Functional TP53 mutations have no impact on response to cytotoxic agents in metastatic colon cancer

To cite this article: Netter, J J, et al. Functional TP53 mutations have no impact on response to cytotoxic agents in metastatic colon cancer. Bull Cancer (2015), http://dx.doi.org/10.1016/j.bulcan.2014.12.010\ Bull Cancer 2015; //: ///

Original article

en ligne sur / on line on www.em-consulte.com/revue/bulcan www.sciencedirect.com

Functional TP53 mutations have no impact on response to cytotoxic agents in metastatic colon cancer Jeanne Netter 1, Jacqueline Lehmann-Che 2,3, Jerome Lambert 4,5, Anne Tallet 2, Nelson Lourenco 1, Hany Soliman 2,3, Philippe Bertheau 6,7, Benjamin Pariente 1,8, Mircea Chirica 9, Marc Pocard 10, Matthieu Allez 1,8, Hugues De The 2,3, Jean-Marc Gornet 1

Received 2 February 2014 Accepted 24 March 2014 Available online:

1. 2. 3. 4. 5. 6. 7. 8. 9.

AP–HP, Saint-Louis Hospital, Department of Gastroenterology, 75010 Paris, France AP–HP, Saint-Louis Hospital, Department of Biochemistry, 75010 Paris, France Inserm/CNRS UMR 944/7212, 75010 Paris, France AP–HP, Saint-Louis Hospital, Department of Biostatistics, 75010 Paris, France Inserm UMR 717, 75010 Paris, France AP–HP, Saint-Louis Hospital, Department of Pathology, 75010 Paris, France University Paris Diderot, UMR-S-728 Inserm, 75013 Paris, France Équipe Avenir Inserm U940, 75010 Paris, France AP–HP, Saint-Louis Hospital, Department of General, Endocrine and Digestive Surgery, 75010 Paris, France 10. Surgical Oncologic and Digestive Unit, Lariboisière Hospital, AP–HP, 75010 Paris, France

Correspondence: Jean-Marc Gornet, Hôpital Saint-Louis, service d'hépato-gastroentérologie, 1, avenue Claude-Vellefaux, 75010 Paris, France. [email protected]

Keywords Metastatic colon cancer TP53 mutation Chemotherapy response

Summary Background > Survival of metastatic colon cancer (mCC) patients has considerably improved with optimization of new drugs regimen. Inactivation of TP53 pathway by TP53 mutations is observed in nearly half of colorectal tumors. The impact of such mutations has been poorly studied in the metastatic setting. Methods > The files of 254 mCC treated in a single institution at Saint-Louis hospital between January 1999 and April 2011 were retrospectively reviewed. Tissue samples for analysis of TP53 mutations were available for 68 patients, performed using FASAY. The prognostic value of TP53 status was evaluated by comparing progression free survival (PFS) and overall survival (OS) in the group of TP53-mutated and wild type patients. Results > PFS was 6.9 months and OS 21.7 months in the whole population. There was no statistical difference in TP53-mutated and wild type groups in term of PFS (HR = 1.04; IC9 5% = 0.6–1.79) and OS (HR = 0.99; IC 95% = 0.53–1.55) whatever the chemotherapy regimen (oxaliplatin- or irinotecan-based). Only BRAF V600 mutation was demonstrated to be a poor prognostic factor for PFS and OS, and CEA level for OS. Conclusions > Routine determination of TP53 mutations, even with a highly sensitive method, cannot be recommended to predict chemotherapy response in mCC.

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tome xx > n8x > mois année http://dx.doi.org/10.1016/j.bulcan.2014.12.010 © 2015 Société Française du Cancer. Published by Elsevier Masson SAS. All rights reserved.

