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INTRODUCTION: Next-generation sequencing has emerged as a clinical tool for the identification of actionable mutations to triage advanced colorectal cancer patients for targeted therapies. The literature is conflicted as to whether primaries or their metastases should be selected for sequencing. Some authors suggest that either site may be sequenced, whereas others recommend sequencing the primary, the metastasis, or even both tumors. Here, we address this issue head on with a meta-analysis and provide for the first time a set of sensible recommendations to make this determination. METHODS: From our own series, we include 43 tumors from 13 patients including 14 primaries, 10 regional lymph node metastases, 17 distant metastases, and two anastomotic recurrences sequenced using the 50 gene Ion AmpliSeq cancer NGS panel v2. RESULTS: Based on our new cohort and a meta-analysis, we found that ~ 77% of patient-matched primary-metastatic pairs have identical alterations in these 50 cancer-associated genes. CONCLUSIONS: Low tumor cellularity, tumor heterogeneity, clonal evolution, treatment status, sample quality, and/or size of the sequencing panel accounted for a proportion of the differential detection of mutations at primary and metastatic sites. The therapeutic implications of the most frequently discordant alterations (TP53, APC, PIK3CA, and SMAD4) are discussed. Our meta-analysis indicates that a subset of patients who fail initial therapy may benefit from sequencing of additional sites to identify new actionable genomic abnormalities not present in the initial analysis. Evidence-based recommendations are proposed.
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Recent work identifies the AKT kinase as a potential mediator of tumor expansion in multiple myeloma. The finding of PTEN mutations in several myeloma cell lines suggests that loss of PTEN function may be one mechanism by which AKT activity is increased in this disease. Because PTEN-deficient myeloma cells may have up-regulated activity of the mammalian target of rapamycin (mTOR), downstream of AKT, they may be particularly sensitive to mTOR inhibition. To test this hypothesis, we challenged myeloma cell lines with CCI-779, a newly developed analogue of rapamycin and an efficient inhibitor of mTOR. Three of four PTEN-deficient cell lines with constitutively active AKT were remarkably sensitive to cytoreduction and G(1) arrest induced by CCI-779 with ID(50) concentrations of <1 nM. In contrast, myeloma cells expressing wild-type PTEN were >1000-fold more resistant. Acute expression of a constitutively active AKT gene in CCI-779-resistant myeloma cells containing wild-type PTEN and quiescent AKT did not convert them to the CCI-779-sensitive phenotype. Conversely, expression of wild-type PTEN in CCI-779-sensitive, PTEN-deficient myeloma cells did not induce resistance. Differential sensitivity did not appear to be due to differences in the ability of CCI-779 to inhibit mTOR and induce dephosphorylation of p70S6kinase or 4E-BP1. However, CCI-779 inhibited expression of c-myc in CCI-sensitive PTEN-null myeloma cells but had no effect on expression in CCI-resistant cells. In contrast, cyclin D1 expression was not altered in either sensitive or resistant cells. These results indicate that PTEN-deficient myeloma cells are remarkably sensitive to mTOR inhibition. Although the results of transfection studies suggest that the level of PTEN and AKT function per se does not regulate sensitivity, PTEN/AKT status may be a good predictive marker of sensitivity.
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Antibióticos Antineoplásicos/farmacología , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/genética , Mutación , Monoéster Fosfórico Hidrolasas/genética , Proteínas Quinasas , Proteínas Serina-Treonina Quinasas , Sirolimus/análogos & derivados , Sirolimus/farmacología , Proteínas Supresoras de Tumor/genética , Proteínas Adaptadoras Transductoras de Señales , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular , Ciclina D1/biosíntesis , Activación Enzimática , Humanos , Mieloma Múltiple/metabolismo , Fosfohidrolasa PTEN , Fosfoproteínas/metabolismo , Monoéster Fosfórico Hidrolasas/deficiencia , Fosforilación , Inhibidores de Proteínas Quinasas , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Proteínas Proto-Oncogénicas c-myc/biosíntesis , Proteínas Quinasas S6 Ribosómicas/metabolismo , Serina-Treonina Quinasas TOR , Proteínas Supresoras de Tumor/deficienciaRESUMEN
Smad proteins transduce signals carried by the transforming growth factor beta (TGF-beta) cytokine superfamily from receptor serine/threonine kinases at the cell surface to the nucleus, thereby affecting cell proliferation, differentiation, as well as pattern formation during early vertebrate development. Smad4/DPC4, located at chromosome 18q21, was identified as a candidate tumor suppressor gene that is inactivated in nearly half of all pancreatic carcinomas. For functional characterization of Smad4, a recombinant adenovirus encoding Smad4 (Ad-Smad4) was generated. When Smad4 was expressed in Smad4-null breast carcinoma cell line MDA-MB-468 using the recombinant adenovirus, TGF-beta signaling was restored as determined by TGF-beta-dependent activity of plasminogen activator inhibitor 1 promoter and p21 expression. Infection with Ad-Smad4 in the presence of TGF-beta1 also resulted in an altered cell morphology that coincided with enhanced beta1 integrin expression and reduced efficiency of colony formation in soft agar. In agreement with increased p21 expression, Smad4-expressing cells showed modest reduction in S phase. However, Smad4 expression did not lead to induction of apoptosis under normal culture conditions. Interestingly, when Smad4-expressing cells were detached and incubated in suspension, they underwent rapid apoptosis in a TGF-beta-dependent manner. Induction of apoptosis caused by loss of anchorage is known as anoikis. Anoikis is believed to prevent colonization elsewhere of detached cells. Additional characterization revealed an increase in the level of focal adhesion kinase 2 (or Pyk2) and activation of caspases 2, 3, 6, and 8 during anoikis because of Smad4 expression and restoration of TGF-beta signaling. Because resistance to anoikis in tumor cells is thought to contribute to metastasis, our data suggest a functional basis for the strong correlation between defects in Smad4 and development of malignancy.
