Randomized Phase 3 Trial of the Hypoxia Modifier Nimorazole Added to Radiation Therapy With Benefit Assessed in Hypoxic Head and Neck Cancers Determined Using a Gene Signature (NIMRAD).

PURPOSE: Tumor hypoxia is an adverse prognostic factor in head and neck squamous cell carcinoma (HNSCC). We assessed whether patients with hypoxic HNSCC benefited from the addition of nimorazole to definitive intensity modulated radiation therapy (IMRT). METHODS AND MATERIALS: NIMRAD was a phase 3, multicenter, placebo-controlled, double-anonymized trial of patients with HNSCC unsuitable for concurrent platinum chemotherapy or cetuximab with definitive IMRT (NCT01950689). Patients were randomized 1:1 to receive IMRT (65 Gy in 30 fractions over 6 weeks) plus nimorazole (1.2 g/m2 daily, before IMRT) or placebo. The primary endpoint was freedom from locoregional progression (FFLRP) in patients with hypoxic tumors, defined as greater than or equal to the median tumor hypoxia score of the first 50 patients analyzed (≥0.079), using a validated 26-gene signature. The planned sample size was 340 patients, allowing for signature generation in 85% and an assumed hazard ratio (HR) of 0.50 for nimorazole effectiveness in the hypoxic group and requiring 66 locoregional failures to have 80% power in a 2-tail log-rank test at the 5% significance level. RESULTS: Three hundred thirty-eight patients were randomized by 19 centers in the United Kingdom from May 2014 to May 2019, with a median follow-up of 3.1 years (95% CI, 2.9-3.4). Hypoxia scores were available for 286 (85%). The median patient age was 73 years (range, 44-88; IQR, 70-76). There were 36 (25.9%) locoregional failures in the hypoxic group, in which nimorazole + IMRT did not improve FFLRP (adjusted HR, 0.72; 95% CI, 0.36-1.44; P = .35) or overall survival (adjusted HR, 0.96; 95% CI, 0.53-1.72; P = .88) compared with placebo + IMRT. Similarly, nimorazole + IMRT did not improve FFLRP or overall survival in the whole population. In total (N = 338), 73% of patients allocated nimorazole adhered to the drug for ≥50% of IMRT fractions. Nimorazole + IMRT caused more acute nausea compared with placebo + IMRT (Common Terminology Criteria for Adverse Events version 4.0 G1+2: 56.6% vs 42.4%, G3: 10.1% vs 5.3%, respectively; P < .05). CONCLUSIONS: Addition of the hypoxia modifier nimorazole to IMRT for locally advanced HNSCC in older and less fit patients did not improve locoregional control or survival.

TORPEdO: A phase III trial of intensity-modulated proton beam therapy versus intensity-modulated radiotherapy for multi-toxicity reduction in oropharyngeal cancer.

•There is a lack of prospective level I evidence for the use of PBT for most adult cancers including oropharyngeal squamous cell carcinoma (OPSCC).•TORPEdO is the UK's first PBT clinical trial and aims to determine the benefits of PBT for OPSCC.•Training and support has been provided before and during the trial to reduce variations of contouring and radiotherapy planning.•There is a strong translational component within TORPEdO. Imaging and physics data along with blood, tissue collection will inform future studies in refining patient selection for IMPT.

Pretreatment Lymphocyte Count Predicts Benefit From Concurrent Chemotherapy With Radiotherapy in Oropharyngeal Cancer.

