October 18, 2021

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What are biomarkers of the first-line treatment for colon cancer?

What are biomarkers of the first-line treatment for colon cancer?

What are biomarkers of the first-line treatment for colon cancer?



What are biomarkers of the first-line treatment for colon cancer?

With the increasingly prominent role of targeted therapy in the treatment of advanced or mCRC disease, the NCCN expert group has expanded its recommendations on biomarker testing.

At present, for patients with mCRC, it is recommended to determine the tumor gene status of KRAS/NRAS and BRAF mutations, as well as HER2 amplification and MSI/MMR status (if not completed in the past). Although no specific method is recommended, it can be tested on individual genes or as part of an NGS group.

The advantage of the NGS group is the ability to detect rare and operable genetic changes, such as neurotrophic tyrosine receptor kinase (NTRK) fusion. For specific information about each of these biomarkers, please see the following sections.


1. KRAS and NRAS mutations

The MAPK pathway of RAS/RAF/MEK/ERK is located downstream of EGFR; mutations in this pathway are currently identified as strong negative predictive markers, which basically rule out the efficacy of these treatments. A large amount of literature has shown that tumors with mutations in exons 2, 3, or 4 of the KRAS or NRAS gene are basically insensitive to cetuximab or panitumumab treatment.

Therefore, the expert group strongly recommends that the tumor tissue (primary tumor or metastasis) of all mCRC patients should be genotyped for RAS (KRAS/NRAS). Patients with known KRAS or NRAS mutations should not be treated with cetuximab or panitumumab, either alone or in combination with other anticancer drugs, because they have little possibility of benefit and are exposed to The toxicity and cost are also unreasonable.

ASCO issued an update of interim clinical opinions on the extended RAS test for patients with mCRC, consistent with the recommendations of the NCCN expert group. The CRC molecular biomarker guidelines developed by ASCP, CAP, AMP and ASCO also recommend that RAS testing should be consistent with NCCN recommendations.

At this point, the recommendation of RAS testing does not imply a preference for first-line treatment. Conversely, this early established RAS status is suitable for planning continuous treatment so that information is obtained in a time-insensitive manner, patients and healthcare providers can discuss the impact of RAS mutations (if any), while other treatment options are still available.

Please note that since anti-EGFR drugs have no effect in the treatment of stage I, II, or III disease, RAS genotyping of CRC in these early stages is not recommended.

KRAS mutation is an early event in the formation of CRC, so there is a very close correlation between the mutation status of the primary tumor and metastasis. Therefore, archival specimens of primary tumors or metastases can be used for RAS genotyping. Unless archived specimens from the primary tumor or metastasis are not available, fresh biopsies should not be obtained just for RAS genotyping.

The expert group recommends that KRAS, NRAS, and BRAF genetic testing can only be performed in laboratories accredited by the 1988 Clinical Laboratory Improvement Amendment (CLIA-88), which are qualified to perform highly complex molecular pathology testing. It is not recommended to use specific detection methods.

These three genes can be tested individually or as part of the NGS group. Regarding the prognostic value of KRAS mutations, the results are mixed. In the Alliance N0147 trial, patients with a KRAS exon 2 mutation had a shorter DFS than patients without this mutation. However, at this time, due to prognostic reasons, this test is not recommended.

A retrospective study conducted by De Roock et al. raised the possibility that the KRAS codon 13 mutation (G13D) may not fully predict non-response. Another retrospective study showed similar results. However, a recent retrospective analysis of three randomized controlled phase III trials concluded that patients with KRAS G13D mutations are unlikely to respond to panitumumab.

The results of a prospective phase II single-arm trial evaluated the benefit of cetuximab monotherapy in 12 patients with refractory mCRC whose tumors contained the KRAS G13D mutation. The primary endpoint of disease-free rate at 4 months was not reached (25%), and no remission was seen.

The preliminary results of the AGITG Phase II ICE CREAM trial also did not find the benefit of cetuximab monotherapy in patients with KRAS G13D mutations. However, after receiving irinotecan plus cetuximab treatment, 9% of irinotecan refractory people experienced partial remission.