BULCAN-15

To cite this article: Netter, J J, et al. Functional TP53 mutations have no impact on response to cytotoxic agents in metastatic colon cancer. Bull Cancer (2015), http://dx.doi.org/10.1016/j.bulcan.2014.12.010\

Original article

J. Netter, J. Lehmann-Che, J. Lambert, A. Tallet, N. Lourenco, H. Soliman, et al.

Mots clés Cancer colique metastatique Mutations TP53 Réponse à la chimiothérapie

Résumé Les mutation du gène TP53 n'ont pas d'impact sur la réponse aux agents cytotoxiques dans le cancer du côlon metastatique Objectifs > Les avancées dans les modalités de traitement du cancer colorectal ont permis une amélioration du pronostic notamment dans le cancer colorectal métastatique (CCRm). Notre étude visait à étudier l'intérêt de la recherche des mutations de p53 dans la réponse à la chimiothérapie en situation métastatique. Patients et méthodes > Il s'agit d'une étude rétrospective monocentrique portant sur 68 patients suivis à l'hôpital Saint-Louis entre janvier 1999 et avril 2011, présentant un cancer colique métastatique (CCm) pour lesquels un statut p53 avait été réalisé par méthode FASAY. Nous avons étudié la survie sans progression et la survie globale de ces patients en fonction du statut p53. Résultats > La survie sans progression (SSP) était de 6,9 mois et la survie globale (SG) de 21,7 mois. Il n'y avait pas de différence significative sur la SSP (HR = 1,04; IC 95 % = 0,6– 1,79) et sur la SG (HR = 0,99; IC 95 % = 0,53–1,55) entre les groupes TP53 mutés et sauvages quelque soit le type de chimiothérapie reçue (oxaliplatine ou irinotecan). La mutation BRAF V600 et le taux d'ACE sont les seuls facteurs de mauvais pronostique retrouvés. Conclusion > La determination du statut p53 ne permet pas de prédire la réponse à la chimiothérapie dans le CCm.

Background In Europe, colorectal cancer (CRC) represents the second most common cause of cancer with 436,000 cases diagnosed in 2008 with a high mortality estimated at 1,212,000 cases [1]. About 40% of patients have a metastatic disease at diagnosis [2]. Metastatic colorectal cancer (mCRC) prognosis has considerably improved with optimization of administration of 5-FU, introduction of oxaliplatin and irinotecan, and more recently of targeted therapies, such as anti-VEGF (vascular endothelial growth factor) (bevacizumab) and anti-EGFR (epidermal growth factor receptor) (cetuximab, panitumumab). Prediction of chemotherapy response using biological markers has emerged as a key point

Glossary

2

mCC CRC mCRC PFS OS FASAY HR PS CI EGFR VEGF MSI 5-FU CEA

metastatic colon cancer colorectal cancer metastatic colorectal cancer progression free survival overall survival functional analysis of separated alleles in yeast hazard ratio performance status confidence interval epidermal growth factor vascular endothelial growth factor microsatellite instability 5-fluorouracil carcinoembryonic antigene

in mCRC. Despite extensive research, there is no predictive biomarker of response to standard therapies and bevacizumab in routine practice. To date, KRAS mutation is the only validated predictive factor of non-response to anti-EGFR therapy leading to contra-indication to its use in such situation [3]. Mechanisms of colorectal carcinogenesis involve multiple tumor suppressor genes (APC, TP53, TGF-b) [4] and oncogenic genes (RAS, BRAF, PTEN and PIK3CA). Inactivation of TP53 pathway by TP53 mutations is present in nearly half of colorectal tumors [5], and is one of the key genetic steps in colorectal carcinogenesis. P53 protein is a transcription factor, which exerts multiple antiproliferative functions. TP53 pathway is activated in stressful situations and leads, depending on the intensity of the damage, to cell cycle arrest to allow DNA repair, senescence or death of the cell by induction of apoptosis [6–8]. Loss of function of TP53 in cells, by mutations, promotes development of cancer. For twenty years, TP53 gene has been extensively studied as a prognostic and predictive marker in colorectal cancer. These studies have focused on located CRC with conflicting results. Several hypotheses can explain those discrepancies. Various methods of detection of TP53 mutations have been reported. The most frequent techniques were immunohistochemistry and TP53 sequencing but they raised some criticism. There is currently no solid data supporting the interest of TP53 mutations analysis regardless of the chemotherapy regimen used. In this study, the analysis of the TP53 status was performed by Functional Analysis of Separated Alleles in Yeast (FASAY), a method allowing the functional evaluation of the p53 protein followed by the characterization of the identified alteration by sequencing [9]. We used routinely this technology in

tome xx > n8x > mois année

To cite this article: Netter, J J, et al. Functional TP53 mutations have no impact on response to cytotoxic agents in metastatic colon cancer. Bull Cancer (2015), http://dx.doi.org/10.1016/j.bulcan.2014.12.010\

