Biomedical subjects

Siva Raja

Publications and source records attributed to Siva Raja.

12 recordsLinked to original sources

PubMed2006

A combination of molecular markers accurately detects lymph node metastasis in non-small cell lung cancer patients.

Occult lymph node metastasis (micrometastasis) is a good prognostic indicator in non-small cell lung cancer (NSCLC) and could be used to direct adjuvant chemotherapy in stage I patients. This study was designed to evaluate molecular markers for detection of occult lymph node metastasis in NSCLC, define the best marker or marker combination to distinguish positive from benign lymph nodes, and evaluate these markers in lymph nodes from pathologically node-negative (pN(0)) NSCLC patients. Potential markers were identified through literature and database searches and all markers were analyzed by quantitative reverse transcription-PCR in a primary screen of six NSCLC specimens and 10 benign nodes. Selected markers were further evaluated on 21 primary NSCLC specimens, 21 positive nodes, and 21 benign nodes, and the best individual markers and combinations were identified. A combination of three markers was further validated on an independent set of 32 benign lymph nodes, 38 histologically positive lymph nodes, and 462 lymph nodes from 68 pN(0) NSCLC patients. Forty-two markers were evaluated in the primary screen and eight promising markers were selected for further analysis. A combination of three markers (SFTPB, TACSTD1, and PVA) was identified that provided perfect classification of benign and positive nodes in all sample sets. PVA and SFTPB are particularly powerful in tumors of squamous and adenocarcinoma histologies, respectively, whereas TACSTD1 is a good general marker for NSCLC metastasis. The combination of these genes identified 32 of 462 (7%) lymph nodes from 20 of 68 (29%) patients as potentially positive for occult metastasis. Long-term follow-up will determine the clinical relevance of these findings.

Antigens, Neoplasm↗

PubMed2006

A rapid, fully automated, molecular-based assay accurately analyzes sentinel lymph nodes for the presence of metastatic breast cancer.

OBJECTIVE: To develop a fully automated, rapid, molecular-based assay that accurately and objectively evaluates sentinel lymph nodes (SLN) from breast cancer patients. SUMMARY BACKGROUND DATA: Intraoperative analysis for the presence of metastatic cancer in SLNs from breast cancer patients lacks sensitivity. Even with immunohistochemical staining (IHC) and time-consuming review, alarming discordance in the interpretation of SLN has been observed. METHOD: A total of 43 potential markers were evaluated for the ability to accurately characterize lymph node specimens from breast cancer patients as compared with complete histologic analysis including IHC. Selected markers then underwent external validation on 90 independent SLN specimens using rapid, multiplex quantitative reverse transcription-polymerase chain reaction (QRT-PCR) assays. Finally, 18 SLNs were analyzed using a completely automated RNA isolation, reverse transcription, and quantitative PCR instrument (GeneXpert). RESULTS: : Following analysis of potential markers, promising markers were evaluated to establish relative level of expression cutoff values that maximized classification accuracy. A validation set of 90 SLNs from breast cancer patients was prospectively characterized using 4 markers individually or in combinations, and the results compared with histologic analysis. A 2-marker assay was found to be 97.8% accurate (94% sensitive, 100% specific) compared with histologic analysis. The fully automated GeneXpert instrument produced comparable and reproducible results in less than 35 minutes. CONCLUSIONS: A rapid, fully automated QRT-PCR assay definitively characterizes breast cancer SLN with accuracy equal to conventional pathology. This approach is superior to intraoperative SLN analysis and can provide standardized, objective results to assist in pathologic diagnosis.

Biomarkers, Tumor↗

PubMed2005

Molecular staging of cervical lymph nodes in squamous cell carcinoma of the head and neck.