A meta-analysis of eight RCTs reached the same conclusion: tumors with KRAS G13D mutations are not more likely to respond to EGFR inhibitors than other tumors with KRAS mutations. The expert group believes that patients with any known KRAS mutations, including G13D mutations, should not be treated with cetuximab or panitumumab.

In the AGITG MAX study, 10% of patients with wild-type KRAS exon 2 had KRAS exon 3 or 4 mutations or NRAS exon 2, 3, and 4 mutations. In the PRIME trial, it was found that 17% of 641 patients without KRAS exon 2 mutations had KRAS exons 3 and 4 mutations or NRAS exons 2, 3, and 4 mutations.

A pre-defined retrospective subgroup analysis of the PRIME trial data showed that among patients with any KRAS or NRAS mutations, the PFS of patients receiving panitumumab plus FOLFOX (HR 1.31; 95% CI 1.07–1.60; P = 0.008) and OS (HR 1.21; 95% CI 1.01–1.45; P = 0.04) compared with patients who received FOLFOX alone. 600 These results indicate that panitumumab is not beneficial to patients with KRAS or NRAS mutations, and may even have harmful effects on these patients.

The latest analysis of the FIRE-3 trial has been published (discussed below in Cetuximab or Panitumumab vs. Bevacizumab in first-line treatment). When all RAS (KRAS/NRAS) mutations were considered, the PFS of patients with RAS mutation tumors who received FOLFIRI + cetuximab was significantly lower than that of patients with RAS mutation tumors who received FOLFIRI + bevacizumab (6.1 months vs. 12.2 Month; P = 0.004).

On the other hand, the PFS of patients with KRAS/NRAS wild-type tumors did not differ between the two regimens (10.4 months vs. 10.2 months; P = 0.54). This result suggests that cetuximab may have harmful effects on patients with KRAS or NRAS mutations.

The FDA updated the indications for panitumumab, stating that panitumumab is not suitable for combination with oxaliplatin-based chemotherapy to treat patients with KRAS or NRAS mutation-positive diseases. The NCCN Colon/Rectal Cancer Expert Group believes that the RAS mutation status should be determined at the time of diagnosis of stage IV disease. Patients with any known RAS mutations should not be treated with cetuximab or panitumumab.


2. BRAF V600E mutation

Although RAS mutations indicate no response to EGFR inhibitors, many tumors that contain the wild-type RAS gene still do not respond to these treatments. Therefore, some studies have used factors downstream of RAS as possible additional biomarkers to predict the response of cetuximab or panitumumab.

About 5%-9% of CRCs are characterized by specific mutations in the BRAF gene (V600E). For all practical purposes, BRAF mutations are limited to tumors without RAS mutations. The activation of the protein product of the non-mutant BRAF gene occurs downstream of the activated KRAS protein in the EGFR pathway.

The mutant BRAF protein product is considered to be constitutively active, which can be speculated to bypass the inhibition of EGFR by cetuximab or panitumumab.

Limited data from an unplanned retrospective subgroup analysis of patients with mCRC (receiving first-line treatment) shows that although BRAF V600E mutations can lead to poor prognosis regardless of treatment, the addition of cetuximab to first-line treatment may have this effect. Patients with mutational characteristics have some benefits.

A planned subgroup analysis of the PRIME trial also found that BRAF mutations suggest a poor prognosis, but cannot predict the benefit of adding panitumumab to FOLFOX in the first-line treatment of mCRC.

On the other hand, the results of the randomized Phase III Medical Research Council (MRC) COIN trial indicate that cetuximab may have no effect on patients with BRAF mutant tumors (first-line treatment with CAPEOX or FOLFOX), and even have harmful effects.

In the follow-up treatment, retrospective evidence shows that in the non-first-line treatment of metastatic disease, mutated BRAF is a sign of resistance to anti-EGFR therapy.

A retrospective study of 773 primary tumor samples from patients with chemotherapy-refractory diseases showed that the response rate (2/24; 8.3%) to cetuximab caused by BRAF mutations was significantly lower than that of wild-type BRAF Tumor (124/326; 38.0%; P = 0.0012).

