Next-generation sequencing has diagnostic utility in challenging small/flat urothelial lesions.

Small/flat urothelial lesions are challenging and currently available ancillary immunohistochemistry testing often cannot reliably distinguish between reactive lesions and urothelial carcinoma (UCa). UCa has a characteristic molecular profile, but small/flat urothelial lesions are typically considered too small to perform next generation sequencing (NGS). Herein, we present our institution's experience with utilizing comprehensive DNA-based NGS to evaluate small/flat urothelial lesions (n = 13 cases). NGS was ordered on 7/13 small/flat urothelial lesions initially diagnosed as urothelial atypia, ordered by the pathologist to aid in further diagnosis; the remaining 6/13 cases were diagnosed as urothelial carcinoma in situ (uCIS), ordered by a treating oncologist. The test was considered as adding value if it yielded pathogenic or likely pathogenic alterations previously associated with urothelial carcinoma in the literature. Macroscopic dissection was determined necessary in all cases and obtained either by scraping (7), punch biopsy (5) or scooping (1) of paraffin tissue blocks. In 4/13 cases, tumor content was considered low (<25%); in 2/13 cases, DNA quantity yield was considered below optimal (<250 ng); all cases met required DNA quantity for testing (>50 ng). Mean target coverage ranged: 498 to 985 (optimal >500 reads). NGS testing identified mutations compatible with urothelial carcinoma in all 7 cases initially diagnosed as atypical; and in one case, the tumor recurred as a lung metastasis. All 6 uCIS had NGS testing results concordant with UCa. In conclusion, despite small sample quantity with low tumor content and DNA concentration yield, NGS testing with appropriate methodology can be considered in the setting of small/flat urothelial lesions to aid in diagnosis or per oncologist request and yield interpretable results.

Variant Classification Concordance using the ACMG-AMP Variant Interpretation Guidelines across Nine Genomic Implementation Research Studies.

Harmonization of variant pathogenicity classification across laboratories is important for advancing clinical genomics. The two CLIA-accredited Electronic Medical Record and Genomics Network sequencing centers and the six CLIA-accredited laboratories and one research laboratory performing genome or exome sequencing in the Clinical Sequencing Evidence-Generating Research Consortium collaborated to explore current sources of discordance in classification. Eight laboratories each submitted 20 classified variants in the ACMG secondary finding v.2.0 genes. After removing duplicates, each of the 158 variants was annotated and independently classified by two additional laboratories using the ACMG-AMP guidelines. Overall concordance across three laboratories was assessed and discordant variants were reviewed via teleconference and email. The submitted variant set included 28 P/LP variants, 96 VUS, and 34 LB/B variants, mostly in cancer (40%) and cardiac (27%) risk genes. Eighty-six (54%) variants reached complete five-category (i.e., P, LP, VUS, LB, B) concordance, and 17 (11%) had a discordance that could affect clinical recommendations (P/LP versus VUS/LB/B). 21% and 63% of variants submitted as P and LP, respectively, were discordant with VUS. Of the 54 originally discordant variants that underwent further review, 32 reached agreement, for a post-review concordance rate of 84% (118/140 variants). This project provides an updated estimate of variant concordance, identifies considerations for LP classified variants, and highlights ongoing sources of discordance. Continued and increased sharing of variant classifications and evidence across laboratories, and the ongoing work of ClinGen to provide general as well as gene- and disease-specific guidance, will lead to continued increases in concordance.

Exome Sequencing for Prenatal Diagnosis in Nonimmune Hydrops Fetalis.

