1. Simoneaux, Richard

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There are a number of cancers of the head and neck, one of which is squamous cell carcinoma of the oropharynx (SCCOP). Although in the past, the use of alcohol and tobacco products have been leading risk factors for SCCOP, an increasing number of patients have developed this disease as a result of their exposure to HPV. One of the most common forms of the virus which is deemed high-risk, or capable of causing cancer in men and women, is HPV16. Although cervical cancer is most commonly associated with HPV, this virus can cause cancers in other mucous membranes, such as the vagina, vulva, anus, or the mouth and throat (including the oropharynx).

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Transforming growth factor-[beta]1 (TGF-[beta]1) is a polypeptide cytokine that plays crucial roles in inflammation and the immune system. Consequently, it is possible that variations in the gene that encodes TGF-[beta]1 (TGFB1) could alter the manner in which SCCOP patients may respond to their disease.


To investigate this possibility, a team of clinicians at The University of Texas MD Anderson Cancer Center, Houston, including Guojun Li, MD, PhD, undertook a study that genotyped 564 incident SCCOP patients for three different polymorphisms of TGFB1 to assess the effect that variation of that gene had on the overall (OS), disease-specific survival (DSS), and disease-free survival (DFS) in this patient subpopulation (Clin Cancer Res 2018; doi:10.1158/1078-0432.CCR-17-1904). The three polymorphisms evaluated in this study included TGFB1 rs1800469, rs1800471, and rs1982073.


When asked to discuss the reasons for this trial, Li replied, "Although several studies have investigated the association between HPV and TGF-[beta]1 expression levels in cervical cancer and assessed their influence on primary risk, few have evaluated their effects on survival." Concerning the results, he noted, "Patients with the TGFB1 rs1982073 CT/CC polymorphism had statistically significantly better OS, DSS, and DFS compared with those with the corresponding common homozygous TT genotype.


"Furthermore, these genotypes were significantly associated with an approximately 5 times reduced risk of overall death, death owing to disease, and recurrence after multivariable adjustment. However, the tumor HPV status stratification data indicated that the significant effects of TGFB1 rs1982073 polymorphism on survival were found among HPV16-positive SCCOP patients only."



The epithelial tissue-based SCCOP is characterized by aggressive tumors that have high local recurrence rates as well as frequently having more than one primary tumor site. Although the frequency of SCCOP has remained fairly constant at about three cases per 200,000 people, an increasing number of these patients are now younger, often as a result of disease arising from HPV infection. In 2016, there were approximately 48,000 new cases of SCCOP and slightly more than 9,500 deaths from this disease. Despite the decrease in the overall incidence of head and neck cancers, there has been a slight uptick in the numbers of SCCOP cases.


Currently, the two main treatments for SCCOP consist of radiotherapy or the combination of chemoradiation. In patients having early-stage disease, the therapy of choice is often radiotherapy alone. However, for later-stage patients, chemoradiation is the preferred treatment modality.


"Specifically, studies have shown that although greater toxicity is observed with chemoradiation than with radiotherapy alone, both DFS and OS tend to be better with the concomitant therapy," Li noted. "In our practice, we often use cisplatin, taxol, and 5-fluorouracil as agents in the chemoradiation combination therapies."



Regarding the specific polymorphisms, Li explained, "We chose to study the TGFB1 polymorphisms rs1800469, rs1982073, and rs1800471 because these appear to affect TGF-[beta]1. The first variant, TGFB1 rs1800469, is located in the promoter region of the gene and is associated with differences in circulating TGF-[beta]1 plasma levels. Prior studies showed that when cytosine is encoded, activator protein 1 (AP1) binds to it, resulting in the downregulation of TGFB1," Li added (Hum Genet 2006;120(4):461-469). Concerning the TGFB1 rs1982073 variant, Li stated, "This polymorphism is located at codon 10 of exon 1 and results in a non-synonymous change from leucine to proline within the signal peptide sequence of TGF-[beta]1. This region of the gene is responsible for directing TGF-[beta]1 into the extracellular matrix and the wild type T allele has been associated with lowered secretion and serum levels of TGF-[beta]1 compared with the variant C allele (Exp Rev Anticancer Ther 2004;4(4):649-661). "TGFB1 rs1800471 is located at codon 25 in exon 1 and results in an arginine to proline change," Li noted regarding the third TGFB1 variant.


"In addition, we previously have reported that TGFB1 rs1982073 was significantly associated with HPV16-positive SCCOP tumors; however, no studies have investigated whether TGFB1 polymorphisms are associated with survival of SCCOP patients, particularly HPV16-positive SCCOP patients. We hypothesized that TGF-[beta]1 polymorphisms are associated with survival of SCCOP patients after radiation or chemoradiation," Li explained.


Study Methodology

From January 2000 to May 2013, 564 newly diagnosed incident SCCOP patients from The University of Texas MD Anderson Cancer Center were recruited to participate in an ongoing molecular epidemiology study. All participants had histologically confirmed disease and were untreated. Patients were not excluded from the study on the basis of age, cancer stage, ethnicity, histology, or sex. Upon enrollment, patients provided a blood sample (~30 mL) for TGFB1 genotyping, as well as completed a questionnaire to provide demographic and risk factor data (e.g., smoking and alcohol status).


Both PCR and in situ hybridization methods were utilized to detect the presence of HPV16 in DNA material obtained from SCCOP patient-derived paraffin-embedded tumor samples. "For some SCCOP patients, tumor HPV16 status was determined by in situ hybridization and p16 immunohistochemical analysis from HPV data in the patient's clinical records, as the pathology laboratory at MD Anderson had begun classifying all SCCOP specimens as a matter of standard clinical practice," Li explained.


