Shuhang Wang

Reproducible copy number variation patterns among single circulating tumor cells of lung cancer patients

Significance In a few milliliters of blood from a cancer patient, one can isolate a few circulating tumor cells (CTCs). Originating from the primary tumor, CTCs seed metastases, which account for the majority of cancer-related deaths. We demonstrate the analyses of the whole genome of single CTCs, which are highly needed for personalized treatment. We discovered that copy number variations (CNVs), one of the major genomic variations, are specific to cancer types, reproducible from cell to cell, and even from patient to patient. We hypothesize that CNVs at certain genomic loci are selected for and lead to metastasis. Our work shows the prospect of noninvasive CTC-based cancer diagnostics. Circulating tumor cells (CTCs) enter peripheral blood from primary tumors and seed metastases. The genome sequencing of CTCs could offer noninvasive prognosis or even diagnosis, but has been hampered by low single-cell genome coverage of scarce CTCs. Here, we report the use of the recently developed multiple annealing and looping-based amplification cycles for whole-genome amplification of single CTCs from lung cancer patients. We observed characteristic cancer-associated single-nucleotide variations and insertions/deletions in exomes of CTCs. These mutations provided information needed for individualized therapy, such as drug resistance and phenotypic transition, but were heterogeneous from cell to cell. In contrast, every CTC from an individual patient, regardless of the cancer subtypes, exhibited reproducible copy number variation (CNV) patterns, similar to those of the metastatic tumor of the same patient. Interestingly, different patients with the same lung cancer adenocarcinoma (ADC) shared similar CNV patterns in their CTCs. Even more interestingly, patients of small-cell lung cancer have CNV patterns distinctly different from those of ADC patients. Our finding suggests that CNVs at certain genomic loci are selected for the metastasis of cancer. The reproducibility of cancer-specific CNVs offers potential for CTC-based cancer diagnostics.

Quantification and dynamic monitoring of EGFR T790M in plasma cell-free DNA by digital PCR for prognosis of EGFR-TKI treatment in advanced NSCLC.

Background Among advanced non-small cell lung cancer (NSCLC) patients with an acquired resistance to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKI), about 50% carry the T790M mutation, but this frequency in EGFR-TKI-naïve patients and dynamic change during therapy remains unclear. This study investigated the quantification and dynamic change of T790M mutation in plasma cell-free DNA (cf-DNA) of advanced NSCLC patients to assess the clinical outcomes of EGFR-TKI therapy. Materials and Methods We retrospectively investigated 135 patients with advanced NSCLC who obtained progression-free survival (PFS) after EGFR-TKI for >6 months for their EGFR sensitive mutations and T790M mutation in matched pre- and post-TKI plasma samples, using denaturing high-performance liquid chromatography (DHPLC), amplification refractory mutation system (ARMS), and digital-PCR (D-PCR). Real-time PCR was performed to measure c-MET amplification. Results Detection limit of D-PCR in assessing the T790M mutation was approximately 0.03%. D-PCR identified higher frequency of T790M than ARMS in pre-TKI (31.3% vs. 5.5%) and post-TKI (43.0% vs. 25.2%) plasma samples. Patients with pre-TKI T790M showed inferior PFS (8.9 vs. 12.1 months, p = 0.007) and overall survival (OS, 19.3 vs. 31.9 months, p = 0.001) compared with those without T790M. In patients harboring EGFR sensitive mutation, high quantities of pre-TKI T790M predicted poorer PFS (p = 0.001) on EGFR-TKI than low ones. Moreover, patients who experienced increased quantity of T790M during EGFR-TKI treatment showed superior PFS and OS compared with those with decreased changes (p = 0.044 and p = 0.015, respectively). Conclusion Qualitative and quantitative T790M in plasma cf-DNA by D-PCR provided a non-invasive and sensitive assay to predict EGFR-TKI prognosis.