BACKGROUND: The cause of most fetal anomalies is not determined prenatally. Exome sequencing has transformed genetic diagnosis after birth, but its usefulness for prenatal diagnosis is still emerging. Nonimmune hydrops fetalis (NIHF), a fetal abnormality that is often lethal, has numerous genetic causes; the extent to which exome sequencing can aid in its diagnosis is unclear. METHODS: We evaluated a series of 127 consecutive unexplained cases of NIHF that were defined by the presence of fetal ascites, pleural or pericardial effusions, skin edema, cystic hygroma, increased nuchal translucency, or a combination of these conditions. The primary outcome was the diagnostic yield of exome sequencing for detecting genetic variants that were classified as either pathogenic or likely pathogenic according to the criteria of the American College of Medical Genetics and Genomics. Secondary outcomes were the percentage of cases associated with specific genetic disorders and the proportion of variants that were inherited. RESULTS: In 37 of the 127 cases (29%), we identified diagnostic genetic variants, including those for disorders affecting the RAS-MAPK cell-signaling pathway (known as RASopathies) (30% of the genetic diagnoses); inborn errors of metabolism and musculoskeletal disorders (11% each); lymphatic, neurodevelopmental, cardiovascular, and hematologic disorders (8% each); and others. Prognoses ranged from a relatively mild outcome to death during the perinatal period. Overall, 68% of the cases (25 of 37) with diagnostic variants were autosomal dominant (of which 12% were inherited and 88% were de novo), 27% (10 of 37) were autosomal recessive (of which 95% were inherited and 5% were de novo), 1 was inherited X-linked recessive, and 1 was of uncertain inheritance. We identified potentially diagnostic variants in an additional 12 cases. CONCLUSIONS: In this large case series of 127 fetuses with unexplained NIHF, we identified a diagnostic genetic variant in approximately one third of the cases. (Funded by the UCSF Center for Maternal-Fetal Precision Medicine and others; ClinicalTrials.gov number, NCT03412760.).

EML4::ALK fusions in complex lymphatic malformations.

Gorham-Stout disease (GSD) and generalized lymphatic anomaly (GLA) are subtypes of complex lymphatic malformations (CLMs) with osseous involvement that cause significant complications, including pain and pathologic fractures. As with other vascular anomalies, somatic mosaic mutations in oncogenes are often present, and the mTOR inhibitor sirolimus alleviates symptoms in some, but not all, patients. We describe two patients, one with GSD and one with GLA, found to have EML4::ALK fusions. This report of a targetable, oncogenic fusion in vascular malformations expands our understanding of the genetic basis for CLMs and suggests additional targeted therapies could be effective.

Further description of two patients with biallelic variants in NADSYN1 in association with cardiac and vertebral anomalies.

Congenital nicotinamide adenine dinucleotide (NAD) deficiency disorders are associated with pathogenic variants in the genes NADSYN1, HAAO, and KYNU. These disorders overlap with the anomalies present in vertebral, anal, cardiac, tracheoesophageal, radial and renal, and limb anomalies (VATER/VACTERL) association and often result in premature death. Children who survive typically have developmental delays or intellectual disability. Here, we describe two patients with compound heterozygous variants in NADSYN1 who presented with cardiac and vertebral defects overlapping with the VATER/VACTERL association, although the patients did not satisfy criteria for the diagnosis of VATER/VACTERL due to their lack of limb anomalies and significant renal anomalies. One patient survived into childhood with developmental delays and may represent an expansion of the survival data for NADSYN1-associated NAD deficiency disorders. Interestingly, one patient had hypoplastic left heart syndrome (HLHS) and one had an aortic coarctation and transverse hypoplasia of the aortic arch, suggesting that NADSYN1 sequencing should be performed in children presenting with congenital anomalies related to VATER/VACTERL association and with HLHS and aortic arch abnormalities.

De novo heterozygous variants in SLC30A7 are a candidate cause for Joubert syndrome.

Joubert syndrome (JS), a well-established ciliopathy, is characterized by the distinctive molar tooth sign on brain MRI, ataxia, and neurodevelopmental features. Other manifestations can include polydactyly, accessory frenula, renal, or liver disease. Here, we report individuals meeting criteria for JS with de novo heterozygous variants in SLC30A7 (Chr1p21.2). The first individual is a female with history of unilateral postaxial polydactyly, classic molar tooth sign on MRI, macrocephaly, ataxia, ocular motor apraxia, neurodevelopmental delay, and precocious puberty. Exome sequencing detected a de novo heterozygous missense variant in SLC30A7: NM_133496.5: c.407 T > C, (p.Val136Ala). The second individual had bilateral postaxial polydactyly, molar tooth sign, macrocephaly, developmental delay, and an extra oral frenulum. A de novo deletion-insertion variant in SLC30A7, c.490_491delinsAG (p.His164Ser) was found. Both de novo variants affect highly conserved residues. Variants were not identified in known Joubert genes for either case. SLC30A7 has not yet been associated with a human phenotype. The SLC30 family of zinc transporters, like SLC30A7, permit cellular efflux of zinc, and although it is expressed in the brain its functions remain unknown. Published data from proteomic studies support SLC30A7 interaction with TCTN3, another protein associated with JS. The potential involvement of such genes in primary cilia suggest a role in Sonic Hedgehog signaling. SLC30A7 is a candidate JS-associated gene. Future work could be directed toward further characterization of SLC30A7 variants and understanding its function.

