The individualisation of Radiation-Therapy: A biological rationale

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The following are the most essential key points:

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Towards isotoxic dose-escalated radiotherapy.
Improve therapeutic ratio. Biological optimisation remains the goal.
The most widely studied tumour tissue is formalin-fixed and paraffin-embedded (FFPE) because relatively large retrospective studies can be carried out in stored material.
A prognostic marker provides information on the risk of local or system outcome (e.g. recurrence) independent of treatment.
A whole blood predictive marker provides information on an outcome which relies on DNA) or separated buffy coat (for lymphocyte assays)

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Genome alterations via comparative genomic hybridisation somatic mutations in KIT for a benefit from imatinib.
Biomarkers for cancer treatment: SNPs for individualisation are now being used clinically, but not investigated for risk prediction.
Groups of SNPs are co-inherited
The genome-wide analysis involves ∼500,000 rather than >10 million SNPs and is SNP profiling can look for germline (e.g. blood or saliva samples) or somatic (tumour samples) variation.
e.g. BRCA1/2 germline for the response to poly(ADP-ribosylation)-polymerase inhibitors; lack of tumour KRAS mutations to predict benefit from cetuximab in EGFR-expressing colorectal cancer.

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An epigenetic variation that includes methylation, acetylation, phosphorylation, ubiquitylation, sumoylation and chromatin modification.
They are usually oligomers (short DNA chains representing the partial sequence of a gene.
Messenger RNA, representing all expressed genes, is extracted including mRNA, rRNA, tRNA and other non-coding RNA.
Signatures are used in breast cancer patients to identify those with a high risk of recurrence (e.g. MammaPrint 70-gene signature) or who are likely to benefit from chemotherapy (e.g. Oncotype DX 21-gene signature)
In situ hybridisation (ISH). ISH uses labelled cDNA (a probe)
Real-time or quantitative polymerase chain reaction (qPCR) is a technique used to amplify and simultaneously to identify a specific DNA sequence in a sample in tissue sections.qPCR detects and quantifies RNA either as an absolute number of copies.
The RNA levels: microRNAs (miRNA), snoRNA and siRNA. miRNA are small non-coding RNAs of 19–24 nucleotides that generally downregulate gene expression by inhibiting protein translation.

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Clonogen/stem cell number), propensity for angiogenesis, metastatic potential and immune response.
Predicts the likelihood of resistant subpopulations
In general, the primary endpoint for tumour predictive assay research should be local control.
Tumour radiosensitivity
Rapid progress is being made, including the development of antibody chips and mass spectrometry approaches.
Intrinsic radiosensitivity is that which is genetically determined. Primary human tumour SF2 was a significant and independent prognostic factor for radiotherapy outcome in both carcinomas of the cervix and head and neck.
The disadvantages of the colony assay are its poor success rate for human tumours (<70%) and the time needed to produce data (often up to 4 weeks).

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Most functional cell-based assays have limited clinical utility as predictivity assays due to poor reproducibility,
The radiosensitivity index (RSI) is calculated by ranking the expression of these 10 genes and using a ranked-based linear algorithm success or failure such as tumour size.
Linear-quadratic model is used to derive a genomic adjusted radiation dose (GARD) model, which was shown to be prognostic in several tumour types.
Hypoxia plays a crucial role in tumour progression by promoting both angiogenesis and metastasis.
Contradictory findings are common in molecular marker studies and highlight the need to develop standardised methods, cut-offs and approaches (e.g. discovery and validation cohorts).
18 F-Fluoroazomycinarabinoside (FAZA) PET is currently of interest as a potential predictor of benefit from hypoxia modification of radiotherapy.
The most widely studied approach for assessing tumour-hypoxia involves measuring hypoxia-inducible proteins
Progress in this area is limited because studies are often small, replication is not carried out, and there are many different imaging intrinsic hypoxia markers).
Markers: CA9, HIF-1α and Glut-1, Osteopontin levels in plasma samples.
Determine the proportion of cells in the S-phase of the cell cycle by DNA flow cytometry, measuring tumour potential doubling time, Tpot, with thymidine analogues such as IdUrd and BrdUrd, and using antibodies to detect proliferation-associated proteins
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Normal tissue radiosensitivity: Tumours negative for p53 and Bcl-2 with a low but organised pattern of Ki-67 staining benefitted from CHART.
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For an assay or biomarker to have clinical utility, it must be precise and accurate.
Several small studies showed correlations between fibroblast radiosensitivity and the severity of late effects.
An excellent, sensitive test: Ability to detect true positives/[number of true positives+number of false negatives]) and specificity (number of true negatives/ [number of true negatives+ number of false positives]) should be determined.
A general problem has been that experimental assay) variability has been relatively large compared with inter-individual differences in radiosensitivity.
RNA and plasma proteomics are very suited to precise repeatability and reproducibility analyses.
Most widely studied biomarker is possibly protein expression using immunohistochemistry on FFPE tumour material. This type of biomarker is particularly challenging to reproduce because of the use of different antigen retrieval methods, antibodies, scoring methods and cut-off values.

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Genome-wide association studies are identifying new candidate genes for radiotherapy toxicity.
Once a predictive marker is discovered and validated, then qualification is required. It must be tested under standardised conditions in new cohorts of patients and preferably prospectively.

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HPV (Human Pappilloma Virus) is usually detected by PCR and/or in situ hybridisation to determine the presence of the virus. PCR can measure DNA or E6/E7 mRNA, which indicate the presence of HPV but do not distinguish integrated and episomal forms of the virus.
HPV is associated in particular with oropharyngeal cancers and a good prognosis.
Although there are some conflicting data, evidence suggests enhanced radiosensitivity in HPV-positive head and neck cancer.
Temozolomide is hydrolysed in the body to form an active metabolite (3-methyl-[triazen-1-yl]imidazole-4- carboxamide) that produces approximately 12 base adducts in DNA including the cytotoxic lesion O6-methylguanine (O6-meG). MGMT encodes for O6-methylguanine-DNA methyltransferase that removes O6-meG from DNA and so the efficacy of temozolomide is increased when the gene is silenced following methylation.

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Patients with very hypoxic tumours could be assigned to treatments which include hypoxia-modifying or hypoxia- exploiting agents (No trials have conferred much benefit and not used in routine clinical practise).
Tumours with fast repopulation potential would-be candidates for accelerated fractionation (including hypofractionation) or radiotherapy combined with drugs designed to combat proliferation (e.g. EGFR inhibitors).
The goal is to be able to obtain a complete genetic picture of each tumour, thereby understanding why a tumour is radioresistant, allowing a rational choice of tumour-specific radiosensitising drugs.
The p16 is considered to be a marker that could be used for individualisation of treatment.
Last, if reliable information were available for predicting the risk of severe normal tissue effects, possible strategies would be to offer surgery rather than radiotherapy for some cancers
Also, therapy intensification could be trialled initially in sub-populations most likely to benefit.

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Steps for individualisation of therapy:

Genetics of that individual and of their tumour.
Well-validated biomarkers that are reproducible.
Availability of alternative treatments and an array of agents which attack specific genes or pathways.
Outcome prediction through genes identified by various molecular techniques.