Catharine M L West

GLUT‐1 and CAIX as intrinsic markers of hypoxia in carcinoma of the cervix: Relationship to pimonidazole binding

The presence of hypoxia in tumours results in the overexpression of certain genes, which are controlled via the transcription factor HIF‐1. Hypoxic cells are known to be radioresistant and chemoresistant, thus, a reliable surrogate marker of hypoxia is desirable to ensure that treatment may be rationally applied. Recently, the HIF‐1‐regulated proteins Glut‐1 and CAIX were validated as intrinsic markers of hypoxia by comparison with pO2 measured using oxygen electrodes. We compare the expression of Glut‐1 and CAIX with the binding of the bioreductive drug hypoxia marker pimonidazole. Pimonidazole was administered to 42 patients with advanced carcinoma of the cervix, 16 hr before biopsy. Sections of single or multiple biopsies were then immunostained for Glut‐1 and CAIX, and the area of staining scored by eye, using a “field‐by‐field” semi‐quantitative averaging system. Using 1 biopsy only, Glut‐1 (r = 0.54, p = <0.001) correlated with the level of pimonidazole binding, and Glut‐1 and CAIX expression also correlated significantly (r = 0.40, p = <0.009). Thus, our study has shown that HIF‐1 regulated genes have potential for future use as predictors of the malignant changes mediated by hypoxia, and warrant further investigation as indicators of response to cancer therapy.

hsa-mir-210 Is a Marker of Tumor Hypoxia and a Prognostic Factor in Head and Neck Cancer

Hypoxia is an important mechanism of treatment resistance in head and neck squamous cell carcinoma (HNSCC). MicroRNAs are short noncoding RNAs that regulate multiple mRNAs and are frequently dysregulated in cancer. The authors have investigated the role of 3 microRNAs, including the hypoxia‐induced hsa‐miR‐210, as potential markers of hypoxia or prognosis.

The small-nucleolar RNAs commonly used for microRNA normalisation correlate with tumour pathology and prognosis.

Background:To investigate small-nucleolar RNAs (snoRNAs) as reference genes when measuring miRNA expression in tumour samples, given emerging evidence for their role in cancer.Methods:Four snoRNAs, commonly used for normalisation, RNU44, RNU48, RNU43 and RNU6B, and miRNA known to be associated with pathological factors, were measured by real-time polymerase chain reaction in two patient series: 219 breast cancer and 46 head and neck squamous cell carcinoma (HNSCC). SnoRNA and miRNA were then correlated with clinicopathological features and prognosis.Results:Small-nucleolar RNA expression was as variable as miRNA expression (miR-21, miR-210, miR-10b). Normalising miRNA PCR expression data to these recommended snoRNAs introduced bias in associations between miRNA and pathology or outcome. Low snoRNA expression correlated with markers of aggressive pathology. Low levels of RNU44 were associated with a poor prognosis. RNU44 is an intronic gene in a cluster of highly conserved snoRNAs in the growth arrest specific 5 (GAS5) transcript, which is normally upregulated to arrest cell growth under stress. Low-tumour GAS5 expression was associated with a poor prognosis. RNU48 and RNU43 were also identified as intronic snoRNAs within genes that are dysregulated in cancer.Conclusion:Small-nucleolar RNAs are important in cancer prognosis, and their use as reference genes can introduce bias when determining miRNA expression.

Intrinsic radiosensitivity and prediction of patient response to radiotherapy for carcinoma of the cervix

The intrinsic radiosensitivity of cervical carcinoma has been measured using a soft agar clonogenic assay. All patients received radical radiotherapy alone with a minimum of 2 years post-treatment follow-up. Only women with stage I, II and III disease were included in the analysis. Values for cell surviving fraction at 2 Gy (SF2) were obtained for 88 tumours with an assay success rate of 73%. The 53 patients alive and well at the time of analysis had tumours with a mean SF2 that was significantly lower than the value from the 22 patients with locoregional failure (P < 0.01). Patients with radioresistant tumours (SF2 > 0.40, the median) had a significantly lower 3 year survival level than those with sensitive tumours (SF2 < or = 0.40) (P = 0.002). Also the frequency of local recurrence was higher (P = 0.001) whether these were central (P = 0.009) or peripheral (P = 0.046). Cell surviving fraction at 3.5 Gy was obtained for 46 tumours and the 3 year patient survival rate was significantly higher for those with SF3.5 values less than the median (P = 0.043). There was, however, no difference in the level of local recurrence (P = 0.24). The ability to grow in culture was not associated with significantly poorer patient survival (P = 0.56) or failure to control the primary disease (P = 0.17). While high colony forming efficiencies were associated with an increased rate of local recurrence (P = 0.029) they did not predict for overall patient survival (P = 0.32). These data suggest that, for cervical carcinoma treated with radical radiotherapy, intrinsic radiosensitivity is important in determining treatment outcome.

