Plasticity of Extrachromosomal and Intrachromosomal BRAF Amplifications in Overcoming Targeted Therapy Dosage Challenges.

Focal amplifications (FA) can mediate targeted therapy resistance in cancer. Understanding the structure and dynamics of FAs is critical for designing treatments that overcome plasticity-mediated resistance. We developed a melanoma model of dual MAPK inhibitor (MAPKi) resistance that bears BRAFV600 amplifications through either extrachromosomal DNA (ecDNA)/double minutes (DM) or intrachromosomal homogenously staining regions (HSR). Cells harboring BRAFV600E FAs displayed mode switching between DMs and HSRs, from both de novo genetic changes and selection of preexisting subpopulations. Plasticity is not exclusive to ecDNAs, as cells harboring HSRs exhibit drug addiction-driven structural loss of BRAF amplicons upon dose reduction. FA mechanisms can couple with kinase domain duplications and alternative splicing to enhance resistance. Drug-responsive amplicon plasticity is observed in the clinic and can involve other MAPK pathway genes, such as RAF1 and NRAS. BRAF FA-mediated dual MAPKi-resistant cells are more sensitive to proferroptotic drugs, extending the spectrum of ferroptosis sensitivity in MAPKi resistance beyond cases of dedifferentiation. SIGNIFICANCE: Understanding the structure and dynamics of oncogene amplifications is critical for overcoming tumor relapse. BRAF amplifications are highly plastic under MAPKi dosage challenges in melanoma, through involvement of de novo genomic alterations, even in the HSR mode. Moreover, BRAF FA-driven, dual MAPKi-resistant cells extend the spectrum of resistance-linked ferroptosis sensitivity. This article is highlighted in the In This Issue feature, p. 873.

Cytoplasmic p53 couples oncogene-driven glucose metabolism to apoptosis and is a therapeutic target in glioblastoma.

Cross-talk among oncogenic signaling and metabolic pathways may create opportunities for new therapeutic strategies in cancer. Here we show that although acute inhibition of EGFR-driven glucose metabolism induces only minimal cell death, it lowers the apoptotic threshold in a subset of patient-derived glioblastoma (GBM) cells. Mechanistic studies revealed that after attenuated glucose consumption, Bcl-xL blocks cytoplasmic p53 from triggering intrinsic apoptosis. Consequently, targeting of EGFR-driven glucose metabolism in combination with pharmacological stabilization of p53 with the brain-penetrant small molecule idasanutlin resulted in synthetic lethality in orthotopic glioblastoma xenograft models. Notably, neither the degree of EGFR-signaling inhibition nor genetic analysis of EGFR was sufficient to predict sensitivity to this therapeutic combination. However, detection of rapid inhibitory effects on [18F]fluorodeoxyglucose uptake, assessed through noninvasive positron emission tomography, was an effective predictive biomarker of response in vivo. Together, these studies identify a crucial link among oncogene signaling, glucose metabolism, and cytoplasmic p53, which may potentially be exploited for combination therapy in GBM and possibly other malignancies.

Extrachromosomal oncogene amplification drives tumour evolution and genetic heterogeneity.

Human cells have twenty-three pairs of chromosomes. In cancer, however, genes can be amplified in chromosomes or in circular extrachromosomal DNA (ecDNA), although the frequency and functional importance of ecDNA are not understood. We performed whole-genome sequencing, structural modelling and cytogenetic analyses of 17 different cancer types, including analysis of the structure and function of chromosomes during metaphase of 2,572 dividing cells, and developed a software package called ECdetect to conduct unbiased, integrated ecDNA detection and analysis. Here we show that ecDNA was found in nearly half of human cancers; its frequency varied by tumour type, but it was almost never found in normal cells. Driver oncogenes were amplified most commonly in ecDNA, thereby increasing transcript level. Mathematical modelling predicted that ecDNA amplification would increase oncogene copy number and intratumoural heterogeneity more effectively than chromosomal amplification. We validated these predictions by quantitative analyses of cancer samples. The results presented here suggest that ecDNA contributes to accelerated evolution in cancer.

