Yongli Shuai

MicroRNA Signature Distinguishes the Degree of Aggressiveness of Papillary Thyroid Carcinoma

BackgroundPapillary thyroid carcinoma (PTC) has relatively indolent behavior, although some tumors recur and disseminate to distant sites. The aggressive biological behavior of PTC is difficult to predict. MicroRNAs (miRNAs) are dysregulated in various tumors types, and some of them serve as markers of poor prognosis. In this study, we evaluated miRNA expression as a marker of more aggressive behavior in PTC.MethodsmiRNA array was used to identify a subset of differentially expressed miRNAs between aggressive and nonaggressive PTC. These miRNAs were further validated by real-time RT-PCR in a cohort of 17 PTC with local tumor recurrence or distant metastases and 15 PTC with no extrathyroidal dissemination and correlated with BRAF,RAS, and RET/PTC mutations and MET expression.ResultsThe miRNA array identified miR-146b, miR-221, miR-222, miR-155, miR-31 upregulation and miR-1, miR-34b, miR-130b, miR-138 downregulation in aggressive compared with nonaggressive PTC. Significant miRNA deregulation was confirmed in the validation cohort, with upregulation of miR-146b and miR-222 and downregulation of miR-34b and miR-130b seen in aggressive PTC. Among BRAF-positive tumors, miR-146b showed strong association with aggressive PTC. MET was identified as a potential target gene for 2 downregulated miRNAs (miR-34b and miR-1), and significantly higher level of MET expression was observed in aggressive PTC.ConclusionsWe demonstrate that miR-146b, miR-222, miR-34b, miR-130b are differentially expressed in aggressive compared with nonaggressive PTC. Among BRAF-positive tumors, overexpression of miR-146b was associated with aggressive behavior, suggesting that it may further refine the prognostic importance of BRAF.

Immune Monitoring of the Circulation and the Tumor Microenvironment in Patients with Regionally Advanced Melanoma Receiving Neoadjuvant Ipilimumab

We evaluated neoadjuvant ipilimumab in patients with surgically operable regionally advanced melanoma in order to define markers of activity in the blood and tumor as assessed at baseline (before ipilimumab) and early on-treatment. Patients were treated with ipilimumab (10 mg/kg intravenously every 3 weeks ×2 doses) bracketing surgery. Tumor and blood biospecimens were obtained at baseline and at surgery. Flow cytometry and immunohistochemistry for select biomarkers were performed. Thirty five patients were enrolled; IIIB (3; N2b), IIIC (32; N2c, N3), IV (2). Worst toxicities included Grade 3 diarrhea/colitis (5; 14%), hepatitis (2; 6%), rash (1; 3%), elevated lipase (3; 9%). Median follow up was 18 months: among 33 evaluable patients, median progression free survival (PFS) was 11 months, 95% CI (6.2–19.2). There was a significant decrease in circulating myeloid derived suppressor cells (MDSC). Greater decrease in circulating monocyte gate MDSC Lin1−/HLA-DR−/CD33+/CD11b+ was associated with improved PFS (p = 0.03). There was a significant increase in circulating regulatory T cells (Treg; CD4+CD25hi+Foxp3+) that, unexpectedly, was associated with improved PFS (HR = 0.57; p = 0.034). Baseline evidence of fully activated type I CD4+ and CD8+ antigen-specific T cell immunity against cancer-testis (NY-ESO-1) and melanocytic lineage (MART-1, gp100) antigens was detected and was significantly potentiated after ipilimumab. In tumor, there was a significant increase in CD8+ T cells after ipilimumab (p = 0.02). Ipilimumab induced increased tumor infiltration by fully activated (CD69+) CD3+/CD4+ and CD3+/CD8+ T cells with evidence of induction/potentiation of memory T cells (CD45RO+). The change in Treg observed within the tumor showed an inverse relationship with clinical benefit and greater decrease in tumor MDSC subset Lin1−/HLA-DR−/CD33+/CD11b+ was associated with improved PFS at one year. Neoadjuvant evaluation revealed a significant immunomodulating role for ipilimumab on Treg, MDSC and effector T cells in the circulation and tumor microenvironment that warrants further pursuit in the quest for optimizing melanoma immunotherapy.