Head and neck cancer—Part 1: Epidemiology, presentation, and prevention

#### Summary points Head and neck cancers include cancers of the upper aerodigestive tract (including the oral cavity, nasopharynx, oropharynx, hypopharynx, and larynx), the paranasal sinuses, and the salivary glands. Cancers at different sites have different courses and variable histopathological types, although squamous cell carcinoma is by far the most common. The anatomical sites affected are important for functions such as speech, swallowing, taste, and smell, so the cancers and their treatments may have considerable functional sequelae with subsequent impairment of quality of life. Decisions about treatment are usually complex, and they must balance efficacy of treatment and likelihood of survival, with potential functional and quality of life outcomes. Patients and their carers need considerable support during and after treatment. #### Sources and selection criteria We used the terms “head and neck”, “larynx”, “oral”, and “oropharynx”—with each limited by “cancer”, “diagnosis”, and “treatment” separately—to search the Medline, Embase, PubMed, Cochrane, CINAHL, and AMED databases. We also used them to cross check national guidelines, reference lists, textbooks, and personal reference lists. We assessed over 1000 identified abstracts for relevance. In this first part of a two article series, we review the common presentations of head and neck cancer. We also discuss common investigations and new …

The independence of intrinsic radiosensitivity as a prognostic factor for patient response to radiotherapy of carcinoma of the cervix.

A study was made of the prognostic value of pretreatment measurements of tumour radiosensitivity (surviving fraction at 2 Gy, SF2) in 128 patients with stage I-III carcinomas of the uterine cervix undergoing radiotherapy. The median follow-up time was 47 months. In a univariate analysis stratifying patients according to the median value, radiosensitivity was a significant prognostic factor for overall survival, local control and metastasis-free survival. The 5-year survival rate for tumours with SF2 values below the median was 81% and was significantly greater than the rate of 51% for those with SF2 values above the median. In bivariate analyses, SF2 was shown to be independent of disease stage, tumour grade, patient age, colony-forming efficiency and tumour diameter. In a multivariate analysis, radiosensitivity was the most important variable and, after allowing for this, only stage was a significant independent predictor of treatment outcome. These data indicate that, in carcinoma of the cervix treated with radiotherapy, pretreatment tumour intrinsic radiosensitivity is an important prognostic parameter and contributes to prognosis independently of other established and putative parameters.

Apoptosis, intrinsic radiosensitivity and prediction of radiotherapy response in cervical carcinoma

Apoptosis is an important mechanism of cell death in tumours and it is seen both prior to and following radiotherapy. In this study patients with proven carcinoma of the cervix had measurement made of the percentage of apoptotic cells (apoptotic index or AI) in pre-therapy biopsies. Measurements of intrinsic radiosensitivity (SF2), already shown to be a predictor of outcome, had previously been made on the same pre-therapy biopsies. Mitotic index (MI) and Ki-67 antigen staining were also recorded as markers for proliferation. Patients were divided into those with an AI above or below the median and in general increasing apoptosis was associated with poor prognosis. The 5-year survival rate for tumours with an AI below the median was 79% and was significantly greater than the rate of 47% for those with an AI above the median (p = 0.003). There was also a significantly increased 5-year local recurrence-free rate for patients with an AI below the median compared with those with an AI above the median (79 versus 61%, p = 0.012). In addition, AI and SF2 acted as independent prognostic indicators. Patients with both an SF2 and AI value above the median did badly (25% 5-year survival, 46% local control) compared with those with an SF2 and AI below the median (80% 5-year survival, 100% local control). Apoptosis showed correlation with MI (n = 66, r = 0.34, p = 0.002) and cell staining for the Ki-67 antigen (n = 57, r = 0.25, p = 0.03), but neither MI nor Ki-67 were related to patient outcome. This suggests that while apoptosis may be a reflection of tumour proliferation this cannot in itself explain the ability of apoptosis to predict clinical outcome for this series of patients. The study raises the possibility of AI and SF2 being used together as predictors of tumour response to radiotherapy.

