Reprogramming of somatic cells to a pluripotent state via expression of Oct4, Klf4, Myc, and Sox2 is a multistep process involving phased changes in gene expression. Here, we focus on the later ...stages of reprogramming, termed maturation and stabilization. We show that the stabilization phase and the acquisition of pluripotency are dependent on the removal of transgene expression late in the maturation phase. Clonal analysis of cells undergoing reprogramming revealed subsets of stabilization-competent (SC) and stabilization-incompetent (SI) cells. SC clones acquire a competency gene-expression signature late in the maturation phase. Functional analysis of SC signature genes identified enhancers of the transition to the stabilization phase and a distinct subset of genes required for the maintenance of pluripotency. Thus, the acquisition and maintenance of pluripotency are regulated by distinct molecular networks, and a specific regulatory program not previously implicated in reprogramming is required for the transition to transgene independence.
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► Transgenes repress phase progression during somatic cell reprogramming ► Over time, cells acquire competency to transition to iPSCs upon transgene withdrawal ► Clonal analysis identifies signatures associated with reprogramming competency ► Distinct networks regulate transition to and maintenance of pluripotency
During reprogramming, only a subset of cells moving through the process are able to complete it, and to do so they require a distinct set of genes separate from the pluripotency network itself.
Mucinous tubular and spindle cell carcinoma (MTSCC) is a rare subtype of renal cell carcinoma with characteristic histologic features and chromosomal alterations. Although typically indolent, a small ...subset of cases has been reported to exhibit aggressive clinical behavior. We retrospectively identified 33 patients with MTSCC, consisting of 10 cases of locally advanced/metastatic MTSCC (pT3 or N1 or M1) and 23 kidney-confined MTSCC (pT1/T2) without disease recurrence or progression. Utilizing a single-nucleotide polymorphism array and a targeted next-generation sequencing platform, we examined genome-wide molecular alterations in 24 cases, including 11 available samples from 8 patients with locally advanced/metastatic MTSCC. Ten patients with locally advanced/metastatic MTSCC were 8 females (80%) and 2 males (20%). At nephrectomy, 7 of these 10 cases (70%) were pT3 or pN1 while the remaining 3 (30%) were pT1/T2. Eight patients (80%) developed metastases and common sites included lymph node (4, 40%), bone (4, 40%), and retroperitoneum (3, 30%). Four patients died of disease (40%) during follow-up. Locally advanced/metastatic MTSCCs shared typical MTSCC genomic profiles with loss of chromosomes 1, 4, 6, 8, 9, 13, 14, 15, and 22, while some exhibited additional complex genomic alterations, most frequently a relative gain of 1q (7/8). Homozygous loss of CDKN2A/B was observed in 3 (38%) locally advanced/metastatic MTSCCs. Tumor necrosis, solid nested/sheet pattern, irregular trabecular/single-file infiltration in a desmoplastic stroma, lymphovascular space invasion, and increased mitotic activity were associated with locally advanced/metastatic MTSCCs (all p < 0.05). Our findings reveal that MTSCCs with aggressive clinical behavior have progressed through clonal evolution; CDKN2A/B deletion and additional complex genomic abnormalities may contribute to this process. Recognizing the morphologic presentation of high-grade MTSCC and evaluating adverse histologic features seen in these tumors can help establish a definitive diagnosis and stratify patients for treatment and prognostication.
