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Prognostic value of microsatellite instability and p53 expression in metastatic colorectal cancer treated with oxaliplatin and fluoropyrimidine-based chemotherapy
Authors
S. Nöpel-Dünnebacke1, K. Schulmann2, A. Reinacher-Schick3, R. Porschen4, W. Schmiegel1, A. Tannapfel5, U. Graeven6
Affiliations
Affiliation addresses are listed at the end of the article.
Schlüsselwörter
Zusammenfassung
Abstract
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Studie: Evaluation des prognostischen Wertes von MSI-H und p53-Überexpression bei metastasierten kolorektalen Karzinomen (mKRK), die mit oxaliplatin- und fluoropyrimidin-basierter Erstlinienchemotherapie behandelt wurden. Methodik: Retrospektiv wurden Tumorgewebeproben von 229 Patienten einer prospektiven randomisierten Phase-III-Studie der kolorektalen Studiengruppe der AIO, welche CAPOX gegen FUFOX bei mKRK verglich, analysiert. Ein immunhistochemischer Nachweis einer p53-Überexpression bzw. der MMR-Proteine sowie Mikrosatellitenanalysen wurden durchgeführt. Ergebnisse: Die Inzidenz von MSI-H und p53Überexpression lag bei 7,9 % bzw. 65,4 %. Der MSH-I-Status korrelierte nicht mit der ORR, dem PFS oder OS. Ein Trend unter MSI-H-Tumoren zu einer geringeren DCR war festzustellen (65 % vs. 85 %). Eine p53-Überexpression korrelierte nicht mit der DCR, ORR, oder dem PFS. Das mediane OS der Patienten mit p53-überexprimierten Tumoren war signifikant länger verglichen mit Tumoren ohne p53-Überexprimierung (19,6 vs. 15,8 Monate; p= 0,05). Das PFS der p53 überexprimierenden Patienten unter der Zweit- und Drittlinienchemotherapie mit Irinotecan und/oder Cetuximab war signifikant länger als bei Patienten ohne p53-Überexpression. Diskussion: MSI-H Tumoren zeigten unter einer Kombinationstherapie aus Oxaliplatin/Fluoropyrimidin tendenziell eine schlechtere DCR. p53 überexprimierte mKRK unter einer irinotecanhaltigen Zweit- bzw. Drittlinienchemotherapie nach Oxaliplatinversagen hatten ein signifikant längeres PFS im Vergleich zu nicht p53 überexprimierenden Tumoren. Um die klinische Bedeutung dieser Beobachtung zu valuieren, werden weitere Studien benötigt.
Purpose: The aim of this study was to evaluate the prognostic value of MSI-H and p53 overexpression in metastatic colorectal cancer (mCRC) treated with oxaliplatin and fluoropyrimidinebased first line chemotherapy. Methods: Tumour samples were retrospectively obtained from 229 patients from a prospective randomised phase III trial of the AIO colorectal study group, comparing CAPOX and FUFOX in mCRC. Immunohistochemistry of p53 and MMR proteins as well as microsatellite analysis were performed. Results: The incidence of MSI-H and p53 overexpression was 7.9 % and 65.4 %, respectively. MSI-H status was not correlated with ORR, PFS and OS. We observed a trend to lower DCR for MSI-H tumours (65 % vs. 85 %, p = 0.055). p53 overexpression was not correlated with DCR, ORR and PFS. The median OS of patients with tumors with p53 overexpression was significantly longer compared to tumors withhout p53 overexpression (19.6 vs. 15.8 months; p = 0.05). The post-progression survival (PPS) of p53-positive patients undergoing 2nd and/or 3rd line chemotherapy with irinotecan and/or cetuximab was significantly longer compared to p53-negative patients. Conclusion: MSI-H tumours tend to have lower disease control rates when treated with an oxaliplatin/fluoropyrmidin combination. mCRC patients with p53 overexpression undergoing an irinotecan containing second- or third-line chemotherapy after oxaliplatin failure have a significantly longer post-progression survival compared to patients without p53 overexpression. To validate the clinical impact of p53 in patients with mCRC treated with irinotecan- and/or cetuximab further studies are needed.