High False-Negative Rate of HER2 Quantitative Reverse Transcription Polymerase Chain Reaction of the Oncotype DX Test: An Independent Quality Assurance Study

PURPOSE HER2 (ERBB2) status is an important prognostic and predictive marker in breast carcinoma. In recent years, Genomic Health (GHI), purveyors of the Oncotype DX test, has been separately reporting HER2 by reverse transcription polymerase chain reaction (RT-PCR) to oncologists. Because of the lack of independent evaluation, this quality assurance study was undertaken to define the concordance rate between immunohistochemistry (IHC)/fluorescent in situ hybridization (FISH) and GHI RT-PCR HER2 assay. METHODS All patients at three participating laboratories (Magee-Womens Hospital [Pittsburgh, PA], Cleveland Clinic [Cleveland, OH], and Riverside Methodist Hospital [Columbus, OH]) with available HER2 RT-PCR results from GHI were included in this study. All IHC-positive and equivocal patient cases were further evaluated and classified by FISH at respective laboratories. RESULTS Of the total 843 patient cases, 784 (93%) were classified as negative, 36 (4%) as positive, and 23 (3%) as equivocal at the three institutions using IHC/FISH. Of the 784 negative patient cases, 779 (99%) were also classified as negative by GHI RT-PCR assay. However, all 23 equivocal patient cases were reported as negative by GHI. Of the 36 positive cases, only 10 (28%; 95% CI, 14% to 45%) were reported as positive, 12 (33%) as equivocal, and 14 (39%) as negative. CONCLUSION There was an unacceptable false-negative rate for HER2 status with GHI HER2 assay in this independent study. This could create confusion in the decision-making process for targeted treatment and potentially lead to mismanagement of patients with breast cancer if only GHI HER2 information is used.

Clinicopathological predictors of EGFR/KRAS mutational status in primary lung adenocarcinomas.

Screening for EGFR and KRAS mutations in patients with lung adenocarcinomas can be used to predict the patients response to EGFR tyrosine kinase inhibitors, but there is a lack of guidelines for testing in clinical practice. We analyzed the morphological and clinicopathological characteristics, including tumor stage, size, presence of scar, inflammatory response, angiolymphatic and pleural invasion, of 345 surgically treated primary lung adenocarcinomas with respect to their EGFR and KRAS mutational profile and EGFR FISH. EGFR and KRAS mutations were found in 33 (10%) and 78 (23%) of lung adenocarcinomas, respectively, whereas 226 (67%) cases were negative for both mutations. There was a large overlap in the analyzed clinicopathological characteristics among the three study groups. Statistically significant predictors for the presence of EGFR mutations included history of never smoking (OR 5.939; 95% Wald confidence limit 1.662–21.223, P=0.0149), mild lymphocytic host response (OR 4.724; 95% Wald confidence limit 1.33–1.776; P=0.0163), female gender (OR 2.571; 95% Wald confidence limit 1.015–6.511, P=0.0463) and absence of solid growth pattern. Statistically significant predictors for the presence of KRAS mutations included older age (OR 1.034; 95% Wald confidence limit 1.007–1.062, P=0.0132), history of smoking (OR 0.617, 95% Wald confidence limit 0.357–1.066, P=0.0412) and mucinous differentiation. EGFR FISH positivity as defined by the Colorado criteria was a significant predictor of EGFR mutations, with high polysomy as the strongest predictive criteria. Despite statistically significant differences among the study groups and because of the large overlap in the analyzed clinicopathological criteria, none of these could be implemented as the selection criteria for molecular testing in clinical practice. The cost-effectiveness of lung carcinoma mutational testing would be improved by initial determination of KRAS mutational status as negative predictor of the patients response to EGFR tyrosine kinase inhibitors, followed by EGFR mutational analysis, if necessary.

Phase II Study of Everolimus (RAD001) in Previously Treated Small Cell Lung Cancer

Purpose: Mammalian target of rapamycin (mTOR) is a promising target in small cell lung cancer (SCLC). We designed a phase II study of everolimus, an mTOR inhibitor, in previously treated, relapsed SCLC. Experimental Design: Patients were treated with everolimus 10 mg orally daily until disease progression. The primary endpoint was disease control rate (DCR) at 6 weeks. PI3K/Akt signaling pathway biomarkers were evaluated on baseline tumor tissue. Results: A total of 40 patients were treated: 23 had 1 prior regimen/sensitive relapse, 4 had 1 prior regimen/refractory, and 13 had 2 prior regimens. Twenty-eight patients received 2 or more cycles of everolimus, 7 received 1 cycle, and 5 did not complete the first cycle. Best response in 35 evaluable patients: 1 (3%) partial response (in sensitive relapse), 8 (23%) stable disease, and 26 (74%) progression; DCR at 6 weeks was 26% (95% CI = 11–40). Median survival was 6.7 months and median time to progression was 1.3 months. Grade 3 toxicities included thrombocytopenia (n = 2), neutropenia (n = 2), infection (n = 2), pneumonitis (n = 1), fatigue (n = 1), elevated transaminases (n = 1), diarrhea (n = 2), and acute renal failure (n = 1). High phosphorylated AKT expression was modestly associated with overall survival (HR = 2.07; 95% CI = 0.97–4.43). Baseline S6 kinase protein expression was significantly higher in patients with disease control versus patients with progression (P = 0.0093). Conclusions: Everolimus was well tolerated but had limited single-agent antitumor activity in unselected previously treated patients with relapsed SCLC. Further evaluation in combination regimens for patients with sensitive relapse may be considered. Clin Cancer Res; 16(23); 5900–7. ©2010 AACR.