In addition, the data of the multicenter randomized controlled PICCOLO trial is consistent with this conclusion, suggesting that in a small number of BRAF mutation patients in non-first-line treatment situations, adding panitumumab to irinotecan can cause harm.

A meta-analysis published in 2015 identified nine phase III trials and one phase II trial that compared cetuximab or panitumumab with standard or best supportive care, including 463 patients with metastatic colorectal cancer with BRAF mutations (first-line, second-line, or refractory treatment).

Compared with the control group, the addition of EGFR inhibitors did not improve PFS (HR 0.88; 95% CI 0.67–1.14; P = 0.33), OS (HR 0.91; 95% CI 0.62–1.34; P = 0.63) or ORR (RR of 1.31; 95% CI of 0.83–2.08; P = 0.25).

Similarly, another meta-analysis identified 7 RCTs and found that cetuximab and panitumumab did not improve PFS (HR 0.86; 95% CI 0.61-1.21) or OS (HR 0.97; 95% CI 0.67-1.41). In addition to being a predictive marker for BRAF targeted therapy, it is clear that BRAF mutations are powerful prognostic markers.

A prospective analysis of tissues from patients with stage II and stage III colon cancer (in the PETACC-3 trial) showed that BRAF mutations have prognostic significance for OS in patients with MSI-L or MSS tumors (HR 2.2; 95% CI is 1.4–3.4; P = 0.0003). In addition, the latest analysis of the CRYSTAL trial showed that patients with metastatic colorectal tumors with BRAF mutations have a worse prognosis than patients with wild-type genes.

In addition, BRAF mutation status can predict OS in the AGITG MAX trial, with an HR of 0.49 (95% CI 0.33–0.73; P = 0.001). In the COIN trial, the OS of patients with BRAF mutations was 8.8 months, while the OS of patients with KRAS exon 2 mutations and wild-type KRAS exon 2 tumors were 14.4 months and 20.1 months, respectively.

In addition, a secondary analysis of the N0147 and C-08 trials found that BRAF mutations are significantly associated with poor survival after resection of stage III colon cancer that has been resected, and a stronger correlation with primary tumors located in the distal colon sex.

The results of a recent systematic review and meta-analysis of 21 studies (including 9885 patients) indicate that BRAF mutations may be accompanied by specific high-risk clinicopathological features.

In particular, BRAF mutations and proximal tumor locations (OR 5.22; 95% CI 3.80–7.17; P <0.001), T4 tumors (OR 1.76; 95% CI 1.16–2.66; P = 0.007) and poor were observed. There was a correlation between differentiation (OR 3.82; 95% CI 2.71–5.36; P <0.001).

In conclusion, the expert group believes that there is increasing evidence that the BRAF V600E mutation is extremely unlikely to respond to panitumumab or cetuximab (as a single drug or in combination with cytotoxic chemotherapy), unless it is used as a BRAF inhibitor Part of the dosage regimen is administered.

The expert group recommends that tumor tissues (primary tumors or metastatic tumors) be genotyped for BRAF when diagnosing stage IV disease. BRAF V600E mutation detection can be performed on formalin-fixed paraffin-embedded tissue, usually by PCR amplification and direct DNA sequence analysis. Allele-specific PCR, NGS, or IHC are other acceptable methods for detecting this mutation.


3. HER2 amplification/overexpression

HER2 is a member of the same signal kinase receptor family as EGFR. It has been successfully targeted to attack breast cancer in advanced and adjuvant treatments. HER2 is rarely amplified/overexpressed in CRC (approximately 3% overall), but has a higher incidence in RAS/BRAF wild-type tumors (reported to be 5%-14%).

It has been proposed to use specific molecular diagnostic methods for the HER2 detection of CRC, and now it is recommended that HER2 targeted therapy be used as a follow-up treatment option for RAS/BRAF wild-type and HER2 overexpression tumor patients (see below for HER2 amplified disease Systemic treatment plan).

Based on this, NCCN Guidelines recommend HER2 amplification testing for mCRC patients. If the tumor is known to have KRAS/NRAS or BRAF mutations, HER2 testing is not required. Since HER2 targeted therapy is still under research, patients are encouraged to participate in clinical trials.

