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Biomedical subjects

Joseph Califano

Publications and source records attributed to Joseph Califano.

At least 19 recordsLinked to original sources
PubMed2006

Increased plasma DNA integrity index in head and neck cancer patients.

Analysis of the length of circulating DNA in plasma has been reported as a marker for solid tumor detection. We assessed the sensitivity and specificity of increased plasma DNA length to identify patients with head and neck squamous cell carcinoma (HNSCC) and monitor posttreatment disease status. Fifty-eight HNSCC patients with paired pre- and postoperative plasma and 47 plasma samples from control subjects were analyzed using quantitative PCR to determine plasma DNA integrity index. We found that the mean DNA integrity index was significantly greater in the plasma from HNSCC patients, 0.24 (95% CI: 0.11, 0.38), when compared to plasma from the control subjects, -2.24 (95% CI: -2.92, -1.56), p < 0.0001 using multivariate analysis. The optimal sensitivity (the value for which sensitivity equals specificity) was found at a plasma DNA integrity index of 0.82: sensitivity, 84.5%; specificity, 83%. However, there was no significant difference noted between pre- and postoperative DNA integrity index in plasma samples from HNSCC patients. This study shows that DNA integrity index in the plasma of the patients with HNSCC is increased in comparison with that in the plasma from non-HNSCC control subjects. Lack of normalization of plasma DNA integrity index after surgical resection implies the persistence of a population of cells with an altered pattern of DNA degradation despite removal of malignancy.

Adult↗
PubMed2006

Phosphodiesterase-5 inhibition augments endogenous antitumor immunity by reducing myeloid-derived suppressor cell function.

Phosphodiesterase-5 (PDE5) inhibitors (sildenafil, tadalafil, and vardenafil) are agents currently in clinical use for nonmalignant conditions. We report the use of PDE5 inhibitors as modulators of the antitumor immune response. In several mouse tumor models, PDE5 inhibition reverses tumor-induced immunosuppressive mechanisms and enables a measurable antitumor immune response to be generated that substantially delays tumor progression. In particular, sildenafil, down-regulates arginase 1 and nitric oxide synthase-2 expression, thereby reducing the suppressive machinery of CD11b+/Gr-1+ myeloid-derived suppressor cells (MDSCs) recruited by growing tumors. By removing these tumor escape mechanisms, sildenafil enhances intratumoral T cell infiltration and activation, reduces tumor outgrowth, and improves the antitumor efficacy of adoptive T cell therapy. Sildenafil also restores in vitro T cell proliferation of peripheral blood mononuclear cells from multiple myeloma and head and neck cancer patients. In light of the recent data that enzymes mediating MDSC-dependent immunosuppression in mice are active also in humans, these findings demonstrate a potentially novel use of PDE5 inhibitors as adjuncts to tumor-specific immune therapy.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed2006

The role of positron emission tomography and computed tomography fusion in the management of early-stage and advanced-stage primary head and neck squamous cell carcinoma.

OBJECTIVE: To evaluate the role of positron emission tomography and computed tomography (PET-CT) fusion in the management of early-stage and advanced-stage primary head and neck squamous cell cancer. DESIGN: Retrospective analysis, with a blinded evaluation of clinical data and formation of a treatment plan. SETTING: Single tertiary academic medical institution. Patients Thirty-six patients with previously untreated head and neck squamous cell carcinoma who underwent staging CT or magnetic resonance imaging of the neck prior to undergoing PET-CT as part of their initial diagnostic evaluation between July 2000 and January 2005. MAIN OUTCOME MEASURES: Confirmation or alteration of the treatment plan with the addition of the PET-CT information compared with traditional clinical and radiological data alone for early-stage and advanced-stage disease. When available, histopathological results were correlated with the PET-CT findings. RESULTS: Among the 36 patients, PET-CT provided additional information that confirmed the treatment plan in 25 patients (69%) and altered the treatment plan in 11 patients (31%). Six of 11 patients in the altered-treatment group had their tumors upstaged. Four of 8 patients with early-stage tumors had their treatment plan altered, compared with 7 of 28 patients with advanced-stage disease. Among 18 patients who underwent a surgical intervention for their primary tumor, PET-CT identified the primary tumor in all 18 patients and, based on histopathological findings, correctly staged the regional nodal disease in 9 of 16 patients who had their nodal disease addressed. CONCLUSION: The use of PET-CT is important in the initial treatment planning of early-stage and advanced-stage head and neck squamous cell carcinoma.

Adolescent↗
PubMed2006

Microsatellite analysis of recurrent vestibular schwannoma (acoustic neuroma) following stereotactic radiosurgery.