● metastasierters kolorektales ● ● ● ● " " " "
Karzinom Mikrosatelliteninstabilität p53 Oxaliplatin Prognose
Key words
● metastatic colorectal cancer ● microsatellite instability ● p53 ● oxaliplatin ● prognosis " " " " "
received accepted
14.11.2013 15.6.2014
Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1366781 Z Gastroenterol 2014; 52: 1394–1401 © Georg Thieme Verlag KG Stuttgart · New York · ISSN 0044-2771 Correspondence Dr. Stefanie NöpelDünnebacke Department of Internal Medicine, Ruhr-University of Bochum, Knappschaftskrankenhaus In der Schornau 23–25 44892 Bochum Germany [email protected]
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Prognostischer Einfluss der Mikrosatelliteninstabilität und p53-Überexpression bei metastasierten kolorektalen Karzinomen unter Oxaliplatin- und 5-FU-haltiger Chemotherapie
Originalarbeit
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Colorectal cancer (CRC) is the fourth common malignant tumor in the world and causes over 500 000 deaths each year [1]. At least three different molecular subtypes of CRC are currently known. The traditional type is characterized by chromosomal instability (CIN) (80 – 85 %). The second type is characterized by high levels of microsatellite instability (MSI-H) (10 – 23 %) and is caused by a deficient mismatch repair (MMR) system. The third type is characterized by widespread hypermethylation of CpG islands in the promoter region of tumor suppressor genes (CpG island methylator phenotype (CIMP)) which overlaps to some amount to CIN and MSI phenotypes [2, 3]. Tumors lacking MSI-H are usually classified as microsatellite stable (MSS). To improve outcomes, reduce toxicities and therefore tailor therapeutic concepts in metastatic CRC (mCRC) prognostic and predictive biomarkers are urgently needed. In addition to its prognostic value, the MSI-H phenotype is a predictive marker for a lack of benefit from 5-FU-based adjuvant chemotherapy in stage II and III [4 – 6], although a recent study suggested that the lack of benefit may be restricted to cases with sporadic MSI whereas patients with Lynch syndrome may benefit from adjuvant 5-FUbased chemotherapy [7]. Few small studies explored the clinical impact of MSI-H status in the adjuvant treatment with FOLFOX. Kim et al., reported no difference in the outcome between MSI CRC and MSS CRC when treated adjuvant with FOLFOX [8]. However, a more recent retrospective analysis of the MOSAIC trial by Flejou demonstrates a benefit from FOLFOX in stage III MSI CRC as compared to an adjuvant 5-FU-based chemotherapy [9]. Another study by Zaanan et al. observed a significant longer 3-year disease free survival (DFS) among MSI-H CRC compared to MSS CRC [10]. Data regarding the value of MSI-H in mCRC undergoing palliative 5-FU based chemotherapy are rare. Des Guetz et al. did not find significant differences in overall survival (OS), progression free survival (PFS) or remission rate (RR) according to MMR status [11]. In a meta-analysis including eight trials in mCRC des Guetz did not find evidence for MSI having impact on 5-FU-based chemotherapy [12]. In a previous report we found evidence that MSI-H mCRC may result in lower disease control rates as compared to MSS mCRC when treated with FUFOX or CAPOX [13]. However the numbers of cases with MSI-H have been small. The largest trial investigating the value of MSI-H in mCRC so far, the FOCUS trial was not able to demonstrate a predictive value of MSI-H in patients randomized to three different chemotherapy regimens [14]. Another putative prognostic marker is the tumor suppressor gene p53. The p53 protein plays a central role in the cell cycle control by transcription regulation [15]. Mutations of p53 are the most common genetic alteration in human cancers [16, 17]. p53 mutations can be identified in approximately 50 % of CRCs [18, 19]. Inactivating point mutations of p53 lead to nuclear accumulation of p53 that can be detected by immunohistochemistry (IHC). Therefore p53 overexpression in a surrogate marker for p53 inactivation and loss of p53 function. The prognostic impact of p53 in CRCs treated with surgery with or out adjuvant chemotherapy remains controversial [20, 21]. A recent meta-analysis failed to demonstrate that p53 alterations influence the prognosis of patients treated with 5-FU-based adjuvant chemotherapy [22]. In addition Munro et al. detected no significant distinction in the outcome between p53 mutated CRCs in different stages and wild type under 5-FU-based chemotherapy [14, 18]. Adding oxaliplatin to 5-FU/leucovorin in adjuvant treated stage
III CRCs resulted in a significantly better disease free survival (DFS) for patients with p53 overexpression [23]. The therapeutic impact of p53 mutation in mCRCs remains unclear. In this study we retrospectively evaluated the prognostic value of p53 overexpression and MSI-H in mCRCs treated with a palliative first line combination therapy with oxaliplatin and fluoropyrimidines within a prospective randomized phase III trial from the German AIO colorectal cancer group comparing the FUFOX (oxaliplatin and 5-FU) and CAPOX (capectabine and oxaliplatin). The clinical results of the trial have been reported previously [24].
Material and Methods !
