Expression of miR-210 in relation to other measures of hypoxia and prediction of benefit from hypoxia modification in patients with bladder cancer.

BACKGROUND: The addition of hypoxia modifiers carbogen and nicotinamide (CON) to radiotherapy (RT) improved overall survival (OS) in bladder cancer patients in the BCON phase III clinical trial. We investigate whether expression of hsa-miR-210 in BCON patient samples reflects hypoxia and predicts benefit from hypoxia modification. METHODS: In all, 183 T1-T4a bladder cancer samples were available for miR-210 analysis. A total of 86 received RT+CON and 97 received RT alone. TaqMan qPCR plates were used to assess miR-210 expression. Patients were classified as low (

Stromal infiltration of CD8 T cells is associated with improved clinical outcome in HPV-positive oropharyngeal squamous carcinoma.

BACKGROUND: Patients with human papillomavirus (HPV)-positive oropharyngeal squamous cell carcinoma (OPSCC) have a better prognosis than those with HPV-negative tumours. There is interest in de-escalating their treatment but strategies are needed for risk stratification to identify subsets with a poor prognosis. This study investigated tumour-infiltrating lymphocytes (TILs) in relation to HPV tumour status and patient survival. METHODS: Biopsies from 218 patients diagnosed with OPSCC between 2002 and 2011, who underwent chemo/radiotherapy were analysed for HPV by PCR, in-situ hybridisation and p16 immunohistochemistry (IHC). One hundred and thirty-nine samples with concordant HPV detection were analysed for CD3, CD4, CD8 and FoxP3 expression in tumour and stromal regions using multiplexIHC and multispectral image analysis. Labelling of smooth muscle actin (SMA) identified activated stroma. RESULTS: Human papillomavirus-positive compared with HPV-negative OPSCC had higher infiltration in both tumour and stromal areas of CD4 and CD8 T cells but not FoxP3 T regulatory cells. Only CD3+CD8+ stromal and not tumour area infiltration was associated with increased survival (P=0.02). There was significantly higher SMA expression in HPV-positive compared with -negative tumours, which did not correlate with survival. CONCLUSIONS: Studies of TILs for risk stratification in OPSCC should assess stromal infiltration.

Enhanced stability of microRNA expression facilitates classification of FFPE tumour samples exhibiting near total mRNA degradation.

BACKGROUND: As degradation of formalin-fixed paraffin-embedded (FFPE) samples limits the ability to profile mRNA expression, we explored factors predicting the success of mRNA expression profiling of FFPE material and investigated an approach to overcome the limitation. METHODS: Bladder (n=140, stored 3-8 years) and cervix (n=160, stored 8-23 years) carcinoma FFPE samples were hybridised to Affymetrix Exon 1.0ST arrays. Percentage detection above background (%DABG) measured technical success. Biological signal was assessed by distinguishing cervix squamous cell carcinoma (SCC) and adenocarcinoma (AC) using a gene signature. As miR-205 had been identified as a marker of SCC, precursor mir-205 was measured by Exon array and mature miR-205 by qRT-PCR. Genome-wide microRNA (miRNA) expression (Affymetrix miRNA v2.0 arrays) was compared in eight newer FFPE samples with biological signal and eight older samples without. RESULTS: RNA quality controls (QCs) (e.g., RNA integrity (RIN) number) failed to predict profiling success, but sample age correlated with %DABG in bladder (R=-0.30, P<0.01) and cervix (R=-0.69, P<0.01). Biological signal was lost in older samples and neither a signature nor precursor mir-205 separated samples by histology. miR-205 qRT-PCR discriminated SCC from AC, validated by miRNA profiling (26-fold higher in SCC; P=1.10 × 10(-5)). Genome-wide miRNA (R=0.95) and small nucleolar RNA (R=0.97) expression correlated well in the eight newer vs older FFPE samples and better than mRNA expression (R=0.72). CONCLUSION: Sample age is the best predictor of successful mRNA profiling of FFPE material, and miRNA profiling overcomes the limitation of age and copes well with older samples.