Commentary on the decision of the American Board of Medical Genetics and Genomics to create a 24-month specialty of Laboratory Genetics and Genomics.

Genet Med 19 3, 294-296.

Pericytic mimicry in well-differentiated liposarcoma/atypical lipomatous tumor.

Pericytes are modified smooth muscle cells that closely enwrap small blood vessels, regulating and supporting the microvasculature through direct endothelial contact. Pericytes demonstrate a distinct immunohistochemical profile, including expression of smooth muscle actin, CD146, platelet-derived growth factor receptor ß, and regulator of G-protein signaling 5. Previously, pericyte-related antigens have been observed to be present among a group of soft tissue tumors with a perivascular growth pattern, including glomus tumor, myopericytoma, and angioleiomyoma. Similarly, malignant tumor cells have been shown to have a pericyte-like immunoprofile when present in a perivascular location, seen in malignant melanoma, glioblastoma, and adenocarcinoma. Here, we examine well-differentiated liposarcoma specimens, which showed some element of perivascular areas with the appearance of smooth muscle (n = 7 tumors). Immunohistochemical staining was performed for pericyte antigens, including smooth muscle actin, CD146, platelet-derived growth factor receptor ß, and regulator of G-protein signaling 5. Results showed consistent pericytic marker expression among liposarcoma tumor cells within a perivascular distribution. MDM2 immunohistochemistry and fluorescence in situ hybridization for MDM2 revealed that these perivascular cells were of tumor origin (7/7 tumors), whereas double immunohistochemical detection for CD31/CD146 ruled out an endothelial cell contribution. These findings further support the concept of pericytic mimicry, already established in diverse malignancies, and its presence in well-differentiated liposarcoma. The extent to which pericytic mimicry has prognostic significance in liposarcoma is as yet unknown.

Src as a novel therapeutic target for endometriosis.

BACKGROUND: Endometriosis is a common condition that is associated with an increased risk of developing ovarian carcinoma. Improved in vitro models of this disease are needed to better understand how endometriosis, a benign disease, can undergo neoplastic transformation, and for the development of novel treatment strategies to prevent this progression. METHODS: We describe the generation and in vitro characterization of novel TERT immortalized ovarian endometriosis epithelial cell lines (EEC16-TERT). RESULTS: Expression of TERT alone was sufficient to immortalize endometriosis epithelial cells. TERT immortalization induces an epithelial-to-mesenchymal transition and perturbation in the expression of genes involved in the development of ovarian cancer. EEC16-TERT was non-tumorigenic when xenografted into immunocompromised mice but grew in anchorage-independent growth assays in an epidermal growth factor and hydrocortisone dependent manner. Colony formation in agar was abolished by inhibition of Src, and the Src pathway was found to be activated in human endometriosis lesions. CONCLUSIONS: This new in vitro model system mimics endometriosis and the early stages of neoplastic transformation in the development of endometriosis associated ovarian cancer. We demonstrate the potential clinical relevance of this model by identifying Src activation as a novel pathway in endometriosis that could be targeted therapeutically, perhaps as a novel strategy to manage endometriosis clinically, or to prevent the development of endometriosis-associated ovarian cancer.

Extranodal Marginal Zone Lymphoma Presenting within the Meckel Diverticulum as Diverticulitis: A Case Report.

Meckel diverticulum is the most common congenital defect of the gastrointestinal tract. It can be asymptomatic or mimic appendicitis and may be complicated by bleeding, diverticulitis, obstruction, and, rarely, neoplasia. We report the first case of extranodal marginal zone lymphoma occupying a Meckel diverticulum. A 44-year-old man with history of colonic diverticulitis presented to the emergency department for evaluation of acute abdominal pain. Radiography showed enteric obstruction, prompting diagnostic laparoscopy. Above the level of mid-ileum an intact Meckel diverticulum was identified. Microscopy showed extensive infiltration of sheets of small lymphocytes with abundant cytoplasm (monocytoid B-cells) prominently in submucosa and focally transmural involving serosal adipose tissue with multiple reactive germinal centers. The immunostains showed positivity for CD20, BCL-2, and CD43 (weak) and negativity for CD3, CD5, BCL-1, CD10, and BCL-6 in monocytoid B-cells. Fluorescence in situ hybridization studies revealed API2-MALT1 fusion signals consistent with t(11;18)(q21;q21), which confirmed the diagnosis of extranodal marginal zone lymphoma, also known as mucosa associated lymphoid tissue lymphoma.