PURPOSE: There is a need to refine the selection of patients with oropharyngeal squamous cell carcinoma (OPSCC) for treatment de-escalation. We investigated whether pretreatment absolute lymphocyte count (ALC) predicted overall survival (OS) benefit from the addition of concurrent chemotherapy to radical radiotherapy. PATIENTS AND METHODS: This was an observational study of consecutive OPSCCs treated by curative-intent radiotherapy, with or without concurrent chemotherapy (n = 791) with external, independent validation from a separate institution (n = 609). The primary end point was OS at 5 years. Locoregional control (LRC) was assessed using competing risk regression as a secondary end point. Previously determined prognostic factors were used in a multivariable Cox proportional hazards model to assess the prognostic importance of ALC and the interaction between ALC and cisplatin chemotherapy use. RESULTS: Pretreatment ALC was prognostic for 5-year OS on multivariable analysis (hazard ratio [HR] 0.64; 95% CI, 0.42 to 0.98; P = .04). It also predicted benefit from the use of concurrent cisplatin chemotherapy, with a significant interaction between cisplatin chemotherapy and pretreatment ALC (likelihood ratio test, P = .04): higher ALC count reduced the 5-year OS benefit compared with radiotherapy alone (HR 2.53; 95% CI, 1.03 to 6.19; P = .043). This was likely driven by an effect on LRC up to 5 years (interaction subdistribution HR 2.29; 95% CI, 0.68 to 7.71; P = .094). An independent validation cohort replicated the OS (HR 2.53; 95% CI, 0.98 to 6.52; P = .055) and LRC findings (interaction subdistribution HR 3.43; 95% CI, 1.23 to 9.52; P = .018). CONCLUSION: For OPSCC, the pretreatment ALC is prognostic for OS and also predicts benefit from the addition of cisplatin chemotherapy to radiotherapy. These findings require prospective evaluation, and could inform the selection of good prognosis patients for a de-escalation trial.

Evaluation of the Clinical Utility of Genomic Profiling to Inform Selection of Clinical Trial Therapy in Salivary Gland Cancer.

For most patients with salivary gland cancer, there are no effective standard systemic therapies. Although clinical trials of biomarker-led drug therapies have delivered significant recent advances, there remains a need to understand the clinical utility of genomic profiling of cancer as a means to match patients with recurrent or metastatic salivary gland cancer to clinical trial therapies. In total, 209 patients with salivary gland cancers were profiled with 24 gene (n = 209)) and >325 gene (n = 32) DNA-based next-generation sequencing panels. A retrospective systematic evaluation was performed to identify the frequency of available matched drug therapies within clinical trials based on the results. The matches were then stratified based upon the level of evidence supporting the drug−biomarker combination being investigated using the ESMO Scale for Clinical Actionability of Molecular Targets (ESCAT) to determine the strength of the clinical rationale for each gene−drug match identified. DNA-based next generation sequencing (NGS) analysis was successful in 175/209 (84%) patients with salivary gland cancer. Using the 24-gene NGS panel, actionable alterations were identified in 27% (48/175) patients. Alterations were most frequent in salivary duct carcinoma (88%) characterized by TP53 and/or PIK3CA mutations, with matched trials available for 63% (10/16). In ACC, biomarker-matched trials were available for 7% (8/115), and no genomic alterations were found in 96/115 (83%) of ACC patients. TP53 was the most frequently altered gene across all subtypes; however, there were no trials recruiting based on TP53 status. In 32 ACC patients with no genomic alterations using the 24-gene panel, a broader (>325 gene) panel identified alterations in 87% (27/32) of cases with biomarker-matched trials available in 40% (13/32) cases. This study identified that genomic profiling using focused (24-gene) NGS panels has potential utility in matching to trial therapies for most patients with non-ACC salivary gland cancer. For patients with ACC, broader genomic profiling has demonstrated added clinical utility. We describe the application of an approach to classification of levels of evidence which may be helpful to inform the clinician and patient decision making around the selection of clinical trial therapies.

The utility of TP53 and PIK3CA mutations as prognostic biomarkers in salivary adenoid cystic carcinoma.

OBJECTIVES: Despite wide excision and post-operative irradiation, loco-regional and/or metastatic recurrence is a significant clinical problem in salivary adenoid cystic carcinoma (SACC). Reliable biomarkers are required to tailor post-treatment surveillance to patients at highest risk of recurrence. We sought to determine the utility of TP53 and PIK3CA mutations as prognostic biomarkers in SACC. MATERIALS AND METHODS: DNA was extracted from archival tumour blocks of 145 SACC patients from 66 UK referral centres and sequenced for TP53 and PIK3CA mutations. Clinical, pathological and outcome data were analysed to determine the impact of the genomic alterations on disease recurrence and overall survival (OS). RESULTS: TP53 and PIK3CA mutations were identified in 8% (10/121 successful analyses) and 2% (3/121) of cases, respectively. There were too few PIK3CA mutations in this cohort for informative further analysis. TP53-mutated SACC had significantly shorter median OS (5.3 vs. 16.3 years, p = 0.019) and lower 10-year survival (48% vs. 81%) compared with TP53 wild-type ACC. Solid-pattern histopathology was more frequent in TP53-mutated SACC (50% vs. 15%, p = 0.27). CONCLUSION: TP53-mutated recurrent and metastatic SACC was associated with shorter OS, which was significant when combined with published genomic data sets. Stratifying by TP53 status, in addition to established clinical, pathological and genomic biomarkers, may usefully inform follow-up strategy.