A replicated association between polymorphisms near TNFα and risk for adverse reactions to radiotherapy.

BACKGROUND: Response to radiotherapy varies between individuals both in terms of efficacy and adverse reactions. Finding genetic determinants of radiation response would allow the tailoring of the treatment, either by altering the radiation dose or by surgery. Despite a growing number of studies in radiogenomics, there are no well-replicated genetic association results. METHODS: We carried out a candidate gene association study and replicated the result using three additional large cohorts, a total of 2036 women scored for adverse reactions to radiotherapy for breast cancer. RESULTS: Genetic variation near the tumour necrosis factor alpha gene is shown to affect several clinical endpoints including breast induration, telangiectasia and overall toxicity. In the combined analysis homozygosity for the rare allele increases overall toxicity (P=0.001) and chance of being in the upper quartile of risk with odds ratio of 2.46 (95% confidence interval 1.52-3.98). CONCLUSION: We have identified that alleles of the class III major histocompatibility complex region associate with overall radiotherapy toxicity in breast cancer patients by using internal replication through a staged design. This is the first well-replicated report of a genetic predictor for radiotherapy reactions.

Effect of Race and Ethnicity on Risk of Radiotherapy Toxicity and Implications for Radiogenomics.

AIMS: Patient factors affect the risk of radiotherapy toxicity, but many are poorly defined. Studies have shown that race affects cancer incidence, survival, drug response, molecular pathways and epigenetics. Effects on radiosensitivity and radiotherapy toxicity are not well studied. The aim of the present study was to identify the effects of race and ethnicity on the risk of radiotherapy toxicity. MATERIALS AND METHODS: A systematic review was carried out of PubMed, Ovid Medline and Ovid Embase with no year limit. PRISMA 2020 guidelines were followed. Two independent assessors reviewed papers. RESULTS: Of 607 papers screened, 46 fulfilled the inclusion criteria. Papers were published between 1996 and 2021 and involved 30-28,354 individuals (median 433). Most involved patients with prostate (33%), breast (26%) and lung (9%) cancer. Both early and late toxicities were studied. Some studies reported a higher risk of toxicity in White men with prostate cancer compared with other races and ethnicities. For breast cancer patients, some reported an increased risk of toxicity in White women compared with other race and ethnic groups. In general, it was difficult to draw conclusions due to insufficient reporting and analysis of race and ethnicity in published literature. CONCLUSIONS: Reporting of race and ethnicity in radiotherapy studies must be harmonised and improved and frameworks are needed to improve the quality of reporting. Further research is needed to understand how ancestral heritage might affect radiosensitivity and risk of radiotherapy toxicity.

Exon-array profiling unlocks clinically and biologically relevant gene signatures from formalin-fixed paraffin-embedded tumour samples.

BACKGROUND: Degradation and chemical modification of RNA in formalin-fixed paraffin-embedded (FFPE) samples hamper their use in expression profiling studies. This study aimed to show that useful information can be obtained by Exon-array profiling archival FFPE tumour samples. METHODS: Nineteen cervical squamous cell carcinoma (SCC) and 9 adenocarcinoma (AC) FFPE samples (10-16-year-old) were profiled using Affymetrix Exon arrays. The gene signature derived was tested on a fresh-frozen non-small cell lung cancer (NSCLC) series. Exploration of biological networks involved gene set enrichment analysis (GSEA). Differential gene expression was confirmed using Quantigene, a multiplex bead-based alternative to qRT-PCR. RESULTS: In all, 1062 genes were higher in SCC vs AC, and 155 genes higher in AC. The 1217-gene signature correctly separated 58 NSCLC into SCC and AC. A gene network centered on hepatic nuclear factor and GATA6 was identified in AC, suggesting a role in glandular cell differentiation of the cervix. Quantigene analysis of the top 26 differentially expressed genes correctly partitioned cervix samples as SCC or AC. CONCLUSION: FFPE samples can be profiled using Exon arrays to derive gene expression signatures that are sufficiently robust to be applied to independent data sets, identify novel biology and design assays for independent platform validation.