EGFR mutations are associated with prognosis but not with the response to front-line chemotherapy in the Chinese patients with advanced non-small cell lung cancer

The study aimed to investigate associations of tumor tissue EGFR mutations with response to the front-line chemotherapy and prognosis in advanced non-small cell lung cancer (NSCLC) patients. EGFR genotypes of 145 chemotherapy-naive patients with Stage IIIB and IV NSCLC were examined by using denaturing high-performance liquid chromatography (DHPLC). All patients received the front-line chemotherapy. There were 69 patients who received gefitinib therapy (32 as second-line and 37 as third-line therapy). About 37.9% (55/145) of the patients was detected to have EGFR mutations in their tumor tissue DNA. The response rate (RR, complete response plus partial response) to the chemotherapy for mutated EGFR carriers was 34.5% (19/55), similar to 33.3% (30/90) for wild-type EGFR carriers (P=0.881). The patients with EGFR mutations had increased median survival time and 1- and 2-year survival rate than those with wild-type EGFR (23 vs 16 months, 86.38% vs 62.64%, 38.78% vs 27.16%, P=0.0273). Among Stage IV NSCLC patients, mutated EGFR carriers had a longer progression-free survival (PFS) than wild-type EGFR carriers (5 vs 3 months, P=0.040). Cox multivariate regression analysis showed that response to the front-line chemotherapy (RR vs PD) and EGFR mutation were independent prognostic factors (HR=0.461, 95% CI: 0.271-0.783, P=0.0042; HR=0.598, 95% CI: 0.372-0.961, P=0.0335, respectively) for patients with advanced NSCLC. We conclude that EGFR mutations in the Chinese patients with advanced NSCLC were not associated with response to the front-line chemotherapy, but Stage IV NSCLC patients with mutated EGFR had a longer PFS after the front-line chemotherapy. EGFR mutation is an independent prognostic factor for Chinese advanced NSCLC.

EML4-ALK Rearrangement and Its Clinical Significance in Chinese Patients with Advanced Non-Small Cell Lung Cancer

Objective: To identify the clinicopathological characteristics and clinical outcomes of Chinese patients with non-small cell lung cancer (NSCLC) and to investigate possible associations of NSCLC with echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) and epidermal growth factor receptor (EGFR) mutations. Methods: Patients with stage IV NSCLC were screened for EML4-ALK rearrangement and EGFR mutations at the Peking University Cancer Hospital. EML4-ALK was identified using fluorescent in situ hybridization and confirmed by immunohistochemistry. EGFR mutations were determined using denaturing high-performance liquid chromatography. Results: The incidence of EML4-ALK was 9.7% (11/113). Patients with EML4-ALK were more likely to present the EGFR wild type (WT; p = 0.033). Response to EGFR-tyrosine kinase inhibitor (TKI) was similar between patients with EML4-ALK rearrangement and EGFR mutation (33.3 vs. 46.9%, p = 0.451), but progression-free survival (PFS) was inferior compared to those with EGFR mutation (2.1 vs. 8.8 months, p = 0.032), and similar to patients with WT/nonrearrangement (2.1 vs. 2.2 months, p = 0.696; and general p = 0.023 between the three cohorts). Moreover, 2 patients with concurrent EML4-ALK and EGFR mutations had superior PFS after EGFR-TKI compared to patients with single EML4-ALK rearrangement. Conclusions: Patients with EML4-ALK conferred similar objective response rates after EGFR-TKI although inferior PFS compared to those with EGFR mutation. Coexistence of EML4-ALK and EGFR mutation might represent a separate NSCLC genotype.

DNA Methylation status of Wnt antagonist SFRP5 can predict the response to the EGFR-tyrosine kinase inhibitor therapy in non-small cell lung cancer