HYPOTHESIS: Genetic and immunohistochemical studies may provide insight into the mechanisms of vestibular schwannoma (VS) recurrence following radiation therapy. BACKGROUND: Stereotactic radiation therapy is an increasingly common alternative to microsurgical resection for the primary management of sporadic VS. The molecular mechanisms associated with recurrent vestibular schwannoma (VS) following radiation therapy are not known. METHODS: Primary or irradiated VS tumors were fresh-frozen at the time of surgical resection and microdissected to undergo DNA extraction. Lymphocytic control DNA was isolated from blood obtained by venipuncture. Paired normal and tumor DNA specimens were analyzed for allelic loss by PCR amplification of polymorphic dinucleotide repeat sequences. Immunohistochemical studies were performed on paraffin-embedded, irradiated surgical specimens. RESULTS: Using 16 polymorphic microsatellite markers, 20 of 26 non-irradiated VS demonstrated loss of heterozygosity (LOH) in at least one locus of chromosome 22q. In contrast, none of the four irradiated recurrent VS demonstrated LOH on chromosome 22q (p = 0.008). No allelic loss was seen in either the primary or irradiated VS utilizing markers mapping to chromosome 10. Deletions on chromosome 10 are seen in both benign and higher-grade meningiomas and intracranial malignancies associated with radiotherapy. Immunohistochemical studies were performed to detect the protein product of the NF2 gene, merlin, in the four irradiated VS. NF2 staining was not observed. CONCLUSION: This study represents the first microsatellite and immunohistochemical analysis of recurrent VS following radiation therapy. Our preliminary observations suggest an alternative mechanism of NF2 inactivation that may correlate with radioresistance in VS.

Base Sequence↗
PubMed2006

Mitochondrial DNA content increase in response to cigarette smoking.

An increase in mitochondrial DNA (mtDNA) content and decline in mitochondrial function occurs with aging and in response to DNA-damaging agents, including tobacco smoke. We did a cross-sectional study and quantified changes in mtDNA content in a population of individuals with varied smoking and alcohol exposure. Age, smoking history, ethanol intake, and other demographic data were characterized for 604 individuals participating in a screening study for smoking-related upper aerodigestive malignancy. Total DNA was extracted from exfoliated cells in saliva. DNA from a nuclear gene, beta-actin, and two mitochondrial genes, cytochrome c oxidase I and II (Cox I and Cox II), were quantified by real-time PCR. mtDNA content was correlated with age, exposure history, and other variables using multivariate regression analyses. A significant increase (P<0.001) in mtDNA content was noted in smokers (31% and 29% increase for Cox I and Cox II, respectively) and former smokers (31% and 34%) when compared with never smokers. This association persisted after adjustment for other significant factors including age, alcohol drinking, and income (P<0.001). Increased mtDNA content was positively associated with pack-years of smoking (P=0.02). Despite an average smoking cessation interval of 21 years in former smokers, tobacco cessation interval was not statistically significantly associated with mtDNA content. Smoking is associated with increased mtDNA content in a dose-dependent fashion. Mitochondrial DNA alterations in response to smoking persist for several decades after smoking cessation, consistent with long-term, smoking-related damage.

Actins↗
PubMed2006

An oligonucleotide microarray for high-throughput sequencing of the mitochondrial genome.

Previously we developed an oligonucleotide sequencing microarray (MitoChip) as an array-based sequencing platform for rapid and high-throughput analysis of mitochondrial DNA. The first generation MitoChip, however, was not tiled with probes for the noncoding D-loop region, a site frequently mutated in human cancers. Here we report the development of a second-generation MitoChip (v2.0) with oligonucleotide probes to sequence the entire mitochondrial genome. In addition, the MitoChip v2.0 contains redundant tiling of sequences for 500 of the most common haplotypes including single-nucleotide changes, insertions, and deletions. Sequencing results from 14 primary head and neck tumor tissues demonstrated that the v2.0 MitoChips detected a larger number of variants than the original version. Multiple coding region variants detected only in the second generation MitoChips, but not the earlier chip version, were further confirmed with conventional sequencing. Moreover, 31 variations in noncoding region were identified using MitoChips v2.0. Replicate experiments demonstrated >99.99% reproducibility in the second generation MitoChip. In seven head and neck cancer samples with matched lymphocyte DNA, the MitoChip v2.0 detected at least one cancer-associated mitochondrial mutation in four (57%) samples. These results indicate that the second generation MitoChip is a high-throughput platform for identification of mitochondrial DNA mutations in primary tumors.

Base Sequence↗
PubMed2005

Feasibility of quantitative PCR-based saliva rinse screening of HPV for head and neck cancer.

