Patterns of HER-2/neu Amplification and Overexpression in Primary and Metastatic Breast Cancer

Background: Only 25% of patients with HER-2/neu-positive metastatic breast tumors respond favorably to trastuzamab (Herceptin) treatment. We hypothesized that a high failure rate of patients on trastuzamab could result if some of the metastases were HER-2 negative and these metastases ultimately determine the course of the disease. Methods: We used tissue microarrays (TMAs) containing four samples each from 196 lymph node-negative primary tumors, 196 lymph node-positive primary tumors, and three different lymph node metastases from each lymph node-positive tumor to estimate HER-2 gene amplification by fluorescence in situ hybridization (FISH) and Her-2 protein overexpression by immunohistochemistry (IHC). Results: FISH and IHC analyses gave the same result with respect to HER-2 status for 93.7% of the tissues contained in the TMAs. Tissue samples were, therefore, considered to be HER-2 positive if they were positive for either HER-2 DNA amplification or Her-2 protein expression and HER-2 negative if both FISH and IHC gave a negative result. The HER-2 status of lymph node-positive primary tumors was maintained in the majority of their metastases. For HER-2-positive primary tumors, 77% (95% confidence interval [CI] = 59% to 90%) had entirely HER-2-positive metastases, 6.5% (95% CI = 8% to 21%) had entirely HER-2-negative metastases, and 16.3% (95% CI = 5% to 34%) had a mixture of HER-2-positive and HER-2-negative metastases. For HER-2-negative primary tumors, 95% (95% CI = 88% to 98%) had metastases that were entirely negative for HER-2. Conclusions: Our data suggest that differences in HER-2 expression between primary tumors and their lymph node metastases cannot explain the high fraction of nonresponders to trastuzamab therap

Detecting Activation of Ribosomal Protein S6 Kinase by Complementary DNA and Tissue Microarray Analysis

Background: Studies by comparative genomic hybridization (CGH) have shown that chromosomal region 17q23 is amplified in up to 20% of primary breast cancers. We used microarray analyses to measure the expression levels of genes in this region and to explore their prognostic importance. Methods: A microarray that contained 4209 complementary DNA (cDNA) clones was used to identify genes that are overexpressed in the MCF-7 breast cancer cell line as compared with normal mammary tissue. Fluorescence in situ hybridization was used to analyze the copy number of one overexpressed gene, ribosomal protein S6 kinase (S6K), and to localize it to the 17q23 region. Northern and western blot analyses were used to measure S6K gene and protein expression, and an enzymatic assay was used to measure S6K activity. Tumor tissue microarray analysis was used to study amplification of S6K and the HER-2 oncogene, another 17q-linked gene, and the relationship between amplification and prognosis was analyzed. The Kaplan-Meier method was used for data analysis, and the log-rank test was used for statistical analysis. All P values are two-sided. Results: S6K was amplified and highly overexpressed in MCF-7 cells relative to normal mammary epithelium, and protein expression and enzyme activity were increased. S6K was amplified in 59 (8.8%) of 668 primary breast tumors, and a statistically significant association between amplification and poor prognosis (P = .0021) was observed. Amplification of both S6K and HER-2 implied particularly poor survival (P = .0001). Conclusions: The combination of CGH information with cDNA and tissue microarray analyses can be used to identify amplified and overexpressed genes and to evaluate the clinical implications of such genes and genomic rearrangements. S6K is likely to be one of the genes at 17q23 that is amplified during oncogenesis and may adversely affect the prognosis of patients with this amplificatio

Role of the medullary perfusion defect in the pathogenesis of ischemic renal failure