Enhancing fraction measured using dynamic contrast-enhanced MRI predicts disease-free survival in patients with carcinoma of the cervix.

BACKGROUND: There is a need for simple imaging parameters capable of predicting therapeutic outcome. METHODS: This retrospective study analysed 50 patients with locally advanced carcinoma of the cervix who underwent dynamic contrast-enhanced MRI before receiving potentially curative radiotherapy. The proportion of enhancing pixels (E(F)) in the whole-tumour volume post-contrast agent injection was calculated and assessed in relation to disease-free survival (DFS). RESULTS: Tumours with high E(F) had a significantly poorer probability of DFS than those with low E(F) (P=0.011). INTERPRETATION: E(F) is a simple imaging biomarker that should be studied further in a multi-centre setting.

The genomics revolution and radiotherapy.

The expansion of our knowledge through the Human Genome Project has been accompanied by the development of new high-throughput techniques, which provide extensive capabilities for the analysis of a large number of genes or the whole genome. These assays can be carried out in various clinical samples at the DNA (genome), RNA (transcriptome) or protein (proteome) level. There is a belief that this genomic revolution, i.e. sequencing of the human genome and developments in high-throughput technology, heralds a future of personalised medicine. For clinical oncology, this progress should increase the possibility of predicting individual patient responses to radiotherapy. This review highlights some of the work involving sparsely ionising radiation and the new technologies.

Hypoxia-regulated glucose transporter Glut-1 may influence chemosensitivity to some alkylating agents: results of EORTC (First Translational Award) study of the relevance of tumour hypoxia to the outcome of chemotherapy in human tumour-derived xenografts.

Tumour hypoxia confers poor prognosis in a wide range of solid tumours, due to an increased malignancy, increased likelihood of metastasis and treatment resistance. Poorly oxygenated tumours are resistant to both radiation therapy and chemotherapy. However, although the link between radiation therapy and hypoxia is well established in a range of clinical studies, evidence of its influence on chemotherapy response is lacking. In this study, a panel of human tumour-derived xenografts that have been characterised previously for in vivo response to a large series of anti-cancer agents, and have been found to show chemosensitivities that correlate strongly with the parent tumour, were used to address this issue. Immunohistochemistry was carried out on formalin-fixed, paraffin-embedded sections of xenograft samples to detect expression of the intrinsic hypoxia marker Glut-1 and adducts of the bioreductive hypoxia marker pimonidazole. Glut-1 scores correlated significantly with T/C values for CCNU sensitivity (r = 0.439, P = 0.036, n = 23) and showed a borderline significant correlation with dacarbazine T/C (r = 0.405, P = 0.076, n = 20). However, there was no correlation between both Glut-1 and pimonidazole scores and T/C obtained for the bioreductive drug mitomycin C. The use of human tumour-derived xenografts offers a potentially useful way of using archival material to determine the influence of hypoxia and other tumour-microenvironmental factors on chemosensitivity without the direct use of human subjects.

Gene Expression Signatures as Biomarkers of Tumour Hypoxia.

