. A newly designed multiplex ligation-dependent probe amplification (MLPA) kit incorporates probes for variants found in both PMS2 and PMS2CL. This provides detection of deletions, but does not allow localization of deletions to the gene or pseudogene. To address this, we have developed a methodology incorporating reference samples with known copy numbers of variants, and paired MLPA results with sequencing of PMS2 and PMS2CL. We tested a subset of clinically indicated samples for which mutations were either unidentified or not fully characterized using existing methods. We identified eight unrelated patients with deletions encompassing exons 9-15, 11-15, 13-15, 14-15, and 15. By incorporating specific, characterized reference samples and sequencing the gene and pseudogene it is possible to identify deletions in this region of PMS2 and provide clinically relevant results. This methodology represents a significant advance in the diagnosis of patients with Lynch syndrome caused by PMS2 mutations.

Molecular Profiling Reveals Limited Targetable Biomarkers in Neuroendocrine Carcinoma of the Cervix.

Neuroendocrine carcinoma of the cervix (NEC) is a rare and highly aggressive cervical malignancy. Given that no targeted therapy has been approved specifically to NEC, we investigated the presence of novel, potentially targetable biomarkers in a large cohort of NEC. Sixty-two NEC were molecularly profiled for biomarkers of targeted therapies including antibody-drug conjugates [delta-like canonical notch ligand 3 (DLL3), a trophoblast cell surface antigen 2 (TROP-2), and folate receptor 1 (FOLR1)], NTRK1-3 gene fusions, and immune checkpoint inhibitors [programmed death-ligand 1 (PD-L1), tumor mutational burden, and microsatellite instability] using immunohistochemistry and DNA/RNA next-generation sequencing assays. A cohort of squamous cell carcinomas of the cervix (n=599) was used for comparison for immune-oncology biomarkers. DLL3 expression was observed in 81% of the cases. DLL3 expression was inversely correlated with commonly observed pathogenic mutations in PIK3CA (17%) (P=0.018) and PTEN (10%) (P=0.006). Other more frequently seen pathogenic mutations (TP53 17%, KRAS 11%, and CTNNB1 5%) were not associated with DLL3 expression. TROP-2 expression was detected in only 1 case and no case expressed FOLR1. Although NTRK protein expression was observed in 21% of the cases, none of these had an NTRK gene fusion. PD-L1 expression (10%) and high tumor mutational burden (3%) were significantly less frequent in NEC compared with the squamous cell carcinoma cohort (79% and 11%, respectively). None of the NEC exhibited high microsatellite instability status. Despite frequent DLL3 expression in NEC, a potential therapeutic benefit of DLL3-targeted drugs remains uncertain given the recent failure of the Rova-T therapeutic trial in small cell lung carcinomas. Small cohorts of NEC enriched in PIK3CA/PTEN/AKT and programmed cell death protein 1/PD-L1 alterations indicate therapeutic roles for their respective inhibitors.

TERT Gene Fusions Characterize a Subset of Metastatic Leydig Cell Tumors.

OBJECTIVE: Metastatic Leydig cell tumors (LCT) are rare, difficult-to-treat malignancies without known underlying molecular-genetic events. An index case of metastatic LCT showed an LDLR-TERT gene fusion upon routine genetic profiling for detection of therapeutic targets, which was then followed by an investigation into a cohort of additional LCTs. PATIENTS AND METHODS: Twenty-nine LCT (27 male and 2 female patients) were profiled using next-generation sequencing and immunohistochemistry. RESULTS: TERT gene fusions were detected only in testicular metastatic LCTs, in 3 of 7 successfully analyzed cases (RMST:TERT, LDLR:TERT, and B4GALT5:TERT). TOP1 and CCND3 amplifications were identified in the case with a B4GALT5:TERT fusion. A TP53 mutation was detected in 1 metastatic tumor without a TERT fusion. Five primary (4 testicular and 1 ovarian) LCTs showed multiple gene amplifications, without a consistent pattern. A single metastatic ovarian LCT showed BAP1 mutation and copy number amplifications affecting the NPM1, PCM1, and SS18 genes. At the protein level, 4 of 7 metastatic and 6 of 10 primary testicular LCTs overexpressed Topo1. Androgen receptor was overexpressed in 10 of 13 primary testicular tumors and 2 of 5 metastatic testicular LCTs (without detectable ARv7 messenger RNA or ARv7 protein). Only 1 metastatic testicular LCT exhibited a high tumor mutational burden; all tested cases were microsatellite instability stable and did not express programmed cell death ligand 1. CONCLUSIONS: Our study for the first time identified TERT gene fusions as a main genetic alteration and a potential therapeutic target in metastatic LCTs. Topo1 and androgen receptor may guide decisions on chemotherapy and/or hormone therapy for selected individual patients.