[Show abstract][Hide abstract] ABSTRACT: to determine whether the favorable outcome associated with human papillomavirus (HPV) 16-positive oropharyngeal cancer is related to a patient's adaptive immunity.
academic medical center.
forty-seven of 66 previously untreated patients (6 of 20 patients with stage III and 41 of 46 with stage IV cancer) in a prospective clinical trial of chemoradiotherapy.
all patients were treated with a single course of neoadjuvant chemotherapy followed by either surgery (for nonresponders) or chemoradiotherapy.
pretreatment levels (percentages and absolute counts) of CD3, CD4, CD8, natural killer, and B cells and overall white blood cell counts were measured by flow cytometry. Correlations of subsets with HPV-16 status, tumor subsite, cancer stage, T class, N class, smoking status, performance status, sex, response to chemoradiotherapy, p53 mutation type, epidermal growth factor receptor expression, and disease-specific and overall survival were determined.
after a median follow-up of 6.6 years, improved survival was associated with an elevated percentage of CD8 cells (P = .04), a low CD4:CD8 ratio (P = .01), low epidermal growth factor receptor expression (P = .002), and HPV status (P = .02). The percentage of CD8 cells was significantly higher (P = .04) and the CD4:CD8 ratio was significantly lower (P = .02) in HPV-16-positive patients. A higher percentage of CD8 cells was associated with response to induction chemotherapy (P = .02) and complete tumor response after chemoradiotherapy (P = .045).
these findings confirm previous correlations of outcome with circulating CD8 cell levels and support the conjecture that improved adaptive immunity may play a role in the favorable prognosis of patients with HPV-16-positive cancers.
Archives of otolaryngology--head & neck surgery 12/2010; 136(12):1267-73. DOI:10.1001/archoto.2010.211 · 2.33 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The goal of this study was to examine the effect of tobacco use on disease recurrence (local/regional recurrence, distant metastasis, or second primary) among patients with human papillomavirus (HPV)-positive squamous cell carcinoma of the oropharynx (SCCOP) following a complete response to chemoradiation therapy.
Between 1999 and 2007, 124 patients with advanced SCCOP (86% with stage IV) and adequate tumor tissue for HPV analysis who were enrolled in one of two consecutive University of Michigan treatment protocols were prospectively included in this study. Patients were categorized as never-, former, or current tobacco users. The primary end points were risk of disease recurrence and time to recurrence; secondary end points were disease-specific survival and overall survival.
One hundred and two patients (82.3%) had HPV-positive tumors. Over two thirds (68%) of patients with HPV-positive tumors were tobacco users. Among HPV-positive patients, current tobacco users were at significantly higher risk of disease recurrence than never-tobacco users (hazard ratio, 5.2; confidence interval, 1.1-24.4; P = 0.038). Thirty-five percent of HPV-positive ever tobacco users recurred compared with only 6% of HPV-positive never users and 50% of HPV-negative patients. All HPV-negative patients were tobacco users and had significantly shorter times to recurrence (P = 0.002), and had reduced disease-specific survival (P = 0.004) and overall survival (P < 0.001) compared with HPV-positive patients. Compared with HPV-positive never-tobacco users, those with a tobacco history showed a trend for reduced disease-specific survival (P = 0.064) but not overall survival (P = 0.221).
Current tobacco users with advanced, HPV-positive SCCOP are at higher risk of disease recurrence compared with never-tobacco users.