miRNA expression profiling of lung adenocarcinomas: correlation with mutational status

MicroRNA (miRNA) expression is deregulated in lung cancer, and some miRNAs are associated with poor prognosis and survival. In this study, we investigated the miRNA expression in lung adenocarcinomas with different oncogenic mutations, including EGFR-positive, KRAS-positive and EGFR/KRAS-negative tumors. The expression of 319 miRNAs was evaluated by Exiqon/Luminex microarray, and expression of individual miRNAs was validated by individual RT-PCR assays (Applied Biosystems). Overall, miRNA expression was similar among three mutationally different groups with most upregulated miRNAs being miR-20a, miR-328, miR-34c and miR-18b and most downregulated miRNAs being miR-32, miR-137 and miR-342. Four miRNAs (miR-155, miR-25, miR-495 and miR-7g) were expressed differently among these tumors. miR-155 was upregulated only in EGFR/KRAS-negative group, miR-25 was upregulated only in EGFR-positive group and miR-495 was upregulated only in KRAS-positive adenocarcinomas. In opposite, let-7g was downregulated in all three groups, with more significant downregulation in EGFR/KRAS-negative adenocarcinomas. Principal component analysis (PCA) revealed significant correlation between miRNA expression patterns and somatic mutations. In this study, we demonstrated that despite the similarity in miRNA expression among lung adenocarcinomas with different somatic mutations, some miRNAs showed unique expression patterns, which were in strong correlation with the mutation type, suggesting different carcinogenic pathway for these tumors. These miRNAs can be further explored for their diagnostic and prognostic use.

Safety and Efficacy of Combination Immunotherapy With Interferon Alfa-2b and Tremelimumab in Patients With Stage IV Melanoma

PURPOSE We tested the hypothesis that the combination of tremelimumab and interferon alfa-2b acting via different and possibly synergistic mechanisms would overcome tumor immune tolerance and lead to significant and durable clinical responses. PATIENTS AND METHODS We conducted a phase II study in which patients were administered tremelimumab 15 mg/kg/course (three cycles [one cycle = 4 weeks]) intravenously every 12 weeks. High-dose interferon alfa-2b (HDI) was administered concurrently, including intravenous induction at 20 MU/m2/d for 5 d/wk for 4 weeks followed by maintenance at 10 MU/m2/d subcutaneously three times a week for 8 weeks per course. From course 2 onward, HDI maintenance was administered subcutaneously. RESULTS Thirty-seven patients with American Joint Committee on Cancer stage IV (9M1a, 6M1b, and 22M1c) were enrolled. Two patients had previously treated brain metastases. Grades 3 and 4 toxicities included neutropenia (six patients; 17%), diarrhea/colitis (four patients; 11%), liver enzyme increase (four patients; 11%), rash (four patients; 11%), fatigue (15 patients; 40%), and anxiety/depression (five patients; 14%). Response data were available for 35 patients. The best objective response rate (RR; Response Evaluation Criteria in Solid Tumors) by intention to treat was 24% (90% CI, 13% to 36%; four complete responses [CRs] and five partial responses [PRs] that lasted 6, 6, > 12, > 14, > 18, 20, > 28, 30, and > 37 months, respectively). Fourteen patients (38%) had stable disease (SD) that lasted 1.5 to 21 months. The median progression-free survival was 6.4 months (95% CI, 3.3 to 12.1 months). The median overall survival (OS) was 21 months (95% CI, 9.5 to not reached). There was a weak association between therapy-induced autoimmunity and clinical benefits (CR/PR/SD; P = .0059), baseline C-reactive protein (CRP) less than or equal to 2.7× the upper limit of normal and clinical benefits (P = .0494) and improved probability of survival (P = .0032), and baseline lymphocyte count of at least 1,000/μL and response (CR/PR; P = .0183) and clinical benefits (CR/PR/SD; P = .0255). Biomarker associations were not significant after adjustment for multiple comparisons. CONCLUSION HDI can be administered combined with tremelimumab with acceptable toxicity and promising durable antitumor efficacy that warrant further testing in a randomized trial.

