Shaohua Lu

Plasma MicroRNA Panel to Diagnose Hepatitis B Virus–Related Hepatocellular Carcinoma

PURPOSE More than 60% of patients with hepatocellular carcinoma (HCC) do not receive curative therapy as a result of late clinical presentation and diagnosis. We aimed to identify plasma microRNAs for diagnosing hepatitis B virus (HBV) -related HCC. PATIENTS AND METHODS Plasma microRNA expression was investigated with three independent cohorts including 934 participants (healthy, chronic hepatitis B, cirrhosis, and HBV-related HCC), recruited between August 2008 and June 2010. First, we used microarray to screen 723 microRNAs in 137 plasma samples for diagnosing HCC. Quantitative reverse-transcriptase polymerase chain reaction assay was then applied to evaluate the expression of selected microRNAs. A logistic regression model was constructed using a training cohort (n = 407) and then validated using an independent cohort (n = 390). Area under the receiver operating characteristic curve (AUC) was used to evaluate diagnostic accuracy. RESULTS We identified a microRNA panel (miR-122, miR-192, miR-21, miR-223, miR-26a, miR-27a and miR-801) that provided a high diagnostic accuracy of HCC (AUC = 0.864 and 0.888 for training and validation data set, respectively). The satisfactory diagnostic performance of the microRNA panel persisted regardless of disease status (AUCs for Barcelona Clinic Liver Cancer stages 0, A, B, and C were 0.888, 0.888, 0.901, and 0.881, respectively). The microRNA panel can also differentiate HCC from healthy (AUC = 0.941), chronic hepatitis B (AUC = 0.842), and cirrhosis (AUC = 0.884), respectively. CONCLUSION We found a plasma microRNA panel that has considerable clinical value in diagnosing early-stage HCC. Thus, patients who would have otherwise missed the curative treatment window can benefit from optimal therapy.

Human miR-1228 as a stable endogenous control for the quantification of circulating microRNAs in cancer patients

Circulating microRNAs are promising biomarkers for non‐invasive testing and dynamic monitoring in cancer patients. However, no consensus exists regarding the normalization of circulating microRNAs in the quantification, making the results incomparable. We investigated global circulating microRNA profiles to identify a stable endogenous control for quantifying circulating microRNAs using three cohorts (n = 544), including 168 control individuals (healthy subjects and those with chronic hepatitis B and cirrhosis) and 376 cancer patients (hepatocellular, colorectal, lung, esophageal, gastric, renal, prostate, and breast cancer patients). GeNorm, NormFinder, and coefficient of variability (CV) were used to select the most stable endogenous control, whereas Ingenuity Pathway Analysis (IPA) was adopted to explore its signaling pathways. Seven candidates (miR‐1225‐3p, miR‐1228, miR‐30d, miR‐939, miR‐940, miR‐188‐5p, and miR‐134) from microarray analysis and four commonly used controls (miR‐16, miR‐223, let‐7a, and RNU6B) from literature were subjected to real‐time quantitative reverse transcription‐polymerase chain reaction validation using independent cohorts. MiR‐1228 (CV = 5.4%) with minimum M value and S value presented as the most stable endogenous control across eight cancer types and three controls. IPA showed miR‐1228 to be involved extensively in metabolism‐related signal pathways and organ morphology, implying that miR‐1228 functions as a housekeeping gene. Functional network analysis found that “hematological system development” was on the list of the top networks that associate with miR‐1228, implying that miR‐1228 plays an important role in the hematological system. The results explained the steady expression of miR‐1228 in the blood. In conclusion, miR‐1228 is a promising stable endogenous control for quantifying circulating microRNAs in cancer patients.

Two MicroRNA Panels to Discriminate Three Subtypes of Lung Carcinoma in Bronchial Brushing Specimens