Clinical Cancer Research 02/2010; 16(4):1226-35. DOI:10.1158/1078-0432.CCR-09-2350 · 8.72 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Knowledge of the central role of high-risk human papillomavirus (HPV) in cervical carcinogenesis, coupled with an emerging need to monitor the efficacy of newly introduced HPV vaccines, warrant development and evaluation of type-specific, quantitative HPV detection methods. In the present study, a prototype PCR and mass spectroscopy (PCR-MS)-based method to detect and quantitate 13 high-risk HPV types is compared to the Hybrid Capture 2 High-Risk HPV DNA test (HC2; Digene Corp., Gaithersburg, MD) in 199 cervical scraping samples and to DNA sequencing in 77 cervical tumor samples. High-risk HPV types were detected in 76/77 (98.7%) cervical tumor samples by PCR-MS. Degenerate and type-specific sequencing confirmed the types detected by PCR-MS. In 199 cervical scraping samples, all 13 HPV types were detected by PCR-MS. Eighteen (14.5%) of 124 cervical scraping samples that were positive for high-risk HPV by HC2 were negative by PCR-MS. In all these cases, degenerate DNA sequencing failed to detect any of the 13 high-risk HPV types. Nearly half (46.7%) of the 75 cervical scraping samples that were negative for high-risk HPV by the HC2 assay were positive by PCR-MS. Type-specific sequencing in a subset of these samples confirmed the HPV type detected by PCR-MS. Quantitative PCR-MS results demonstrated that 11/75 (14.7%) samples contained as much HPV copies/cell as HC2-positive samples. These findings suggest that this prototype PCR-MS assay performs at least as well as HC2 for HPV detection, while offering the additional, unique advantages of type-specific identification and quantitation. Further validation work is underway to define clinically meaningful HPV detection thresholds and to evaluate the potential clinical application of future generations of the PCR-MS assay.
[Show abstract][Hide abstract] ABSTRACT: To test induction chemotherapy (IC) followed by concurrent chemoradiotherapy (CRT) or surgery/radiotherapy (RT) for advanced oropharyngeal cancer and to assess the effect of human papilloma virus (HPV) on response and outcome.
Sixty-six patients (51 male; 15 female) with stage III to IV squamous cell carcinoma of the oropharynx (SCCOP) were treated with one cycle of cisplatin (100 mg/m(2)) or carboplatin (AUC 6) and with fluorouracil (1,000 mg/m(2)/d for 5 days) to select candidates for CRT. Those achieving a greater than 50% response at the primary tumor received CRT (70 Gy; 35 fractions with concurrent cisplatin 100 mg/m(2) or carboplatin (AUC 6) every 21 days for three cycles). Adjuvant paclitaxel was given to patients who were complete histologic responders. Patients with a response of 50% or less underwent definitive surgery and postoperative radiation. Pretreatment biopsies from 42 patients were tested for high-risk HPV.
Fifty-four of 66 patients (81%) had a greater than 50% response after IC. Of these, 53 (98%) received CRT, and 49 (92%) obtained complete histologic response with a 73.4% (47 of 64) rate of organ preservation. The 4-year overall survival (OS) was 70.4%, and the disease-specific survival (DSS) was 75.8% (median follow-up, 64.1 months). HPV16, found in 27 of 42 (64.3%) biopsies, was associated with younger age (median, 55 v 63 years; P = .016), sex (22 of 30 males [73.3%] and five of 12 females [41.7%]; P = .08), and nonsmoking status (P = .037). HPV titer was significantly associated with IC response (P = .001), CRT response (P = .005), OS (P = .007), and DSS (P = .008).
Although the numbers in this study are small, IC followed by CRT is an effective treatment for SCCOP, especially in patients with HPV-positive tumors; however, for patients who do not respond to treatment, alternative treatments must be developed.
[Show abstract][Hide abstract] ABSTRACT: To prospectively identify markers of response to therapy and outcome in an organ-sparing trial for advanced oropharyngeal cancer.
Pretreatment biopsies were examined for expression of epidermal growth factor receptor (EGFR), p16, Bcl-xL, and p53 as well as for p53 mutation. These markers were assessed for association with high-risk human papillomavirus (HPV), response to therapy, and survival. Patient variables included smoking history, sex, age, primary site, tumor stage, and nodal status.
EGFR expression was inversely associated with response to induction chemotherapy (IC) (P = .01), chemotherapy/radiotherapy (CRT; P = .055), overall survival (OS; P = .001), and disease-specific survival (DSS; P = .002) and was directly associated with current smoking (P = .04), female sex (P = .053), and lower HPV titer (P = .03). HPV titer was significantly associated with p16 expression (P < .0001); p16 was significantly associated with response to IC (P = .008), CRT (P = .009), OS (P = .001), and DSS (P = .003). As combined markers, lower HPV titer and high EGFR expression were associated with worse OS (rho(EGFR) = 0.008; rho(HPV) = 0.03) and DSS (rho(EGFR) = 0.01; rho(HPV) = 0.016). In 36 of 42 biopsies, p53 was wild-type, and only one HPV-positive tumor had mutant p53. The combination of low p53 and high Bcl-xL expression was associated with poor OS (P = .005) and DSS (P = .002).
