Jianfei Wang

Effectors of Epidermal Growth Factor Receptor Pathway: The Genetic Profiling of KRAS, BRAF, PIK3CA, NRAS Mutations in Colorectal Cancer Characteristics and Personalized Medicine

Mutations in KRAS oncogene are recognized biomarkers that predict lack of response to anti- epidermal growth factor receptor (EGFR) antibody therapies. However, some patients with KRAS wild-type tumors still do not respond, so other downstream mutations in BRAF, PIK3CA and NRAS should be investigated. Herein we used direct sequencing to analyze mutation status for 676 patients in KRAS (codons 12, 13 and 61), BRAF (exon 11 and exon 15), PIK3CA (exon 9 and exon 20) and NRAS (codons12, 13 and 61). Clinicopathological characteristics associations were analyzed together with overall survival (OS) of metastatic colorectal cancer patients (mCRC). We found 35.9% (242/674) tumors harbored a KRAS mutation, 6.96% (47/675) harbored a BRAF mutation, 9.9% (62/625) harbored a PIK3CA mutation and 4.19% (26/621) harbored a NRAS mutation. KRAS mutation coexisted with BRAF, PIK3CA and NRAS mutation, PIK3CA exon9 mutation appeared more frequently in KRAS mutant tumors (P = 0.027) while NRAS mutation almost existed in KRAS wild-types (P<0.001). Female patients and older group harbored a higher KRAS mutation (P = 0.018 and P = 0.031, respectively); BRAF (V600E) mutation showed a higher frequency in colon cancer and poor differentiation tumors (P = 0.020 and P = 0.030, respectively); proximal tumors appeared a higher PIK3CA mutation (P<0.001) and distant metastatic tumors shared a higher NRAS mutation (P = 0.010). However, in this study no significant result was found between OS and gene mutation in mCRC group. To our knowledge, the first large-scale retrospective study on comprehensive genetic profile which associated with anti-EGFR MoAbs treatment selection in East Asian CRC population, appeared a specific genotype distribution picture, and the results provided a better understanding between clinicopathological characteristics and gene mutations in CRC patients.

Antitumor Effects of a Novel Chromosome Region Maintenance 1 (CRM1) Inhibitor on Non-Small Cell Lung Cancer Cells In Vitro and in Mouse Tumor Xenografts

Background Chromosome Region Maintenance 1 (CRM1) is a nuclear exporter and its inhibitor has anti-tumor activity in various cancers. This study assessed the therapeutic efficiency of the novel CRM1 inhibitor KPT-185 on non-small cell lung cancer (NSCLC). Methods NSCLC cell lines were treated with KPT-185 to assess changes in cell viability, cell cycle, apoptosis, and protein expression. NOD-SCID mice carrying NSCLC cell xenografts were orally treated with KPT-276, a clinical analog of KPT-185, to examine the efficacy and side effects of KPT-276 in vivo. Results KPT-185 significantly reduced the viability of six NSCLC cell lines in a time- and dose-dependent manner, including epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI)-resistant H1975 and H1650GR cell lines. In addition, KPT-185 induced these NSCLC cells to arrest at G1 phase of the cell cycle and caused apoptosis in a dose-dependent manner. KPT-185 treatment also reduced CRM1 protein levels in six NSCLC cell lines, and the reduction could be completely abolished by the proteasome inhibitor bortezomib. KPT-185 activated caspase 3, 8, and 9, but inhibited survivin expression in NSCLC cells. In a mouse H1975 cell xenograft model, tumor growth was significantly inhibited by oral KPT-276 administration, and there was no significant mouse body weight loss or other side effects. Conclusions The current study demonstrated the anti-tumor effects of KPT-185 in NSCLC cells, including EGFR-TKI-resistant NSCLC cell lines. Further studies will assess anti-tumor activity of KPT-185 in a clinical trial for NSCLC patients.

Direct sequencing is a reliable assay with good clinical applicability for KRAS mutation testing in colorectal cancer

BACKGROUND The analysis of KRAS mutations in colorectal cancer (CRC) has made the need urgent for a reliable and easy to implement assay in daily practice. OBJECTIVE This study was designed to compare the different assays for KRAS testing and elucidate its mutation status in Chinese CRC patients. METHODS Direct sequencing was conducted to detect mutations in KRAS codons 12, 13 and 61 using 574 colorectal paraffin embedded clinical samples. And a subset of 66 samples was further detected independently by a commercial kit for comparison of different assays. RESULTS KRAS codons 12 and 13 mutations were detected in 40.9 and 42.4% of 66 CRC samples using the kit and direct sequencing methods, respectively. The concordance between two methods reached 95.5% (Kappa=0.907, P < 0.001). Workload and time to results were comparable for both. Moreover, KRAS mutations were detected in the total 574 CRC tumors by direct sequencing as followed: 25.3% in codon 12, 6.8% in codon 13, and 2.1% in codon 61. Notably, the mutations were more frequent in females than males, and patients older than 60 years exhibited higher rates of mutation (P < 0.05). CONCLUSIONS Direct sequencing showed similar mutation rate as the detection kit and hence can be used effectively and reliably for clinical screening of somatic tumor gene mutations.