Clinical staging of cervical lymph nodes from patients with squamous cell carcinoma of the head and neck (SCCHN) has only 50% accuracy compared with definitive pathologic assessment. Consequently, both clinically positive and clinically negative patients frequently undergo neck dissections that may not be necessary. To address this potential overtreatment, sentinel lymph node (SLN) biopsy is currently being evaluated to provide better staging of the neck. However, to fully realize the potential improvement in patient care afforded by the SLN procedure, a rapid and accurate SLN analysis is necessary. We used quantitative reverse transcription-PCR (QRT-PCR) to screen 40 potential markers for their ability to detect SCCHN metastases to cervical lymph nodes. Seven markers were identified with good characteristics for identifying metastatic disease, and these were validated using a set of 26 primary tumors, 19 histologically positive lymph nodes, and 21 benign nodes from patients without cancer. Four markers discriminated between positive and benign nodes with accuracy >97% but only one marker, pemphigus vulgaris antigen (PVA), discriminated with 100% accuracy in both the observed data and a statistical bootstrap analysis. A rapid QRT-PCR assay for PVA was then developed and incorporated into a prototype instrument capable of performing fully automated RNA isolation and QRT-PCR. The automated analysis with PVA provided perfect discrimination between histologically positive and benign lymph nodes and correctly identified two lymph nodes with micrometastatic tumor deposits. These assays were completed (from tissue to result) in approximately 30 minutes, thus demonstrating the feasibility of intraoperative staging of SCCHN SLNs by QRT-PCR.

Adult↗

PubMed2005

Technology for automated, rapid, and quantitative PCR or reverse transcription-PCR clinical testing.

BACKGROUND: PCR-based assays can improve clinical care, but they remain technically demanding and labor-intensive. We describe a new instrument, the GeneXpert, that performs automated nucleic acid isolation, reverse transcription, and fluorescence-based quantitative PCR in approximately 35 min. METHODS: Yield and integrity of RNA isolated on the GeneXpert were compared with Qiagen-based extraction for parallel samples (5-microm frozen tissue sections). The reproducibility of automated RNA isolation, reverse transcription, and quantitative PCR was determined by replicate (n = 10) analysis of 10 tissues, using duplex (target and endogenous control) reverse transcription-PCR reactions for two gene combinations. The GeneXpert was then used to perform rapid analysis of lymph nodes from melanoma, breast cancer, and lung cancer patients and analysis of melanoma metastatic to the lung, primary lung adenocarcinoma, and healthy lung tissue. RESULTS: On the GeneXpert, RNA was recovered in slightly over 6 min, and the yield was approximately 70% of that from parallel Qiagen reactions. The RNA integrity was comparable to that of Qiagen-isolated RNA as determined by gel electrophoresis. For the melanoma samples, the 95% prediction interval for the deltaCt for a new measurement was +/-1.54 cycles, and for breast cancer samples, the interval for a newly observed deltaCt was +/-1.40 cycles. GeneXpert assays successfully detected the presence of metastatic melanoma, breast cancer, and lung cancer in lymph nodes and also differentiated among metastatic melanoma, lung adenocarcinoma, and healthy lung. CONCLUSIONS: RNA yield and integrity on the GeneXpert are comparable to benchtop methods. Reproducibility of the GeneXpert data is similar to that seen with manual methods in our hands but may need improvement for some applications. The GeneXpert can perform RNA isolation, reverse transcription, and quantitative PCR in approximately 35 min and could therefore be used for intraoperative testing when applicable.

Adenocarcinoma↗

PubMed2005

Molecular staging of lymph nodes from patients with esophageal adenocarcinoma.

PURPOSE: This study was designed to evaluate molecular markers for the detection of micrometastasis in esophageal adenocarcinoma, define algorithms to distinguish positive from benign lymph nodes and to validate these findings in an independent tissue set and in patients with p(N0) esophageal adenocarcinoma. EXPERIMENTAL DESIGN: Potential markers were identified through literature and database searches. All markers were analyzed by quantitative reverse transcription (QRT)-PCR on a limited set of primary tumors and benign lymph nodes. Selected markers were further evaluated on a larger tissue set and classification algorithms were generated for individual markers and combinations. Algorithms were statistically validated internally as well as externally on an independent set of lymph nodes. Selected markers were then used to identify occult disease in lymph nodes from 34 patients with p(N0) esophageal adenocarcinoma. RESULTS: Thirty-nine markers were evaluated, six underwent further analysis and five were analyzed in the external validation study. Two markers provided perfect classification in both the screening and validation sets, although parametric bootstrap analysis estimated 2% to 3% optimism in the observed classification accuracy. Several marker combinations also gave perfect classification in the observed data sets, and estimates of optimism were lower, implying more robust classification than with individual markers alone. Five of thirty-four patients with esophageal adenocarcinoma had positive nodes by multimarker QRT-PCR analysis and disease-free survival was significantly worse in these patients (P = 0.0023). CONCLUSIONS: We have identified novel QRT-PCR markers for the detection of occult lymph node disease in patients with esophageal adenocarcinoma. The objective nature of QRT-PCR results, and the ability to detect occult metastases, make this an attractive alternative to routine pathology.