Targeted therapy resistance mediated by dynamic regulation of extrachromosomal mutant EGFR DNA.

Intratumoral heterogeneity contributes to cancer drug resistance, but the underlying mechanisms are not understood. Single-cell analyses of patient-derived models and clinical samples from glioblastoma patients treated with epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) demonstrate that tumor cells reversibly up-regulate or suppress mutant EGFR expression, conferring distinct cellular phenotypes to reach an optimal equilibrium for growth. Resistance to EGFR TKIs is shown to occur by elimination of mutant EGFR from extrachromosomal DNA. After drug withdrawal, reemergence of clonal EGFR mutations on extrachromosomal DNA follows. These results indicate a highly specific, dynamic, and adaptive route by which cancers can evade therapies that target oncogenes maintained on extrachromosomal DNA.

Deletions of chromosomes 3p and 14q molecularly subclassify clear cell renal cell carcinoma.

BACKGROUND: The short arm of chromosome 3 (3p) harbors the von Hippel-Lindau (VHL) tumor suppressor gene, and the long arm of chromosome 14 (14q) harbors the hypoxia-inducible factor 1α (HIF-1α) gene. The objective of this study was to evaluate the significance of 3p loss (loss VHL gene) and 14q loss (loss HIF-1α gene) in clear cell renal cell carcinoma (ccRCC). METHODS: In total, 288 ccRCC tumors underwent a prospective cytogenetic analysis for alterations in chromosomes 3p and 14q. Tumors were assigned to 1 of 4 possible chromosomal alterations: VHL +3p/+14q (VHL wild type [VHL-WT]), VHL +3p/-14q (VHL-WT plus HIF2α [WT/H2]), -3p/+14q (HIF1α and HIF2α [H1H2]), and -3p/-14q (HIF2α [H2]). RESULTS: Among patients who had loss of 3p, tumors with -3p/-14q (H2) alterations were larger (P = .002), had higher grade (P = .002) and stage (P = .001), and more often were metastatic (P = .029) than tumors that retained 14q (H1H2). All patients who had tumors with -3p/-14q (H2) had worse cancer-specific survival (P = .014), and patients who had localized disease (P = .012) and primary T1 (pT1) tumors (P = .008) had worse recurrence-free survival. In patients who had pT1 tumors, combined 3p/14q loss was an independent predictor of recurrence-free survival (hazard ratio, 11.19; 95% confidence interval, 1.91-65.63) and cancer-specific survival (hazard ratio, 15.93; 95% confidence interval, 3.09-82.16). The current investigation was limited by its retrospective design, single-center experience, and a lack of confirmatory protein analyses. CONCLUSIONS: Loss of chromosome 3p (the VHL gene) was associated with improved survival in patients with ccRCC, whereas loss of chromosome 14q (the HIF-1α gene) was associated with worse outcomes. The results of the current study support the hypothesis that HIF-1α functions as an important tumor suppressor gene in ccRCC.

Gain of chromosome 8q is associated with metastases and poor survival of patients with clear cell renal cell carcinoma.