Prognostic impact of mutation profiling in patients with stage II and III colon cancer.

Development of colorectal cancer (CRC) associates with accumulation of genetic mutations include the epidermal growth factor receptor (EGFR) signaling pathway. However, whether mutations in KRAS together with downstream factors BRAF, PIK3CA and NRAS impact prognosis is still unclear for stage II-III colon cancer. In the present study a total of 228 stage II-III colon cancer samples were retrospectively collected, KRAS (codons 12, 13 and 61), BRAF (exon 11 and exon 15), PIK3CA (exon 9 and exon 20) and NRAS (codons 12, 13 and 61) status was detected by Sanger sequencing, 37.89% (86/227) tumors harbored a KRAS mutation, 7.02% (16/228) harbored a BRAF mutation, 13.18% (29/220) harbored a PIK3CA mutation and 0.89% (2/224) harbored a NRAS mutation. NRAS mutations existed only in stage II colon cancer. Older groups harbored a higher KRAS and BRAF mutation (P < 0.05), PIK3CA (exon9) mutations appeared more common in worse differentiation tumors (P = 0.032). Moreover, PIK3CA (E545K) mutation was significantly associated with tumor recurrence (P = 0.031) and acted independently prognostic for poor OS (P = 0.044), while only in stage III colon cancer. KRAS, BRAF and NRAS mutations do not have major prognostic value in stage II and III colon cancer, subtypes of gene mutations should be further investigated for a better understanding in CRC.

Oral topotecan: Bioavailability, pharmacokinetics and impact of ABCG2 genotyping in Chinese patients with advanced cancers

Oral topotecan (Hycamtin(®)) has been recently approved for the treatment of relapsed small cell lung cancer (SCLC) in 2007, however, the bioavailability and pharmacokinetic data of topotecan for Chinese patients is still limited. Xinze(®) is a new and the only capsule formulation of topotecan used in China that is similar to Hycamtin(®). The current study aimed to investigate the absolute bioavailability and pharmacokinetics of Xinze(®) in Chinese patients with advanced cancers. On day 1, an IV dose of 1.5 mg/m(2)/d as a 30 min continuous infusion was administered. Patients took the oral topotecan at one of two dose levels: 1.5 mg/m(2)/d (six patients) or 1.9 mg/m(2)/d (seven patients) on day 2. Plasma pharmacokinetics of total topotecan and topotecan in the lactone form were performed on both days using ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Single-nucleotide polymorphisms (SNPs) identified in exon 5 (421C>A) and in exon 2 (34G>A) in ATP-binding cassette sub-family G member 2 (ABCG2) were analyzed by direct sequencing. Safety assessments were performed throughout the study. The maximum plasma concentration (Cmax) reached at 1-2 h and the elimination half-life time (T1/2) was approximately 4.2 h after oral administration. The absolute bioavailability of total topotecan in the 1.5 mg/m(2)/d and 1.9 mg/m(2)/d groups averaged 41.23 ± 11.8% and 36.00 ± 14.8%, respectively. The patients with heterozygous SNPs had essentially the same bioavailability and pharmacokinetics. The bioavailability of topotecan after oral administration illustrates good systemic exposure at dosages of 1.5 mg/m(2)/d and 1.9 mg/m(2)/d over a five-day schedule in Chinese patients. On a dose-normalized basis, the values of Cmax and AUC0-t for total topotecan in Chinese patients were higher than in Caucasians following oral and intravenous administration, while the T1/2 was consistent.

The prognostic utility and the association of serum light chains (free and total) and absolute lymphocyte count in patients with newly diagnosed diffuse large B-cell lymphoma

In this study, serum free and total light chains (sFLC/sTLC) were measured in 108 serum samples of therapy-naïve patients with DLBCL. Clinicopathologic data and survival outcomes were analyzed according to the results of sFLC/sTLC measurements. Moreover, the association of sFLC/sTLC with absolute monocyte count (AMC) and absolute lymphocyte count (ALC) was evaluated. Elevated sFLC and abnormal κ/λ ratio was present in 42.6% (51/108) and 4.6% (5/108) of patients, respectively. sTLC was successfully measured in 107 serum samples, abnormal sTLC and abnormal κ/λ ratio was found in 28.0% (30/107) and 26.2% (28/107) of patients, respectively. Patients with elevated sFLC more frequently displayed adverse clinical characteristics, including age (P=0.001), B symptoms (P=0.022), low ALC (P=0.024) and hyperglobulinemia (P=0.012). Patients with elevated sFLC had an inferior overall survival (OS) (P=0.012) and tended to have shorter progression-free survival (PFS) (P=0.061) compared to patients with normal sFLC. Abnormal sTLC or abnormal sTLC ratio showed no significant association with clinical outcomes, with exception of abnormal concurrent κ and λ. Only association of sFLC and ALC with survival remained significant after adjusting for the International Prognostic Index (IPI). The measurement of sFLC and ALC at diagnosis might be useful for the prognostic stratification of patients and sTLC measurement was of little prognostic utility in DLBCL.