The utility of pathologic examination and comprehensive phenotyping for accurate diagnosis with perinatal exome sequencing.

OBJECTIVE: Exome sequencing (ES) offers the ability to assess for variants in thousands of genes and is particularly useful in the setting of fetal anomalies. However, the ES pipeline relies on a thorough understanding of an individual patient's phenotype, which may be limited in the prenatal setting. Additional pathology evaluations in the pre- and postnatal settings can add phenotypic details important for clearly establishing and characterizing a diagnosis. METHODS: This is a case series of prenatal ES performed at our institution in which pathology evaluations, including autopsy, dysmorphology examination, histology, and peripheral blood smear, augmented the understanding of the fetal phenotype. ES was performed at our institution and a multidisciplinary panel reviewed and classified the variants for each case. RESULTS: We present four cases wherein pathology evaluations were beneficial for supporting a perinatal diagnosis identified with ES. In each of these cases, pathology findings provided additional data to support a more complete understanding of the relationship between the perinatal phenotype and variants identified with ES. CONCLUSION: These cases highlight challenges of perinatal ES related to incomplete prenatal phenotyping, demonstrate the utility of pathology evaluations to support diagnoses identified with ES, and further characterize the disease manifestations of specific genetic variants.

Preference for secondary findings in prenatal and pediatric exome sequencing.

OBJECTIVE: We aimed to determine the frequency of accepting secondary findings in families undergoing exome sequencing in prenatal and pediatric settings. METHODS: This was a secondary analysis of prospectively enrolled patients undergoing trio exome sequencing for congenital anomalies or developmental disorders in prenatal and pediatric settings, in which families were offered receiving secondary findings (initially assessed in the proband and, if identified, then in the parents). The primary outcome was frequency of accepting secondary findings. Secondary outcomes included frequency of acceptance in prenatal versus pediatric settings, and sociodemographic differences between those who accepted versus declined secondary findings. RESULTS: There were 682 families included in the cohort (289 prenatal and 393 pediatric). Overall, 84% (576/682) of families accepted secondary findings: 86.2% (249/289) of families undergoing prenatal versus 83.2% (327/393) pediatric (p = 0.30) testing. Secondary findings were identified in 2.6% (15/576) of cases, with no difference between prenatal and pediatric settings. There were no differences in sociodemographics between families that accepted versus declined secondary findings. CONCLUSION: The majority of families undergoing exome sequencing accepted secondary findings; this did not differ in prenatal versus pediatric settings. This highlights the need for guidance surrounding the offer of secondary findings in the prenatal setting.

De novo variants in SNAP25 cause an early-onset developmental and epileptic encephalopathy.

PURPOSE: This study aims to provide a comprehensive description of the phenotypic and genotypic spectrum of SNAP25 developmental and epileptic encephalopathy (SNAP25-DEE) by reviewing newly identified and previously reported individuals. METHODS: Individuals harboring heterozygous missense or loss-of-function variants in SNAP25 were assembled through collaboration with international colleagues, matchmaking platforms, and literature review. For each individual, detailed phenotyping, classification, and structural modeling of the identified variant were performed. RESULTS: The cohort comprises 23 individuals with pathogenic or likely pathogenic de novo variants in SNAP25. Intellectual disability and early-onset epilepsy were identified as the core symptoms of SNAP25-DEE, with recurrent findings of movement disorders, cerebral visual impairment, and brain atrophy. Structural modeling for all variants predicted possible functional defects concerning SNAP25 or impaired interaction with other components of the SNARE complex. CONCLUSION: We provide a comprehensive description of SNAP25-DEE with intellectual disability and early-onset epilepsy mostly occurring before the age of two years. These core symptoms and additional recurrent phenotypes show an overlap to genes encoding other components or associated proteins of the SNARE complex such as STX1B, STXBP1, or VAMP2. Thus, these findings advance the concept of a group of neurodevelopmental disorders that may be termed "SNAREopathies."

