Linda Wiens

DNA Methylation in Tumor and Matched Normal Tissues from Non-Small Cell Lung Cancer Patients

We used MethyLight assays to analyze DNA methylation status of 27 genes on 49 paired cancerous and noncancerous tissue samples from non-small cell lung cancer (NSCLC) patients who underwent surgical resection. Seven genes (RARB, BVES, CDKN2A, KCNH5, RASSF1, CDH13, and RUNX) were found to be methylated significantly more frequently in tumor tissues than in noncancerous tissues. Only methylation of CCND2 and APC was frequently detected in both cancerous and noncancerous tissues, supporting the hypothesis that the methylation of these two genes is a preneoplastic change and may be associated with tobacco smoking exposure. Methylation of any one of eight genes (RASSF1, DAPK1, BVES, CDH13, MGMT, KCNH5, RARB, or CDH1) was present in 80% of NSCLC tissues but only in 14% of noncancerous tissues. Detection of methylation of these genes in blood might have utility in monitoring and detecting tumor recurrence in early-stage NSCLC after curative surgical resection. (Cancer Epidemiol Biomarkers Prev 2008;17(3):645–54)

Fluorodeoxyglucose Uptake of Primary Non-Small Cell Lung Cancer at Positron Emission Tomography: New Contrary Data on Prognostic Role

Purpose: This prospective study evaluated the prognostic significance of 18F-fluorodeoxyglucose (18F-FDG) uptake in primary non-small cell lung cancer (NSCLC) at positron emission tomography, in a carefully staged population, while correcting for partial volume effects. Experimental Design: Two hundred eight potentially resectable NSCLC patients were referred for FDG positron emission tomography staging after thoracic computed tomography. Each tumor stage was confirmed surgically, or for some stage IV tumors by additional imaging. The tumor maximum pixel-standardized uptake value (maxSUV) and the maxSUV partial volume corrected for lesion size (PVCmaxSUV) were compared with overall survival and disease-free survival using Cox proportional hazards regression. Results: Stage distribution: stage I, 36%; stage II, 15%; stage III, 30%; stage IV, 19%. Patients were followed for a median of 33.6 months, with 90 deaths from NSCLC (median survival for all stages, 43.3 months). With respect to overall survival, the most significant cutoff value for both maxSUV and PVCmaxSUV was 7. MaxSUV ≥7 was significantly associated with an increased risk of death from NSCLC in univariable analysis, whereas PVCmaxSUV ≥7 was only marginally associated. However, in multivariable analyses, neither maxSUV ≥7 nor PVCmaxSUV ≥7 provided significant additional prognostic information over stage, tumor size, and age. In the 103 patients who underwent surgical resection only, surgical stage, but not maxSUV or PVCmaxSUV, was univariably associated with survival or recurrence. SUV definitions based on lean body mass, body surface area, and plasma glucose correction yielded identical results. Conclusions: As expected, tumor stage is prognostic in NSCLC. However, tumor FDG uptake does not provide additional prognostic information. This prospective study contradicts prior reports.

Relationship Between Non-small Cell Lung Cancer FDG Uptake at PET, Tumor Histology, and Ki-67 Proliferation Index

Introduction: We compared primary non-small cell lung cancer (NSCLC) 18F-fluorodeoxyglucose (FDG) uptake at positron emission tomography (PET) to tumor histologic features and Ki-67 proliferation index. This large, prospectively-recruited patient cohort has previously been analyzed based on differences in FDG uptake across stage groups; the current analysis adds further dimensions to this characterization. Materials and Methods: One hundred seventy-eight patients with potentially-resectable NSCLC were scanned with FDG PET before therapy. A partial volume correction algorithm was used to correct FDG uptake values for their dependence on tumor size. Primary tumor resection specimens, core biopsies, and biopsies of metastatic lymph nodes were used to assess each tumor’s NSCLC histologic subtype, degree of differentiation, and Ki-67 proliferation index. Results: Bronchioalveolar carcinomas were found to have lower FDG uptake at PET and lower Ki-67 scores than any other histologic subtype. Non-bronchioalveolar adenocarcinomas had lower FDG uptake and Ki-67 scores than squamous cell carcinomas or large cell undifferentiated carcinomas. Better differentiated NSCLCs had lower FDG uptake and Ki-67 scores than more poorly differentiated NSCLCs. There was a significant positive correlation between FDG uptake and Ki-67 scores. Partial volume correction increased the strength of this correlation, while also diminishing the strong positive correlation between FDG uptake and tumor size. Conclusions: There are significant differences in NSCLC FDG uptake across histologic subtypes and differentiation groups. These differences parallel nearly identical differences in Ki-67 scores, implying that differences in NSCLC tumor cell proliferation may give rise to commensurate differences in tumor glucose metabolism.

DNA hypermethylation of tumors from non-small cell lung cancer (NSCLC) patients is associated with gender and histologic type.