Low EGFR and high p16 (or higher HPV titer) expression are markers of good response to organ-sparing therapy and outcome, whereas high EGFR expression, combined low p53/high Bcl-xL expression, female sex, and smoking are associated with a poor outcome. Smoking cessation and strategies to target EGFR and Bcl-xL are important adjuncts to the treatment of oropharyngeal cancer.
[Show abstract][Hide abstract] ABSTRACT: Induction chemotherapy and concurrent chemoradiation for responders or immediate surgery for non-responders is an effective treatment strategy head and neck squamous cell carcinoma (HNSCC) of the larynx and oropharynx. Biomarkers that predict outcome would be valuable in selecting patients for therapy. In this study, the presence and titer of high risk human papilloma virus (HPV) and expression of epidermal growth factor receptor (EGFR) in pre-treatment biopsies, as well as smoking and gender were examined in oropharynx cancer patients enrolled in an organ sparing trial. HPV16 copy number was positively associated with response to therapy and with overall and disease specific survival, whereas EGFR expression, current or former smoking behavior, and female gender (in this cohort) were associated with poor response and poor survival in multivariate analysis. Smoking cessation and strategies to target EGFR may be useful adjuncts for therapy to improve outcome in the cases with the poorest biomarker profile.
International Journal of Radiation OncologyBiologyPhysics 02/2007; 69(2 Suppl):S109-11. DOI:10.1016/j.ijrobp.2007.05.072 · 4.26 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In previous work, a reasonably large number of genes was found to be on chromosome 21. This frequency demands the creation of new techniques to investigate the transcription of this small human autosome, and to relate these findings to the phenotype of Down syndrome. Here we describe the elaboration of new vectors and hosts, which in conjunction with flow-sorted cosmid libraries of chromosome 21 and genic (cDNA) libraries of relevant human tissues, will permit us to examine transcription of chromosome 21.
American Journal of Medical Genetics 06/2005; 37(S7):115 - 119. DOI:10.1002/ajmg.1320370723 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Chromosome heteromorphisms and restriction fragment length polymorphisms were used to study the origin of the extra chromosome in 54 trisomy 21 conceptuses. The parental origin was determined in 43 cases, with 39 (91%) being maternally and 4 (9%) parentally derived. Analysis of recombination demonstrated the presence of one or two cross-overs in most cases for which sufficient information was available, suggesting that failure to pair/exchange at meiosis I is relatively unimportant in the genesis of trisomy 21.
American journal of medical genetics. Supplement 06/2005; 7(S7):175-81. DOI:10.1002/ajmg.1320370735
[Show abstract][Hide abstract] ABSTRACT: We assayed for the presence of human papilloma virus (HPV) DNA in serum and/or peripheral blood fraction (PBF) of individuals with cervical, head/neck, or bladder cancer due to schistosomiasis. Using mass spectroscopy coupled with competitive PCR, HPV DNA was detected at the individual molecule level by using "MassARRAY" assays. The resultant sensitivity was superior to real-time fluorescent PCR-based assays, while specificity was maintained. Our principal findings were: (i) Virtually all tested cervical cancers and schistosomiasis-associated bladder cancers, and a plurality of head/neck cancers, are associated with HPV DNA in the tumor. (ii) All 27 bladder cancers due to schistosomiasis were associated with the presence of HPV-16 DNA, which can be detected in tumor and serum but not in PBF. In contrast, no serum HPV-16 DNA signal was detected in seven individuals with schistosomiasis-associated bladder cancers after surgical removal of the tumor. (iii) Among the head/neck cancers we studied, anterior tumors were more often associated with HPV DNA in tumor, serum, and/or PBF than posterior tumors. (iv) In cervical cancer, where all tumors contain HPV DNA, viral DNA could be detected often in serum and/or PBF. Further, HPV-16 DNA was detected in serum and/or PBF of most patients with untreated high-grade cervical dysplasia but disappeared if the dysplasia was eliminated. The sensitive, specific, and quantitative MassARRAY technique should make it feasible to monitor cancer occurrence and treatment and recurrence of malignancies and dysplasias associated with HPV DNA.