Next-Generation Sequencing of Retinoblastoma Identifies Pathogenic Alterations beyond RB1 Inactivation That Correlate with Aggressive Histopathologic Features.

PURPOSE: To determine the usefulness of a comprehensive, targeted-capture next-generation sequencing (NGS) assay for the clinical management of children undergoing enucleation for retinoblastoma. DESIGN: Cohort study. PARTICIPANTS: Thirty-two children with retinoblastoma. METHODS: We performed targeted NGS using the UCSF500 Cancer Panel (University of California, San Francisco, San Francisco, CA) on formalin-fixed, paraffin-embedded tumor tissue along with constitutional DNA isolated from peripheral blood, buccal swab, or uninvolved optic nerve. Peripheral blood samples were also sent to a commercial laboratory for germline RB1 mutation testing. MAIN OUTCOME MEASURES: Presence or absence of germline RB1 mutation or deletion, tumor genetic profile, and association of genetic alterations with clinicopathologic features. RESULTS: Germline mutation or deletion of the RB1 gene was identified in all children with bilateral retinoblastoma (n = 12), and these NGS results were 100% concordant with commercial germline RB1 mutation analysis. In tumor tissue tested with NGS, biallelic inactivation of RB1 was identified in 28 tumors and focal MYCN amplification was identified in 4 tumors (2 with wild-type RB1 and 2 with biallelic RB1 inactivation). Additional likely pathogenic alterations beyond RB1 were identified in 13 tumors (41%), several of which have not been reported previously in retinoblastoma. These included focal amplifications of MDM4 and RAF1, as well as damaging mutations involving BCOR, ARID1A, MGA, FAT1, and ATRX. The presence of additional likely pathogenetic mutations beyond RB1 inactivation was associated with aggressive histopathologic features, including higher histologic grade and anaplasia, and also with both unilateral and sporadic disease. CONCLUSIONS: Comprehensive NGS analysis reliably detects relevant mutations, amplifications, and chromosomal copy number changes in retinoblastoma. The presence of genetic alterations beyond RB1 inactivation correlates with aggressive histopathologic features.

Pediatric bithalamic gliomas have a distinct epigenetic signature and frequent EGFR exon 20 insertions resulting in potential sensitivity to targeted kinase inhibition.

Brain tumors are the most common solid tumors of childhood, and the genetic drivers and optimal therapeutic strategies for many of the different subtypes remain unknown. Here, we identify that bithalamic gliomas harbor frequent mutations in the EGFR oncogene, only rare histone H3 mutation (in contrast to their unilateral counterparts), and a distinct genome-wide DNA methylation profile compared to all other glioma subtypes studied to date. These EGFR mutations are either small in-frame insertions within exon 20 (intracellular tyrosine kinase domain) or missense mutations within exon 7 (extracellular ligand-binding domain) that occur in the absence of accompanying gene amplification. We find these EGFR mutations are oncogenic in primary astrocyte models and confer sensitivity to specific tyrosine kinase inhibitors dependent on location within the kinase domain or extracellular domain. We initiated treatment with targeted kinase inhibitors in four children whose tumors harbor EGFR mutations with encouraging results. This study identifies a promising genomically-tailored therapeutic strategy for bithalamic gliomas, a lethal and genetically distinct brain tumor of childhood.

The expanding spectrum of NFIB-associated phenotypes in a diverse patient population-A report of two new patients.