Hypoxia is a feature of most solid tumours and is associated with a poor prognosis. The hypoxic environment can reduce the efficacy of radiotherapy and some chemotherapeutics, and has been investigated extensively as a therapeutic target. The clinical use of hypoxia-targeting treatment will benefit from the development of a biomarker to assess tumour hypoxia. There are several possible techniques that measure either the level of oxygen or the tumour molecular response to hypoxia. The latter includes gene expression profiling, which measures the transcriptional response of a tumour to its hypoxic microenvironment. A systematic review identified 32 published hypoxia gene expression signatures. The methods used for their derivation varied, but are broadly classified as: (i) identifying genes with significantly higher or lower expression in cancer cells cultured under hypoxic versus normoxic conditions; (ii) using either previously characterised hypoxia-regulated genes/biomarkers to define hypoxic tumours and then identifying other genes that are over- or under-expressed in the hypoxic tumours. Both generated gene signatures useful in furthering our understanding of hypoxia biology. However, signatures derived using the second method seem to be superior in terms of providing prognostic information. Here we summarise all 32 published hypoxia signatures, discuss their commonalities and differences, and highlight their strengths and limitations. This review also highlights the importance of reproducibility and gene annotation, which must be accounted for to transfer signatures robustly for clinical application as biomarkers.

Incorporating Genetic Biomarkers into Predictive Models of Normal Tissue Toxicity.

There is considerable variation in the level of toxicity patients experience for a given dose of radiotherapy, which is associated with differences in underlying individual normal tissue radiosensitivity. A number of syndromes have a large effect on clinical radiosensitivity, but these are rare. Among non-syndromic patients, variation is less extreme, but equivalent to a ±20% variation in dose. Thus, if individual normal tissue radiosensitivity could be measured, it should be possible to optimise schedules for individual patients. Early investigations of in vitro cellular radiosensitivity supported a link with tissue response, but individual studies were equivocal. A lymphocyte apoptosis assay has potential, and is currently under prospective validation. The investigation of underlying genetic variation also has potential. Although early candidate gene studies were inconclusive, more recent genome-wide association studies are revealing definite associations between genotype and toxicity and highlighting the potential for future genetic testing. Genetic testing and individualised dose prescriptions could reduce toxicity in radiosensitive patients, and permit isotoxic dose escalation to increase local control in radioresistant individuals. The approach could improve outcomes for half the patients requiring radical radiotherapy. As a number of patient- and treatment-related factors also affect the risk of toxicity for a given dose, genetic testing data will need to be incorporated into models that combine patient, treatment and genetic data.

Exploiting biological and physical determinants of radiotherapy toxicity to individualize treatment.

The recent advances in radiation delivery can improve tumour control probability (TCP) and reduce treatment-related toxicity. The use of intensity-modulated radiotherapy (IMRT) in particular can reduce normal tissue toxicity, an objective in its own right, and can allow safe dose escalation in selected cases. Ideally, IMRT should be combined with image guidance to verify the position of the target, since patients, target and organs at risk can move day to day. Daily image guidance scans can be used to identify the position of normal tissue structures and potentially to compute the daily delivered dose. Fundamentally, it is still the tolerance of the normal tissues that limits radiotherapy (RT) dose and therefore tumour control. However, the dose-response relationships for both tumour and normal tissues are relatively steep, meaning that small dose differences can translate into clinically relevant improvements. Differences exist between individuals in the severity of toxicity experienced for a given dose of RT. Some of this difference may be the result of differences between the planned dose and the accumulated dose (DA). However, some may be owing to intrinsic differences in radiosensitivity of the normal tissues between individuals. This field has been developing rapidly, with the demonstration of definite associations between genetic polymorphisms and variation in toxicity recently described. It might be possible to identify more resistant patients who would be suitable for dose escalation, as well as more sensitive patients for whom toxicity could be reduced or avoided. Daily differences in delivered dose have been investigated within the VoxTox research programme, using the rectum as an example organ at risk. In patients with prostate cancer receiving curative RT, considerable daily variation in rectal position and dose can be demonstrated, although the median position matches the planning scan well. Overall, in 10 patients, the mean difference between planned and accumulated rectal equivalent uniform doses was -2.7 Gy (5%), and a dose reduction was seen in 7 of the 10 cases. If dose escalation was performed to take rectal dose back to the planned level, this should increase the mean TCP (as biochemical progression-free survival) by 5%. Combining radiogenomics with individual estimates of DA might identify almost half of patients undergoing radical RT who might benefit from either dose escalation, suggesting improved tumour cure or reduced toxicity or both.

