Impact of Smad4 and p53 mutations on the prognosis of patients with pancreatic ductal adenocarcinoma undergoing chemotherapy.

BACKGROUND: This prospective cohort study evaluated the feasibility of using endoscopic ultrasound-guided fine-needle biopsy (EUS-FNB) samples for comprehensive mutational analysis of cancer-related genes using microtissues. METHODS: Fifty patients with suspected pancreatic cancer presenting consecutively at the Kindai University Hospital between January 2018 and January 2019 were enrolled. Cancerous tissues from EUS-FNB were obtained from each tumor and subjected to histological examination and mutational analysis. The primary endpoint was the collection rate of EUS-FNB specimens suitable for comprehensive cancer panels using deep sequencing. Clinical history and genetic variations between the disease control and progressive disease groups of patients on chemotherapy were evaluated as secondary endpoints. RESULTS: The collection rate of EUS-FNB specimens suitable for comprehensive cancer panels using deep sequencing was 93.6%. The cancer panel was sequenced for 25 patients with pancreatic cancer treated initially with systemic chemotherapy. Mutation in p53 and Smad4 were positively and negatively associated, respectively, with disease control at the initial evaluation. The median time to progression in 15 patients with p53 and without Smad4 mutations was 182.0 days; whereas, it was 92.5 days in other 10 patients; this difference was significant (p = 0.020). CONCLUSIONS: Tissue samples from EUS-FNB were suitable for mutational analysis. Pancreatic cancers with p53 and without Smad4 mutations responded better to chemotherapy and had a better prognosis than those others.

Impact of coexisting gene mutations in EGFR-mutated non-small cell lung cancer before treatment on EGFR T790M mutation status after EGFR-TKIs.

OBJECTIVES: The T790M secondary mutation of epidermal growth factor receptor gene (EGFR) is the most common mechanism of acquired resistance to first- or second-generation EGFR tyrosine kinase inhibitors (TKIs). We investigated the association between gene mutation profile in EGFR mutation-positive non-small cell lung cancer (NSCLC) before EGFR-TKI treatment and T790M status after EGFR-TKI treatment. MATERIALS AND METHODS: A total of 57 EGFR mutation-positive NSCLC patients who had undergone a repeat biopsy (tissue or liquid) after failure of treatment with a first- or second-generation EGFR-TKI and who had sufficient tumor tissue available from before treatment for genetic analysis was enrolled. The gene mutation profile of tumor tissue obtained before EGFR-TKI treatment was evaluated by next-generation sequencing with a comprehensive cancer gene panel (409 genes). The number of potentially damaging nonsynonymous mutations was predicted with PolyPhen-2 software. RESULTS: Progression-free survival during EGFR-TKI treatment did not differ significantly between patients who developed T790M-mediated resistance and those who developed T790M-independent resistance. The predicted number of damaging nonsynonymous mutations in pretreatment tumor tissue was significantly lower in patients who developed T790M-mediated resistance than in those with T790M-independent resistance (P =  0.049). CONCLUSIONS: Coexisting mutations in tumor tissue before EGFR-TKI treatment may contribute to the emergence of cell clones responsible for development of T790M-dependent or T790M-independent TKI resistance in patients with EGFR-mutated NSCLC. Multiplex genomic testing of pretreatment tumor tissue may thus provide a means of identifying patients likely to develop T790M-mediated TKI resistance and therefore inform treatment selection.

Patients with SMARCA4-deficient thoracic sarcoma and severe skeletal-related events.

OBJECTIVES: SMARCA4-deficient thoracic sarcoma(DTS) is a recently identified new entity of thoracic malignancies characterized by inactivation of SMARCA4. Patients with SMARCA4-DTS have a particulary aggresive clinical course and no effective treatments. However, the detailed clinical features of SMARCA4-DTS remain unclear. Here, we report the clinical courses and molecular profiles of two cases of SMARCA4-DTS. MATERIALS AND METHODS: We experienced strikingly similar two patients of SMARCA4-DTS. The clinicopathologic features were reviewed, and detailed immunohistochemical and comprehensive cancer panel analysis with next generation sequencing confirmed the diagnosis. RESULTS: Our cases had many clinical and radiological observations characteristic of SMARCA4-DTS in common. Immunohistochemical staing showed complete loss of SMARCA4 in tumor cells. Loss of function mutations were detected in SMARCA4. We found that severe SREs comprise a new significant clinical feature of SMARCA4-DTS. CONCLUSION: Integrated clinico-radiologic-pathologic-genetic diagnosis is essential for SMARCA4-DTS and physicians should pay attention to severe SREs during the clinical course of this disease.