Methods Patients We retrospectively reviewed 362 consecutive medical charts of patients treated for metastatic colorectal cancer at Saint-Louis University Hospital (Paris, France) between January 1999 and April 2011. We excluded all the patients with rectal cancer (n = 108) to have a homogeneous profile of treatment. We included only patients with complete clinical data and available TP53 status. In cases of incomplete molecular data, if possible, frozen tissue samples were retrieved from the pathology department and TP53 status and other molecular analyses were performed retrospectively. Finally, we included 68 patients with TP53 status available by FASAY method on resected primary tumor or metastasis. Samples were provided by the biological resource center after approval of the Saint-Louis hospital ethical review board (Paris, France: agreement no DC 2009-929), following the Ethics and Legal National French rules for the patients' information and consent (ANAES, HAS and INCa). All patients were informed of the study and did not oppose to it, according to our Institutional Review Board recommendations.

TP53 mutation analysis Mutational analysis of TP53 was performed using FASAY, in accordance to the method described by Flaman et al. [9]. This method requires frozen tumor samples to obtain high quality of RNA to evaluate the transactivation activity of the corresponding p53 protein on a p53-responsive promoter stably integrated in the yeast genome. RNA was extracted by phenol– chloroform method, reverse transcribed and p53 transcripts were amplified by PCR and co-transfected into yeast with the Gap repair plasmid. The corresponding TP53 sequence is expressed in the yeast and directs the growth and the color of the yeast colonies. Yeast colonies transformed with wild type or mutated TP53 sequences appear white and large, or red and small, respectively. TP53 status was considered non-functional if more than 15% of the yeast colonies were red and if confirmed by the split versions of the test (defect in the 50 and/or 30 part of the gene). After identification of non-functional TP53 samples or in cases of "borderline'' FASAY results (between 10% and 15% of red colonies), red colonies were cloned and sequenced by Sanger method to characterize the TP53 mutation. Finally, we classified the mutations into two groups using the IARC p53 database according to their impact on the protein:

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presence of a modified p53 protein (p-MOD) and absence of a p53 protein (p-NO) [13].

Other mutations analysis DNA was extracted from selected sections of the tumor samples (presence of > 30% tumor cells) after overnight proteinase K digestion, using the QIAamp DNA minikit (Qiagen) according to manufacturer's instructions. The seven KRAS mutations on codon 12 and 13 were analyzed by allelic discrimination: specific probes for each allele (mutated or wild type) were labeled with fluorescent reporter dyes at their 50 end and analysed by real-time PCR on a LC480 (Roche) [14]. The BRAF V600E mutation status was also assessed by allelic discrimination on a LC480 (Roche) [15]. PIK3CA (exon 20) gene was analyzed by HRM (LC480 Roche) and the defect was identified by sequencing on a sequencer 3130 (Applied) in case of abnormal HRM profile. For all samples and all genotyping, a second independent analysis was performed to confirm the final result. Primers, probes and PCR conditions are available upon request. To evaluate microstatellite instability (MSI), a pentaplex PCR, comprising five quasimonomorphic mononucleotide repeats (BAT-25, BAT-26, NR-21, NR-24, and NR-27) was used [16]. Tumors with instability at  3 markers were defined as MSIH, whereas those with < 3 as MSI-L or MSS if any instability.

Statistical methods Results were expressed as frequency with percentages for categorical variables, and median with interquartile range (IQR) for quantitative variables. Patients' characteristics according to p53 status were compared using Fisher's exact test and Wilcoxon rank sum test. Overall survival (OS) was defined as the time between diagnosis of metastatic disease and death from any cause. Progression free survival (PFS) was defined as the time between first metastatic chemotherapy cure and first documentation of tumor progression (date of CT scan or surgery) or death from any cause. The closing date for the analysis was 2012/07/01. OFS and PFS according to p53 status were plotted using the Kaplan–Meier survival estimator and compared using the log-rank test. The effect of several candidate prognostic factors on overall survival was assessed using a Cox proportional hazard regression model and presented by the hazard ratio (HR) and its 95% confidence interval. Analysis was performed using R 2.10.1. All statistical tests were two-sided with P-values lower than 0.05 considered as significant.