Proceedings of the National Academy of Sciences 06/2005; 102(21):7683-8. DOI:10.1073/pnas.0406904102 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Treatment strategies for squamous cell carcinoma of the head and neck (SCCHN) are based on the TNM classification. Biological markers that can predict the response to therapy have so far not been introduced. The objective of this study was to investigate cyclin D1 deregulation relative to sensitivity to cisplatin.
This was a laboratory study of 23 established University of Michigan SCC cell lines. Chemosensitivity was assessed by means of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The cyclin D1 amplification status was evaluated by real-time polymerase chain reaction (PCR; data were verified by differential and conventional PCR) using a chromosome 18q microsatellite marker probe (D18S70) as an internal control. Cyclin D1 protein expression was tested using Western blotting.
Cyclin D1 amplification was seen in 9/23 (39%) and cyclin D1 overexpression in 12/19 (63%) of the cell lines. As expected, all cell lines showing amplification also showed overexpression of cyclin D1 (p=0.004; Fisher's exact test). The mean cisplatin concentration inhibiting growth of 50% of the cells (ID50) was 9.8 microM in all cell lines (range 2.7-36.7 microM). Five of nine cell lines showing cyclin D1 amplification were highly sensitive to cisplatin (ID50 3-4.8 microM) and the remaining four revealed intermediate sensitivity. Five cell lines that strongly overexpressed cyclin D1 protein responded better to cisplatin than cell lines that showed any other expression (ID50 5.1 vs 11.2 microM; p=0.025; Student's t-test).
This in vitro study suggests that overexpression of cyclin D1 is associated with a good response to cisplatin in SCC cell lines. Our results support the hypothesis that overexpression of cyclin D1 is one of the molecular factors that can be used to predict sensitivity to chemotherapy, thus enabling individualization of treatment of head and neck cancer.
[Show abstract][Hide abstract] ABSTRACT: Obscurin and obscurin myosin light chain kinase (MLCK) are two recently identified muscle proteins encoded by the same gene cluster. The production of obscurin, which contains a Rho-guanine exchange factor (GEF)-like sequence, and obscurin-MLCK by this cluster suggests that these novel genes may be involved in signal transduction cascades that control adaptive and compensatory responses of the heart. The goal of the present study was to investigate the transcriptional response of the obscurin gene cluster to the initiation of myocardial hypertrophy induced in mice by aortic constriction. The transcriptional activity of the obscurin genes was examined using reverse-transcriptase primed quantitative PCR. We found that the transcripts encoding the obscurin Rho-GEF and the obscurin-MLCK internal serine-threonine kinase II (SK II) domains were significantly upregulated following aortic constriction. The expression of Rho-GEF-containing transcripts at different stages of the hypertrophic growth exceeded the control levels by 2- to 6-fold. Following the induction of hypertrophy, the quantity of the SK II-encoding transcripts increased 10-fold by 24h and 16-fold by 48h, then decreased by day 7, and returned to the control level by day 56. The quantity of the carboxy terminal obscurin-MLCK transcripts encoding for SK I increased 2-fold by day 2 and returned to the control values at later stages. Immunolocalization of obscurin, which contains Rho-GEF domain, in cardiomyocytes during pharmacologically induced hypertrophic growth in vitro demonstrated that the expression was topographically associated with the growing myofibrils and with the sites of initiation and progression of myofibrillogenesis at the periphery of the sarcoplasm. This suggests that upregulation of obscurin synthesis is associated with the formation of additional amounts of contractile structures during cardiac hypertrophy. Thus, the obscurin gene cluster represents a new example of an operon that encodes differentially regulated structural and signaling proteins implicated in the control of assembly and adaptive remodeling of myofibrils during normal and hypertrophic growth.