Cytotoxicity of the bioreductive agent RH1 and its lack of interaction with radiation.

BACKGROUND AND PURPOSE: RH1 is a new bioreductive agent that was developed as a cytotoxic agent with selectivity for tumour cells expressing high levels of the enzyme DT-diaphorase (DTD). The aim of the present study was to investigate the cytotoxicity of RH1 in relation to cellular levels of reducing enzymes and any interaction of RH1 with ionizing radiation under oxic and hypoxic conditions. PATIENTS AND METHODS: The MB-MDA231 human breast cancer cell line (WT) and WT cells transfected with the NQO1 gene encoding DTD (the D7 cell line) were used to examine the dependency of RH1's cytotoxicity on cellular DTD activity. The role of the 1-electron reducing enzyme P450 reductase was also studied using a P450 reductase-transfected isogenic cell line (R4). A clonogenic assay was used to investigate the cytotoxicity of RH1 with and without irradiation in air and in nitrogen. In all cases drug exposure was for 3 h. RESULTS: DTD levels were around 300-fold higher in D7 compared to WT and R4 cells. RH1 was cytotoxic at nanomolar concentrations to all the cell lines, and was 2-3 times more toxic in the D7 cells with high DTD than in the other two cell lines. Doses of RH1 was around 2-fold more effective in hypoxic than in oxic WT cells, but not by as much in D7 cells. RH1 did not radiosensitise the cells but showed an additive effect when combined with irradiation under oxic and hypoxic conditions. CONCLUSIONS: RH1 shows high clonogenic cytotoxicity to MDA231 cells with high DTD activity but its selectivity based on the presence of DTD is much less than as shown in previous reports. RH1 showed an additive cell killing effect when combined with irradiation under both oxic and hypoxic conditions.

Evaluation of multiple biologic parameters in cervical carcinoma: high macrophage infiltration in HPV-associated tumors.

A number of diverse biologic parameters have been assessed prior to treatment in a series of patients with cervical carcinoma. Factors analyzed were HPV DNA presence, MHC class I expression, expression of the oncofetal antigen 5T4, the proportions of macrophages, lymphocytes and granulocytes in cell suspensions prepared from tumors, in vitro colony-forming efficiency (CFE) and a measure of intrinsic radiosensitivity, surviving fraction at 2 Gy. Several associations were found. First, HPV DNA-negative tumors contained a small, but significant, decreased number of tumor infiltrating macrophages compared with HPV DNA-positive tumors. Secondly, patients with HPV-positive tumors were significantly younger than those where no HPV was detected. Thirdly, loss of one or more specific alleles in MHC class I positive tumors resulted in higher numbers of tumor lymphocytes and CFEs. Finally, strong expression of the 5T4 antigen was related to a reduction in the proportion of macrophages in tumor cell suspensions. In addition, for stage I and II patients expression of the MHC class I molecule was associated with improved survival compared with patients with tumors where loss of expression was seen.

Late radiation morbidity in a patient with carcinoma of the cervix.

The Grand Round was held at the Christie Hospital on the 26 November 2001. It followed a talk by Professor Kate Vallis of the Princess Margaret Hospital in Toronto on, "A functional genomics approach to understanding normal issue response and ionizing radiation".

The prognostic significance of the biomarker p16 in oropharyngeal squamous cell carcinoma.