BackgroundIt is well known that genetic alternation of epidermal growth factor receptor (EGFR) plays critical roles in tumorgenesis of lung cancer and can predict outcome of non-small-cell lung cancer treatment, especially the EGFR tyrosine-kinase inhibitors (EGFR-TKIs) therapy. However, it is unclear whether epigenetic changes such as DNA methylation involve in the response to the EGFR-TKI therapy.MethodsTumor samples from 155 patients with stages IIIB to IV NSCLC who received EGFR-TKI therapy were analyzed for DNA methylation status of Wnt antagonist genes, including SFRP1, SFRP2, SFRP5, DKK3, WIF1, and APC, using methylation specific PCR (MSP) method. EGFR mutations detections were performed in the same tissues samples using Denaturing High Performance Liquid Chromatography (DHPLC).ResultsWe found that Wnt antagonists tend to methylate simultaneously. Methylation of sFRP1 and sFRP5 are reversely correlated with EGFR mutation (P = 0.005, P = 0.011). However, no correlations of methylations of other Wnt antagonist genes with EGFR mutation were found. The patients with methylated SFRP5 have a significant shorter progression free survival than those with unmethylated SFRP5 in response to EGFR-TKI treatment (P = 0.002), which is independent of EGFR genotype.ConclusionsPatients with unmethylated SFRP5 are more likely to benefit from EGFR-TKI therapy.

Phosphorylated EGFR expression may predict outcome of EGFR-TKIs therapy for the advanced NSCLC patients with wild-type EGFR

BackgroundEGFR mutation is a strong predictive factor of EGFR-TKIs therapy. However, at least 10% of patients with EGFR wild-type are responsive to TKIs, suggesting that other determinants of outcome besides EGFR mutation might exist. We hypothesized that activation of phosphorylated EGFR could be a potential predictive biomarker to EGFR-TKIs treatment among patients in wild-type EGFR.MethodTotal of 205 stage IIIb and IV NSCLC patients, tissue samples of whom were available for molecular analysis, were enrolled in this study. The phosphorylation of EGFR at tyrosine 1068 (pTyr1068) and 1173 (pTyr1173) were assessed by immunohistochemistry, and EGFR mutations were detected by denaturing high performance liquid chromatograph (DHPLC).ResultsAmong 205 patients assessable for EGFR mutation and phosphorylation analysis, 92 (44.9%) were EGFR mutant and 165 patients (57.6%) had pTyr1173 expression. Superior progression-free survival (PFS) was seen after EGFR-TKIs therapy in patients with pTyr1068 expression compared to pTyr1068 negative ones (median PFS 7.0 months vs. 1.2 months, P < 0.001). Inversely, patients with pTyr1173 had a shorter PFS (4.8 months VS. 7.7 months, P = 0.016). In subgroup of patients with wild-type EGFR, pTyr1068 expression positive ones had a significantly prolonged PFS (4.2 months vs.1.2 months P < 0.001) compared with those without pTyr1068 expression. Sixteen patients with both wild-type EGFR and pTyr1068 who responded to EGFR-TKIs had median PFS of 15.6 months (95%CI: 7.28-23.9).ConclusionpTyr1068 may be a predictive biomarker for screening the population for clinical response to EGFR-TKIs treatment; especially for patients with wild-type EGFR.

The detection of EGFR mutation status in plasma is reproducible and can dynamically predict the efficacy of EGFR-TKI

Background:  The validity of epidermal growth factor receptor (EGFR) mutation in serum and plasma DNA as a surrogate of tumor tissue has been comprehensively explored. However, the concordance between peripheral blood and tumor tissue samples in EGFR mutation detection remains variable. The question as to whether real‐time samples for EGFR mutation analysis are required before epidermal growth factor receptor tyrosine kinase inhibitor (EGFR‐TKI) therapy remains unanswered.

Carcinosarcoma of pancreas with liver metastasis combined with gastrointestinal stromal tumour of the stomach: is there a good prognosis with the complete resection?

We report a carcinosarcoma of the pancreas with liver metastasis combined with gastrointestinal stromal tumour (GIST) of the stomach in a 72-year-old woman who presented with right upper quadrant pain, nausea and vomiting. A radical resection including pancreaticoduodenectomy, left hepatic lobe resection and local resection of the gastric mass was performed. The tumour in the head of pancreas was found to be grossly yellow-white, and it infiltrated the common bile duct and duodenum; the mass of the liver metastasis is solitary. Pathologic examination showed two components separated from each other: one component was a conventional infiltrating pancreatic ductal adenocarcinoma, and the other component showed sarcomatous growth pattern composed of pleomorphic spindle cells. The neoplasm of the stomach was confirmed a low malignant potential GIST by histology and immunohistochemistry. The patient was obliged to be in hospital because of abnormal bowel function; moreover, surveillance CT scans performed at 1.5 months post-operatively showed multiple liver metastasis and recurrence in the tail of pancreas. Unfortunately, the patient died of multiple organ failure at 2 months post-operatively. To our knowledge, this is the first experience report about surgical treatment of carcinosarcoma of pancreas with liver metastasis combined with GIST of the stomach. The patient performed a radical surgery for the metastatic carcinosarcoma even if that could be resected completely did not have a good consequence.