Biochemical and Biophysical Research Communications 11/2003; 310(3):910-8. DOI:10.1016/j.bbrc.2003.09.035 · 2.30 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We recently showed that rap1 regulates growth and proliferation in normal keratinocytes, which provoked us to investigate its expression and regulation in malignant cells. Rap1 is variably expressed in whole cell lysates of squamous cell carcinoma (SCC) cell lines. Immunoblot analysis of nuclear and cytosolic fractions and immunohistochemistry revealed that in addition to cytoplasmic expression, SCC cells also exhibit prominent punctate rap1 expression in the nucleus. This unexpected nuclear distribution was confirmed by the evaluation of human oral cancer specimens by immunohistochemistry, which showed both nuclear and cytoplasmic localization. Cytoplasmic rap1 expression was observed mostly in large differentiated cells, whereas nuclear localization was found in morphologically less differentiated cells. Quantitative reverse transcriptase polymerase chain reaction and Northern blot analysis showed that both rap1A and rap1B are expressed in SCC cell lines although rap1B signals are more prominent. Transfection with enhanced GFP-tagged constitutively active and inactive forms of rap1B demonstrated that the active GTP-bound form translocates to the nucleus whereas inactive rap1B(GDP) is retained in the cytoplasm, much of which is in a perinuclear distribution. Furthermore, growth factors induce nuclear translocation of rap1 in oral cancer cells. This novel discovery that active, GTP-bound rap1 translocates to the nucleus makes it only the second of over 100 small GTP-binding proteins to be identified in the nucleus, and the striking prominence of rap1 expression in the nucleus of SCC cells suggests that activated rap1 plays a role in the malignant process.
[Show abstract][Hide abstract] ABSTRACT: Tumor-Node-Metastasis classification does not fully predict outcome of treatment and prognosis in patients with squamous cell carcinoma of the head and neck. Different biomarkers have been suggested to yield additional prognostic information, but no single marker has thus far been introduced in the clinic. The objective of the present study was to analyze the copy number of the frequently amplified oncogenes CCND1 and c-MYC in relation to the commonly deleted tumor suppressor gene cyclin-dependent kinase (CDK)N2A (p16) to enhance the clinical significance.
Extracted DNA from diagnostic biopsies of 78 untreated patients were analyzed by real-time PCR with specific primers for c-MYC, CCND1, and CDKN2A. Gene copy number ratios were calculated by dividing the copy number of c-MYC or CCND1 with CDKN2A. Ratios > 2 were defined as enhanced. These data were related to disease-free interval and disease-specific survival.
Enhanced gene ratio of c-MYC:CDKN2A was detected in 35 of 78 (45%) and enhanced ratio of CCND1:CDKN2A in 36 of 78 (46%) of the cases. The c-MYC:CDKN2A and CCND1:CDKN2A ratios correlated with disease-specific survival with respect to death (P = 0.042 and 0.049, respectively; Log-rank test). Furthermore, enhancement of c-MYC:CDKN2A was associated with a shorter disease-free interval as marked by the development of recurrences or metastases (P = 0.014; Log-rank test).
We conclude that CCND1 and/or c-MYC amplification, when combined with CDKN2A deletion, yield additional prognostic information as compared with analysis of single genetic aberrations. These gene ratios, as analyzed by a sensitive method like real-time PCR on diagnostic biopsies, might help clinicians to individualize the treatment of squamous cell carcinoma of the head and neck as they reflect the biological properties of the tumors. This could be used as an adjunct to the Tumor-Node-Metastasis classification system.
Clinical Cancer Research 05/2003; 9(5):1750-5. · 8.72 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We used oligonucleotide microarrays to analyze comprehensively hypothalamic gene expression changes that correlate with energy homeostasis. We compared the hypothalamic gene expression profiles of freely fed and 48-h fasted rats using 26,379 oligonucleotide probe sets. Expression of 96 genes was up-regulated and expression of 73 genes was down-regulated in a statistically significant manner with fasting. The gene encoding the enzyme minoxidil sulfotransferase, an enzyme that catalyzes the transfer of sulfonate groups to biogenic amines and other substrates, was foremost among a set of genes whose mRNAs were uniformly detectable and displaying the greatest transcriptional changes with fasting. Northern blot analysis indicated that minoxidil sulfotransferase mRNA is up-regulated in the fasted rat and mouse, ob/ob mouse, and fa/fa rat. Results of reverse transcription quantitative PCR indicated that minoxidil sulfotransferase mRNA is also up-regulated in the microdissected arcuate and paraventricular nuclei of the fasted rat. Several index genes known to be either up-regulated (neuropeptide Y) or down-regulated (amphetamine-regulated transcript and proopiomelanocortin) with fasting were also found to be present among our set of "differentially expressed" genes. This study identifies a novel gene induced by fasting and demonstrates the feasibility of using oligonucleotide microarrays for the study of complex neuronal processes.