BACKGROUND: The aim of this study was to evaluate the prevalence of chromosome 8q gain in clear cell renal cell carcinoma (CCRCC) and to correlate the findings with tumor phenotype and disease-specific survival (DSS). METHODS: The tumor karyotypes of 336 consecutive patients with CCRCC were prospectively evaluated with classical cytogenetic analysis. Chromosome 8q status was correlated with clinicopathological variables, and its impact on DSS was evaluated. RESULTS: Gain of 8q occurred in 28 tumors (8.3%). Gain of 8q was associated with a higher risk of regional lymph node (21.4% vs 6.2%, P = .011) and distant metastases (50.0% vs 24.4%, P = .006), and greater tumor sizes (P = .030). Patients with gain of 8q had a 3.22-fold increased risk of death from CCRCC (P < .001). In multivariable analysis, gain of 8q was identified as an independent prognostic factor (hazard ratio, 2.37; P = .006). The concordance index of a multivariable base model increased significantly following inclusion of 8q gain (P = .0015). CONCLUSIONS: Gain of chromosome 8q occurs in a subset of CCRCCs and is associated with an increased risk of metastases and death from CCRCC. Because the proto-oncogene c-MYC is among the list of candidate genes located on 8q, our data suggest that these tumors may have unique pathways activated, which are associated with an aggressive tumor phenotype. If confirmed, defining tumors with gain of 8q may assist in identifying patients who would benefit for specific c-MYC inhibitors or agents that target the MAPK/ERK (mitogen-activated protein kinase) pathway.

Clinical, molecular, and genetic correlates of lymphatic spread in clear cell renal cell carcinoma.

BACKGROUND: While it is well known that clear cell renal cell carcinoma (ccRCC) that presents with lymphatic spread is associated with an extremely poor prognosis, its molecular and genetic biology is poorly understood. OBJECTIVE: Define the clinicopathologic, molecular, and genetic biological characteristics of these tumors in comparison to nonmetastatic (N0M0) renal cell carcinomas. DESIGN, SETTING, AND PARTICIPANTS: A retrospective study defined clinicopathologic features, expression of 28 molecular markers, and occurrence of chromosomal aberrations for their correlation with lymphatic spread in three cohorts of 502, 196, and 272 patients, respectively. MEASUREMENTS: Fisher exact test or the χ(2) test were used to compare categorical variables; continuous variables were compared with the Mann-Whitney U test or student t test. Cut-off values were calculated based on receiver operating characteristic curves and the Youden Index. Uni- and multivariate regression analyses were used to investigate the correlation with lymphatic spread. RESULTS AND LIMITATIONS: In clinical analyses, a predictive model consisting of smoking history (p=0.040), T stage (p<0.0001), Fuhrman grade (p<0.0001), Eastern Cooperative Oncology Group performance status (p<0.0001), and microvascular invasion (p<0.0001) was independently associated with lymphatic spread. After adjustment with these clinical variables, low carbonic anhydrase IX (CAIX) (p=0.043) and high epithelial vascular endothelial growth factor receptor 2 (p=0.033) protein expression were associated with a higher risk of lymphatic spread, and loss of chromosome 3p (p<0.0001) with a lower risk. The current study is limited by its retrospective design, small sample size, and single-center experience. CONCLUSIONS: The low rates of CAIX expression and loss of chromosome 3p suggest that lymphatic spread in ccRCC occurs independently of von Hippel-Lindau tumor suppressor inactivation.

Solid tumor cytogenetics: current perspectives.

Conventional cytogenetics in conjunction with Fluorescence in Situ Hybridization (FISH) continues to remain an important and integral component in the diagnosis and management of solid tumors. The ability to effectively detect the vast majority of clinically relevant chromosomal aberrations with a rapid-to-acceptable turnaround time makes them the most cost-effective screening/detection tool currently available in modern pathology. In this review, we describe a representative set of solid tumors in which chromosomal analysis and/or FISH plays a significant role in the routine clinical management of solid tumors.

Frequent EVI1 translocations in myeloid blast crisis CML that evolves through tyrosine kinase inhibitors.

