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The era of stratified medicine is upon us and it is important that we choose the right drug for the right patient and reduce treatment failure rates. Colorectal cancer is a complex disease characterised by multiple lesions in key molecular and genetic pathways. Some of these pathways are clinically useful, in particular we are now familiar with treatment pathways such as the EGFR signalling pathway and drugs which target these pathways, such as Cetuximab, are used successfully in advanced disease. However, knowledge of the molecular and genetic makeup of a tumour may also help decide how a particular patient may respond to a certain drug and so called ‘predictive’ pathways are probably more useful than ‘prognostic’ pathways.

32 At present, the only biomarker sufficiently validated for routine clinical use for determining treatment is KRAS in the setting of metastatic colorectal cancer, with patients who are KRAS wild-type being offered EGFR targeted therapy. But, as will be discussed in section 1.1.9.2, our increasing knowledge of the EGFR-signalling pathways is adding increasing complexity to such decisions, especially in light of recent data regarding the value of other downstream mutations such as BRAF, NRAS and PIK3CA.(122)

An ideal biomarker would be simple, sensitive, specific, inexpensive and reproducible for it to achieve use in routine clinical practise. The Cancer Research UK Biomarker Discovery and Development Committee have defined several categories of biomarkers:

1. Risk assessment/predisposition biomarkers: e.g. APC gene mutations in the

diagnosis of FAP.

2. Screening/early detection biomarkers: e.g. FOB testing.

3. Diagnostic biomarkers: e.g. CEA, PSA, CA125 to be used alongside standard

imaging.

4. Pharmacological biomarkers: to assess pharmokinetics or demonstrate clinical

effects of the drug, e.g. thiopurine methyltransferase (TMPT) gene and azathioprine treatment in Crohn’s disease.

5. Predictive biomarkers: e.g. KRAS and response to EGFR therapy in metastatic

colorectal cancer.

6. Prognostic biomarkers: to predict the course of disease and may guide treatment

33 This thesis examines the potential of GRP78 as a predictive and prognostic biomarker. The Cancer Research UK Biomarker Discovery and Development Committee have suggested ‘roadmaps’ which define a chronological research pathway for identifying clinically useful predictive biomarkers. The roadmap is illustrated in Figure 1.1.9-1.(123)

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35 1.1.9.1 Current status of biomarkers and decision making in the adjuvant setting

The reported lack of benefit from molecular targeted therapy in the adjuvant setting,(124) as discussed in section 1.1.8.4, means that 5-FU-based regimes (FOLFOX) remain the backbone of adjuvant treatment.(87) Below is a summary regarding some of the most promising potential biomarkers to date.

1.1.9.1.1 Thymidylate synthase

Thymidylate synthase (TS) is a target of 5-FU. A number of preclinical biochemical and clinical immunohistochemical and RT-PCR studies have consistently shown that high TS expression predicts a poor response to 5-FU based chemotherapy.(73) However a recent meta-analysis cast doubt on the usefulness of TS as a biomarker for response as the association between high TS and survival was better for those treated by surgery alone than for those who received adjuvant treatment. Furthermore, controversy exists as to the relationship between expression of TS and its relationship to resistance to 5-FU therapy.(31)

1.1.9.1.2 p53 mutation

A potential mode of action of 5-FU may be due to its ability to stabilise p53 function leading to apoptosis.(125) Indeed, a number of studies have shown that disruption of both alleles of TP53 in vitro can make colon cell lines resistant to 5-FU.(126, 127) However, although there is in vitro evidence for p53 involvement in the down-stream response to 5-FU,(73) and p53 mutation has been associated with worse prognosis, reviews of p53 as a potential predictive marker suggest p53 mutation has no effect on outcome in patients treated with 5-FU based chemotherapy.(128)

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1.1.9.1.3 18q-LOH/DCC

Loss of heterozygosity on the long arm of chromosome 18 or deleted in colon cancer protein determination by PCR has been implicated as an important step in the development of many colorectal cancers.(24) Some evidence suggests it might be associated with worse prognosis and reduced response rates to chemotherapy.(129, 130) However, it is difficult to draw conclusions from studies investigating chromosome 18q allelic instability as different methodologies and different genetic markers are employed to examine different regions on the chromosome. Additionally, the Pan European Trials in Adjuvant Colon Cancer (PETACC)-3 study identified a stage-specific effect of this biomarker, it showing a prognostic effect in stage III but not stage II disease, as well as revealing that 18q LOH status lost significance when MSI was included in the multivariate analysis suggesting that these markers do not act independently.(131) Therefore, there is insufficient evidence at present to support its routine use.