Prediction of the Oncotype DX recurrence score: use of pathology-generated equations derived by linear regression analysis

Oncotype DX is a commercial assay frequently used for making chemotherapy decisions in estrogen receptor (ER)-positive breast cancers. The result is reported as a recurrence score ranging from 0 to 100, divided into low-risk (<18), intermediate-risk (18–30), and high-risk (≥31) categories. Our pilot study showed that recurrence score can be predicted by an equation incorporating standard morphoimmunohistologic variables (referred to as original Magee equation). Using a data set of 817 cases, we formulated three additional equations (referred to as new Magee equations 1, 2, and 3) to predict the recurrence score category for an independent set of 255 cases. The concordance between the risk category of Oncotype DX and our equations was 54.3%, 55.8%, 59.4%, and 54.4% for original Magee equation, new Magee equations 1, 2, and 3, respectively. When the intermediate category was eliminated, the concordance increased to 96.9%, 100%, 98.6%, and 98.7% for original Magee equation, new Magee equations 1, 2, and 3, respectively. Even when the estimated recurrence score fell in the intermediate category with any of the equations, the actual recurrence score was either intermediate or low in more than 80% of the cases. Any of the four equations can be used to estimate the recurrence score depending on available data. If the estimated recurrence score is clearly high or low, the oncologists should not expect a dramatically different result from Oncotype DX, and the Oncotype DX test may not be needed. Conversely, an Oncotype DX result that is dramatically different from what is expected based on standard morphoimmunohistologic variables should be thoroughly investigated.

The Diagnostic Utility of p16 FISH and GLUT-1 Immunohistochemical Analysis in Mesothelial Proliferations

Two promising ancillary tests used in the diagnosis of mesothelioma include GLUT-1 immunohistochemical analysis and fluorescence in situ hybridization (FISH) testing for the p16 deletion. This study compared the diagnostic usefulness of p16 FISH and GLUT-1 immunohistochemical analysis in the diagnosis of mesothelial proliferations in 158 cases with a diagnosis of benign (45.4%), atypical (10.4%), or malignant/mesothelioma (44.2%). Of the 70 benign cases, none had a deletion of p16 and 5 cases (7%) were positive for GLUT-1. Of the 68 mesotheliomas, 40 (59%) had a deletion of p16 (sensitivity, 59%; specificity, 100%) and 27 (40%) were positive for GLUT-1 (sensitivity, 40%; specificity, 93%). GLUT-1 showed lower sensitivity in pleural (56% vs 70%) and peritoneal (29% vs 51%) mesotheliomas (P = .004). Our results demonstrate that p16 FISH is a more sensitive and specific test than GLUT-1 immunohistochemical analysis and can be a more reliable ancillary tool to support the diagnosis of mesothelioma.

Differing Patterns of Circulating Regulatory T-Cells and Myeloid Derived Suppressor Cells in Metastatic Melanoma Patients Receiving Anti-CTLA4 Antibody and Interferon-α or TLR-9 Agonist and GM-CSF with Peptide Vaccination

Changes in the biomarkers of host suppressor immune response were evaluated in patients with melanoma enrolled in 2 trials. Two similar cohorts of patients participating in the 2 studies were evaluated. The first (IFN/treme) tested interferon (IFN)-&agr;2b and tremelimumab in metastatic melanoma and reported a response rate of 24%, 6.4 months median progression-free survival, and 21 months median overall survival. The second [toll-like receptor 9 (TLR)/GM] tested vaccination with MART-1, gp100, tyrosinase given with TLR-9 agonist and granulocyte-macrophage colony-stimulating factor and reported 9% response rate, median progression-free survival of 1.9 months, and median overall survival of 13.4 months. We monitored circulating T regulatory cells (T-reg) and myeloid-derived suppressor cells (MDSC) utilizing multicolor flow cytometry. In “IFN/treme,” changes in circulating T-reg and MDSC were compared between baseline, day 29 (end of IFN-&agr; induction) and day 85 (1 course). The CD4+CD25hi+CD39+ T-reg percentage was increased most at day 85 (P=0.018) and less significantly at day 29 (P=0.09). There was a decrease in the percentage of MDSC populations taken in aggregate, which was most significant for monocytic MDSC (HLA-DR+ low/CD14+) at day 29 (P<0.0001) and day 85 (P=0.001). In “TLR-9/GM,” changes in T-reg and MDSC were compared between baseline and day 50 (4 vaccinations) and day 90 (8 vaccinations). There were no significant changes in T-reg or MDSC, except for a trend towards decreased (HLA-DR+ low/CD14+) MDSC at day 50 (P=0.07). Therefore, IFN/treme significantly downregulated MDSC suggesting a role on the significant clinical activity observed in this trial. T-reg findings suggest that IFN/treme induced clinically significant antitumor responses by inhibiting CTLA4 suppressive effects on T effectors, and less so by affecting T-reg.