Adenocarcinoma↗

PubMed2005

The HGF receptor c-Met is overexpressed in esophageal adenocarcinoma.

The hepatocyte growth factor (HGF) receptor, Met, has established oncogenic properties; however, its expression and function in esophageal adenocarcinoma (EA) remain poorly understood. We aimed to determine the expression and potential alterations in Met expression in EA. Met expression was investigated in surgical specimens of EA, Barrett's esophagus (BE), and normal esophagus (NE) using immunohistochemistry (IHC) and quantitative reverse transcriptase polymerase chain reaction. Met expression, phosphorylation, and the effect of COX-2 inhibition on expression were examined in EA cell lines. IHC demonstrated intense Met immunoreactivity in all (100%) EA and dysplastic BE specimens. In contrast, minimal immunostaining was observed in BE without dysplasia or NE specimens. Met mRNA and protein levels were increased in three EA cell lines, and Met protein was phosphorylated in the absence of serum. Sequence analysis found the kinase domain of c-met to be wild type in all three EA cell lines. HGF mRNA expression was identified in two EA cell lines. In COX-2-overexpressing cells, COX-2 inhibition decreased Met expression. Met is consistently overexpressed in EA surgical specimens and in three EA cell lines. Met dysregulation occurs early in Barrett's dysplasia to adenocarcinoma sequence. Future study of Met inhibition as a potential biologic therapy for EA is warranted.

Adenocarcinoma↗

PubMed2004

Quantitative analysis of circulating plasma DNA as a tumor marker in thoracic malignancies.

BACKGROUND: Increased plasma DNA has been found in cancer patients and may have potential as a tumor marker. The objectives of this study were to develop a controlled, quantitative PCR (QPCR) assay to measure plasma DNA and then evaluate plasma DNA concentrations as a tumor marker in patients with thoracic malignancies. METHODS: We developed a QPCR assay for DNA, using the human beta-actin gene. Plasma samples were analyzed from 58 patients with esophageal cancer (EC; 20 banked samples and 38 prospectively collected samples) and 25 patients with lung cancer (LC; all prospectively collected). Control groups consisting of 51 patients with gastroesophageal reflux disease (GERD; 23 banked samples and 28 prospectively collected) and 11 healthy volunteers were also analyzed. RESULTS: The assay had an experimental variability <4%. In our banked samples, the mean concentration of plasma DNA in EC was 819.0 microg/L (range, 46.2-4738.0 microg/L) vs 432.0 microg/L (6.0-2888.0 microg/L) in GERD (P = 0.02). However, the prospectively collected samples had lower DNA concentrations, and there was no difference between cancer patients and controls. The mean DNA concentration was 10.6 microg/L (range, 7.0-14.0 microg/L) in healthy volunteers and 10.5 microg/L (range, 4.0-23.5 microg/L) in GERD controls vs 13.0 microg/L (range, 4.5-46.5 microg/L) in EC and 14.6 microg/L (range, 3.0-30.0 microg/L) in LC. CONCLUSIONS: Our data indicate that plasma DNA concentrations are of limited diagnostic value when samples are prospectively collected and uniformly handled. This is in contrast to previously published results. Qualitative analysis of DNA may be needed if plasma nucleic acids are to be used as a diagnostic tool in cancer screening.

Biomarkers, Tumor↗

PubMed2004

Characterization of amplifiable, circulating RNA in plasma and its potential as a tool for cancer diagnostics.