Tubulocystic renal cell carcinoma, a unique tumor, was recently included as a new entity in the World Health Organization classification of renal tumors. It has variably been reported to be related ...to other renal cell carcinomas, including papillary renal cell carcinoma, fumarate hydratase-deficient carcinoma, and others, likely because many such carcinomas may show variable amounts of tubulocystic architecture. The published data characterizing the molecular features of these tumors are inconsistent. We studied nine "pure" tubulocystic renal cell carcinomas, as defined by International Society of Urologic Pathologists (ISUP) and World Health Organization (WHO), by targeted next-generation sequencing, and fluorescence in situ hybridization for X and Y chromosomes, to investigate if these show any unique characteristics or any overlap with known mutational/molecular profiles or copy number alterations in other subtypes of renal cell carcinoma. All nine tubulocystic carcinomas demonstrated combined losses at chromosome 9 and gains at chromosome 17, as well as, loss of chromosome Y (in 5/5). None of the tumors showed mutational profiles characteristic of other renal neoplasms, including those seen in fumarate hydratase-deficient renal cell carcinoma. Recurrent mutations in chromatin-modifying genes, KMT2C and KDM5C, were detected in two of nine tumors. Thus, tubulocystic renal cell carcinoma, if defined strictly, at the clinical and pathologic level, demonstrates genomic features distinct from other subtypes of renal cell carcinoma. These findings support the contention that tubulocystic renal cell carcinoma should be diagnosed only using strict morphological criteria and only when presenting in a "pure" form; presence of variable papillary, poorly differentiated, or other architectural patterns most likely do not belong to the category of tubulocystic renal cell carcinoma.
Cell free DNA (cfDNA) and circulating tumor cell free DNA (ctDNA) from blood (plasma) are increasingly being used in oncology for diagnosis, monitoring response, identifying cancer causing mutations ...and detecting recurrences. Circulating tumor RB1 DNA (ctDNA) is found in the blood (plasma) of retinoblastoma patients at diagnosis before instituting treatment (naïve). We investigated ctDNA in naïve unilateral patients before enucleation and during enucleation (6 patients/ 8 mutations with specimens collected 5-40 minutes from severing the optic nerve) In our cohort, following transection the optic nerve, ctDNA RB1 VAF was measurably lower than pre-enucleation levels within five minutes, 50% less within 15 minutes and 90% less by 40 minutes.
Genomic profiling of hematologic malignancies has augmented our understanding of variants that contribute to disease pathogenesis and supported development of prognostic models that inform disease ...management in the clinic. Tumor only sequencing assays are limited in their ability to identify definitive somatic variants, which can lead to ambiguity in clinical reporting and patient management. Here, we describe the MSK-IMPACT Heme cohort, a comprehensive data set of somatic alterations from paired tumor and normal DNA using a hybridization capture-based next generation sequencing platform. We highlight patterns of mutations, copy number alterations, and mutation signatures in a broad set of myeloid and lymphoid neoplasms. We also demonstrate the power of appropriate matching to make definitive somatic calls, including in patients who have undergone allogeneic stem cell transplant. We expect that this resource will further spur research into the pathobiology and clinical utility of clinical sequencing for patients with hematologic neoplasms.
Inherited mutations are easily detected factors that influence the disease courses and optimal treatment strategies of some cancers. Germline mutations in BRCA1 associated protein 1 (BAP1) are ...associated with unique disease profiles in mesothelioma, atypical spitz nevi, and uveal melanoma, but the patient characteristics of an unselected population of BAP1 carriers identified by an ascertainment prevalence study are unknown.
We collected blood samples, cancer histories, and occupational exposures from 183 unselected patients with BAP1-related diseases. Clinical information for each patient was obtained from medical records. Germline DNA was extracted from blood samples and sequenced using a next-generation sequencing assay. We tested screening criteria developed to identify patients with a possible germline BAP1 mutation.
Pathogenic or likely pathogenic germline BAP1 mutations were observed in 5 of 180 sequenced specimens and were exclusively found in patients identified by our screening criteria. Several patients with characteristics suspicious for a heritable deleterious mutation did not have a germline BAP1 mutation. The prevalence of pathogenic germline BAP1 mutations in patients with mesothelioma was 4.4% (95% confidence interval 1.1–11.1).
Results from the first unselected prevalence ascertainment study of germline BAP1 alterations suggest that the frequency of this mutation is low among patients with mesothelioma. The proposed screening criteria successfully identified all patients with germline BAP1-mutant mesothelioma. These screening guidelines may assist physicians in selecting patients who would benefit from genetic testing. Future efforts should validate and refine these criteria and search for other germline mutations associated with mesothelioma and related diseases.