NFIB (Nuclear Factor I B) haploinsufficiency has recently been identified as a cause of intellectual disability and macrocephaly. Here we describe two patients with pathogenic variants in NFIB. The first is a 6-year-old Latino male with developmental delays, mild hypotonia, facial anomalies, and brain magnetic resonance imaging findings comprising mild thinning of the corpus callosum, with more marked thinning of the splenium and blunting of the rostrum and cavum septum pellucidum. Exome sequencing identified a previously described de novo variant in NFIB, c.265C>T, predicting p.Arg89Ter. The second is a 5-year-old Latino male with developmental delays, hypotonia, dysmorphic features, a preauricular tag and pit, a small ventricular septal defect, and brain magnetic resonance imaging findings including a dysmorphic corpus callosum and a small posterior fossa. A single nucleotide polymorphism microarray identified a 92 kb interstitial deletion at 9p23 including several exons of NFIB and no other known genes. Our two patients add to the knowledge of this rare condition through our addition of new brain MRI findings and dysmorphic features. Additionally, these are the first known Latino patients to be described with NFIB haploinsufficiency, expanding our understanding of the associated facial features in diverse populations. Further data are needed to determine genotype-phenotype relationships for NFIB.

A novel truncating variant in ring finger protein 113A (RNF113A) confirms the association of this gene with X-linked trichothiodystrophy.

We describe an 11-year old boy with severe global developmental delays, failure to thrive and growth retardation, refractory seizures with recurrent status epilepticus, hypogammaglobulinemia, hypergonadotropic hypogonadism, and duodenal strictures. He had facial and skin findings compatible with trichothiodystrophy, including sparse and brittle hair, thin eyebrows, and dry skin. Exome sequencing showed a hemizygous, truncating variant in RNF113A, c.903_910delGCAGACCA, predicting p.(Gln302fs*12), that was inherited from his mother. Although his clinical features overlap closely with features described in the two previously reported male first cousins with RNF113A loss of function mutations, the duodenal strictures seen in this patient have not been reported. Interestingly, the patient's mother had short stature and 100% skewed X-inactivation as seen in other obligate female carriers. A second male with developmental delays, microcephaly, seizures, ambiguous genitalia, and facial anomalies that included sparse and brittle hair, thin eyebrows and dry skin was recently reported to have c.897_898delTG, predicting p.(Cys299*) in RNF113A and we provide additional clinical details for this patient. This report further supports deleterious variants in RNF113A as a cause of a novel trichothiodystrophy syndrome.

Molecular Insights in Transmission of Cancer From an Organ Donor to Four Transplant Recipients.

Organ donors are systematically screened for infection, whereas screening for malignancy is less rigorous. The true incidence of donor-transmitted malignancies is unknown due to a lack of universal tumor testing in the posttransplant setting. Donor-transmitted malignancy may occur even when not suspected based on donor or recipient factors, including age and time to cancer diagnosis. We describe the detection of a gastrointestinal adenocarcinoma transmitted from a young donor to 4 transplant recipients. Multidimensional histopathologic and genomic profiling showed a CDH1 mutation and MET amplification, consistent with gastric origin. At the time of writing, one patient in this series remains alive and without evidence of cancer after prompt organ explant after cancer was reported in other recipients. Because identification of a donor-derived malignancy changes management, our recommendation is to routinely perform short tandem repeat testing (or a comparable assay) immediately upon diagnosis of cancer in any organ transplant recipient. Routine testing for a donor-origin cancer and centralized reporting of outcomes are necessary to establish a robust evidence base for the future development of clinical practice guidelines.

Well-differentiated papillary mesothelioma of the peritoneum is genetically defined by mutually exclusive mutations in TRAF7 and CDC42.