Increased FGF19 copy number is frequently detected in hepatocellular carcinoma with a complete response after sorafenib treatment.

The multi-kinase inhibitor sorafenib is clinically approved for the treatment of patients with advanced hepatocellular carcinoma (HCC). We previously reported that fibroblast growth factor 3 and 4 (FGF3/FGF4) amplification is a predictor of a response to sorafenib. This study aims to analyze the relationship between FGF-FGF receptor (FGFR) genetic alterations and the response to sorafenib. Formalin-fixed, paraffin-embedded tissue specimens from HCC patients who had achieved a complete response (CR, N=6) or non-CR (N=39) to sorafenib were collected and were examined for FGF-FGFR gene alterations using next generation sequencing and copy number assay. FGFR mutations were detected in 5 of 45 (11.1%) cases. There was no significant association between FGFR mutation status and the response to sorafenib. We detected no increase in the FGF3/FGF4 copy number in CR cases. An FGF19 copy number gain was detected more frequently among CR cases (2/6, 33.3%) than among non-CR cases (2/39, 5.1%) (P = 0.024, Chi-squared test). In conclusion, a copy number gain for FGF19 may be a predictor of a response to sorafenib, in addition to FGF3/FGF4 amplification.

[Kinase inhibitors and their resistance].

Kinase cascades are involved in all stages of tumorigenesis through modulation of transformation and differentiation, cell-cycle progression, and motility. Advances in molecular targeted drug development allow the design and synthesis of inhibitors targeting cancer-associated signal transduction pathways. Potent selective inhibitors with low toxicity can benefit patients especially with several malignancies harboring an oncogenic driver addictive signal. This article evaluates information on solid tumor-related kinase signals and inhibitors, including receptor tyrosine kinase or serine/threonine kinase signals that lead to successful application in clinical settings. In addition, the resistant mechanisms to the inhibitors is summarized.

Targeted DNA and RNA sequencing of fine-needle biopsy FFPE specimens in patients with unresectable hepatocellular carcinoma treated with sorafenib.

The multi-kinase inhibitor sorafenib is now used as standard therapy for advanced hepatocellular carcinoma (HCC). Predictive biomarkers of response to sorafenib are thus necessary. The purpose of this study was to assess the feasibility of using targeted DNA and RNA sequencing to elucidate candidate biomarkers of sorafenib response using fine-needle biopsy, formalin-fixed paraffin-embedded (FFPE) specimens in patients with HCC. Targeted DNA and RNA deep sequencing were feasible for the evaluation of fine-needle biopsy FFPE specimens obtained from 46 patients with HCC treated with sorafenib. Frequent mutations of suppressor genes, such as CTNNB1 (34.8%) and TP53 (26.1%), were detected in the HCC tumors. After excluding these suppressor genes, the average numbers of detected oncogene mutations differed significantly between the non-PD and PD groups (P = 0.0446). This result suggests that the oncogene mutational burden in the tumor might be associated with the clinical response to sorafenib. We have identified candidate gene expression (TGFa, PECAM1, and NRG1) in tumor for the prediction of sorafenib response and PFS by RNA sequencing. Our findings provide new insights into biomarkers for sorafenib therapy and allow us to discuss future therapeutic strategies.

Evaluation of in vivo responses of sorafenib therapy in a preclinical mouse model of PTEN-deficient of prostate cancer.