Mapping Hypoxia in Renal Carcinoma with Oxygen-enhanced MRI: Comparison with Intrinsic Susceptibility MRI and Pathology.

Purpose To cross-validate T1-weighted oxygen-enhanced (OE) MRI measurements of tumor hypoxia with intrinsic susceptibility MRI measurements and to demonstrate the feasibility of translation of the technique for patients. Materials and Methods Preclinical studies in nine 786-0-R renal cell carcinoma (RCC) xenografts and prospective clinical studies in eight patients with RCC were performed. Longitudinal relaxation rate changes (∆R1) after 100% oxygen inhalation were quantified, reflecting the paramagnetic effect on tissue protons because of the presence of molecular oxygen. Native transverse relaxation rate (R2*) and oxygen-induced R2* change (∆R2*) were measured, reflecting presence of deoxygenated hemoglobin molecules. Median and voxel-wise values of ∆R1 were compared with values of R2* and ∆R2*. Tumor regions with dynamic contrast agent-enhanced MRI perfusion, refractory to signal change at OE MRI (referred to as perfused Oxy-R), were distinguished from perfused oxygen-enhancing (perfused Oxy-E) and nonperfused regions. R2* and ∆R2* values in each tumor subregion were compared by using one-way analysis of variance. Results Tumor-wise and voxel-wise ∆R1 and ∆R2* comparisons did not show correlative relationships. In xenografts, parcellation analysis revealed that perfused Oxy-R regions had faster native R2* (102.4 sec-1 vs 81.7 sec-1) and greater negative ∆R2* (-22.9 sec-1 vs -5.4 sec-1), compared with perfused Oxy-E and nonperfused subregions (all P < .001), respectively. Similar findings were present in human tumors (P < .001). Further, perfused Oxy-R helped identify tumor hypoxia, measured at pathologic analysis, in both xenografts (P = .002) and human tumors (P = .003). Conclusion Intrinsic susceptibility biomarkers provide cross validation of the OE MRI biomarker perfused Oxy-R. Consistent relationship to pathologic analyses was found in xenografts and human tumors, demonstrating biomarker translation. Published under a CC BY 4.0 license. Online supplemental material is available for this article.

Dose intensified hypofractionated intensity-modulated radiotherapy with synchronous cetuximab for intermediate stage head and neck squamous cell carcinoma.

BACKGROUND: For stage II and III head and neck squamous cell carcinoma (HNSCC) treated with radiotherapy alone, loco-regional recurrence is the main cause of treatment failure. Strategies to improve loco-regional control should not be at the expense of increased late normal tissue toxicity. We investigated dose-intensified hypofractionated intensity-modulated radiotherapy (IMRT) with synchronous cetuximab. MATERIAL AND METHODS: In a phase I/II trial, 27 patients with stage III or high risk stage II HNSCC were recruited. They received three dose level simultaneous integrated boost IMRT, 62.5 Gy in 25 daily fractions to planning target volume one over five weeks with synchronous cetuximab. The primary endpoint was acute toxicity. Secondary endpoints included: late toxicity and quality of life; loco-regional control, cause-specific and overall survival. RESULTS: Radiotherapy was completed by 26/27 patients; for one (4%) the final fraction was omitted due to skin toxicity. All cycles of cetuximab were received by 23/27 patients. Grade 3 acute toxicities included: pain (81%), oral mucositis (78%) and dysphagia (41%). There were few grade 3 physician-recorded late toxicities, including: pain (11%), problems with teeth (8%) and weight loss (4%). At 12 months, only one (4%) patient required a feeding tube, inserted prior to treatment due to dysphagia. The maximal/peak rates of patient-reported late toxicities included: severe pain (11%), any dry mouth (89%) and swallowing dysfunction that required a soft/liquid diet (23%). At 12 months, all quality of life and most symptoms mean scores had resolved to baseline or were only a little worse; dry mouth, sticky saliva and dentition scores remained very much worse. At a median follow-up of 47 months, there were five (18.5%) loco-regional recurrences and the overall cause-specific survival was 79% (95% CI 53-92). CONCLUSIONS: This regimen is safe with acceptable acute toxicity, low rates of late toxicity and impact on quality of life at 12 months following treatment. Further evaluation is recommended.