Well-differentiated papillary mesothelioma is an uncommon mesothelial neoplasm that most frequently arises in the peritoneal cavity of women of reproductive age. Whereas malignant mesothelioma is an aggressive tumor associated with poor outcome, well-differentiated papillary mesothelioma typically exhibits indolent behavior. However, histologically differentiating between these two entities can be challenging, necessitating the development of distinguishing biomarkers. While the genetic alterations that drive malignant mesothelioma have recently been determined, the molecular pathogenesis of well-differentiated papillary mesothelioma is unknown. Here we performed genomic profiling on a cohort of ten well-differentiated papillary mesothelioma of the peritoneum. We identified that all tumors harbored somatic missense mutations in either the TRAF7 or CDC42 genes, and lacked alterations involving BAP1, NF2, CDKN2A, DDX3X, SETD2, and ALK that are frequent in malignant mesothelioma. We recently identified that another mesothelial neoplasm, adenomatoid tumor of the genital tract, is genetically defined by somatic missense mutations in the TRAF7 gene, indicating a shared molecular pathogenesis between well-differentiated papillary mesothelioma and adenomatoid tumors. To the best of our knowledge, well-differentiated papillary mesothelioma is the first human tumor type found to harbor recurrent mutations in the CDC42 gene, which encodes a Rho family GTPase. Immunohistochemistry demonstrated intact BAP1 expression in all cases of well-differentiated papillary mesothelioma, indicating that this is a reliable marker for distinguishing well-differentiated papillary mesothelioma from malignant mesotheliomas that frequently display loss of expression. Additionally, all well-differentiated papillary mesothelioma demonstrated robust expression of L1 cell adhesion molecule (L1CAM), a marker of NF-kB pathway activation, similar to that observed in adenomatoid tumors. In contrast, we have previously shown that L1CAM staining is not observed in normal mesothelial cells and malignant mesotheliomas of the peritoneum. Together, these studies demonstrate that well-differentiated papillary mesothelioma is genetically defined by mutually exclusive mutations in TRAF7 and CDC42 that molecularly distinguish this entity from malignant mesothelioma.

The genetic landscape of gliomas arising after therapeutic radiation.

Radiotherapy improves survival for common childhood cancers such as medulloblastoma, leukemia, and germ cell tumors. Unfortunately, long-term survivors suffer sequelae that can include secondary neoplasia. Gliomas are common secondary neoplasms after cranial or craniospinal radiation, most often manifesting as high-grade astrocytomas with poor clinical outcomes. Here, we performed genetic profiling on a cohort of 12 gliomas arising after therapeutic radiation to determine their molecular pathogenesis and assess for differences in genomic signature compared to their spontaneous counterparts. We identified a high frequency of TP53 mutations, CDK4 amplification or CDKN2A homozygous deletion, and amplifications or rearrangements involving receptor tyrosine kinase and Ras-Raf-MAP kinase pathway genes including PDGFRA, MET, BRAF, and RRAS2. Notably, all tumors lacked alterations in IDH1, IDH2, H3F3A, HIST1H3B, HIST1H3C, TERT (including promoter region), and PTEN, which genetically define the major subtypes of diffuse gliomas in children and adults. All gliomas in this cohort had very low somatic mutation burden (less than three somatic single nucleotide variants or small indels per Mb). The ten high-grade gliomas demonstrated markedly aneuploid genomes, with significantly increased quantity of intrachromosomal copy number breakpoints and focal amplifications/homozygous deletions compared to spontaneous high-grade gliomas, likely as a result of DNA double-strand breaks induced by gamma radiation. Together, these findings demonstrate a distinct molecular pathogenesis of secondary gliomas arising after radiation therapy and identify a genomic signature that may aid in differentiating these tumors from their spontaneous counterparts.

CRTC1-MAML2 fusion in mucoepidermoid carcinoma of the breast.

AIMS: Mucoepidermoid carcinomas (MEC) are the most common malignant neoplasms of salivary glands, but are uncommon in other sites. Salivary gland MEC are most frequently associated with CRTC1-MAML2 translocations. Exceedingly rare MEC of the breast demonstrate a basal-like and often triple (oestrogen and progesterone receptor, HER2)-negative immunophenotype, with a single case previously reported to show MAML2 rearrangement, although the fusion partner was not known. Comprehensive genomic studies of breast MEC are lacking. In this study, we analysed the immunophenotype and molecular landscape of two breast MEC to elucidate the pathogenesis of these rare tumours. METHODS AND RESULTS: Two breast MEC were subjected to capture-based next-generation DNA sequencing of 479 cancer-related genes. The presence of the CRTC1-MAML2 fusion transcript was interrogated by reverse transcriptase-polymerase chain reaction. In addition, the immunoprofiles of breast MEC were compared to salivary gland MEC. Both breast MEC harboured CRTC1-MAML2 fusions. In contrast to most triple-negative breast carcinomas of no special type, the mutational burden of MEC was very low, with one case demonstrating only an inactivating SETD2 mutation, and the other harbouring no somatic variants in genes on the panel. No copy number alterations were identified. The immunoprofiles of breast and salivary gland MEC were overlapping, but not identical. CONCLUSIONS: The findings highlight MEC as a breast cancer subtype more closely related to its salivary gland counterpart than to basal-like/triple-negative breast cancers of no special type.

