Changes in gene expression induced by chemoradiation in advanced cervical carcinoma: a microarray study of RTOG C-0128.

PURPOSE: To evaluate gene expression patterns in patients with advanced cervix cancer before and during chemoradiation in a multi-institutional cooperative group setting. METHODS: RTOG C0128 was designed as a Phase II trial of radiation therapy with concomitant chemotherapy and Celecoxib at 400 mg twice daily for one year. Tumor samples were obtained for microarray gene expression analysis before treatment and at the time of the first implant (paired sample). RNA was extracted, linearly amplified, and purity was assessed by gel electrophoresis. Each sample was hybridized against a universal RNA mixture on a customized spotted array consisting of >10,000 genes. Gene expression pre-treatment was compared with clinical characteristics. Changes in gene expression following radiation were assessed within the paired samples (same patient) and then compared across all paired samples. Data were normalized using the AROMA software, and clustering analysis was performed using Ward's method in Spotfire. Differences in paired samples were calculated with Significance Analysis of Microarrays (SAM). RESULTS: From August 2001 to March 2004, 84 patients were accrued to the trial. Tissue was obtained prior to initiation of therapy from 34 patients (40%). FIGO stages of the patients providing tissue were IB (23%), II (57%), and IIIA-IVA (20%). RNA quality was sufficient in 22 pre-treatment and 14 post-treatment samples. Among pre-treatment samples, no significant differences in gene expression were observed by FIGO stage, age, or race. However, between comparison of histologic subtypes (adenocarcinoma, n=5; squamous cell carcinoma, n=17) demonstrated 45 genes differentially expressed with a false discovery rate of 0.018. Cluster analysis segregated unpaired samples into 2 groups: 18/22 comprising pre-treatment samples and 10/14 in group 2 representing post-treatment samples. In all 13 paired samples, gene expression after chemoradiation was significantly upregulated in 91 genes and downregulated in 251 genes (false discovery rate of 0.0018). Genes significantly upregulated included bax, cdk inhibitor 1, MMP2, and adhesion molecules PECAM1, VCAM1, and ICAM2. Genes significantly downregulated included topoisomerase II alpha, myc, H2AX, MSH2, RAD51, RAD53, PCNA, and cell cycle-regulating molecules chk1, CDK2, cyclinB1, cyclin D3, cdc2, and cdc25. CONCLUSIONS: Microarray analysis was successfully performed in a multi-institutional cooperative group trial. Gene expression significantly correlated with histology, but not stage, age or race. Cluster analysis identified two groups of gene expression profiles correlating with pre or post-treatment acquisition of tissue. Notably, paired samples showed significant changes in gene expression following chemoradiation, including several downregulated radiation response genes. Further analysis comparing gene expression to clinical outcomes, acute and late toxicities awaits maturation of clinical data. Hopefully, this data will lead to the development of molecularly based therapies.

Feasibility of RNA collection for micro-array gene expression analysis in the treatment of cervical carcinoma: a scientific correlate of RTOG C-0128.

PURPOSE: To determine the feasibility of RNA collection in a multi-institutional cooperative group setting to be utilized for micro-array gene expression analysis, and to describe the methodology. METHODS: RTOG C0128, a phase I-II, protocol was designed to look at the safety and efficacy of external beam radiation therapy to 45 Gy with concomitant 5-FU and cisplatin chemotherapy, brachytherapy to deliver 85 Gy to point A, and Celecoxib at 400 mg twice daily for 1 year. Patients had the option of participating in a tissue collection portion of the protocol to be utilized for micro-array gene expression analysis before treatment and at the time of the first implant. RNA quality was determined by two parameters: the absorbance ratio at 260 nm/280 nm, and by the ratio of the integrated peak of 28S RNA to 18S RNA after gel electrophoresis. RESULTS: From August 2001 to March 2004, 84 patients were accrued to the trial, and tissue was obtained prior to initiation of therapy on 34 patients (40%). FIGO stages for the patients who provided tissue were IB (23%), II (57%), and IIIA-IVA (20%). Additionally, biopsies were obtained at the time of the first implant from 22 of the accrued patients making paired samples available on 26% for RNA extraction and micro-array gene expression analysis. The mean +/- SEM amount of tissue obtained pretreatment was 97 +/- 13 mg compared with 51 +/- 8 mg for tissue obtained at the time of the first implant (P = 0.009). The mean total RNA extracted from the samples prior to treatment was 119 +/- 19 microg versus 35 +/- 6 microg at the time of the first procedure (P = 0.001). The RNA quality was assessed via the absorbance ratio at 260 nm divided by 280 nm. The mean values pretreatment and at first implant were 1.87 +/- 0.07 versus 1.66 +/- 0.11, respectively (P = 0.002); however, the integrated peak of 28S RNA to 18S RNA after gel electrophoresis was not significantly different (P = 0.26). CONCLUSIONS: RNA extraction for gene expression analysis can be successfully performed in the multi-institutional cooperative group setting. Fresh tissue samples were obtained on 40% of accrued patients prior to treatment. The amount of biopsy material and the quantity of RNA extracted were greater prior to treatment compared with the first implant. The quality of RNA was superior prior to treatment as measured by the ratio of absorbance at 260/280 nm. These results indicate that gene expression analysis is feasible in the cooperative group setting utilizing amplification techniques for the RNA. Hopefully, this will allow for improvement in prognosis, therapeutic development, and correlation with acute and late toxicities in patients with cancer.