RATIONALE Effective treatment for lung cancer requires accuracy in subclassification of carcinoma subtypes. OBJECTIVES To identify microRNAs in bronchial brushing specimens for discriminating small cell lung cancer (SCLC) from non-small cell lung cancer (NSCLC) and for further differentiating squamous cell carcinoma (SQ) from adenocarcinoma (AC). METHODS Microarrays were used to screen 723 microRNAs in laser-captured, microdissected cancer cells from 82 snap-frozen surgical lung specimens. Quantitative reverse-transcriptase polymerase chain reaction was performed on 153 macrodissected formalin-fixed, paraffin-embedded (FFPE) surgical lung specimens to evaluate seven microRNA candidates discovered from microarrays. Two microRNA panels were constructed on the basis of a training cohort (n = 85) and validated using an independent cohort (n = 68). The microRNA panels were applied as differentiators of SCLC from NSCLC and of SQ from AC in 207 bronchial brushing specimens. MEASUREMENTS AND MAIN RESULTS Two microRNA panels yielded high diagnostic accuracy in discriminating SCLC from NSCLC (miR-29a and miR-375; area under the curve [AUC], 0.991 and 0.982 for training and validation data set, respectively) and in differentiating SQ from AC (miR-205 and miR-34a; AUC, 0.977 and 0.982 for training and validation data set, respectively) in FFPE surgical lung specimens. Moreover, the microRNA panels accurately differentiated SCLC from NSCLC (AUC, 0.947) and SQ from AC (AUC, 0.962) in bronchial brushing specimens. CONCLUSIONS We found two microRNA panels that accurately discriminated between the three subtypes of lung carcinoma in bronchial brushing specimens. The identified microRNA panels may have considerable clinical value in differential diagnosis and optimizing treatment strategies based on lung cancer subtypes.

Long non-coding RNAs: novel prognostic biomarkers for liver metastases in patients with early stage colorectal cancer.

Liver metastasis is the primary cause of death for colorectal cancer (CRC) patients. To investigate the prognostic value of long non-coding RNAs (lncRNAs) on colorectal liver metastases, quantitative reverse-transcriptase PCR (quantitative RT-PCR) was performed on 15 lncRNAs in 51 stage IV CRC with liver metastases and 57 stage I/II CRC specimens. The expression levels of four lncRNAs (GAS5, H19, MEG3 and Yiya) were significantly different between liver metastases and primary tumors of stage IV CRC patients. Furthermore, the high expression levels of GAS5 and Yiya were significantly associated with future occurrence of liver metastases in early stage CRC patients. Kaplan-Meier analysis showed that the high expression levels of GAS5 or Yiya were correlated with poor prognosis of early stage CRC patients (p = 0.0206 and 0.0005 for GAS5 and Yiya, respectively). Yiya expression was proved to be an independent prognostic indicator of colorectal liver metastases in a multivariate analysis (relative risk = 10.7; p < 0.0001). Our study revealed that GAS5 and Yiya were promising prognostic biomarkers of liver metastases for early stage CRC patients.

Quantitative assessment of short amplicons in FFPE-derived long-chain RNA

Formalin-fixed paraffin-embedded (FFPE) tissues are important resources for molecular medical research. However, long-chain RNA analysis is restricted in FFPE tissues due to high levels of degradation. To explore the possibility of long RNA quantification in FFPE tissues, we selected 14 target RNAs (8 mRNAs and 6 long noncoding RNAs) from literatures, and designed short (~60 bp) and long (~200 bp) amplicons for each of them. Colorectal carcinomas with adjacent normal tissues were subjected to quantitative reverse-transcription PCR (quantitative RT-PCR) in 3 cohorts, including 18 snap-frozen and 83 FFPE tissues. We found that short amplicons were amplified more efficiently than long amplicons both in snap-frozen (P = 0.0006) and FFPE (P = 0.0152) tissues. Nonetheless, comparison of colorectal carcinomas with their adjacent normal tissues demonstrated that the consistency of fold-change trends in a single short amplicon between snap-frozen and FFPE tissues was only 36%. Therefore, we innovatively performed quantitative RT-PCR with 3 non-overlapping short amplicons for 14 target RNAs in FFPE tissues. All target RNAs showed a concordance of 100% of fold-change trends in at least two short amplicons, which offers sufficient information for accurate quantification of target RNAs. Our findings demonstrated the possibility of long-chain RNA analysis with 3 non-overlapping short amplicons in standardized-preserved FFPE tissues.

The Expression of miR-375 Is Associated with Carcinogenesis in Three Subtypes of Lung Cancer