BACKGROUND: Despite recent advances in the treatment for advanced prostate cancer, outcomes remain poor. This lack of efficacy has prompted the development of alternative treatment strategies. In the present study we investigate the effects of the multikinase inhibitor sorafenib in a genetically engineered mouse model of prostate cancer and explore the rational combination with the mTOR inhibitor everolimus. METHODS: Conditional prostate specific PTEN-deficient knockout mice were utilized to determine the pharmacodynamic and chemopreventive effects of sorafenib. This mouse model was also used to examine the therapeutic efficacy of sorafenib alone or in combination with everolimus. Preclinical efficacy was assessed by comparing the reduction of tumor burden, proliferation, angiogenesis and the induction of apoptosis. Molecular responses were assessed by immunohistochemical, TUNEL and western blot assays. RESULTS: Pharmacodynamic analysis revealed that a single dose of sorafenib decreased activation of the PI3K/AKT/mTOR signaling axis at doses of 30-60 mg/kg, but activated JAK/STAT3 signaling. Levels of cleaved casapase-3 increased in a dose dependent manner. Chemoprevention studies showed that chronic sorafenib administration was capable of inhibiting tumor progression through the reduction of cancer cell proliferation, angiogenesis and the induction of apoptosis. In intervention models of established castration-naïve and castration-resistant prostate cancer, treatment with sorafenib provided modest but statistically insignificant reduction in tumor burden. However, sorafenib significantly inhibited cancer cell proliferation and MVD but had minimal effects on the induction of apoptosis. Interestingly, the administration of sorafenib increased the expression levels of the androgen receptor, p-GSK3ß and p-ERK1/2 in castration-resistant prostate cancers. In both intervention models, combination therapy demonstrated a clear tendency of enhanced antitumor effects over monotherapy. Notably, the treatment combination of sorafenib and everolimus overcame therapeutic escape from single agent therapy in castration-resistant prostate cancers. CONCLUSIONS: In summary, we provide insights into the molecular responses of sorafenib therapy in a clinically relevant model of prostate cancer and present preclinical evidence for the development of targeted treatment strategies based on the use of multikinase inhibitors in combination with mTOR inhibitors for the treatment of advanced prostate cancer.

Chemoradiation provides a physiological selective pressure that increases the expansion of aberrant TP53 tumor variants in residual rectal cancerous regions.

Neoadjuvant chemoradiotherapy has been introduced in patients with surgically resected rectal cancer and reduced the local recurrence. Heterogeneity exists in rectal cancer, and we hypothesized that there are subclones resistant to chemoradiotherapy within the cancer mass. We performed DNA-targeted sequencing of pre- and post-treatment tumor tissues obtained from 20 rectal cancer patients who received chemoradiotherapy. The variant frequency of the mutant clones was compared between pre- and post-treatment samples of nine non-responder patients. RNA-targeted sequencing of 57 genes related to sensitivity to chemotherapy and radiotherapy was performed for the paired samples. Immunohistochemical analyses of p53 expression were also performed on the paired samples from the nine non-responder patients. DNA-sequencing detected frequent mutations of suppressor genes including TP53, APC and FBXW7 in the post-treatment samples of the nine non-responders. The frequency of TP53 mutations showed significant increases after chemoradiotherapy. RNA-targeted sequencing of 29 tumor tissues demonstrated that decreased expression of three genes and increased expression of four genes were detected in the post-treatment samples. Significantly increased expression of TP53 was observed in the post-treatment samples. Immunohistochemical staining for p53 revealed that increased p53 intensity scores were observed after chemoradiotherapy. These results suggest that the tumors with TP53 mutations tend to accumulate through chemoradiotherapy.

FGF3/FGF4 amplification and multiple lung metastases in responders to sorafenib in hepatocellular carcinoma.

UNLABELLED: The response rate to sorafenib in hepatocellular carcinoma (HCC) is relatively low (0.7%-3%), however, rapid and drastic tumor regression is occasionally observed. The molecular backgrounds and clinico-pathological features of these responders remain largely unclear. We analyzed the clinical and molecular backgrounds of 13 responders to sorafenib with significant tumor shrinkage in a retrospective study. A comparative genomic hybridization analysis using one frozen HCC sample from a responder demonstrated that the 11q13 region, a rare amplicon in HCC including the loci for FGF3 and FGF4, was highly amplified. A real-time polymerase chain reaction-based copy number assay revealed that FGF3/FGF4 amplification was observed in three of the 10 HCC samples from responders in which DNA was evaluable, whereas amplification was not observed in 38 patients with stable or progressive disease (P = 0.006). Fluorescence in situ hybridization analysis confirmed FGF3 amplification. In addition, the clinico-pathological features showed that multiple lung metastases (5/13, P = 0.006) and a poorly differentiated histological type (5/13, P = 0.13) were frequently observed in responders. A growth inhibitory assay showed that only one FGF3/FGF4-amplified and three FGFR2-amplified cancer cell lines exhibited hypersensitivity to sorafenib in vitro. Finally, an in vivo study revealed that treatment with a low dose of sorafenib was partially effective for stably and exogenously expressed FGF4 tumors, while being less effective in tumors expressing EGFP or FGF3. CONCLUSION: FGF3/FGF4 amplification was observed in around 2% of HCCs. Although the sample size was relatively small, FGF3/FGF4 amplification, a poorly differentiated histological type, and multiple lung metastases were frequently observed in responders to sorafenib. Our findings may provide a novel insight into the molecular background of HCC and sorafenib responders, warranting further prospective biomarker studies.