Clinical data, which were obtained at both initial presentation and throughout follow-up examinations, included the following: stage of the index tumor at presentation, site of the index tumor, and treatment. Dichotomization was done for both index cancer stage (early-stage (stage I or II disease) or late-stage (stage III or IV)) and treatment (radiotherapy alone or chemoradiation) parameters. Comorbidity classification was performed using a modified Kaplan-Feinstein index (Adult Comorbidity Evaluation 27), in which related comorbidities were categorized as none, mild, moderate, or severe.


Primary endpoints for the study included overall deaths, deaths due to disease, and recurrence. Differences in OS, DFS, and DSS among the SCCOP patients were determined. Time to recurrence was defined as the time between the end of treatment and the date of last follow-up or clinical detection of recurrent cancer (local, regional, or distant). Those patients who did not have disease recurrence or who were lost to follow-up were censored from the results. Survival criteria were defined as follows: OS-the time between first appointment and death from any cause or date of last follow-up; DSS-time between first appointment and death from disease or date of last follow-up. Participants who were alive at the end of the study period or lost to follow-up were censored for both OS and DSS calculations.


Result Findings

For the 564 patients included in this study, the median age at diagnosis was 55 years with a range of 28-82 years. Additionally, 491 (87.1%) were male; 518 (91.8%) had white, non-Hispanic ethnicity; and 528 (93.6%) had late-stage (III or IV) index cancer. Histories of smoking and alcohol use were noted in 284 (50.3%) and 377 (66.8%) patients, respectively. In terms of treatments, only 23.6 percent had radiotherapy alone while 76.4 percent had radiochemotherapy combination. During the course of this study, there were only 78 deaths from all causes;, however, only 44 were considered disease-related. Positive HPV tumor status was noted in 85 percent of the participants in this study.


When calculating HRs, these figures were adjusted for the following factors: age, sex, HPV status, treatment, comorbidity, smoking status, alcohol use. HR values for the TGFB1 rs1982073 variant participants were determined utilizing data from the 170 patients having the TT genotype as the reference (i.e., HR defined as 1.0) for evaluating the 394 patients having the CT+CC variant genotypes. For CT+CC genotypes, the OS, DSS, and DFS data afforded HR values of 0.2 (95% CI: 0.1-0.5; p< 0.001), 0.2 (95% CI: 0.1-0.4; p< 0.001), and 0.2 (95% CI: 0.1-0.4; p< 0.001), respectively.


When stratification of data was performed using tumor HPV16 status, as before, data from the TGFB1 rs1982073 variant participants with the TT genotype were utilized as the reference. For the participants with HPV16-positive tumors having the CT+CC variant genotypes (n=327), OS, DSS, and DFS data yielded HR values of 0.1 (95% CI: 0.1-0.3; p< 0.001), 0.1 (95% CI: 0.1-0.3; p< 0.001), and 0.2 (95% CI: 0.1-0.3; p< 0.001), respectively.


Correspondingly, for HPV16-negative CT+CC variant genotype participants (n=67), the OS, DSS, and DFS data gave these HR values: 2.1 (95% CI: 0.4-11.4; p=0.585), 4.2 (95% CI: 0.3-56.4; p=0.667), and 0.8 (95% CI: 0.1-4.7; p=0.772).


Summing up their findings concerning the polymorphisms investigated, Li observed, "We found that TGFB1 rs1982073 had statistically significant associations with survival, while TGFB1 rs1800469 and TGFB1 rs1800471 did not."


Further Discussion

Regarding the TGF-[beta]1 levels in patients, Li stated, "In this study, we found significantly higher expression of TGF-[beta]1 in serum among HPV16-positive patients than among HPV16-negative patients. Furthermore, the patients with the variant genotypes of TGFB1 rs1982073 were significantly correlated with increased serum TGF-[beta]1 expression as compared to those with the corresponding common TT genotype; this similar correlation was also seen among HPV16 positive patients only.


"Although the functional relevance of this polymorphism has not yet been elucidated, our results might partially suggest a functional correlation between this polymorphism and expression of TGF-[beta]1, providing preliminary evidence of biological plausibility for the observed association in this study," he further commented.


When asked about some of the limitations of their study, Li replied, "Some limitations [include the following]:


* As the majority (>90%) of the SCCOP cases in our study were non-Hispanic whites, our current results for generalizability to other ethnic populations may be limited.


* Because of relatively limited number of outcome events (death or recurrence) and duration of follow-up in this study, the significant associations could be found by chance.


* Some significant findings in the stratified analysis may have limited the interpretation due to the relatively small subgroup numbers in each subgroup.


* It is possible that misclassification of HPV status occurred in the HPV-negative group due to presence of other high-risk HPV types, leading to a bias of our results away from the null. However, this is unlikely to have occurred as we did not find a significant association in the HPV negative group."



When asked what direction some of the short-term research in this area may take, Li stated, "First, since the TGFB1 rs1982073 polymorphism was significantly associated with survival in HPV16-positive SCCOP, that variant, together with other significant markers and details on radiation dosages, may help stratify HPV16-positive SCCOP patients for appropriate treatment strategies. Second, in order to validate our findings, larger, well-designed prospective studies may be required to more accurately evaluate the clinical validity and utility of this biomarker before implementation.


"Finally, future studies for exploring the molecular mechanisms underlying the observed associations are also needed," Li added concerning long-term research goals.


Richard Simoneaux is a contributing writer.


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