Neuroendocrine carcinomas: role of immunocytochemistry and electron microscopy.

Neuroendocrine tumors occur in many sites of the body and can present significant diagnostic problems when poorly differentiated. To identify a tumor as neuroendocrine, pathologists commonly use either immunocytochemistry or electron microscopy. In this report, the various immunocytochemical reagents are reviewed along with the ultrastructural features of neuroendocrine tumors. Site-specific variations in neuroendocrine tumors are discussed. A cost-effectiveness evaluation was performed on tumors from one laboratory which showed that electron microscopy was a less expensive diagnostic modality if more than three antibodies were necessary to arrive at the correct pathological diagnosis.

Clinically relevant breast cancer reporting: using process measures to improve anatomic pathology reporting.

OBJECTIVE: Breast cancer reports form an important part of the basis of clinical decision making for patients. Our objectives were to improve breast cancer reporting in the Urban Central Region in Utah of Intermountain Health Care in a clinically relevant manner and to show that the method chosen actually improved information transfer among physicians of breast cancer patients and led to durable changes in pathologist behavior. METHODS/INTERVENTION: Pathologists designed a synoptic report based on interviews with oncologists about what data were meaningful. The report format was piloted with one hospital pathology group, modified, and implemented in three hospitals. A report evaluation of missing information was done before, immediately after, and 2 years after the intervention. Oncologists were surveyed after 2 years to evaluate satisfaction with report format. RESULTS: Changing breast cancer reporting to a synoptic format significantly decreased information missing from pathology reports. Prior to implementation, 32 of 365 reports lacked some item(s) of pathology information desirable to clinicians; after the intervention, 8 of 250 reports contained missing information. After 2 years, 1 in 190 reports contained missing data elements. Synoptic breast cancer reports continued to be used by pathologists throughout the reporting period. Oncologists responding to a survey reported uniform satisfaction with the new reporting format. SUMMARY: Pathologists are important members of the clinical oncology team. They provide patient-specific information crucial to patient care. Activities designed to improve the quality of reporting processes should use clinically relevant indicators of process improvement, such as measurement of missing information and satisfaction of clinical colleagues with format/quality of information.

The diagnosis of thymoma: a review.

Thymoma is the most common tumor of the anterior-superior mediastinum, especially in middle-aged or older adults. Microscopically, thymomas can be differentiated from other tumors with which they can be confused by the finding of a mixed population of cells, including neoplastic thymic epithelial cells with numerous processes surrounding activated-appearing lymphocytes. Thymomas can be classified as benign or malignant, and the majority of those that are malignant appear cytologically benign and are locally invasive. Cytologically benign thymomas have been classified as being lymphocyte rich, epithelial cell rich, or spindle cell type. This classification has not been found to be prognostically useful. A new method classifies these tumors as being cortical, medullary, or mixed. This new classification appears to have prognostic significance. Malignant thymomas that are cytologically malignant are uncommon. Such tumors usually are squamous cell carcinomas. Other types include sarcomatoid carcinoma, clear cell carcinoma, basaloid carcinoma, and mucoepidermoid carcinoma. Because other tumors can occur in the anterior mediastinum, electron microscopy and/or immunocytochemistry is helpful in making the distinction. The differential features by ultrastructural and immunocytochemical analysis are reviewed.

Gastrointestinal stromal tumor of the duodenum: a case report.

The report describes a malignant gastrointestinal stromal tumor occurring in the duodenum in a 71-year-old woman. The neoplasm showed both epithelioid and spindle cell patterns by light microscopy. The ultrastructural features were diagnostic of nerve sheath origin. The tumor had numerous wrapping processes joined by junctions and surrounded by axons. No features of smooth muscle differentiation were identified. Immunocytochemistry was inconclusive. The differential diagnosis of such neoplasms is discussed.

Thyrolipoma: diagnostic pitfalls in the cytologic diagnosis and review of the literature.

Thyrolipoma, or adenolipoma of the thyroid gland, is an uncommon neoplasm and thus not recognized by many cytopathologists. This is the first article to describe the fine-needle aspirate findings of a histologically confirmed thyrolipoma. The gross morphologic and computerized tomographic features of this entity are also discussed, and the literature is reviewed.

Concurrent occurrence of adenocarcinoma and carcinoid tumor in the stomach: a composite tumor or collision tumors?

A morphologic curiosity is presented in a polypoid gastric tumor combined with adenocarcinoma and carcinoid tumor. It is likely that those two represent a composite tumor; however, the authors think it is more likely that they are two concurrent, independent collided tumors.