Role of the medullary perfusion defect in the pathogenesis of ischemic renal failure. Experiments were performed on rats to investigate the significance of the medullary hyperemia known to follow renal ischemia. To this end, its frequency was determined, its severity was quantified, and its relation to renal function was examined early (1 to 3 hr) and later (18 hr) after 45 min of warm ischemia. All kidneys were found to have a hyperemic outer medulla early after ischemia, which was shown to develop during the period of ischemia itself, but which was found to be highly variable in its severity. The degree of hyperemia was assessed both subjectively by grading and by histometric determinations of inner stripe capillary volume. One to three hours after ischemia, the severity of medullary hyperemia was reflected in all indices of renal function, the least congested kidneys showing the best function. Eighteen hours after ischemia, the degree of medullary hyperemia was reflected in all indices of renal function, except urine flow rate; the non-congested kidneys showed functional recovery and the still-congested kidneys showed worsening function. Glomerular blood flow, known to be preferentially reduced in deep nephrons 1 to 3 hr after ischemia, had normalized 18 hr after ischemia in the non-congested kidneys but was still severely and unevenly depressed in the congested kidneys. It is concluded that congestion of the outer medulla is a key event in ischemic renal failure, its occurrence is coincidental with the reduction in deep nephron perfusion and urinary concentrating power in the early and maintenance phase and its disappearance heralds the restoration of deep nephron perfusion and urinary concentrating ability in the recovery phase.Rôle du défaut de perfusion médullaire dans la pathogénie de l'insuffisance rénale ischémique. Des expériences ont été effectuées chez le rat pour étudier la signification de l'hyperémie médullaire connue pour faire suite à l'ischémie rénale. Dans ce but, sa fréquence a été déterminée, sa sévérité quantifiée et sa relation avec la fonction rénale examinée précocément (1 à 3 hr) et plus tardivement (18 hr) après une ischémie chaude de 45 min. Tous les reins se sont avérés avoir une hyperémie de la médullaire externe précocement après l'ischémie, qui se développait pendant la période d'ischémie ellemême, mais dont la sévérité était très variable. Le degré d'hyperémie a été apprécié subjectivement par gradation et par des déterminations histométriques du volume capillaire de la couche interne. Une à trois heures après ischémie, la sévérité de l'hyperémie médullaire était reflétée par tous les paramètres de la fonction rénale, les reins les moins congestifs ayant la meilleure fonction. Dix-huit heures après ischémie, le degré d'hyperémie médullaire était reflété par tous les paramètres de la fonction rénale, sauf le débit urinaire, les reins non congestifs présentant une récupération fonctionnelle et les reins encore congestifs une dégradation fonctionnelle. Le débit sanguin glomérulaire, connu pour être préférentiellement réduit dans les nephrons profonds 1 à 3 hr après ischémie, s'était normalisé 18 hr après l'ischémie dans les reins non congestifs mais restait encore sévèrement et irrégulièrement diminué dans les reins congestifs. On concult que la congestion de la médullaire externe constitue un phénomène-clé dans l'insuffisance rénale ischémique, sa survenue coïncide avec la réduction de la perfusion des néphrons profonds et du pouvoir de concentration des urines à la phase de début et d'entretien, et sa disparition annonce la restauration de la perfusion des néphrons profonds et du pouvoir de concentration des urines à la phase de récupération

The contribution of vascular obstruction to the functional defect that follows renal ischemia

The contribution of vascular obstruction to the functional defect that follows renal ischemia. Experiments were performed on rats subjected to renal ischemia and various treatment procedures to determine the origin and functional consequences of vascular obstruction. To this end, its occurrence and severity was assessed qualitatively and quantitatively in the outer medulla, where it is particularly prominent. The incidence of medullary hyperemia was not influenced by inhibiting thrombocyte aggregation with 5 or 70 mg/kg of acetyl salicylic acid or preventing fibrin deposition with 100 IE/kg of heparin before ischemia, and these substances produced no improvement renal function. The incidence and degree of hyperemia, however, could be substantially reduced or completely eliminated by acutely raising blood pressure after ischemia or by decreasing the number of circulating erythrocytes before ischemia. These procedures were effective in raising filtration rate and tubular reabsorption from 20% to 60% of normal, in restoring renal blood flow and vascular resistance to completely normal, and in diminishing epithelial damage both three and 18 hours after ischemia. The following conclusions are drawn: first, vascular obstruction, which is not lessened by inhibiting thrombus formation but is easily reversed or prevented by raising perfusion pressure or decreasing hematocrit, is probably caused by erythrocyte aggregation during ischemia. Second, vascular obstruction, which appears to raise renal vascular resistance and lower blood flow and filtration rate, cannot be limited to the medulla but must also be present in the cortex. Finally, reversing or preventing vascular obstruction can fully restore renal perfusion, partially restore glomerular and tubular function, greatly reduce tubular necrosis and thus prevent renal failure