BACKGROUND We previously identified a number of genes which were methylated significantly more frequently in the tumor compared to the non-cancerous lung tissues from non-small cell lung cancer (NSCLC) patients. Detection of methylation profiles of genes in NSCLC could provide insight into differential pathways to malignancy and lead to strategies for better treatment of individuals with NSCLC. METHODS We determined the DNA methylation status of 27 genes using quantitative MethyLight assays in lung tumor samples from 117 clinically well-characterized NSCLC patients. RESULTS Hypermethylation was detected in one of more of the genes in 106 (91%) of 117 cases and was detected at high levels (percentage methylation reference (PMR)> or =4%) in 79% of NSCLC cases. Methylation of APC, CCND2, KCNH5 and, RUNX was significantly more frequent in adenocarcinomas compared to squamous cell carcinomas (SCC), while methylation of CDKN2A was more common in SCC. Hypermethylation of KCNH5, KCNH8, and RARB was more frequent in females compared to males. Hypermethylation of APC and CCND2 was inversely associated with proliferation score assessed by Ki-67 level. CONCLUSIONS Our findings of differential gene hypermethylation frequencies in tumor tissues from patients with adenocarcinoma or squamous cell cancers and in females compared to males suggests that further investigation is warranted in order to more fully understand the potential disparate pathways and/or risk factors for NSCLC associated with histologic type and gender.

Tumor 3′-Deoxy-3′-18F-Fluorothymidine (18F-FLT) Uptake by PET Correlates with Thymidine Kinase 1 Expression: Static and Kinetic Analysis of 18F-FLT PET Studies in Lung Tumors

We report the first, to our knowledge, findings describing the relationships between both static and dynamic analysis parameters of 3′-deoxy-3′-18F-fluorothymidine (18F-FLT) PET and the expression of the proliferation marker Ki-67, and the protein expression and enzymatic activity of thymidine kinase-1 (TK1) in surgically resected lung lesions. Methods: Static and dynamic analyses (4 rate constants and 2 compartments) of 18F-FLT PET images were performed in a cohort of 25 prospectively accrued, clinically suspected lung cancer patients before surgical resection (1 lesion was found to be benign after surgery). The maximal and overall averaged expression of Ki-67 and TK1 were determined by semiquantitative analysis of immunohistochemical staining. TK1 enzymatic activity was determined by in vitro assay of extracts prepared from flash-frozen samples of the same tumors. Results: Static 18F-FLT uptake (partial-volume–corrected maximum-pixel standardized uptake value from 60- to 90-min summed dynamic data) was significantly correlated with the overall (ρ = 0.57, P = 0.006) and maximal (ρ = 0.69, P < 0.001) immunohistochemical expressions of Ki-67 and TK1 (overall expression: ρ = 0.65, P = 0.001; maximal expression: ρ = 0.68, P < 0.001) but not with TK1 enzymatic activity (ρ = 0.34, P = 0.146). TK1 activity was significantly correlated with TK1 protein expression only when immunohistochemistry was scored for maximal expression (ρ = 0.52, P = 0.029). Dynamic analysis of 18F-FLT PET revealed correlations between the flux constant (KFLT) and both overall (ρ = 0.53, P = 0.014) and maximal (ρ = 0.50, P = 0.020) TK1 protein expression. KFLT was also associated with both overall (ρ = 0.59, P = 0.005) and maximal (ρ = 0.63, P = 0.002) Ki-67 expression. We observed no significant correlations between TK1 enzyme activity and KFLT. In addition, no significant relationships were found between TK1 expression, TK1 activity, or Ki-67 expression and any of the compartmental rate constants. Conclusion: The absence of observable correlations of the imaging parameters with TK1 activity suggests that 18F-FLT uptake and retention within cells may be complicated by a variety of still undetermined factors in addition to TK1 enzymatic activity.

Relationship between Non-Small Cell Lung Cancer Fluorodeoxyglucose Uptake at Positron Emission Tomography and Surgical Stage with Relevance to Patient Prognosis

Purpose: Because the tumor stage is the most significant prognostic factor for non-small cell lung cancer (NSCLC) and given that NSCLC [18F]fluorodeoxyglucose (18F-FDG) uptake appears to have prognostic significance, we examined the relationship between NSCLC 18F-FDG uptake and surgical stage. Experimental Design: One hundred seventy-eight patients with a proven diagnosis of NSCLC were enrolled, then imaged with 18F-FDG positron emission tomography and their disease thoroughly staged. Primary tumor size at computed tomography and 18F-FDG uptake were compared to overall tumor stage and to T, N, and M stage descriptors. Tumor uptake was quantitated by maximum pixel-standardized uptake value (maxSUV) and then partial volume corrected for lesion size using recovery coefficients. Results: A significant difference in tumor size was associated with tumors of different TNM stage, T status, N status, or M status. Similarly, the primary tumor maxSUV was significantly associated with TNM stage, T status, and M status. However, we observed no significant difference in the partial-volume-corrected tumor maxSUV for different stages; different T, N, or M descriptors; tumors without evidence of spread (N0M0) versus tumors with nodal spread (N1,2,3M0); or tumors without spread (N0M0) versus all others. Conclusions: We found an association between tumor stage and 18F-FDG maxSUV, but this relationship disappeared after correction of tumor uptake for lesion size. Therefore, if partial-volume-corrected 18F-FDG uptake is prognostic of NSCLC outcome, it is not on the basis of a relationship with tumor stage but through a different mechanism.