AIMS: There is an increasing incidence of human papillomavirus (HPV)-positive oropharyngeal squamous cell cancers (OPSCC) mostly associated with favourable outcomes. p16 immunohistochemistry is a surrogate marker for HPV positivity in OPSCC. The prognostic strength of p16 over traditional prognostic factors is not fully characterised. In this study, we evaluated the clinical and demographic differences between p16-positive and -negative OPSCC and characterised its prognostic strength versus traditional prognostic factors. MATERIALS AND METHODS: Formalin-fixed, paraffin-embedded blocks and clinical information from 217 OPSCC patients, treated with radiotherapy (alone or in combination with other therapies) between 2000 and 2010 were collected retrospectively. Immunohistochemistry for p16 protein was carried out; cancer-specific survival (CSS), recurrence-free survival (RFS) and locoregional control (LRC) were calculated for both univariate and multivariate analyses. RESULTS: Ninety-two per cent of the OPSCC originated from tonsil and tongue base sites, 61% were p16 positive. Patients with p16-positive OPSCC were younger (P < 0.0001), with lower alcohol (P = 0.0002) and tobacco (P = 0.0001) exposure. The tumours were less differentiated (P = 0.0069), had a lower T stage (P = 0.0027), higher nodal status (P = 0.014) and higher American Joint Committee on Cancer (AJCC) prognostic group (P = 0.0036). AJCC prognostic group was significant for RFS (P = 0.0096) and CSS (P = 0.018) in patients with p16-negative OPSCC, but not those with p16-positive tumours (P = 0.30 and 0.54). Other significant factors for CSS and RFS in univariate analysis were: pretreatment haemoglobin (P < 0.0001 and <0.0001), chemoradiotherapy (P = 0.005 and 0.03) and P16 status (P < 0.0001 and 0.0001). In multivariate analysis, p16 positivity was the strongest independent prognostic variable for both CSS, RFS and LRC (P < 0.0001, hazard ratio 4.15; 95% confidence interval 2.43-7.08), (P < 0.0001, hazard ratio 6.15; 95% confidence interval 3.57-10.61) and (P = 0.001, hazard ratio 3.74; confidence interval 1.76-7.95). CONCLUSION: This study shows that p16 is the single most important prognostic variable in OPSCC, surpassing traditional prognostic factors for both CSS and RFS. Furthermore, disease stage has no prognostic significance in p16-positive patients, highlighting the need for routine p16 assessment in OPSCC.

Lack of prognostic effect of carbonic anhydrase-9, hypoxia inducible factor-1α and bcl-2 in 286 patients with early squamous cell carcinoma of the glottic larynx treated with radiotherapy.

AIMS: To evaluate the prognostic significance of potential tumour markers of hypoxia and apoptosis in early squamous cell carcinoma of the glottic larynx managed with radiotherapy. MATERIALS AND METHODS: In total, 382 patients with T1 and T2 squamous cell carcinoma of the glottic larynx (vocal cords) received radical radiotherapy (50-55 Gy, in 16 fractions in 98% of cases). Pre-treatment haemoglobin was available for 328 patients; biopsy samples were available for 286. Immunohistochemistry was carried out for carbonic anhydrase-9 (CA-9), hypoxia inducible factor-1α (HIF-1α) and Bcl-2. RESULTS: At 5 years, locoregional control was achieved in 88.2%, cancer-specific survival in 95.0% and overall survival in 78.7%. Adverse prognostic factors for locoregional tumour recurrence were pre-treatment haemoglobin <13.0 g/dl (P = 0.035, Log rank test; sensitivity 0.28, specificity 0.84) and stage T2 rather than T1 (P = 0.002). The effect of haemoglobin level on locoregional control was not significant when stratified by the median of 14.2 g/dl (P = 0.43) or as a continuous variable (P = 0.59). High CA-9 (P = 0.11), HIF-1α (P = 0.67) and Bcl-2 (P = 0.77) expression had no prognostic significance. CONCLUSIONS: High CA-9, HIF-1α and Bcl-2 do not add to the prognostic significance of tumour stage and lower haemoglobin in predicting failure of local control in early glottic larynx squamous cell carcinoma managed with radiotherapy. The effect of haemoglobin was not strong enough to be useful as a prognostic biomarker.