Results Patient's population and tissue specimen Study population consisted of 68 patients treated for metastatic colon cancer. Patients were predominantly males (51%), and median age at diagnosis was 62 years (range 53–74 years) with performance status (PS)  2 in 90% of the cases. Primary tumor

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breast cancer, [10,11] and we were able to detect, with a good sensitivity, over 90% of TP53 mutations [12]. The aim of our study was to evaluate the predictive value of TP53 mutations using FASAY in mCC. We have planned a subgroup analysis according to the functional implication of each mutation. The other biological markers currently known to be involved in colorectal carcinogenesis have been also studied to determine if a subgroup of molecular profile emerged.

Original article

Functional TP53 mutations have no impact on response to cytotoxic agents in metastatic colon cancer

To cite this article: Netter, J J, et al. Functional TP53 mutations have no impact on response to cytotoxic agents in metastatic colon cancer. Bull Cancer (2015), http://dx.doi.org/10.1016/j.bulcan.2014.12.010\

Original article

J. Netter, J. Lehmann-Che, J. Lambert, A. Tallet, N. Lourenco, H. Soliman, et al.

was left-sided in 54% and well differentiated in 49% of the cases. Metastases were synchronous in 68% of the cases. Seventy-one percent of patients had one metastatic site, mainly hepatic (69%) (table I). First-line of chemotherapy contained 5fluoro-uracile (5-FU) in all cases with oxaliplatin (59%) or irinotecan (16%) (table II). Only 11 patients received cetuximab (6%) or bevacizumab (11%) in association with irinotecan- or oxaliplatin-based regimen. Seven patients received exclusive base supportive care because of poor condition. Other histological prognostic data (vascular, lymphocytic and perineural invasion) collected from pathology reports could not be analyzed due to the large number of missing data. TP53 status was mostly obtained in primary tumor site (n = 62) and rarely metastasis (n = 6).

TABLE II Metastatic chemotherapy regimensa n = 61 patients

Chemotherapy regimens First-line regimens 5-FU + oxaliplatin

36

59%

5-FU + irinotecan

10

16%

5-FU

4

7%

Cetuximab (5-FU + oxaliplatin or irinotecan)

4

7%

Bevacizumab (5-FU+ oxaliplatin or irinotecan)

7

12%

61

100%

Oxaliplatin

53

87%

TABLE I

Irinotecan

43

70%

Patient and tumor characteristics.

Anti-EGFR therapies

21

34%

Anti-VEGF therapies

25

40%

1

17

25%

All lines regimens 5-FU

n = 68 patients

Caracteristics Age (years)

62.4

[53.5–74]

Number of lines

Gender Male

35

51%

2

18

27%

Female

33

49%

3

16

24%

4 or more

10

15%

PS 0–2

61

90%

>2

7

10%

Synchronous

46

68%

Metachronous

22

32%

1

49

72%

>1

19

28%

Liver

47

69%

Lung

7

10%

19

28%

Poor/moderate

29

51%

Well

28

49%

45

69%

a

Base supportive care alone: n = 7.

Metastasis

Number of metastatic sites

Metastasis

Carcinomatosis Differentiation

a

Data are presented as frequency (percentage) or median [25th–75th percentiles]. PS: performance status; CEA: carcinoembryonic antigene. a Missing data: n = 10.

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TP53 status could be determined for 68 patients. A non-functional TP53 status was found in 38 patients (56%). There was no evidence of heterogeneity in patients' characteristics (age, sex, PS status, tumor differentiation, number and type of metastasis, molecular factors) between the TP53-mutated and the TP53wild type group of tumors (table III). We identified the TP53 alteration leading to non-functional FASAY by sequencing in 36 cases, and recorded the types of mutation according to the IARC p53 database. Six patients presented a tumor with a mutation leading to absence of p53 protein (P-NO), and 30 patients a mutation leading to the presence of a mutated p53 protein with potential new functions (P-MOD). We failed to perform sequencing for 2 patients.