Human papilloma virus (HPV) 16 is likely to be an etiologic factor in a subset of head and neck squamous cell carcinomas (HNSC). We investigated the ability of a quantitative PCR-based assay to detect HPV 16 in salivary rinses as a screening method for HNSC. Real time quantitative PCR was applied to detect HPV16 E6 and E7 DNA level in 92 primary tumors and salivary rinses from patients with HNSC and 604 control subjects without HNSC. A total of 45.6% (42/92) of primary HNSC and 32.6% (30/92) of saliva rinse samples from HNSC patients had any detectable HPV 16 DNA, 30.4% (28/92) of tumors had HPV 16 levels >0.1 copies/cell. HPV16 was detected in 50% (confidence interval [CI] = 34.2-65.8%) (21/42) of the saliva rinse samples from HNSC patients with detectable HPV16 level in their tissue, 18% (CI = 8.4-30.9%) (9/50) of saliva rinse samples from patients with HPV 16 negative primary HNSC, in 2.8% (CI = 1.7-4.5%) (17/604) of the normal controls (p < 0.001). Using a cutoff of HPV 16 >0.001 copies/cell in saliva rinse yielded a sensitivity of 30.4% and a specificity of 98.3%. Nonsmokers had significant higher HPV 16 level than smokers in the cohort of cancer patients. HPV 16 DNA in saliva rinses can reflect HPV 16 status of primary HNSC. Quantitative analysis of HPV 16 DNA in salivary rinses allows for detection of HPV-related HNSC, however, specific limitations exist that prevent the application of this as a screening technique for a broad population.

Adolescent↗
PubMed2005

Differential DNA hypermethylation of critical genes mediates the stage-specific tobacco smoke-induced neoplastic progression of lung cancer.

Promoter DNA methylation status of six genes in samples derived from 27 bronchial epithelial cells and matching blood samples from 22 former/current smokers and five nonsmokers as well as 49 primary non-small cell lung cancer samples with corresponding blood controls was determined using methylation-specific PCR (MSP). Lung tumor tissues showed a significantly higher frequency of promoter DNA methylation in p16, MGMT, and DAPK (P < 0.05; Fisher's exact test). p16 promoter DNA methylation in tumors was observed at consistently higher levels when compared with all the other samples analyzed (P = 0.001; Fisher's exact test). ECAD and DAPK exhibited statistically insignificant differences in their levels of DNA methylation among the tumors and bronchial epithelial cells from the smokers. Interestingly, similar levels of methylation were observed in bronchial epithelial cells and corresponding blood from smokers for all four genes (ECAD, p16, MGMT, and DAPK) that showed smoking/lung cancer-associated methylation changes. In summary, our data suggest that targeted DNA methylation silencing of ECAD and DAPK occurs in the early stages and that of p16 and MGMT in the later stages of lung cancer progression. We also provide preliminary evidence that peripheral lymphocytes could potentially be used as a surrogate for bronchial epithelial cells to detect altered DNA methylation in smokers.

Apoptosis Regulatory Proteins↗
PubMed2005

Increased mitochondrial DNA content in saliva associated with head and neck cancer.

Alterations of the mitochondrial DNA (mtDNA) have been described in human tumors and in other tissues in association with smoking exposure. We did quantitative PCR of cytochrome c oxidase I (Cox I) and cytochrome c oxidase II (Cox II) genes on oral rinse samples obtained from 94 patients with primary head and neck squamous cell carcinoma (HNSC) and a control group of 656 subjects. Mitochondrial DNA/nuclear DNA in saliva from HNSC patients and controls in relationship to smoking exposure, ethanol intake, and tumor stage were examined. Mean levels of Cox I and Cox II in saliva samples were significantly higher in HNSC patients: Cox I, 0.076 [95% confidence interval (95% CI), 0.06-0.09] and Cox II, 0.055 (95% CI, 0.04-0.07) in comparison with controls Cox I, 0.054 (95% CI, 0.05-0.06), P < 0.0001 and Cox II, 0.046 (95% CI, 0.04-0.05), P = 0.003 (t test). MtDNA levels were elevated in primary tumors when compared with matched, pretreatment saliva and significant correlation was noted (Cox I, r = 0.30, P = 0.005 and Cox II r = 0.33, P = 0.002, respectively, Pearson's correlation). On univariate analysis, smoking, age, HNSC diagnosis, and advanced stage of HNSC were associated with higher level of mtDNA content in saliva. Multivariate analysis showed a significant and independent association of HNSC diagnosis, age, and smoking with increasing mtDNA/nuclear DNA for Cox I and Cox II. mtDNA content alteration is associated with HNSC independently of age and smoking exposure, can be detected in saliva, and may be due to elevation in mtDNA content in primary HNSC.

