Prognostic significance of K-Ras mutation rate in metastatic colorectal cancer patients

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Prognostic significance of K-Ras mutation rate in metastatic colorectal cancer patients

Bruno Vincenzi

Università Campus Bio-Medico di Roma

(2)

Colorectal cancer



3

^rd

most common cancer worldwide



Approximately 25% of patients present with metastatic colorectal cancer (mCRC)



Metastatic disease develops in 30-45% of newly diagnosed patients



Treatment is typically by surgery and chemotherapy



Now includes EGFR targeted monoclonal antibodies e.g. cetuximab & panitumumab

Karapetis, NEJM, 2009

(3)

Normanno et al. Nat Rev Clin Oncol, 2009

RAS

RAF PI3K

Key Drivers in Colorectal Cancer

(4)

RAS, RAF and PIK3CA Mutations in Colorectal Cancer



Mutation frequency

◦ KRAS: 40%

◦ NRAS: 3%

◦ BRAF: 5%

◦ PIK3CA: 15%



BRAF and

KRAS/NRAS mutations are mutually exclusive



KRAS and NRAS

mutations are mutually exclusive

De Roock, Lancet Oncol 2010

(5)

K-RAS Status and Prognosis in CRC



RASCAL I. KRAS mutation associated with increased risk of recurrence and death

⁽¹⁾



RASCAL II. RASCAL II (2001) confirmed but only in Dukes C (Stage 3) tumors

⁽²⁾

•

posthoc PETACC-3, EORTC 40993, SAKK 60-00. KRAS mutation was not a prognostic marker

⁽³⁾

•

posthoc PETACC-8. MSS, KRAS mut and BRAF mut associated with worse outcomes

1. Andreyev HJ, J Natl Cancer Inst.

1998

2. Andreyev HJ et al. Br J Cancer.

2001

3. Roth AD, J Clin Oncol. 2010 4. Taieb J , JAMA Oncol. 2016

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Is KRAS Gene Mutation a Prognostic Factor for Colorectal Cancer: A Systematic Review and Meta-analysis.

Ren, JiaoJiao; Li, GuangXiao; Ge, Jie; Li, Xia; Zhao, YaShuang

JJ Ren, Diseases of the Colon & Rectum.

2012

RESULTS: The pooled HR for the association between KRAS gene mutations and overall survival in patients with colorectal cancer was 1.04 (95% CI: 0.99–1.10, p = 0.11). Subgroup analysis showed significant reductions in the overall survival associated with mutations at KRAS codon 12, the articles that reported HR directly, and the studies published before and after 2005, although publication bias was present. All the associations disappeared after adjustment with the trim- and-fill method. The pooled HR of 3 studies examining mutations at KRAS codon 13 was 1.47 (95% CI: 1.09–1.97, p = 0.02), and no publication bias was observed.

No significant association was observed in different study regions.

CONCLUSIONS: Overall KRAS gene mutations seem not to correlate with the prognosis of patients with colorectal cancer. The association remains to be confirmed with a more precise analysis of a large sample.

STUDY SELECTION: The prognostic value of KRAS gene mutations was examined in patients with colorectal cancer who did not receive preoperative chemotherapy or radiation. After exclusion of duplicate and irrelevant studies, 23 eligible published studies that included 25 sets of data were finally retrieved for the meta- analysis.

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K-RAS Status and Prognosis in mCRC

Modest DP, Ann Oncol 2016

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K-RAS Status and Prognosis in mCRC

Modest DP, Ann Oncol 2016

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K-Ras mutation status has been always considered a dichotomic information, so far.

BUT…

In the cancers’ genetic heterogeneity scenario is important to take into account the percentage of K-Ras mutated

cells into the tumour

Mutation rate

defined as a measure of the

rate of mutant alleles

(10)

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Aim:

Evaluation of the prognostic significance of K-RAS mutation rate in mCRC patients treated with bevacizumab-containing chemotherapy

Network

:

Policlinico Universitario Campus Bio-Medico di Roma Azienda Ospedaliero-Universitaria Pisana

Ospedale Niguarda Ca’ Granda, Milano Oncologia Medica, Università di Palermo Università Cattolica del Sacro Cuore, Roma Azienda Ospedaliero-Universitaria, Udine

Università “G. D’Annunzio”, Chieti

Methods

: Pirosequencing on FFPE tumor tissue (>60% tumor cells)

Endpoints :

PFS and OS

Statistical analyses

: Univariate Analyses Kaplan-Meier

(KRAS Mutation Rate dichotomized >40% vs <40%)