Many studies demonstrated unique microRNA profiles in lung cancer. Nonetheless, the role and related signal pathways of miR-375 in lung cancer are largely unknown. Our study investigated relationships between carcinogenesis and miR-375 in adenocarcinoma, squamous cell carcinoma and small cell lung carcinoma to identify new molecular targets for treatment. We evaluated 723 microRNAs in microdissected cancerous cells and adjacent normal cells from 126 snap-frozen lung specimens using microarrays. We validated the expression profiles of miR-375 and its 22 putative target mRNAs in an independent cohort of 78 snap-frozen lung cancer tissues using quantitative reverse-transcriptase PCR. Moreover, we performed dual luciferase reporter assay and Western blot on 6 targeted genes (FZD8, ITGA10, ITPKB, LRP5, PIAS1 andRUNX1) in small cell lung carcinoma cell line NCI-H82. We also detected the effect of miR-375 on cell proliferation in NCI-H82. We found that miR-375 expression was significantly up-regulated in adenocarcinoma and small cell lung carcinoma but down-regulated in squamous cell carcinoma. Among the 22 putative target genes, 11 showed significantly different expression levels in at least 2 of 3 pair-wise comparisons (adenocarcinoma vs. normal, squamous cell carcinoma vs. normal or small cell lung carcinoma vs. normal). Six targeted genes had strong negative correlation with the expression level of miR-375 in small cell lung carcinoma. Further investigation revealed that miR-375 directly targeted the 3’UTR of ITPKB mRNA and over-expression of miR-375 led to significantly decreased ITPKB protein level and promoted cell growth. Thus, our study demonstrates the differential expression profiles of miR-375 in 3 subtypes of lung carcinomas and finds thatmiR-375 directly targets ITPKB and promoted cell growth in SCLC cell line.

Poor efficacy response to trastuzumab therapy in advanced gastric cancer with homogeneous HER2 positive and non-intestinal type

Introduction Factors affecting trastuzumab efficacy in advanced gastric cancer (GC) are largely unknown. Heterogeneity is a notable feature of HER2 in GC. Whether the heterogeneity influences trastuzumab efficacy is still unknown. Results The HER2homogeneous group and HER2heterogeneous group showed no statistical difference in RR (46.4% vs 55.0%, P = 0.558), PFS (5.80 vs 6.30 months, P = 0.804) and OS (16.00 vs 16.00 months, P = 0.787). The Laurenintestinal group and Laurennon-intestinal group demonstrated no discrepancy in PFS (6.00 vs 6.00 months, P = 0.912) and OS (16.50 vs 14.00 months, P = 0.227). However, by combining HER2 heterogeneity and Lauren classification, PFS and OS of HER2homogeneous/Laurennon-intestinal subgroup was the shortest among the 4 subgroups (P = 0.012 and P = 0.037), which was much shorter than the other patients (PFS:3.00 vs 6.30 months, P = 0.003; OS: 4.50 vs 16.50 months, P = 0.004). Univariate and multivariate analysis showed that HER2 heterogeneity combined with Lauren classification was an independent prognostic factor in both PFS (P = 0.031 and P = 0.002) and OS (P = 0.039 and P = 0.013). Materials and Methods 48 patients with HER2 positive advanced GCs accepting trastuzumab treatment were retrospectively analyzed. Based on HER2 heterogeneity, the patients were divided into a HER2homogeneous group and a HER2heterogeneous group. Response rate (RR), progression free survival (PFS), and overall survival (OS) were compared. Main clinicopathological factors including Lauren classification were subjected to subgroup analysis. Conclusions HER2 heterogeneity alone may not correlate with trastuzumab efficacy in HER2 positive advanced GCs. HER2 heterogeneity combined with Lauren classification may help to identify a subgroup with poor response to trastuzumab which is homogeneous HER2 positive and non-intestinal type.

Procedure-Specific Risk Prediction for Recurrence in Patients Undergoing Lobectomy or Sublobar Resection for Small (≤2 cm) Lung Adenocarcinoma: An International Cohort Analysis

Introduction: This work was performed to develop and validate procedure‐specific risk prediction for recurrence following resection for early‐stage lung adenocarcinoma (ADC) and investigate risk prediction utility in identifying patients who may benefit from adjuvant chemotherapy (ACT). Methods: In patients who underwent resection for small (≤2 cm) lung ADC (lobectomy, 557; sublobar resection, 352), an association between clinicopathologic variables and risk of recurrence was assessed by a competing risks approach. Procedure‐specific risk prediction was developed based on multivariable regression for recurrence. External validation was conducted using cohorts (N = 708) from Japan, Taiwan, and Germany. The accuracy of risk prediction was measured using a concordance index. We applied the lobectomy risk prediction approach to a propensity score–matched cohort of patients with stage II‐III disease (n = 316, after matching) with or without ACT and compared lung cancer–specific survival between groups among low‐ or high‐risk scores. Results: Micropapillary pattern, solid pattern, lymphovascular invasion, and necrosis were involved in the risk prediction following lobectomy, and micropapillary pattern, spread through air spaces, lymphovascular invasion, and necrosis following sublobar resection. Both internal and external validation showed good discrimination (concordance index in lobectomy and sublobar resection: internal, 0.77 and 0.75, respectively; and external, 0.73 and 0.79, respectively). In the stage II‐III propensity score–matched cohort, among high‐risk patients, ACT significantly reduced the risk of lung cancer–specific death (subhazard ratio 0.43, p = 0.001), but not among low‐risk patients. Conclusions: Procedure‐specific risk prediction for patients with resected small lung ADC can be used to better prognosticate and stratify patients for further interventions.