FGFR2, HER2 and cMet in gastric adenocarcinoma: detection, prognostic significance and assessment of downstream pathway activation.

Receptor tyrosine kinase pathways are potential therapeutic targets in gastric adenocarcinoma patients. We evaluated HER2 and cMet protein expression, and FGFR2 gene amplification to assess their prognostic significance, and downstream mediators pS6 and pERK for their potential utility as pharmacodynamic biomarkers in patients with gastric adenocarcinoma. Tissue microarrays were constructed from resection samples of 184 patients who underwent surgery for gastric/gastro-oesophageal junction adenocarcinoma. Tissue cores were obtained from the tumour body (TB), luminal surface (LS) and invasive edge (IE), and immunohistochemical and fluorescence in situ hybridisation (FGFR2) analysis was performed. FGFR2 amplification was identified in 2 % of cases and associated with worse survival (P = 0.005). HER2 overexpression was observed in 10 % of cases and associated with increased survival (P = 0.041). cMet overexpression was observed in 4 % of cases and associated with worse survival (P < 0.001). On multivariate analysis, only cMet retained significance (P = 0.006). pS6 and pERK expression were observed in 73 % and 30 % of tumours, respectively, with no association with survival. HER2 (P = 0.004) and pERK (P = 0.001) expression differed between tumour regions with HER2 expression increased in the LS compared with the TB and IE. These findings confirm subpopulations in gastric adenocarcinoma with poor outcome that may benefit from specific therapeutic strategies. However, we found heterogeneous HER2, pS6 and pERK overexpression, which presents challenges for their use as predictive biomarkers in gastric biopsies. The potential downstream pharmacodynamic markers pS6 and pERK were expressed across tumour regions, providing evidence that resections and biopsies would yield comparative results in clinical trials.

A 26-gene hypoxia signature predicts benefit from hypoxia-modifying therapy in laryngeal cancer but not bladder cancer.

PURPOSE: Tumor hypoxia is associated with a poor prognosis, hypoxia modification improves outcome, and hypoxic status predicts benefit from treatment. Yet, there is no universal measure of clinical hypoxia. The aim of this study was to investigate whether a 26-gene hypoxia signature predicted benefit from hypoxia-modifying treatment in both cancer types. EXPERIMENTAL DESIGN: Samples were available from 157 T2-T4 laryngeal cancer and 185 T1-T4a bladder cancer patients enrolled on the accelerated radiotherapy with carbogen and nicotinamide (ARCON) and bladder carbogen nicotinamide (BCON) phase III randomized trials of radiotherapy alone or with carbogen and nicotinamide (CON) respectively. Customized TaqMan low density arrays (TLDA) were used to assess expression of the 26-gene signature using quantitative real-time PCR. The median expression of the 26 genes was used to derive a hypoxia score (HS). Patients were categorized as TLDA-HS low (≤median) or TLDA-HS high (>median). The primary outcome measures were regional control (RC; ARCON) and overall survival (BCON). RESULTS: Laryngeal tumors categorized as TLDA-HS high showed greater benefit from ARCON than TLDA-HS low tumors. Five-year RC was 81% (radiotherapy alone) versus 100% (CON) for TLDA-HS high (P=0.009). For TLDA-HS low, 5-year RC was 91% (radiotherapy alone) versus 90% (CON; P=0.90). TLDA-HS did not predict benefit from CON in bladder cancer. CONCLUSION: The 26-gene hypoxia signature predicts benefit from hypoxia-modifying treatment in laryngeal cancer. These findings will be evaluated in a prospective clinical trial.

An in vivo hypoxia metagene identifies the novel hypoxia inducible factor target gene SLCO1B3.