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Anoicis/fisiología , Neoplasias de la Mama/patología , Proteínas de Unión al ADN/fisiología , Transactivadores/fisiología , Factor de Crecimiento Transformador beta/fisiología , Adenoviridae/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Caspasas/metabolismo , División Celular/fisiología , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/genética , Activación Enzimática , Quinasa 2 de Adhesión Focal , Humanos , Integrina beta1/biosíntesis , Proteínas Tirosina Quinasas/biosíntesis , Transducción de Señal/fisiología , Proteína Smad4 , Transactivadores/biosíntesis , Transactivadores/genética , Transfección , Células Tumorales CultivadasRESUMEN
Approximately 30-40% of malignant glial tumors exhibit mutations in the tumor suppressor gene, PTEN/MMAC. Additionally, these tumors are associated with (a) mutations in epidermal growth factor receptor (EGFR), leading to a pro-oncogenic constitutive activation, as well as amplification of its gene, and/or (b) mutations in p53, disrupting normal cellular homeostatic processes. Whereas PTEN/MMAC has been shown to possess antiangiogenic action, constitutively active EGFR or p53 gene defects have been associated with proangiogenic action. In this article, we asked if PTEN/MMAC gene transfer into human glioma cells that possess inactivating mutations of the PTEN/MMAC gene but also express either constitutively active EGFR (U87DeltaEGFR cells) or possess an inactivating mutation of p53 (U251 cells) still display inhibited angiogenesis in orthotopic and ectopic models of gliomas. Human glioma xenografts treated with PTEN/MMAC gene transfer exhibited significantly decreased vascularity both in an orthotopic and in an ectopic model. Taken in combination, these results provide strong evidence of PTEN/MMAC's role in regulating glioma angiogenesis even in the presence of strong proangiogenic signals provided by constitutive EGFR activation or p53 inactivation.
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Neoplasias Encefálicas/irrigación sanguínea , Terapia Genética/métodos , Glioma/irrigación sanguínea , Neovascularización Patológica/terapia , Monoéster Fosfórico Hidrolasas/genética , Proteínas Supresoras de Tumor/genética , Animales , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/terapia , Receptores ErbB/biosíntesis , Receptores ErbB/genética , Receptores ErbB/fisiología , Femenino , Vectores Genéticos/genética , Glioma/genética , Glioma/terapia , Humanos , Neovascularización Patológica/genética , Neovascularización Patológica/metabolismo , Fosfohidrolasa PTEN , Monoéster Fosfórico Hidrolasas/biosíntesis , Ratas , Ratas Desnudas , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/fisiología , Proteínas Supresoras de Tumor/biosíntesis , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Human tumor xenografts established in athymic rat brains were used to determine the feasibility of intravascular delivery of tumor suppressor genes to brain tumors. Both tumor size and number were compared to characterize the effect of tumor burden on tumor transduction efficacy by a control LacZ-containing adenoviral vector. Experiments with tumors grown in vivo for either 3, 5, or 7 days demonstrated that 5-day-old tumors provided the best target for vector infection and transgene expression by this mode of administration. Intra-arterial mannitol facilitated transduction efficiency. Tumor burden did not seem to affect transduction, while tumor location appeared to be an important factor. Based on these results, intra-arterial infusion of a p53-containing adenoviral vector was carried out and resulted in significant retardation of brain tumor growth 3 days after administration. Effects at longer time points were not as significant. These findings indicate that intra-arterial administration of adenoviral vectors containing p53 is efficient and can result in changes in tumor size, but that long-term control of tumor growth may require multiple adenoviral treatments.