Clinical variables associated with ecotropic viral integration site 1 (EVI1) translocations were evaluated in 42 consecutive chronic myeloid leukemia (CML) patients in myeloid blast crisis (MBC). Translocations were confirmed with fluorescence in situ hybridization, and Western blot analysis demonstrated EVI1 expression. Translocations of EVI1 were present in 3 of 24 (12%) patients whose disease evolved MBC before tyrosine kinase inhibitor (TKI) exposure, and 7 of 18 (39%) patients who had received one or more TKIs. Univariate analysis showed that prior TKI therapy was the only clinical variable that was significantly associated with EVI1 translocation (P = 0.047). TKI-resistant BCR-ABL1 mutations were present in 71% of MBC patients with EVI1 translocations at the time of disease progression. These observations suggest that EVI1 overexpression collaborates with BCR-ABL1 in the evolution of TKI-resistant MBC. Inhibition of c-ABL kinase-mediated DNA double-strand repair by TKIs may predispose to EVI1 translocation in this setting.

Chromosomal study for prognostic grouping in chronic lymphocytic leukemia.

OBJECTIVE: To determine the frequency of various cytogenetic aberrations in newly diagnosed chronic lymphocytic leukemia (CLL) patients, and their detection rate by cytogenetic and fluorescent In situ hybridization (FISH) technique separately. STUDY DESIGN: A case series. PLACE AND DURATION OF STUDY: Clinical and Molecular Cytogenetics Laboratories, University of California, Los Angeles, USA, from November 2007 to July 2008. METHODOLOGY: Analysis was made on 100 diagnosed chronic lymphocytic leukemia patients. Cytogenetics and FISH technique were performed on blood or bone marrow samples. RESULTS: Nineteen out of 100 cases (19%) showed karyotype abnormalities; whereas 55 showed abnormalities using the CLL-specific FISH probes. The most frequent abnormality detected by standard cytogenetics was trisomy 12. The most common abnormality detected by FISH was a deletion of 13q14 (40 out of 55 cases; 72% of the abnormal). CONCLUSION: For prognostic grouping of CLL patients, FISH must always be requested which may even replace standard karyotyping. These chromosomal markers help in choosing the therapeutic options.

Pathological, immunohistochemical and cytogenetic features of papillary renal cell carcinoma with clear cell features.

PURPOSE: Papillary renal cell carcinoma is characterized histologically by tumors with cells arranged in a papillary pattern. Typically the cells have a chromophilic appearance but areas may show cells with clear cytoplasm, similar to those in clear cell renal cell carcinoma. MATERIALS AND METHODS: We re-reviewed the histological slides of 148 patients with papillary renal cell carcinoma who underwent nephrectomy for the presence of clear cells. Results were correlated with other pathological features, immunohistochemical expression of 22 protein markers, cytogenetic analysis and overall survival. RESULTS: Papillary renal cell carcinoma with clear cells was identified in 57 patients (39%). Clear cells were associated with higher T classification and grade, vascular invasion and type 2 papillary renal cell carcinoma. On immunohistochemistry these tumors revealed higher expression of epithelial vascular endothelial growth factor receptor-2 than papillary renal cell carcinoma without clear cells. All papillary renal cell carcinomas with clear cells expressed α-methylacyl-coenzyme A racemase and 76% expressed cytokeratin 7. Six of 8 tumors (75%) with chromosome 3p loss had clear cell features. The presence of clear cells was retained as an independent prognostic factor on multivariate analysis. In cases of papillary renal cell carcinoma with clear cells the loss of 3p material and absent cytokeratin 7 expression were associated with a worse outcome. CONCLUSIONS: Papillary renal cell carcinoma with clear cells is a novel entity with a unique clinical, immunohistochemical and cytogenetic phenotype. The presence of clear cells is associated with aggressive pathological characteristics and poorer prognosis.

Genomic instability in mice is greater in Fanconi anemia caused by deficiency of Fancd2 than Fancg.