Adolescent↗
PubMed2005

Molecular margin analysis predicts local recurrence after sublobar resection of lung cancer.

Sublobar resection for early-stage lung cancer has been used for patients who are not candidates for lobar resection. However, sublobar resection is associated with high local recurrence rates in the context of tumor-free parenchymal margins. The mechanism underlying this high recurrence rate is not well understood. We hypothesized that this elevated risk of local recurrence is due to undetected tumor cells present at parenchymal margins thought to be negative by conventional light microscopy. Thirteen of 44 patients who underwent sublobar resection for lung cancer were found to have a k-ras mutation at codon 12.1. A novel fluorescence-based assay for detection of rare copies of mutant DNA in a background of wild-type DNA, fluorescent gap ligase chain reaction, was used to quantitate the mutant/wild-type DNA in a range of 1 to 1/10,000 in histologically normal margins from these resections. Nine of 13 patients had at least one margin with the number of mutant cells over or equal to a threshold of 1/5,000, and of these, 6/9 (67%) recurred locally. None of the remaining 4 patients without mutant DNA in any surgical margin had evidence of recurrence. The higher rate of local recurrence associated with sublobar resection of lung cancer is likely due to the occult presence of tumor cells at resection margins. These occult tumor cells can be quantitated using a novel fluorescence-based assay and define a group of patients at high risk for local recurrence who are candidates for adjuvant therapy or more extensive resection. This methodology may be adaptable to a real-time format for intraoperative use.

Carcinoma, Non-Small-Cell Lung↗
PubMed2005

Loss of heterozygosity patterns provide fingerprints for genetic heterogeneity in multistep cancer progression of tobacco smoke-induced non-small cell lung cancer.

Dilution end point loss of heterozygosity (LOH) analysis, a novel approach for the analysis of LOH, was used to evaluate allelic losses with the use of 21 highly polymorphic microsatellite markers at nine chromosomal sites most frequently affected in smoking-related non-small cell lung cancers. Allelotyping was done for bronchial epithelial cells and matching blood samples from 23 former and current smokers and six nonsmokers as well as in 33 adenocarcinomas and 25 squamous cell carcinomas (SCC) and corresponding matching blood from smokers. Major conclusions from these studies are as follows: (a) LOH at chromosomal sites 8p, 9p, 11q, and 13q (P >0.05, Fisher's exact test) are targeted at the early stages, whereas LOH at 1p, 5q, 17p, and 18q (P <0.05, Fisher's exact test) occur at the later stages of non-small cell lung cancer progression; (b) LOH at 1p, 3p, 5q, 8p, 9p, 11q, 13q, 17p, and 18q occurs in over 45% of the tobacco smokers with SCC and adenocarcinoma; (c) compared with bronchial epithelial cells from smokers, there is a significantly higher degree of LOH at 1p, 5q, and 18q in adenocarcinoma and at 1p, 3p, and 17p in SCC (P <0.05, Fisher's exact test). We propose that lung cancer progression induced by tobacco smoke occurs in a series of target gene inactivations/activations in defined modules of a global network. The gatekeeper module consists of multiple alternate target genes, which is inclusive of but not limited to genes localized to chromosomal loci 8p, 9p, 11q, and 13q.

Adenocarcinoma↗
PubMed2004

Effect of human papillomavirus-16 infection on CD8+ T-cell recognition of a wild-type sequence p53264-272 peptide in patients with squamous cell carcinoma of the head and neck.

PURPOSE: Wild-type sequence (wt) p53 peptides are attractive candidates for broadly applicable cancer vaccines, currently considered primarily for patients whose tumors overexpress p53. Circumstances exist, however, where increased p53 degradation may result in appreciable presentation of p53-derived peptides, despite low p53 expression. Squamous cell carcinoma of the head and neck is associated with oncogenic human papillomavirus (HPV) subtypes, which inactivate p53 through proteasomal degradation. The criterion of p53 overexpression would exclude these individuals from wt p53-based immunotherapy. EXPERIMENTAL DESIGN: We tested the correlation of HPV infection with enhanced antigenicity of the p53 protein and postulated that removal of HPV-16(+) tumors with enhanced p53(264)-(272) peptide presentation might lead to a drop in T cells specific for this peptide in vivo. Circulating frequencies of T cells specific for the HLA A*0201:p53(264)-(272) complex were measured ex vivo using dimeric HLA:peptide complexes in 15 head and neck cancer patients before and 6 months after tumor excision. RESULTS: CD8+ T-cell recognition of HLA A*0201 restricted wt p53(264)-(272) peptide presented by HPV-16(-) squamous cell carcinoma of the head and neck lines was enhanced by HPV-16 E6 expression, sometimes exceeding that of a naturally transformed, HPV-16(+) wt p53 expressing squamous cell carcinoma of the head and neck cell line. In patients with HPV-16(-) tumors, the frequency of wt p53(264-272)-specific T cells remained largely unchanged after tumor removal. However, a significant decline in frequency of anti-p53(264-272) T cells was observed postoperatively in HPV-16(+) patients (P < 0.005). CONCLUSIONS: Recognition of HPV-associated squamous cell carcinoma of the head and neck appears associated with levels of wt p53-specific T cells and inversely with p53 expression. p53 peptides may be useful tumor antigens for squamous cell carcinoma of the head and neck immunotherapy in addition to viral gene products.