A hypoxia-associated gene signature (metagene) was previously derived via in vivo data-mining. In this study, we aimed to investigate whether this approach could identify novel hypoxia regulated genes. From an initial list of nine genes, three were selected for further study (BCAR1, IGF2BP2 and SLCO1B3). Ten cell lines were exposed to hypoxia and interrogated for the expression of the three genes. All three genes were hypoxia inducible in at least one of the 10 cell lines with SLCO1B3 induced in seven. SLCO1B3 was studied further using chromatin immunoprecipitation and luciferase assays to investigate hypoxia inducible factor (HIF) dependent transcription. Two functional HIF response elements were identified within intron 1 of the gene. The functional importance of SLCO1B3 was studied by gene knockdown experiments followed by cell growth assays, flow cytometry and Western blotting. SLCO1B3 knockdown reduced cell size and 3-dimensional spheroid volume, which was associated with decreased activation of the mammalian target of rapamycin (mTOR) pathway. Finally, Oncomine analysis revealed that head and neck and colorectal tumours had higher levels of SLCO1B3 compared to normal tissue. Thus, the knowledge based approach for deriving gene signatures can identify novel biologically relevant genes.

Prospective technical validation and assessment of intra-tumour heterogeneity of a low density array hypoxia gene profile in head and neck squamous cell carcinoma.

BACKGROUND AND PURPOSE: Tumour hypoxia is associated with a poor prognosis in head and neck squamous cell carcinoma (HNSCC), however there is no accepted method for assessing hypoxia clinically. We aimed to conduct a technical validation of a hypoxia gene expression signature using the TaqMan Low Density Array (TLDA) platform to investigate if this approach reliably identified hypoxic tumours. MATERIALS AND METHODS: Tumour samples (n=201) from 80 HNSCC patients were collected prospectively from two centres. Fifty-three patients received pimonidazole prior to surgery. TaqMan Low Density Array-Hypoxia Scores (TLDA-HS) were obtained by quantitative real-time PCR (qPCR) using a 25-gene signature and customised TLDA cards. Assay performance was assessed as coefficient of variation (CoV). RESULTS: The assay was sensitive with linear reaction efficiencies across a 4 log(10) range of inputted cDNA (0.001-10 ng/µl). Intra- (CoV=6.9%) and inter- (CoV=2.0%) assay reproducibility were excellent. Intra-tumour heterogeneity was lower for TLDA-HS (23.2%) than for pimonidazole (67.2%) or single gene measurements of CA9 (62.2%), VEGFA (45.0%) or HIG2 (39.4%). TLDA-HS in HNSCC cell lines increased with decreasing pO(2). TLDA-HS correlated with Affymetrix U133 Plus 2.0 microarray HS (p<0.01) and positive pimonidazole scores (p=0.005). CONCLUSIONS: Gene expression measurements of hypoxia using a 25-gene signature and TLDA cards are sensitive, reproducible and associated with lower intra-tumour heterogeneity than assaying individual genes or pimonidazole binding. The approach is suitable for further assessment of prognostic and predictive capability in clinical trial material.

Perfusion estimated with rapid dynamic contrast-enhanced magnetic resonance imaging correlates inversely with vascular endothelial growth factor expression and pimonidazole staining in head-and-neck cancer: a pilot study.

PURPOSE: To analyze, in a pilot study, rapidly acquired dynamic contrast-enhanced (DCE)-MRI data with a general two-compartment exchange tracer kinetic model and correlate parameters obtained with measurements of hypoxia and vascular endothelial growth factor (VEGF) expression in patients with squamous cell carcinoma of the head and neck. METHODS AND MATERIALS: Eight patients were scanned before surgery. The DCE-MRI data were acquired with 1.5-s temporal resolution and analyzed using the two-compartment exchange tracer kinetic model to obtain estimates of parameters including perfusion and permeability surface area. Twelve to 16 h before surgery, patients received an intravenous injection of pimonidazole. Samples taken during surgery were used to determine the level of pimonidazole staining using immunohistochemistry and VEGF expression using quantitative real-time polymerase chain reaction. Correlations between the biological and imaging data were examined. RESULTS: Of the seven tumors fully analyzed, those that were poorly perfused tended to have high levels of pimonidazole staining (r = -0.79, p = 0.03) and VEGF expression (r = -0.82, p = 0.02). Tumors with low permeability surface area also tended to have high levels of hypoxia (r = -0.75, p = 0.05). Hypoxic tumors also expressed higher levels of VEGF (r = 0.82, p = 0.02). CONCLUSIONS: Estimates of perfusion obtained with rapid DCE-MRI data in patients with head-and-neck cancer correlate inversely with pimonidazole staining and VEGF expression.

Spectral clustering of microarray data elucidates the roles of microenvironment remodeling and immune responses in survival of head and neck squamous cell carcinoma.