Loss of activating EGFR mutant gene contributes to acquired resistance to EGFR tyrosine kinase inhibitors in lung cancer cells.

Non-small-cell lung cancer harboring epidermal growth factor receptor (EGFR) mutations attains a meaningful response to EGFR-tyrosine kinase inhibitors (TKIs). However, acquired resistance to EGFR-TKIs could affect long-term outcome in almost all patients. To identify the potential mechanisms of resistance, we established cell lines resistant to EGFR-TKIs from the human lung cancer cell lines PC9 and11-18, which harbored activating EGFR mutations. One erlotinib-resistant cell line from PC9 and two erlotinib-resistant cell lines and two gefitinib-resistant cell lines from 11-18 were independently established. Almost complete loss of mutant delE746-A750 EGFR gene was observed in the erlotinib-resistant cells isolated from PC9, and partial loss of the mutant L858R EGFR gene copy was specifically observed in the erlotinib- and gefitinib-resistant cells from 11-18. However, constitutive activation of EGFR downstream signaling, PI3K/Akt, was observed even after loss of the mutated EGFR gene in all resistant cell lines even in the presence of the drug. In the erlotinib-resistant cells from PC9, constitutive PI3K/Akt activation was effectively inhibited by lapatinib (a dual TKI of EGFR and HER2) or BIBW2992 (pan-TKI of EGFR family proteins). Furthermore, erlotinib with either HER2 or HER3 knockdown by their cognate siRNAs also inhibited PI3K/Akt activation. Transfection of activating mutant EGFR complementary DNA restored drug sensitivity in the erlotinib-resistant cell line. Our study indicates that loss of addiction to mutant EGFR resulted in gain of addiction to both HER2/HER3 and PI3K/Akt signaling to acquire EGFR-TKI resistance.

Identification of c-Src as a potential therapeutic target for gastric cancer and of MET activation as a cause of resistance to c-Src inhibition.

Therapeutic strategies that target c-Src hold promise for a wide variety of cancers. We have now investigated both the effects of dasatinib, which inhibits the activity of c-Src and several other kinases, on cell growth as well as the mechanism of dasatinib resistance in human gastric cancer cell lines. Immunoblot analysis revealed the activation of c-Src at various levels in most gastric cancer cell lines examined. Dasatinib inhibited the phosphorylation of extracellular signal-regulated kinase (ERK) and induced G(1) arrest, as revealed by flow cytometry, in a subset of responsive cell lines. In other responsive cell lines, dasatinib inhibited both ERK and AKT phosphorylation and induced apoptosis, as revealed by an increase in caspase-3 activity and cleavage of poly(ADP-ribose) polymerase. Depletion of c-Src by RNA interference also induced G(1) arrest or apoptosis in dasatinib-responsive cell lines, indicating that the antiproliferative effect of dasatinib is attributable to c-Src inhibition. Gastric cancer cell lines positive for the activation of MET were resistant to dasatinib. Dasatinib had no effect on ERK or AKT signaling, whereas the MET inhibitor PHA-665752 induced apoptosis in these cells. The subsets of gastric cancer cells defined by a response to c-Src or MET inhibitors were distinct and nonoverlapping. Our results suggest that c-Src is a promising target for the treatment of gastric cancer and that analysis of MET amplification might optimize patient selection for treatment with c-Src inhibitors.

Differential gene expression profiles and identification of the genes relevant to clinicopathologic factors in colorectal cancer selected by cDNA array method in combination with principal component analysis.

The clinical outcome of patients with colorectal cancer frequently varies even if they are at the same clinicopathologic stage. Alternative superior tumor markers of colorectal cancer are needed for prediction of clinical outcome. To clarify the regulatory factors in colorectal cancers, we examined differential expression profiles using cDNA macroarray technique with surgically resected specimens obtained from the patients with colorectal cancer. The gene profiles by an average-linkage hierarchical clustering analysis were found to be almost separable into two groups: tumor group and normal mucosa group. The relationship between several clinicopathologic factors and cancer related genes were investigated by using statistical analyses including principal component analysis (PCA). c-myc-binding protein MM-1, and c-jun proto-oncogene were identified as possible markers of tumor histology and clinical prognosis and early growth response protein 1 (EGR1) was selected to play an important role in progression of clinical stage. We conclude that, with PCA method, we successfully selected the genes relevant to clinicopathologic factors using limited population of clinical samples.