Other mutation analysis

CEA Informative

TP53 mutations of the tumors

Microsatellite instability was found in 23% of patients (12 MSI-L patients, 4 patients MSI-H), KRAS mutation in 48% of patients (mutations found: 7 G12A, 7 G12V, 6 G12D, 4 G12C, 2 G12S, 9 G13D), BRAF mutation in 9% of patients, and PIK3CA mutation

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To cite this article: Netter, J J, et al. Functional TP53 mutations have no impact on response to cytotoxic agents in metastatic colon cancer. Bull Cancer (2015), http://dx.doi.org/10.1016/j.bulcan.2014.12.010\

TABLE III Patient and tumor characteristics according to p53 status. TP53-mutated (n = 38) Age (years)

TP53-wild type (n = 30)

P value

62

[55–75]

63

[55–72]

0.956

Male

21

(55%)

14

(47%)

0.626

Female

17

(45%)

16

(53%)

0–2

35

(92%)

26

(87%)

>2

3

(8%)

4

(13%)

Synchronous

24

(63%)

22

(73%)

Metachronous

14

(37%)

8

(27%)

Poor

0

(0%)

2

(8%)

Moderate

16

(50%)

11

(44%)

Well

16

(50%)

12

(48%)

Liver

26

(69%)

21

(70%)

1

Lung

2

(5%)

5

(17%)

0.227

Carcinomatosis

10

(26%)

9

(30%)

0.79

Lymph nodes

4

(10%)

3

(10%)

1

Other

9

(24%)

3

(10%)

0.0723

KRAS mutated

17

(46%)

16

(53%)

0.627

BRAF mutated

2

(6%)

4

(13%)

0.407

PIK3CA mutated

1

(3%)

4

(13%)

0.177

MSI-H and L

8

(22%)

8

(28%)

0.773

24

(69%)

20

(69%)

1

Original article

Functional TP53 mutations have no impact on response to cytotoxic agents in metastatic colon cancer

Gender

PS 0.691

Metastasis 0.44

Differentiation 0.364

Metastasis

Molecular factor

CEA Informative

Data are presented as frequency (percentage) or median [25th–75th percentiles]. PS: performance status; CEA: carcinoembryonic antigene; KRAS mutated: Kirsten rat sarcoma 2 viral oncogene homolog (c-Ki-ras2) codons 12 and 13, BRAF mutated: v-Raf murine sarcoma viral oncogene homolog B1 V600E; PIK3CA: mutated: phosphatidylinositol-4,5-bisphosphonate3-kinase exon 20; MSI-H and L: microsatellite instability-high and low.

Progression free survival analysis At the time of statistical analysis, 5 patients were still in first-line chemotherapy and 49 patients had progressed. Seven patients

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had stopped chemotherapy in first-line because of an impaired general condition. Median PFS was 6.9 months. PFS was not significantly different between the TP53-wild type (WT) and mutated groups (MUT) (HR = 1.04; IC 95% = 0.6–1.79), and between the 3 groups wild type, p-MOD (HR = 0.92; IC 95% = 0.51–1.64) and p-NO (HR = 1.62; IC 95% = 0.65–4.04) (figure 1). We did not find any mutation effect on PFS according to the type of chemotherapy received (oxaliplatin- or irinotecanbased) (HR = 1.45; IC 95% = 0.76–2.75) (figure 2).

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(exon 20) in 8% of patients. The molecular status could not be determined in some cases because of a lack of material (MSI status in 2 patients, KRAS in 1 patient, BRAF in 4 patients, and PIK3CA in 4 patients).

To cite this article: Netter, J J, et al. Functional TP53 mutations have no impact on response to cytotoxic agents in metastatic colon cancer. Bull Cancer (2015), http://dx.doi.org/10.1016/j.bulcan.2014.12.010\

Original article

J. Netter, J. Lehmann-Che, J. Lambert, A. Tallet, N. Lourenco, H. Soliman, et al.

Figure 1 Log-rank curve of progression free survival according to p53 status A. Progression free survival according to p53 status. B. Progression free survival according to p53 mutation type. P53 WT: wild type p53 protein. P53-MOD: gain of function or dominant. P53-NO: absence of protein p53.