Thymidine Kinase 1 and Thymidine Phosphorylase Expression in Non-Small-cell Lung Carcinoma in Relation to Angiogenesis and Proliferation

The thymidine salvage pathway enzymes thymidine kinase 1 (TK1) and thymidine phosphorylase (TP) compete for thymidine as a substrate and catalyze opposing synthetic and catabolic reactions that have been implicated in the control of proliferation and angiogenesis, respectively. We investigated the relationship between the expression of TK1 and TP as they relate to proliferation (Ki-67 labeling index) and angiogenesis (Chalkley count of CD31-stained blood vessels) in a series of 110 non-small-cell lung cancer (NSCLC) tumors from patients prospectively enrolled in an imaging trial. TK1 and TP exhibited similar patterns of immunohistochemical distribution, in that each was found in both the nucleus and the cytoplasm of tumor cells. Each enzyme exhibited a significant positive correlation between its levels of nuclear and cytoplasmic expression. A significant positive correlation between TK1 expression and the Ki-67 labeling index (r = 0.53, p<0.001) was observed. TP was significantly positively correlated with Chalkley scoring of CD31 staining in high vs low Chalkley scoring samples (mean TP staining of 115.8 vs 79.9 scoring units, p<0.001), respectively. We did not observe a substantial inverse correlation between the TP and TK1 expression levels in the nuclear compartment (r = −0.17, p=0.08). Tumor size was not found to be associated with TK1, TP, Ki-67, or Chalkley score. These findings provide additional evidence for the role of thymidine metabolism in the complex interaction of proliferation and angiogenesis in NSCLC.

Functional inactivation of p53 by HPV-E6 transformation is associated with a reduced expression of radiation-induced potentially lethal damage

PURPOSE To examine the effect of functional loss of p53 on radiation sensitivity and potentially lethal damage repair (PLDR). MATERIALS AND METHODS Radiation sensitivity and PLDR were examined in an isogenic pair of human tumour cell lines created by HPV-E6 transformation. RESULTS Inactivation of p53 by E6 transformation resulted in a cell line that was more resistant to killing by radiation but showed little enhancement in survival (PLDR) when plateau-phase cells were held non-cycling after radiation exposure. Holding p53-normal cells in plateau-phase after radiation exposure not only led to enhanced survival, but also to a reduction in the proportion of cells that blocked in G1 subsequent to release. CONCLUSIONS These results suggest that p53 expression influences that component of radiation sensitivity associated with PLDR.

Synthesis and biodistribution of R- and S-isomers of [18F]-fluoropropranolol, a lipophilic ligand for the β-adrenergic receptor

The S and R isomers of [18F]-fluoropropranolol (1-[1-fluoro-2-isopropylamino]-3-naphthalen-1-yloxy-propan-2 -ol) have been prepared by reductive alkylation of the appropriate aminoalcohols. The radiosynthesis provides a reasonable yield (approximately 25%) to give products of 99% enantiomeric excess and specific activities of 1-3 Ci/micromol. The dissociation constants for the beta2 adrenergic receptor are 0.5 and 2.5 nM for the S and the R isomers, respectively. The biodistribution data in rats show that uptake and egress of the tracer is rapid but that the result of blocking studies and the difference between the R and the S isomers suggest receptor-mediated uptake in receptor-rich tissue.

Hypermethylation of CCND2 May Reflect a Smoking-Induced Precancerous Change in the Lung

It remains unknown whether tobacco smoke induces DNA hypermethylation as an early event in carcinogenesis or as a late event, specific to overt cancer tissue. Using MethyLight assays, we analyzed 316 lung tissue samples from 151 cancer-free subjects (121 ever-smokers and 30 never-smokers) for hypermethylation of 19 genes previously observed to be hypermethylated in nonsmall cell lung cancers. Only APC (39%), CCND2 (21%), CDH1 (7%), and RARB (4%) were hypermethylated in >2% of these cancer-free subjects. CCND2 was hypermethylated more frequently in ever-smokers (26%) than in never-smokers (3%). CCND2 hypermethylation was also associated with increased age and upper lobe sample location. APC was frequently hypermethylated in both ever-smokers (41%) and never-smokers (30%). BVES, CDH13, CDKN2A (p16), CDKN2B, DAPK1, IGFBP3, IGSF4, KCNH5, KCNH8, MGMT, OPCML, PCSK6, RASSF1, RUNX, and TMS1 were rarely hypermethylated (<2%) in all subjects. Hypermethylation of CCND2 may reflect a smoking-induced precancerous change in the lung.