BACKGROUND: Several recent reports have described the detection of circulating, cancer-related RNA molecules in serum or plasma from cancer patients, but little is known about the biology of this extracellular RNA. We aimed to determine how RNA is protected against degradation in serum, to optimize RNA isolation from large volumes of serum, and to test our optimized assays for serum-based cancer detection. METHODS: We used quantitative reverse transcription-PCR (QRT-PCR) analysis to investigate the isolation and biology of extracellular plasma RNA. We then examined the presence of amplifiable RNA transcripts in plasma and serum from controls and from patients with esophageal cancer and malignant melanoma. RESULTS: We found that extracellular RNA in plasma is highly degraded and can be isolated most efficiently by guanidinium-phenol extraction followed by precipitation. Extracellular RNA is stable in serum for up to 3 h but is destroyed immediately by addition of detergents. Extracellular RNA can be captured on 0.2 microm filters, allowing concentration of RNA from several milliliters of plasma. When we concentrated RNA from up to 4 mL of serum, detection of cancer-related transcripts in serum from cancer patients and controls was infrequent and inconsistent. CONCLUSIONS: Extracellular RNA is most likely protected within protein or lipid vesicles, possibly apoptotic bodies, which can be disrupted by detergents. Despite optimizing many aspects of plasma RNA detection, we were unable to reproducibly detect cancer-related transcripts. Our data suggest that measurement of circulating RNA may not be a good approach to early cancer diagnosis.

Animals↗

PubMed2003

Widespread molecular alterations present in stage I non-small cell lung carcinoma fail to predict tumor recurrence.

Stage I non-small cell carcinoma (NSCLC) of the lung is typically treated with surgery alone, but with a 30 to 40% recurrence rate. Prognostic factors to stratify these patients into high- and low-risk groups would be of significant clinical value, but published data are conflicting. We studied 39 Stage I NSCLC treated with resection alone, followed for a minimum of 5 years, and divided into recurrent (RC) and non-recurrent (NRC) groups (n = 12 and 27, respectively). Allelic imbalance (loss of heterozygosity, LOH) involving genomic regions containing L-myc (1p32), hOGG1 (3p26), APC/MCC (5q21), c-fms (5q33.3), p53 (17p13), and DCC (18q21), and point mutational change in K-ras-2 (12p12) were studied by PCR-based microsatellite analysis and DNA sequencing. Mutations in k-ras-2 were seen in 25% and 19% of RC and NRC tumors, respectively, most frequently in adenocarcinomas. LOH in the RC and NRC respectively were 50% and 37% for L-myc, 60% and 33% for hOGG1, 60% and 50% for APC, 38% and 35% for c-fms, 78% and 75% for p53, and 17% and 45% for DCC. No statistical significance was seen comparing any of the allelic alterations with recurrence. LOH for hOGG1 and L-myc were more commonly seen in squamous cell carcinomas. Stage I NSCLC are genetically heterogeneous with respect to mutation acquisition. The approach of investigating a panel of genes for alterations can be applied to any given tumor type, and provides information on patterns of mutations/LOH that can help us better understand the molecular biology of tumorigenesis.

Aged↗

PubMed2002

High-resolution mapping of the 11q13 amplicon and identification of a gene, TAOS1, that is amplified and overexpressed in oral cancer cells.

Amplification of chromosomal band 11q13 is a common event in human cancer. It has been reported in about 45% of head and neck carcinomas and in other cancers including esophageal, breast, liver, lung, and bladder cancer. To understand the mechanism of 11q13 amplification and to identify the potential oncogene(s) driving it, we have fine-mapped the structure of the amplicon in oral squamous cell carcinoma cell lines and localized the proximal and distal breakpoints. A 5-Mb physical map of the region has been prepared from which sequence is available. We quantified copy number of sequence-tagged site markers at 42-550 kb intervals along the length of the amplicon and defined the amplicon core and breakpoints by using TaqMan-based quantitative microsatellite analysis. The core of the amplicon maps to a 1.5-Mb region. The proximal breakpoint localizes to two intervals between sequence-tagged site markers, 550 kb and 160 kb in size, and the distal breakpoint maps to a 250 kb interval. The cyclin D1 gene maps to the amplicon core, as do two new expressed sequence tag clusters. We have analyzed one of these expressed sequence tag clusters and now report that it contains a previously uncharacterized gene, TAOS1 (tumor amplified and overexpressed sequence 1), which is both amplified and overexpressed in oral cancer cells. The data suggest that TAOS1 may be an amplification-dependent candidate oncogene with a role in the development and/or progression of human tumors, including oral squamous cell carcinomas. The approach described here should be useful for characterizing amplified genomic regions in a wide variety of tumors.

Amino Acid Sequence↗

PubMed2002

Rapid, quantitative reverse transcriptase-polymerase chain reaction: application to intraoperative molecular detection of occult metastases in esophageal cancer.