Figure 2 Log-rank curve of disease free survival according to p53 status and type of first-line chemotherapy regimen

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A. Oxaliplatin-based chemotherapy. B. Irinotecan-based chemotherapy.

tome xx > n8x > mois année

To cite this article: Netter, J J, et al. Functional TP53 mutations have no impact on response to cytotoxic agents in metastatic colon cancer. Bull Cancer (2015), http://dx.doi.org/10.1016/j.bulcan.2014.12.010\

At the time of statistical analysis, 57 patients (83%) had died. The median OS was 21.7 months. There was no significant difference in OS between TP53-mutated and wild type groups (HR = 0.99; IC 95% = 0.53–1.55). We did not find any mutation effect on OS depending on the type of chemotherapy received (oxaliplatin versus irinotecan) (HR = 0.9; IC 95% = 0.47–1.7).

Univariate analysis of prognostic factors Prognostic factors studied in univariate analysis were: PS status, degree of differentiation of the primary tumor, number of metastatic sites at metastasis diagnosis, type of chemotherapy, CEA, KRAS, PIK3CA and BRAF mutation, TP53 and MSI status. Among molecular factors, only BRAF V600 mutation was a poor prognostic factor for PFS (HR = 3.3, 95% CI = 1.12–6.56) and OS (HR = 3.5; 95% CI = 1.45–8.44, P = 0,015). Elevated level of CEA (upper than normal value of analysis laboratory) at diagnosis was also a poor prognostic factor for OS (HR = 1.94; 95% CI = 1.01–3.75) (table IV).

Discussion Determination of prognostic and predictive factors of response to colorectal cancer treatment has been studied extensively for several years. Some genetic polymorphisms have been correlated to irinotecan and 5-FU toxicities. A recent study suggests a correlation between some of these polymorphisms and tumor response to 5-FU and oxaliplatin [17]. However, the most significant advances have occurred in the molecular biology field. KRAS mutation has been widely shown as predictive of anti-EGFR inefficacy [3]. More recently, BRAF mutation has been clearly shown to be a poor prognostic factor for patients with mCRC [18]. Thus, in a recent randomized trial comparing 3 arms of treatment with 5-FU and oxaliplatin plus or minus cetuximab, BRAF mutation was associated with significantly poorer OS (9.5 vs 22.0 months) [19]. As well, double mutations of exon 9 and 20 of PIK3CA have been shown to be a poor prognostic factor of survival in 2 large prospective cohorts of colorectal patients [20]. Additionally, null expression of PTEN has been correlated with poor overall survival in the subgroup of KRAS wild type patients [18]. TP53 tumor suppressor gene is involved in the development of many human cancers and crucial in response to DNA damages. Understanding various functions and interactions of p53 with other intracellular pathways is still unclear despite several decades of research on this topic. In addition, there are over 900 mutations with different functional impact making interpretation of the mutational status difficult. Many studies have investigated prognostic and predictive impact of the TP53 status on different cancer contexts. Especially, we have previously shown, in breast cancer, that the presence of TP53 mutation improved response to high doses of anthracyclines and cyclophosphamide as neoadjuvant therapy in locally advanced non-inflammatory breast cancer [21]. However,

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TABLE IV Univariate analysis of prognostic factors for overall survival. HR

95% CI

P value

1.1

[0.47–2.59]

0.826

1 vs > 1

1.19

[0,68–2,08]

0.539

Function of sites number

1.15

[0.76–1.74]

0.524

1.79

[0,88–3,65]

0,126

1.94

[1,01–3.75]

0.037

1.73

[0.96–3.14]

0.069

1.54

[0.83–2.87]

0.184

0.99

[0.53–1.55]

0.73

1.17

[0.69–1.99]

0.561

3.5

[1.45–8.44]

0.0153

0.79

[0.28–2.21]

0.646

1.84

[0.65–5,18]

0.286

Irinotecan

1.08

[0.34–3,4]

0.933

Oxaliplatin

0.95

[0.32–2,81]

Variables PS 0–2 vs > 2 Number of metastatic sites

Chemotherapy Mono/bitherapy vs tritherapy CEA Informative Differentiation Poor/moderate vs well Metastasis Metachronous vs synchronous P53 Mutated vs wild type KRAS Mutated vs wild type BRAF Mutated vs wild type PIK3CA Mutated vs wild type MSI MSI-H vs MSI-L/MSS Type of chemotherapy

PS: performance status; CEA: carcinoembryonic antigene; KRAS mutated: Kirsten rat sarcoma 2 viral oncogene homolog (c-Ki-ras2) codons 12 and 13, BRAF mutated: vRaf murine sarcoma viral oncogene homolog B1 V600E; PIK3CA: mutated: phosphatidylinositol-4,5-bisphosphonate3-kinase exon 20; MSI-H and L: microsatellite instability-high and low.