Fanconi anemia (FA) results from mutations in the FANC genes and is characterized by bone marrow failure, birth defects, and a high incidence of cancer. FANCG is a part of the FA core complex that is responsible for monoubiquitination of FANCD2 and FANCI. The precise role of the FA pathway is not well understood, although it may be involved in homologous recombination (HR), nonhomologous end joining, and translesion synthesis (TLS). Fancd2(-/-) mice have a more severe phenotype than Fancg(-/-), and other FA core complex-deficient mice, although both Fancg and Fancd2 belong to the same FA pathway. We hypothesized that Fancd2 deficiency results in a more severe phenotype because Fancd2 also has a FA pathway-independent function in the maintenance of genomic integrity. To test this hypothesis, we determined the level of DNA damage and genomic instability in Fancd2(-/-), Fancg(-/-), and wild-type controls. Fancd2(-/-) mice displayed a higher magnitude of chromosomal breakage and micronucleus formation than the wild-type or Fancg(-/-) mice. Also, DNA strand breaks were increased in Fancd2(-/-) but not in Fancg(-/-) mice. In addition, Fancd2(-/-) mice displayed an elevated frequency of DNA deletions, resulting from HR at the endogenous p(un) locus. In contrast, in Fancg(-/-) mice, the frequency of DNA deletions was decreased. Thus, Fancd2 but not Fancg deficiency results in elevated chromosomal/DNA breakage and permanent genome rearrangements. This provides evidence that Fancd2 plays an additional role in the maintenance of genomic stability than Fancg, which might explain the higher predisposition to cancer seen in the Fancd2(-/-) mice.

Section E6.5 of the ACMG technical standards and guidelines: chromosome studies for solid tumor abnormalities.

PURPOSE: : Cytogenetic analysis of tumor tissue detects clonal abnormalities. The information obtained from these studies is utilized for diagnosis, prognosis, and patient management. METHODS: : The Working Group of the Laboratory Quality Assurance Committee of the American College of Medical Genetics provides these Standards and Guidelines for chromosome studies for solid tumors abnormalities as a resource for clinical cytogenetic laboratories. RESULTS: : The guidelines incorporate aspects of sample procurement, handling, processing, harvesting, analysis, quality control, and quality assurance. It is recommended that all pediatric solid tumors be studied by cytogenetic analysis when feasible due to the clinical and therapeutic implications of the genetic abnormalities. Cytogenetic analysis of certain adult solid tumors also provides information that impacts diagnosis and therapeutics. Molecular cytogenetic analysis or fluorescence in situ hybridization (FISH) may be a primary or secondary method of evaluation of the tumor tissue. FISH can document a specific molecular event, e.g. gene rearrangement, provide a rapid result to aid in the differential diagnosis or planning of therapy, clarify chromosome anomalies, or assess gene amplification. CONCLUSION: : Genetic analysis adds valuable information to the understanding of and therapeutic approach to solid tumors. Laboratories may use their professional judgment to make modifications or additions to these guidelines.

Comparison of fluorescent in situ hybridization HER-2/neu results on core needle biopsy and excisional biopsy in primary breast cancer.

HER-2/neu status is critical for the therapy for breast carcinoma. Fluorescent in situ hybridization for gene amplification and immunohistochemical stains for protein expression are widely used methods to detect HER-2/neu status. Multiple studies have shown fluorescent in situ hybridization and immunohistochemical stain results to have high concordance rates. To our knowledge, a comparison between fluorescent in situ hybridization results for core needle biopsy and the subsequent excisional biopsy specimens has not yet been studied. We retrospectively evaluated the fluorescence in situ hybridization and immunohistochemical results in both the breast core needle and the excisional biopsy of 125 patients with invasive breast carcinoma from 2002 to 2005. There was complete concordance with respect to both immunohistochemical and fluorescence in situ hybridization results for core needle biopsy and excisional biopsy specimens in 87% of the patients evaluated. Comparison of fluorescent in situ hybridization results of the 129 core needle biopsies to the 131 excisional biopsies of all 125 patients showed a concordance rate of 92%. The immunohistochemical stain results of the same core needle and excisional biopsies showed a concordance rate of 98%. Comparison of the immunohistochemical stain results with the fluorescent in situ hybridization results for all 260 cases examined showed 95% concordance. On the basis of our study, we observed that repeating HER-2/neu testing by immunohistochemical stain and/or fluorescent in situ hybridization methods on excisional biopsy is not unreasonable, in particular in cases of intratumoral heterogeneity, indeterminate/borderline HER-2/neu results and after neoadjuvant chemotherapy.