Prospective controlled study of the safety and oncological outcomes of ELAPE procure with definitive anatomic landmarks versus conventional APE for lower rectal cancer

BACKGROUND The use of extra-levator abdominoperineal resection (ELAPE) procedure for lower rectal cancer is controversial. It is unclear whether the ELAPE procedure could improve surgical safety and lead to better oncological outcomes. METHODS Sixty-nine lower rectal cancer patients who underwent ELAPE (36 cases) or conventional abdominoperineal resection (APE; 33 cases) between June 2011 and February 2013 were prospectively investigated. Clinicopathological variables including blood loss, intraoperative perforation (IOP) rate, circumferential resection margin (CRM) involvement, lymph node harvest, the postoperative complications, urinary and sexual function, quality of life (QOL), local recurrence rate and survival were recorded and compared. RESULTS Blood loss (P = 0.021), perineal wound complication (P = 0.039), IOP rate (P = 0.028), local recurrence (P = 0.034) were significantly less frequent in the ELAPE group. There was greater CRM involvement in the conventional APE group but no statistical difference between the two groups. Urinary function, sexual function and QOL were not significantly different between the two groups. Overall survival and progression-free survival were not significantly different between two groups, even when survival was analyzed according to TNM stage, T stage, N stage, and with or without neoadjuvant chemoradiotherapy. In patients who underwent ELAPE there was no statistical difference in postoperative complications between younger and elderly patients (age ≥60). CONCLUSIONS ELAPE procedure with definitive anatomic landmarks demonstrated surgical safety and decreased local recurrence for lower rectal cancer patients including the elderly, but there were no survival improvements in compared to conventional APE procedure.

Alterations in EGFR and Related Genes following Neo-Adjuvant Chemotherapy in Chinese Patients with Non-Small Cell Lung Cancer

Introduction Genetic aberrancies within epidermal growth factor receptor (EGFR) pathway are associated with therapeutic outcomes of EGFR-tyrosine kinase inhibitors (TKIs) in advanced non-small cell lung cancer (NSCLC). However, the impact of chemotherapy on EGFR-related genes alterations has not been defined in NSCLC. Our study aims to investigate the impact of neoadjuvant chemotherapy (Neoadj-Chemo) on EGFR activating mutations and associated EGFR-TKIs resistance-related genes. Patients and Methods Matched tumor samples were obtained retrospectively from 66 NSCLC patients (stages IIb–IIIb) corresponding to pre- and post- Neoadj-Chemo. EGFR mutations were detected by denaturing high performance liquid chromatography (DHPLC) and confirmed by Amplification Refractory Mutation System technology (ARMS), KRAS mutations, T790M mutation and c-MET amplification were identified using Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP), ARMS, and real-time PCR, respectively. Results Before Neoadj-Chemo, EGFR mutations were identified in 33.3% (22/66) of NSCLC patients. Only 18.2% (12/66) of patients carried EGFR mutations after Neoadj-Chemo (p = 0.013). The median peak value of EGFR 19 exon mutations decreased non-significantly after Neoadj-Chemo. KRAS mutation rate decreased from 4.6% (3/66) to 3.0% (2/66) with Neoadj-Chemo. Although the overall percentage of patients exhibiting c-MET amplifications (6.1% [4/66]) did not change with Neoadj-Chemo, two patients transitioned from negative to positive c-MET amplification, and two patients reversed these changes post-Neoadj-Chemo. T790M mutations were absent from all samples. Conclusion Neoadjuvant chemotherapy tends to decrease the mutation frequency of EGFR mutation and downstream genes, which suggests that real-time samples analysis for genetic aberrancies within EGFR pathways have important value to delineate specific patient populations and facilitate individualized treatment.