impact of TP53 gene mutation on CRC natural history and its possible interaction with response to chemotherapy remains controversial. One explanation is the variability of methods used to determine the p53 status. The two most common techniques used are immunohistochemistry (IHC) and sequencing. IHC is a technique that evaluates the TP53 status by detecting p53 protein stabilization as consequence of some, but not all, TP53 mutations. IHC positivity correlates with TP53 mutations in only 80% of cases. On the other hand, sequencing is usually performed only for a part of TP53 gene (usually exon

7

Overall survival analysis

Original article

Functional TP53 mutations have no impact on response to cytotoxic agents in metastatic colon cancer

To cite this article: Netter, J J, et al. Functional TP53 mutations have no impact on response to cytotoxic agents in metastatic colon cancer. Bull Cancer (2015), http://dx.doi.org/10.1016/j.bulcan.2014.12.010\

Original article

J. Netter, J. Lehmann-Che, J. Lambert, A. Tallet, N. Lourenco, H. Soliman, et al.

4 to 8) and reveals nucleotide modifications without prejudging of their impact on the protein functionality. Results of a large number of studies about prognostic impact of TP53 mutations on survival and predicting response to adjuvant chemotherapy are still debatable [22–24]. As well, results of the few studies about the predictive value of TP53 mutations in mCRC are disappointing [25,26]. Discordant data were reported in old series regarding the impact of TP53 mutation in 5-FU treated patients. TP53 mutations do not seem to be predictive of tumor response in folfox regimen and bevacizumab-based treatment [27,28]. One study has suggested that TP53 mutation was correlated with tumor response in WT KRAS patients receiving cetuximab-based chemotherapy [29]. There is no data on the potential role of TP53 mutation in patients treated with irinotecan-based regimen. One strength of our work is represented by determination of TP53 mutation with a sensitive and functional assay (FASAY) and the systematic characterization of TP53 mutation by sequencing. We evaluated the impact of the presence of a mutated p53 protein with potentially new functions (p53-MOD) or the absence of the protein (p53-NO) on the prognostic and the response to treatment. No implication of the TP53 status has been highlighted, but we cannot exclude that our relatively small patient cohort do not allow final conclusions. Finally, our study does not support the interest of determining TP53 status in mCC whatever the chemotherapy administered. These negative results are not influenced by patient's heterogeneity. There was no difference in the characteristics of the population in the two groups of TP53-mutated and wild type patients.

The proportion of mutations of KRAS, BRAF, PIK3CA, TP53 and MSI were consistent with those usually observed. The study population is rather small. However, we choose a homogeneous population of solely colon cancer and the PFS and OS reported are concordant with literature. Despite the retrospective design and the number of patients analyzed, the systematic use of two complementary techniques to better characterize TP53 mutation allow us to hypothesize that routine determination of p53 status doesn't seem useful. The high number of chemotherapy regimens used (n = 8) led us to separate analysis in two subgroups of patients whether they received oxaliplatin- or irinotecan-based first-line chemotherapy. This point could be a limitation of the study but despite the numerous possibilities of combinations in mCRC, either oxaliplatin or irinotecan remain constantly used in first-line setting. Thus, the impact of TP53 status in these two groups is clinically relevant. Contrariwise, there were not enough patients treated with targeted therapy in our study to draw any conclusion. BRAF mutation and CEA level are the only prognostic factors found, in agreement with literature data [30,31].

Conclusions Complexity of p53 functions remains a barrier to TP53 routine use as prognostic or predictive factor of response to chemotherapy in mCRC. To date, determination of TP53 status in such situation is not recommended in clinical practice. Disclosure of interest: the authors declare that they have no conflicts of interest concerning this article.

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To cite this article: Netter, J J, et al. Functional TP53 mutations have no impact on response to cytotoxic agents in metastatic colon cancer. Bull Cancer (2015), http://dx.doi.org/10.1016/j.bulcan.2014.12.010\

tome xx > n8x > mois année

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Original article

Functional TP53 mutations have no impact on response to cytotoxic agents in metastatic colon cancer