[Show abstract][Hide abstract] ABSTRACT: The unfolded protein response (UPR) is a transcriptional and translational intracellular signaling pathway activated by the accumulation of unfolded proteins in the lumen of the endoplasmic reticulum (ER). We have used C. elegans as a genetic model system to dissect UPR signaling in a multicellular organism. C. elegans requires ire-1-mediated splicing of xbp-1 mRNA for UPR gene transcription and survival upon ER stress. In addition, ire-1/xbp-1 acts with pek-1, a protein kinase that mediates translation attenuation, in complementary pathways that are essential for worm development and survival. We propose that UPR transcriptional activation by ire-1 as well as translational attenuation by pek-1 maintain ER homeostasis. The results demonstrate that the UPR and ER homeostasis are essential for metazoan development.
[Show abstract][Hide abstract] ABSTRACT: Van der Woude syndrome (VWS) is an orofacial clefting disorder with an autosomal dominant pattern of inheritance. In our efforts to clone the VWS gene, 900 kb of genomic sequence from the VWS candidate region at chromosome 1q32-q41 was analyzed for new DNA sequence variants. We observed that in clone CTA-321i20 a 7922 bp sequence is absent relative to the sequence present in PAC clone RP4-782d21 at positions 1669-9590, suggesting the presence of a deletion/insertion (del/ins) polymorphism. Embedded in this 7922 bp region was a TTCC short tandem repeat (STR). Genotype analysis showed that both the internal STR and the (del/ins) mutation were true polymorphisms. This is a novel example of intraallelic variation, a polymorphism within a polymorphism, and we suggest that it be termed a "Matroshka" polymorphism. Further genetic and DNA sequence analysis indicated that the ancestral state of the 1669-9590 del/ins polymorphism was the insertion allele and that the original deletion mutation probably occurred only once. A second class of novel DNA sequence variation was discovered on chromosome 5 that shared a 328 bp identical sequence with this region on chromosome 1. A single nucleotide polymorphism (SNP) was detected by SSCP using a pair of primers derived from the chromosome 1 sequence. Surprisingly, these primers also amplified the identical locus on chromosome 5, and the SNP was only located on chromosome 5. Since the probe unexpectedly detected alleles from another locus, we suggest that this type of sequence variant be termed an "ectopic" polymorphism. These two novel classes of DNA sequence polymorphisms have the potential to confound genetic and DNA sequence analysis and may also contribute to variation in disease phenotypes.
Human Mutation 11/2001; 18(5):422-34. DOI:10.1002/humu.1213 · 5.14 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Astrocytomas are heterogeneous intracranial glial neoplasms ranging from the highly aggressive malignant glioblastoma multiforme (GBM) to the indolent, low-grade pilocytic astrocytoma. We have investigated whether DNA microarrays can identify gene expression differences between high-grade and low-grade glial tumors. We compared the transcriptional profile of 45 astrocytic tumors including 21 GBMs and 19 pilocytic astrocytomas using oligonucleotide-based microarrays. Of the approximately 6800 genes that were analyzed, a set of 360 genes provided a molecular signature that distinguished between GBMs and pilocytic astrocytomas. Many transcripts that were increased in GBM were not previously associated with gliomas and were found to encode proteins with properties that suggest their involvement in cell proliferation or cell migration. Microarray-based data for a subset of genes was validated using real-time quantitative reverse transcription-PCR. Immunohistochemical analysis also localized the protein products of specific genes of interest to the neoplastic cells of high-grade astrocytomas. Our study has identified a large number of novel genes with distinct expression patterns in high-grade and low-grade gliomas.
Cancer Research 10/2001; 61(18):6885-91. · 9.33 Impact Factor