Genomic aberrations in invasive cervical cancer

Purpose: Cervical carcinomas are almost always squamous cell carcinomas, whereas adenocarcinomas are rare. There is evidence that specific genetic events are involved in initiation and progression of invasive cervical cancer. The genotype-phenotype correlations in cervical cancer are unclear. Material and Methods: Comparative genomic hybridization was applied to screen for DNA copy number changes in 62 squamous cell carcinomas and 22 adenocarcinomas of clinical stage 1B. Immunohistochemistry was performed in adenocarcinoma samples to determine the HER-2 expression (HercepTest(TM)). Results: In both cancer types, DNA sequence losses were most prevalent at chromosomes 4 q, Xq and 18 q. Losses were the most frequent alterations in squamous cell carcinoma, whereas DNA sequence gains of chromosome 17 q (54%) represented the most frequent copy number alterations in cervical adenocarcinomas. HER-2 overexpression was detected in only two adenocarcinomas. The total number of DNA aberrations per tumor (P > 0.02), and the number of DNA sequence losses per tumor (P > 0.04) were associated with disease-specific survival in squamous cell carcinoma. 9 p deletions were significantly more frequent in squamous cell carcinomas with lymph node metastasis than in node negative tumors (P > 0.03). Losses of chromosome 11 p (P > 0.0001) and 18 q (P > 0.01) were associated with poor prognosis in squamous cell carcinomas without lymph node metastasis. In analogy to squamous cell carcinoma, DNA sequence losses of chromosome 18 q were significantly associated with poor prognosis in cervical adenocarcinoma (P > 0.01). Conclusion: DNA sequence copy number gains on 17 q are not associated with HER-2 expression in adenocarcinomas. The inactivation of tumor suppressor genes on chromosome 18 q might be responsible for the progression of both cervical adenocarcinoma and squamous cell carcinoma

DNA copy number changes in cervical adenocarcinoma

PURPOSE: There is evidence that specific genetic events are involved in the initiation and progression of squamous cell carcinoma of the uterine cervix. The genotype-phenotype correlations in cervical adenocarcinoma (AC) are unclear. Experimental Design: Comparative genomic hybridization was applied to screen for DNA copy number gains and losses in 22 cervical ACs of clinical stage IB. IHC was performed in all of the samples to determine HER-2/neu expression (HercepTest). RESULTS: The most frequent copy number alterations were DNA sequence gains of chromosome 17q (54%). HER-2/neu expression (score 2+) was immunohistochemically detected in 2 of 22 tumors. DNA sequence losses were most prevalent on chromosomes Xq (50%), Xp (36%), 18q (36%), and 4q (36%). DNA sequence losses of chromosome 18q were associated significantly with poor prognosis in cervical AC (P > 0.01). CONCLUSIONS: DNA sequence copy number gains of chromosome 17q are frequent events in ACs of the cervix. However, gains on 17q are not associated with HER-2/neu expression in cervical ACs. The inactivation of tumor suppressor genes on chromosome 18q might be responsible for the progression of both cervical AC and cervical squamous cell carcinoma

DNA sequence losses on chromosomes 11p and 18q are associated with clinical outcome in lymph node-negative ductal breast cancer

PURPOSE: Histological and other markers alone cannot predict the risk of disease progression in node-negative breast cancer. Several genomic aberrations have been linked to clinical outcome in breast cancer. EXPERIMENTAL DESIGN: In this study, comparative genomic hybridization was applied to screen for specific DNA copy number gains and losses in 20 pT1/pT2 node-negative invasive ductal carcinomas with no disease recurrence with at least 8 years of follow-up and in 20 pT1/pT2 node-negative tumors with distant disease recurrence. RESULTS: The number of genomic aberrations (copy number gains and losses) per tumor was significantly higher in tumors with disease recurrence (P > 0.05). The number of genomic aberrations was associated with histological grade (P > 0.02). Within the group of tumors with disease recurrence, the total number of genetic aberrations per tumor (P > 0.02) and the number of DNA sequence losses per tumor (P > 0.01) were significantly associated with poor survival. Of the individual loci involved, only losses at chromosomes 11p (P > 0.002) and 18q (P > 0.004) were associated with poor survival in the recurrence group. Histological grade and loss of 18q were independent prognostic variables in multivariate analysis. CONCLUSIONS: This genome-wide analysis by comparative genomic hybridization suggests that node-negative ductal breast cancers with a high number of genomic aberrations have an increased risk of disease recurrence. The number of DNA sequence losses, particularly losses of chromosomes 11p and 18q, were associated with poor prognosis. Genes on chromosomes 11p and 18q may play a role in the progression of ductal breast carcinoma