PURPOSE: To identify functionally related prognostic gene sets for head and neck squamous cell carcinoma (HNSCC) by unsupervised statistical analysis of microarray data. PATIENTS AND METHODS: Microarray analysis was performed on 14 normal oral epithelium and 71 HNSCCs from patients with outcome data. Spectral clustering (SC) analysis of the data set identified multiple vectors representing distinct aspects of gene expression heterogeneity between samples. Gene ontology (GO) analysis of vector gene lists identified gene sets significantly enriched within defined biologic pathways. The prognostic significance of these was established by Cox survival analysis. RESULTS: The most influential SC vectors were V2 and V3. V2 separated normal from tumor samples. GO analysis of V2 gene lists identified pathways with heterogeneous expression between HNSCCs, notably focal adhesion (FA)/extracellular matrix remodeling and cytokine-cytokine receptor (CR) interactions. Similar analysis of V3 gene lists identified further heterogeneity in CR pathways. V2CR genes represent an innate immune response, whereas high expression of V3CR genes represented an adaptive immune response that was not dependent on human papillomavirus status. Survival analysis demonstrated that the FA gene set was prognostic of poor outcome, whereas classification for adaptive immune response by the CR gene set was prognostic of good outcome. A combined FA&CR model dramatically exceeded the performance of current clinical classifiers (P < .001 in our cohort and, importantly, P = .007 in an independent cohort of 60 HNSCCs). CONCLUSION: The application of SC and GO algorithms to HNSCC microarray data identified gene sets highly significant for predicting patient outcome. Further large-scale studies will establish the usefulness of these gene sets in the clinical management of HNSCC.

hsa-mir-210 is a marker of tumor hypoxia and a prognostic factor in head and neck cancer.

BACKGROUND: 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. METHODS: Three hypoxia-related microRNAs, hsa-miR-210, hsa-miR-21, and hsa-miR-10b, were measured in 46 samples from patients with HNSCC. Expression levels were correlated with clinicopathological variables and other markers of hypoxia: a published 99-gene hypoxia metagene, individual hypoxia-related genes such as TWIST1, and immunohistochemical expression of hypoxia-inducible factor 1 and its target gene carbonic anhydrase 9. We then performed survival analyses to investigate the prognostic significance of these microRNAs. RESULTS: Only the level of hsa-miR-210 was significantly correlated with other markers of hypoxia, including the 99-gene hypoxia metagene (rho = 0.67, P < .001). We found no association between hsa-miR-210, hsa-miR-21, or hsa-miR-10b and clinicopathological variables such as tumor size, differentiation, and stage. However, high levels of hsa-miR-210 were associated with locoregional disease recurrence (P = .001) and short overall survival (P = .008). hsa-miR-21 and hsa-miR-10b had no prognostic significance. CONCLUSIONS: Expression of hsa-miR-210 in head and neck cancer correlates with other approaches for assessing hypoxia and is associated with prognosis. This warrants further study as a classification marker of patients for therapies involving modulation of hypoxia.

Exon array analysis of head and neck cancers identifies a hypoxia related splice variant of LAMA3 associated with a poor prognosis.

The identification of alternatively spliced transcript variants specific to particular biological processes in tumours should increase our understanding of cancer. Hypoxia is an important factor in cancer biology, and associated splice variants may present new markers to help with planning treatment. A method was developed to analyse alternative splicing in exon array data, using probeset multiplicity to identify genes with changes in expression across their loci, and a combination of the splicing index and a new metric based on the variation of reliability weighted fold changes to detect changes in the splicing patterns. The approach was validated on a cancer/normal sample dataset in which alternative splicing events had been confirmed using RT-PCR. We then analysed ten head and neck squamous cell carcinomas using exon arrays and identified differentially expressed splice variants in five samples with high versus five with low levels of hypoxia-associated genes. The analysis identified a splice variant of LAMA3 (Laminin alpha 3), LAMA3-A, known to be involved in tumour cell invasion and progression. The full-length transcript of the gene (LAMA3-B) did not appear to be hypoxia-associated. The results were confirmed using qualitative RT-PCR. In a series of 59 prospectively collected head and neck tumours, expression of LAMA3-A had prognostic significance whereas LAMA3-B did not. This work illustrates the potential for alternatively spliced transcripts to act as biomarkers of disease prognosis with improved specificity for particular tissues or conditions over assays which do not discriminate between splice variants.