Aged↗
PubMed2004

Gene expression alterations over large chromosomal regions in cancers include multiple genes unrelated to malignant progression.

In solid tumors, the relationship between DNA copy number and global expression over large chromosomal regions has not been systematically explored. We used a 12,626-gene expression array analysis of head and neck squamous cell carcinoma and normal oral mucosa and annotated gene expression levels to specific chromosomal loci. Expression alterations correlated with reported data using comparative genomic hybridization. When genes with significant differences in expression between normal and malignant lesions, as defined by significance analysis of microarrays (SAM), were compared to nonsignificant genes, similar chromosomal patterns of alteration in expression were noted. Individual tumors underwent microsatellite analysis and chi(2) analysis of expression at 3p and 22q. Significant 3p underexpression and 22q overexpression were found in all primary tumors with 3p and 22q allelic imbalance, respectively, whereas no tumor without allelic imbalance on these chromosomal arms demonstrated expression differences. Loss and gain of chromosomal material in solid cancers can alter gene expression over large chromosomal regions, including multiple genes unrelated to malignant progression.

Allelic Imbalance↗
PubMed2004

The V599E BRAF mutation is uncommon in biliary tract cancers.

Activating point mutations of the BRAF oncogene have been identified in several solid tumors, most commonly in cutaneous melanomas and papillary carcinomas of the thyroid. A specific point mutation--V599E--accounts for the overwhelming majority of these mutational events. We explored the frequency of the V599E BRAF mutation in biliary tract cancers. In all, 62 archival biliary tract cancers, including 15 gallbladder cancers, 15 extrahepatic, and 10 intrahepatic cholangiocarcinomas from the United States, and 22 gallbladder carcinomas from Chile were analyzed for the V599E mutation of the BRAF gene using three distinct methods: direct sequencing, a primer extension method (Mutector assay), and the highly sensitive quantitative Gap Ligase Chain Reaction. The common V599E mutation was not identified in any of the 62 biliary cancer samples using these three methods of detection. The V599E somatic mutation of the BRAF gene is absent in biliary tract cancers, at least in the two geographic populations (United States and Chile) examined. Activation of the RAS/RAF/MAP kinase pathway in biliary tract cancers is likely to be secondary to oncogenic RAS mutations, or due to mutations of the BRAF gene at nucleotide positions not explored in the current study.

Biliary Tract Neoplasms↗
PubMed2004

The Human MitoChip: a high-throughput sequencing microarray for mitochondrial mutation detection.

Somatic mitochondrial mutations are common in human cancers, and can be used as a tool for early detection of cancer. We have developed a mitochondrial Custom Reseq microarray as an array-based sequencing platform for rapid and high-throughput analysis of mitochondrial DNA. The MitoChip contains oligonucleotide probes synthesized using standard photolithography and solid-phase synthesis, and is able to sequence >29 kb of double-stranded DNA in a single assay. Both strands of the entire human mitochondrial coding sequence (15,451 bp) are arrayed on the MitoChip; both strands of an additional 12,935 bp (84% of coding DNA) are arrayed in duplicate. We used 300 ng of genomic DNA to amplify the mitochondrial coding sequence in three overlapping long PCR fragments. We then sequenced >2 million base pairs of mitochondrial DNA, and successfully assigned base calls at 96.0% of nucleotide positions. Replicate experiments demonstrated >99.99% reproducibility. In matched fluid samples (urine and pancreatic juice, respectively) obtained from five patients with bladder cancer and four with pancreatic cancer, the MitoChip detected at least one cancer-associated mitochondrial mutation in six (66%) of nine samples. The MitoChip is a high-throughput sequencing tool for the reliable identification of mitochondrial DNA mutations from primary tumors in clinical samples.

Body Fluids↗