1.1.9.1.4 DNA mismatch repair and microsatellite instability

As discussed earlier, DNA MMR repairs DNA polymerase mistakes that commonly occur during DNA replication. Affected cells accumulate mutations that drive tumourigenesis and manifest the phenotype of microsatellite instability (MSI), a predisposition to right sided tumours and an unusual histopathological appearance. DNA MMR may also recognise drugs that intercalate with DNA and act as a trigger for apoptosis and thus, alkylation damage as a result of 5-FU incorporation into DNA would not be recognised by a deficient mismatch repair system. MSI may therefore act as a predictor of response to chemotherapy however studies investigating the role

37 of MSI in response to 5-FU have produced some conflicting data. Despite evidence to demonstrate better prognosis for patients with MSI, there is a body of evidence reporting lack of benefit and worse overall survival following 5-FU.(132, 133) Equally, there are reports that to suggest that benefit from 5-FU is maintained in patients with MSI, although some data suggest that benefit may be limited to those with germline rather than sporadic MSI tumour.(134) Subsequently, at present, the routine use of MSI status to predict response to 5-FU is not supported.(87)

1.1.9.2 Current status of biomarkers and decision making in advanced colorectal

cancer

KRAS is an important intermediary in signalling via a number of growth factor receptors, especially epidermal growth factor (EGF) receptor signalling. Epidermal growth factor receptor (EGFR), is an attractive target for cancer treatment because its activation stimulates key processes involved in tumour growth and progression,

including proliferation, angiogenesis, invasion, and metastasis.(135) EGFR-targeted

monoclonal antibodies, such as Cetuximab and Panitumumab, have been extensively studied in metastatic colorectal cancer and shown to provide modest improvement in overall survival.(136, 137) Positive EGFR expression was initially a criterion for entry into studies evaluating EGFR antibodies, however it soon became apparent that positive EGFR expression was a poor marker for response to treatment as responses were observed in patients with low or negative expression of EGFR.(107, 138-140)

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Figure 1.1.9-2: Representation of the EGFR signalling pathway.

Activating KRAS mutations, present in approximately 40% of colorectal cancers,(141-143) result in activation of the EGF signalling pathway via the mitogen activated protein (MAP) kinase pathway at a point downstream of the EGFR (Figure 1.1.9-2). A number of studies have shown that benefit from EGFR antibodies is confined to patients with wild-type KRAS tumours,(143-145) and based on these results, it is now a requirement for patients deemed suitable for EGFR antibodies to undergo KRAS testing and therapy is only approved for KRAS wild-type tumours at present. This is an excellent example of genetic tailoring of treatment in colorectal cancer.

However, not all KRAS mutant tumours are the same. The reality is that response to EGFR targeted therapies based upon KRAS status is actually quite variable and KRAS mutant tumours represent a very heterogenous biological subgroup. Data is

RAS PI3K RAF MEK MAPK mTOR AKT PTEN EGFR Membrane Other effectors Growth factors

Cell growth, proliferation and survival

Common sites of mutation in

39 emerging that some KRAS mutant tumours do respond and some KRAS wild-type tumours do not respond to EGFR targeted therapy.

A study of a large cohort of patients treated with cetuximab in the pre-KRAS selection era (2001-2008) reveals a number of other mutations in the EGFR signalling pathway downstream of KRAS that are associated with low response rates. De Roock et al.,(122) report that KRAS mutation was present in 40% of tumours, 14.5% had a PIK3CA mutation, 4.7% had a BRAF mutation and 2.6% had an NRAS mutation. In this cohort, outcome in KRAS wild-type patients was worse in the presence of BRAF, NRAS and PIK3CA exon 20 mutations. The PICCOLO trial (Panitumumab, Irinotecan & Ciclosporin in COLOrectal cancer therapy)(146, 147) was a randomised clinical trial of treatment for fluorouracil-resistant advanced colorectal cancer comparing standard single-agent irinotecan versus irinotecan plus panitumumab and versus irinotecan plus ciclosporin. Following an amendment to protocol in 2008 to include prospective KRAS testing, the trial released results this year which showed a failure to meet the primary endpoint of improved overall survival in KRAS wild-type patients; however a planned biomarker analysis revealed some interesting findings. As expected, progression free survival was improved in patients with KRAS/BRAF wild-type tumours who received panitumumab, with no benefit seen in those patients with KRAS or BRAF mutated tumours. Interestingly, subset analysis revealed that nearly a third of the KRAS wild-type patients were found to have other mutations, thereby conferring drug resistance. Patients with a broadly wild-type profile for KRAS, NRAS, BRAF and PI3K had a good response from panitumumab, but those with a mutation in any of these kinases did not fare as well.

40 The variable response is further highlighted by additional work by De Roock et al.,(148) revealing better outcomes with cetuximab in patients with p.G13D-mutated tumours than with other KRAS-mutated tumours. In the present context of patients with KRAS codon 12-or KRAS codon 13-mutated tumours being excluded from treatment with cetuximab, this poses some serious questions regarding ongoing randomised controlled trials that are using KRAS status as a discriminator for treatment decisions and suggests that biomarker testing needs to be extended beyond wild-type or mutant KRAS to avoid treatment failure or resistance to EGFR therapy developing.

Whilst many authors have investigated defects in particular molecular pathways, the role of the tumour microenvironment in the behaviour of colorectal cancer is relatively understudied.