Multivariate Analyse Cox-regression analyses

(KRAS Mutation Rate as continue variable)

Type:

Multicentric Retrospective Study

Study Design

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Methods: Patients’ population

Absolute

number %

Number of evaluable patients 263 100

Available data about Bevacizumab-chemotherapy 263 100

Gender (male/female) 144/119 54.6/43.4

K-Ras determination (primary vs met) 194/69 73.8/26.2

Second Line 191 72.6

Third Line 122 46.4

Irinotecan based/Oxaliplatin based 201/62 76.4/23.6

Number of involved organs 1 2

3 or more

178 49 36

67.7 18.6 13.7

Cumulative response rate (cPR + cCR) 49% -

PFS 8.25 months 7.6-8.9

OS 26 months 22-29.9

B.Vincenzi, Oncotarget 2016

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Methods: Patients’ population

Diagram of the patients’ enrolled

397 patients (K-Ras mutated/beva in 1line)

92 out (<60% cancer cells)

305 patients

27 out (incomplete FUP data)

278 patients

15 out (miscellaneous)

263 evaluable patients

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Laboratory methods

• FFPE tumor samples selection and dissection by pathologist

(presence of tumor cells at least 60%)

• RNA extraction and purification

• Pyrosequencing analysis of K-Ras status

(codons 12, 13, 61, 117 and 146)

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Results: univariate survival analysis

A mutation rate higher than 40% in all codons analyzed was significantly associated with a worse survival

POPULATION

(K-Ras mutated codons)

PFS(95% CI) [mts]

OS(95% CI) [mts]

Codon 12,

all patients (pts) 8.25 (95% CI:7.48-9.01) 28 (95% CI:23.67-32.32) Codon 12,

Pts with >40% vs ≤40% mutant alleles

7.45 (95% CI:5.78-9.21) vs 9.45 (95% CI:8.47-10.4)

23 (95% CI:19.79-26.20) vs 31 (95% CI:27.00-34.99)

Codon 13,

all patients (pts) 8.90 (95% CI:7.69-10.10) 21 (95% CI:16.63-25.36) Codon 13,

7.25 (95% CI:3.6-10.89) vs 10.25 (95% CI:8.15-12.32)

17 (95% CI:11.61-22.78) vs 24 (95% CI:19.41-28.58)

Rare codons,

all patients (pts) 5.50 (95% CI:2.95-8.04)

Rare codons,

Pts with >40% vs ≤40% mutant alleles

5.5 (95% CI:3.75-8.55) vs 7.25 (95% CI:2.44-12.05)

All K-Ras mutated codons,

7.25 (95% CI:5.76-8.73) vs 9 (95% CI:7.96-10.03)

21 (95% CI:18.35-23.64) vs 31 (95% CI:26.66-35.33)

Log Rank P P=0.000 P=0.002

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Results: Kaplan-Meyer analysis

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Results: multivariate survival analysis

Only codon 12 mutation rate remained significantly associated with PFS and OS in multivariate analyses

PFS OS

Survival Analysis (unadjusted) HR: 0.63 (0.46 – 0.80) P<0.001

HR: 0.66 (0.44 – 0.84) P=0.004

Survival Analysis (adjusted) HR: 0.67 (0.53 – 0.82) P=0.003

HR: 0.66 (0.52 – 0.85) P=0.012

Codon 12 HR: 0.71 (0.52 – 0.85) P=0.003

HR: 0.69 (0.32 – 0.89) P=0.011

Codon 13 HR: 0.62 (0.25 – 1.04) P=0.066

HR: 0.579 (0.26 – 1.28) P=0.179

Rare codons HR: 0.19 (0.02 – 2.08) P=0.397

HR: 0.43 (0.06 – 3.05) P=0.397

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Conclusions

• Despite of the well known importance of K-Ras mutations as predictive factor to anti-EGFR drugs administration, there is no consensus about the prognostic value of mutational status of K-Ras in CRC patients

• One of the reasons of these inconclusive results can be also attributed to cancer heterogeneity

• The use of pyrosequencing technology allow us to identify a novel parameter the “mutation rate” defined as the measure of the rate of mutant alleles

• We demonstrated for the first time a correlation between the K-Ras mutation rate (presence of more or less than 40% mutant alleles) in advanced CRC population treated with bevacizumab containing first-line regimens

• The limit of the study derives from its retrospective nature, thus our data need to be confirmed in a validation prospective set

• The cut-off of 40% (median value) was arbitrarily chosen and should be validated

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