Point mutations in the TERT gene promoter occur at high frequency in multiple cancers, including urothelial carcinoma (UC). However, the relationship between TERT promoter mutations and UC patient ...outcomes is unclear due to conflicting reports in the literature. In this study, we examined the association of TERT alterations, tumor mutational burden per megabase (Mb), and copy number alteration (CNA) burden with clinical parameters and their prognostic value in a cohort of 398 urothelial tumors. The majority of TERT mutations were located at two promoter region hotspots (chromosome 5, 1 295 228 C>T and 1 295 250 C>T). TERT alterations were more frequently present in bladder tumors than in upper tract tumors (73% vs 53%; p=0.001). ARID1A, PIK3CA, RB1, ERCC2, ERBB2, TSC1, CDKN1A, CDKN2A, CDKN2B, and PTPRD alterations showed significant co-occurrence with TERT alterations (all p<0.0025). TERT alterations and the mutational burden/Mb were independently associated with overall survival (hazard ratioHR 2.31, 95% confidence interval CI 1.46–3.65; p<0.001; and HR 0.96, 95% CI 0.93–0.99; p=0.002), disease-specific survival (HR 2.23, 95% CI 1.41–3.53; p<0.001; and HR 0.96, 95% CI 0.93–0.99; p=0.002), and metastasis-free survival (HR 1.63, 95% CI 1.05–2.53; p=0.029; and HR 0.98, 95% CI 0.96–1.00; p=0.063) in multivariate models.
The majority of TERT gene mutations that we detected in urothelial carcinoma are located at two promoter hotspots. Urothelial tumors with TERT alterations had worse prognosis compared to tumors without TERT alterations, whereas tumors with a higher mutational burden had more favorable outcome compared to tumors with low mutational burden.
The majority of TERT mutations are located at two promoter region hotspots. Urothelial tumors with TERT alterations had worse prognosis compared to tumors without TERT alterations, whereas tumors with a higher mutational burden/Mb had a more favorable outcome compared to tumors with a low mutational burden/Mb.
Analysis of circulating tumor DNA (ctDNA) in the plasma of patients with retinoblastoma and simulating lesions.
Retrospective cross-sectional study of the association of plasma ctDNA from ...retinoblastoma and simulating lesions with disease course.
Fifty-eight Memorial Sloan Kettering Cancer Center patients with retinoblastoma comprising 68 plasma ctDNA samples and 5 with retinoblastoma-simulating lesions.
The ctDNA analyzed with hybridization capture and next-generation sequencing in blood (plasma) of patients who had retinoblastoma or simulating lesions were evaluated for association with clinical course of the disease.
Presence or absence of molecular aberrations in the RB1 gene and correlations with clinical features.
RB1 cell-free DNA (cfDNA) was detected in 16 of 19 patients with newly diagnosed, untreated intraocular retinoblastoma and in 3 of 3 patients with newly diagnosed, untreated metastatic disease. It was also present in 3 patients with recurrent intraocular disease before therapy, but was not present in patients with recurrent disease who received intra-arterial chemotherapy, nor in 21 patients who had undergone enucleation for unilateral disease. In 1 patient who had delayed treatment (insurance reasons) and showed rapid growth of the intraocular tumor, the variant allele frequency increased in 1 month from 0.34% to 2.48%. No RB1 mutations were detected in the cfDNA from plasma of patients with simulating lesions (3 with Coats disease and 1 with persistent fetal vasculature PFV). In 2 patients, we identified 2 independent RB1 mutations in plasma.
Mutations in RB1 were found in the cfDNA from blood of patients with newly diagnosed, untreated retinoblastoma and in patients who showed disease recurrence in the eye after prior treatment, but not in unilateral retinoblastoma after enucleation Levels of ctDNA increase in patients with progressive disease who did not receive any treatment. High plasma cfDNA levels were detected in patients with newly diagnosed metastatic disease, and these levels decreased after systemic chemotherapy was administered. Further validation is needed for measuring the somatic alterations in cfDNA from blood in retinoblastoma that could provide a promising method of monitoring patients in the future.
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