The evidence does not support the prognostic role of HER2 overexpression. In addition to being a predictive marker for HER2 targeted therapy, preliminary results indicate that HER2 amplification/overexpression may indicate resistance to EGFR-targeted monoclonal antibodies. For example, in the 98 RAS/BRAF wild-type mCRC patient group, the median PFS without EGFR inhibitor treatment was similar regardless of HER2 status.

However, in treatment with EGFR inhibitors, the PFS of patients with HER2 amplification was significantly shorter than that of patients without HER2 amplification (2.8 months vs. 8.1 months; HR was 7.05; 95% CI, 3.4–14.9; P <0.0001 ).


4. dMMR/MSI-H status

In clinical trials, the proportion of stage IV colorectal tumors with MSI-H (dMMR) characteristics ranged from 3.5% to 5.0%, while in nurse health studies and health professional follow-up studies, the proportion was 6.5%. dMMR tumors contain countless mutations that can encode mutant proteins, which may be recognized and targeted by the immune system.

However, the programmed death ligands 1 and 2 (PD-L1 and PD-L2) on tumor cells can inhibit the immune response by binding to the programmed cell death protein 1 (PD-1) receptors on T effector cells. This system will evolve to protect the host from uninhibited immune responses.

Many tumors up-regulate PD-L1, thereby avoiding the immune system. Therefore, it is hypothesized that dMMR tumors may be sensitive to PD-1 inhibitors. Subsequently, this hypothesis was confirmed in clinical trials, leading to the addition of recommendations for dMMR/MSI-H disease checkpoint inhibitors.

The NCCN Guidelines recommend general MMR or MSI testing for all patients with a personal history of colon or rectal cancer.

In addition to being used as a predictive marker for immunotherapy in advanced CRC, MMR/MSI status can also help identify patients with Lynch syndrome and provide a basis for adjuvant treatment decisions for patients with stage II disease.


5. NTRK integration

Three NTRK genes encode tropomyosin receptor kinase (TRK) proteins. The expression of TRK is mainly in the nervous system. These kinases help regulate pain, perception of movement/position, appetite and memory. NTRK gene fusion causes overexpression of TRK fusion protein, resulting in constitutive activity of downstream signals.

Recent studies estimate that approximately 0.2% to 1% of CRCs carry NTRK gene fusion. A study of 2314 CRC specimens (0.35% of which had NTRK fusion) found that NTRK fusion was limited to wild-type KRAS, NRAS and BRAF cancers.

In addition, most CRCs containing NTRK fusion also have MMR defects. These results may support the limitation of NTRK fusion testing to patients with wild-type KRAS, NRAS, and BRAF. TRK inhibitors are the treatment of choice for patients with mCRC who are positive for NTRK gene fusion.


6. Tumor mutation burden (TMB)

TMB measures the total amount of somatically encoded mutations in a given coding region of the tumor genome and can be quantified using NGS technology. Studies have determined that TMB is a potential biomarker of immunotherapy response, and the FDA has approved pembrolizumab for unresectable or metastatic high TMB (TMB-H ) Patients with solid tumors.

The FDA-approved test in the label defines TMB-H as 10 or more mutations per megabase. The approval is based on the results of the Phase 2 KEYNOTE-158 study enrolling patients with advanced solid tumors. Patients with TMB-H tumors who received pembrolizumab had an ORR of 29%, while those with non-TMB-H tumors had an ORR of 6%.

However, none of the 796 patients who underwent the efficacy evaluation in this study had colorectal cancer. The phase II TAPUR basket study summarized the results of 27 patients with advanced CRC of TMB-H who were treated with pembrolizumab. 1 case of partial remission and 7 cases of stable disease for at least 16 weeks were reported, with a disease control rate of 28% and an ORR of 4%.

Based on limited data in the colorectal cancer population, the NCCN expert group currently does not recommend TMB biomarker testing unless it is measured as part of a clinical trial.

Source: NCCN Guidelines for Colon Cancer 2021.v2

(source:internet, reference only)


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