Independent validation of genes and polymorphisms reported to be associated with radiation toxicity: a prospective analysis study

BACKGROUND Several studies have reported associations between radiation toxicity and single nucleotide polymorphisms (SNPs) in candidate genes. Few associations have been tested in independent validation studies. This prospective study aimed to validate reported associations between genotype and radiation toxicity in a large independent dataset. METHODS 92 (of 98 attempted) SNPs in 46 genes were successfully genotyped in 1613 patients: 976 received adjuvant breast radiotherapy in the Cambridge breast IMRT trial (ISRCTN21474421, n=942) or in a prospective study of breast toxicity at the Christie Hospital, Manchester, UK (n=34). A further 637 received radical prostate radiotherapy in the MRC RT01 multicentre trial (ISRCTN47772397, n=224) or in the Conventional or Hypofractionated High Dose Intensity Modulated Radiotherapy for Prostate Cancer (CHHiP) trial (ISRCTN97182923, n=413). Late toxicity was assessed 2 years after radiotherapy with a validated photographic technique (patients with breast cancer only), clinical assessment, and patient questionnaires. Association tests of genotype with overall radiation toxicity score and individual endpoints were undertaken in univariate and multivariable analyses. At a type I error rate adjusted for multiple testing, this study had 99% power to detect a SNP, with minor allele frequency of 0·35, associated with a per allele odds ratio of 2·2. FINDINGS None of the previously reported associations were confirmed by this study, after adjustment for multiple comparisons. The p value distribution of the SNPs tested against overall toxicity score was not different from that expected by chance. INTERPRETATION We did not replicate previously reported late toxicity associations, suggesting that we can essentially exclude the hypothesis that published SNPs individually exert a clinically relevant effect. Continued recruitment of patients into studies within the Radiogenomics Consortium is essential so that sufficiently powered studies can be done and methodological challenges addressed. FUNDING Cancer Research UK, The Royal College of Radiologists, Addenbrookes Charitable Trust, Breast Cancer Campaign, Cambridge National Institute of Health Research (NIHR) Biomedical Research Centre, Experimental Cancer Medicine Centre, East Midlands Innovation, the National Cancer Institute, Joseph Mitchell Trust, Royal Marsden NHS Foundation Trust, Institute of Cancer Research NIHR Biomedical Research Centre for Cancer.

Prediction of radiotherapy outcome using dynamic contrast enhanced MRI of carcinoma of the cervix

PURPOSE To investigate whether analysis of MRI enhancement data using a pharmacokinetic model improved a previously found correlation between contrast enhancement and tumor oxygenation measured using PO2 histograph. To evaluate the prognostic value of gadolinium enhancement data for radiotherapy outcome, and to study the efficacy of combined enhancement and MRI volume data. METHODS AND MATERIALS Fifty patients underwent dynamic gadolinium-enhanced MRI as part of their initial staging investigations before treatment. Gadolinium enhancement was analyzed using the Brix pharmacokinetic model to obtain the parameters amplitude and rate of contrast enhancement. Pretreatment tumor oxygen measurements (Eppendorf PO2 histograph) were available for 35 patients. RESULTS Both standard and pharmacokinetic-derived enhancement data correlated with tumor oxygenation measurements, and poorly enhancing tumors had low tumor oxygen levels. However, only the pharmacokinetic-analyzed data correlated with patient outcome and patients with poorly (amplitude less than median) vs. well-enhancing tumors had significantly worse disease-specific survival (p = 0.024). For the 50 patients studied, no relationship was found between enhancement and volume data. Combining MRI volume and enhancement information highlighted large differences in outcome (p = 0.0054). At the time of analysis, only 55% of patients with large, poorly enhanced tumors were alive compared with 92% of patients with small, well-enhanced tumors. CONCLUSION These preliminary results suggest that pharmacokinetic modeling of dynamic contrast-enhanced MRI provides data that reflect tumor oxygenation and yields useful prognostic information in patients with locally advanced carcinoma of the cervix. Combining MRI-derived enhancement and volume data delineates large differences in radiotherapy outcome.

Vascular endothelial growth factor (VEGF) expression is a prognostic factor for radiotherapy outcome in advanced carcinoma of the cervix.

The aim of the study was to evaluate VEGF expression in tumour biopsies as a prognostic factor for radiotherapy outcome in advanced carcinoma of the cervix. A retrospective study was carried out on 100 patients. Pre-treatment tumour VEGF expression was examined immunohistochemically in formalin-fixed, paraffin-embedded biopsies using a widely available commercial antibody. A semi-quantitative analysis was made using a scoring system of 0, 1, 2, and 3, for increasing intensity of staining. High VEGF expression was associated with a poor prognosis. A univariate log rank analysis found a significant relationship with overall survival (P = 0.0008) and metastasis-free survival (P = 0.0062), but not local control (P = 0.23). There was no correlation between VEGF expression and disease stage, tumour differentiation, patient age, or tumour radiosensitivity (SF2). In a Cox multivariate analysis of survival VEGF expression was the most significant independent prognostic factor (P = 0.001). After allowing for VEGF only SF2 was a significant prognostic factor (P = 0.003). In conclusion, immunohistochemical analysis of VEGF expression is a highly significant and independent prognostic indicator of overall and metastasis-free survival for patients treated with radiotherapy for advanced carcinoma of the cervix. It is also a rapid and easy method that could be used in the clinical setting, to identify patients at high risk of failure with conventional radiotherapy who may benefit from novel approaches or chemoradiotherapy.