Adenomatoid tumors of the male and female genital tract are defined by TRAF7 mutations that drive aberrant NF-kB pathway activation.

Adenomatoid tumors are the most common neoplasm of the epididymis, and histologically similar adenomatoid tumors also commonly arise in the uterus and fallopian tube. To investigate the molecular pathogenesis of these tumors, we performed genomic profiling on a cohort of 31 adenomatoid tumors of the male and female genital tracts. We identified that all tumors harbored somatic missense mutations in the TRAF7 gene, which encodes an E3 ubiquitin ligase belonging to the family of tumor necrosis factor receptor-associated factors (TRAFs). These mutations all clustered into one of five recurrent hotspots within the WD40 repeat domains at the C-terminus of the protein. Functional studies in vitro revealed that expression of mutant but not wild-type TRAF7 led to increased phosphorylation of nuclear factor-kappa B (NF-kB) and increased expression of L1 cell adhesion molecule (L1CAM), a marker of NF-kB pathway activation. Immunohistochemistry demonstrated robust L1CAM expression in adenomatoid tumors that was absent in normal mesothelial cells, malignant peritoneal mesotheliomas and multilocular peritoneal inclusion cysts. Together, these studies demonstrate that adenomatoid tumors of the male and female genital tract are genetically defined by TRAF7 mutation that drives aberrant NF-kB pathway activation.

Genomic profiling of malignant phyllodes tumors reveals aberrations in FGFR1 and PI-3 kinase/RAS signaling pathways and provides insights into intratumoral heterogeneity.

Malignant phyllodes tumors of the breast are poorly understood rare neoplasms with potential for aggressive behavior. Few efficacious treatment options exist for progressed or metastatic disease. The molecular features of malignant phyllodes tumors are poorly defined, and a deeper understanding of the genetics of these tumors may shed light on pathogenesis and progression and potentially identify novel treatment approaches. We sequenced 510 cancer-related genes in 10 malignant phyllodes tumors, including 5 tumors with liposarcomatous differentiation and 1 with myxoid chondrosarcoma-like differentiation. Intratumoral heterogeneity was assessed by sequencing two separate areas in 7 tumors, including non-heterologous and heterologous components of tumors with heterologous differentiation. Activating hotspot mutations in FGFR1 were identified in 2 tumors. Additional recurrently mutated genes included TERT promoter (6/10), TP53 (4/10), PIK3CA (3/10), MED12 (3/10), SETD2 (2/10) and KMT2D (2/10). Together, genomic aberrations in FGFR/EGFR PI-3 kinase and RAS pathways were identified in 8 (80%) tumors and included mutually exclusive and potentially actionable activating FGFR1, PIK3CA and BRAF V600E mutations, inactivating TSC2 mutation, EGFR amplification and PTEN loss. Seven (70%) malignant phyllodes tumors harbored TERT aberrations (six promoter mutations, one amplification). For comparison, TERT promoter mutations were identified by Sanger sequencing in 33% borderline (n=12) and no (0%, n=8) benign phyllodes tumors (P=0.391 and P=0.013 vs malignant tumors, respectively). Genetic features specific to liposarcoma, including CDK4/MDM2 amplification, were not identified. Copy number analysis revealed intratumoral heterogeneity and evidence for divergent tumor evolution in malignant phyllodes tumors with and without heterologous differentiation. Tumors with liposarcomatous differentiation revealed more chromosomal aberrations in non-heterologous components compared with liposarcomatous components. EGFR amplification was heterogeneous and present only in the non-heterologous component of one tumor with liposarcomatous differentiation. The results identify novel pathways involved in the pathogenesis of malignant phyllodes tumors, which significantly increase our understanding of tumor biology and have potential clinical impact.