Implications of the Eighth Edition of the TNM Proposal: Invasive Versus Total Tumor Size for the T Descriptor in Pathologic Stage I-IIA Lung Adenocarcinoma

Introduction: The eighth edition of the TNM staging system included the proposal that the T descriptor be determined according to the invasive component, excluding lepidic component, for nonmucinous lung adenocarcinomas. We sought to conduct a clinicopathologic comparative analysis of the newly proposed classification using invasive size versus total tumor size. Methods: Patients who underwent lung resection for primary lung adenocarcinoma with pathologic stage (p‐Stage) I‐IIA (based on total size [t]) were reviewed (n = 1704). Pathologic invasive size was measured, and tumors were reclassified using invasive size (i). Cumulative incidence of recurrence and lung cancer–specific cumulative incidence of death were analyzed using a competing‐risks approach. Prognostic discrimination by p‐Stage(t) and p‐Stage(i) was evaluated using a concordance index (C‐index). Results: The use of invasive size resulted in downstaging in 377 of 1704 patients (22%), with twice as many patients with p‐Stage IA1 (IA1[i] versus IA1[t]: 389 [23%] versus 195 [11%]). However, outcomes were similar between the two groups (IA1[i] versus IA1[t]: 5‐year cumulative incidence of recurrence, 11% versus 13%; 5‐year lung cancer–specific cumulative incidence of death, 5% versus 7%). Prognostic discrimination by p‐Stage(i) was better than by p‐Stage(t) (C‐index for p‐Stage[i] versus p‐Stage[t]: recurrence, 0.614 versus 0.593; lung cancer–specific death, 0.634 versus 0.621). Conclusions: When invasive size, rather than total size, was used for the T descriptor, a larger number of patients were classified with a favorable prognosis (p‐Stage IA1) and better prognostic discrimination of p‐Stage I‐IIA nonmucinous lung adenocarcinomas was achieved.

Lobectomy Is Associated with Better Outcomes than Sublobar Resection in Spread through Air Spaces (STAS)-Positive T1 Lung Adenocarcinoma: A Propensity Score–Matched Analysis

Introduction: Spread through air spaces (STAS) is a form of invasion wherein tumor cells extend beyond the tumor edge within the lung parenchyma. In lung adenocarcinoma (ADC), we investigated the (1) association between STAS and procedure‐specific outcomes (sublobar resection and lobectomy), (2) effect of surgical margin‐to‐tumor diameter ratio in STAS‐positive patients, and (3) potential utility of frozen sections (FSs) for detecting STAS intraoperatively. Methods: We investigated 1497 patients who underwent lobectomy (n = 970) or sublobar resection (n = 527) for T1N0M0 lung ADC after propensity score matching. Outcomes were analyzed by using a competing risks approach. The effect of margin‐to‐tumor ratio on recurrence pattern (locoregional and distant) was investigated in patients who underwent sublobar resection. Five pathologists evaluated the feasibility of intraoperatively identifying STAS by using FSs (sensitivity, specificity, and interrater reliability). Results: On multivariable analysis after propensity score matching (349 pairs/procedure), sublobar resection was significantly associated with recurrence (subhazard ratio = 2.84 [p < 0.001]) and lung cancer–specific death (subhazard ratio = 2.63 [p = 0.021]) in patients with STAS but not in those without STAS. Patients with STAS who underwent sublobar resection had a higher risk of locoregional recurrence regardless of margin‐to‐tumor ratio (for a margin‐to‐tumor ratio of ≥1 versus <1, the 5‐year cumulative incidence of recurrence rates were 16% and 25%, respectively); among patients without STAS, locoregional recurrences occurred in patients with margin‐to‐tumor ratio lower than 1 (a 5‐year cumulative incidence of recurrence rate of 7%). The sensitivity and specificity for detecting STAS by use of FSs were 71% and 92%, with substantial interrater reliability (Gwets AC1, 0.67). Conclusions: In patients with T1 lung ADC with STAS, lobectomy was associated with better outcomes than sublobar resection was. Pathologists can recognize STAS on FSs.