OBJECTIVE: Our earlier data showed that quantitative reverse transcriptase-polymerase chain reaction can discriminate patients with node-negative cancer who are at high risk for recurrence. The objective of this study was to determine whether a new, more rapid quantitative reverse transcriptase-polymerase chain reaction assay could provide this information in a time frame suitable for intraoperative decision making. METHODS: We studied formalin-fixed, archived lymph nodes from 30 patients with histologically determined node-negative esophageal cancer with rapid quantitative reverse transcriptase-polymerase chain reaction to measure expression of carcinoembryonic antigen messenger RNA. We also performed rapid quantitative reverse transcriptase-polymerase chain reaction on 37 snap-frozen lymph nodes from 23 patients. Eleven of the 23 patients had benign esophageal disorders (negative control group). The other 12 had esophageal cancer, 6 with histologically determined positive lymph nodes and 6 with histologically determined negative lymph nodes. RESULTS: In the retrospective analysis of archival tissue from 30 patients with esophageal cancer with histologically determined negative lymph nodes, rapid quantitative reverse transcriptase-polymerase chain reaction predicted disease recurrence with a sensitivity and a specificity of 90% and 80%, respectively, and was comparable to conventional quantitative reverse transcriptase-polymerase chain reaction. In the frozen-tissue analysis rapid quantitative reverse transcriptase-polymerase chain reaction detected significantly higher levels of carcinoembryonic antigen expression in all 12 of the histologically determined positive lymph nodes than in the benign nodes. For 2 of these 12 nodes the intraoperative frozen-section analysis had negative histologic results, and N1 status was determined only on final pathologic examination. Rapid (intraoperative) quantitative reverse transcriptase-polymerase chain reaction discriminated both nodes as positive. Among the 14 histologically determined negative nodes, 1 of 3 nodes from 1 patient showed increased carcinoembryonic antigen according to rapid quantitative reverse transcriptase-polymerase chain reaction, and this patient had a clinical recurrence. CONCLUSIONS: In our study we were able to rapidly discriminate patients with node negative-esophageal cancer who had a high risk of recurrence. In frozen tissues rapid quantitative reverse transcriptase-polymerase chain reaction correlated with final pathologic report for 11 of 12 patients. In the 1 discordant case, the quantitative reverse transcriptase-polymerase chain reaction result was positive and may have detected microscopically occult metastasis, because this patient did have disease recurrence. Rapid quantitative reverse transcriptase-polymerase chain reaction was more sensitive than intraoperative frozen sections for detecting metastatic disease. These data suggest that rapid quantitative reverse transcriptase-polymerase chain reaction may have a prognostic role and could guide intraoperative decisions.

Aged↗

PubMed2002

Temperature-controlled primer limit for multiplexing of rapid, quantitative reverse transcription-PCR assays: application to intraoperative cancer diagnostics.

BACKGROUND: Rapid-cycling, real-time PCR instruments bring the opportunity for improved intraoperative detection of metastasis to sentinel lymph nodes. Rapid, standardized, and internally controlled assays need to be developed that are sensitive and accurate. METHODS: We describe rapid, multiplexed, internally controlled, quantitative reverse transcription-PCR (QRT-PCR) assays for tyrosinase and carcinoembryonic antigen mRNAs on the SmartCycler (Cepheid). We used a temperature-controlled primer-limiting approach to eliminate amplification of the endogenous control gene as soon as its signal had reached threshold. Positive-control oligonucleotide mimics were incorporated into all reactions to differentiate failed reactions from true negative samples. RESULTS: The optimized assays for rapid QRT-PCR yielded results with threshold cycle values that were only 1-2 cycles higher than slower, more conventional protocols. In rapid PCR, the temperature-controlled multiplex assay was quantitative over a dynamic range of at least 15 cycles, compared with only 6 cycles for conventional multiplexing methods. All histologically positive lymph nodes examined were also QRT-PCR positive for the appropriate marker, and the exogenous, internal positive-control mimics produced signals in all negative samples. CONCLUSION: Internally controlled, rapid QRT-PCR assays can be performed in an intraoperative time frame and with sufficient sensitivity to detect histologically identified metastases to lymph nodes.

Base Sequence↗