Study design and patient’s characteristics Tumor tissue samples from patients with mCRC participating in a prospective randomized phase III first-line chemotherapy trial of the AIO Group (Arbeitsgemeinschaft Internistische Onkologie of the German Cancer Society) were collected retrospectively. 474 patients were randomized to be treated with either FUFOX (oxaliplatin, 50 mg/m2; FA, 500 mg/m2; continuous 5-FU, 2000 mg/m2/22h; on day 1, 8, 15, 22; q day 36) or CAPOX (oxaliplatin, 70 mg/m2 on day 1 and 8; capecitabine, 2 × 1000 mg/m2/ day consecutively for 2 weeks, q day 22). Primary and secondary endpoints, as well as inclusion and exclusion criteria and eligibility are reported elsewhere [24]. The ethical committee of the Central Hospital Bremen and of the Medical Faculty of the RuhrUniversity Bochum approved the study. This analysis included a subgroup of 229 patients (48 % of all patients) with available tumor tissue. Overall response rate (ORR) was defined as partial remission (PR) plus complete remission (CR), and disease control rate (DCR) included CR, PR and stable disease (SD).
Pathological processing Formalin-fixed paraffin embedded samples containing tumor tissue and from tumor free resection margins (as a negative control) were collected from primary pathologists at the study sites. 219 (95 %) blocks were derived from primary tumors and 10 (5 %) from metastases. Blocks were cut in 1 µm thick sections and stained with hematoxylin and eosin (H&E) to identify tumor tissue and estimate tumor cell cellularity.
p53 immunohistochemistry As primary monoclonal antibody the mouse Do-7-antibody (DAKO, Glostrup, DK, 1:50) was applied. The determination of positive stained nuclei was accomplished semi-quantitatively and classified as follows: < 1 % lack of p53 overexpression, 1 – 10 %, 10 – 50 % and > 50 % of nuclei stained positively. When more than 10 % of tumor cells showed a nuclear staining for p53, we classified them according to previous studies as p53 overexpression positive [18, 24]. p53 overexpression is a surrogate for loss of p53 function. Staining intensity was scored as low, intermediate and strong. However almost all slides with positive staining revealed a strong staining intensity.
Immunhistochemistry of MMR proteins MMR immunhistochemistry was performed as described preciously [26]. The determination of stained nuclei was assessed semiquantitatively: < 1 % represented a lack of expression of the MMR proteins; 1 – 10 % indicated a reduction of expression; > 10 % was classified as retained expression. These criteria were adopted from the German HNPCC Consortium studies [27, 28].
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Primary mouse monoclonal antibodies for MLH1, MLH2 and MSH 6 (BD Bioscience Heidelberg, Germany, 1:20) were applied. As positive internal control, staining was considered informative when a nuclear staining in adjacent normal tissue or tumor infiltrating lymphocytes was observed.
Microsatellite analysis Tumor and normal tissue was microdissected. Tumor cell cellularity was required to be at least 70 %. Genomic DNA was isolated with the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany). All samples were analyzed for instability of the microsatellite marker Bat-26 which was performed as described before [26]. In cases with instability of Bat-26, a testing with the complete National Institute of Health reference marker panel (Bat-25, D5S346, D17S250 and D2S123) via PCR was performed [29]. The application of Bat-26 alone has a sensitivity of 95 % to detect MSI-H [30]. If at least two of five markers exhibited instability tumor tissue was considered MSI-H, low microsatellite instability (MSI-L) was defined if only one marker was instable. Tumors without instabilities were classified as MSS. The combination of two different methods to detect MSI-H increased the sensitivity and specificity.
Statistical analyses For all test p values < 0.05 were considered as significant. We used only two sided tests. To calculate the relationship between dichotomous and continuous variables Student´s Test was used. Fisher´s exact test was performed to estimate the associations between MSI-H / p53 and other dichtotomous variables. KruskalWallis test exhibited significant differences between continuous variables. To analyze overall survival (OS) progression-free survival (PFS) and post-progression survival (PPS) the Kaplan-Meier method was applied. PPS was defined as the time interval from disease progression until the date of death or date of last followup (whatever occurred first). To detect differences between groups the log-rank test was applied.
Results !
Patient’s characteristics This analysis included a subgroup of 229 patients (48 % of all trial patients) with available tumor tissue. The median PFS among this cohort was 7.5 months (95 % CI, 6.7 – 8.3 months) and the median OS was 17.5 months (95 % CI, 15.3 – 19.7 months), which did not differ from the whole clinical trial population reported previously [23]. Patients´ characteristics were: 142 male (62 %), with a median age of 64.2 years (range 35 – 82). 77 of 229 (33.6 %) patients had their primary tumor located in the rectum, in 141 of 229 (61.6 %) the primary tumor was located in the colon, in eleven patients the localization was not known. 116 (50.7 %) patients had synchronous metastases and 91 (39.7 %) metachronous metastases, in 22 cases data was missing. Synchronous metastasis was defined as diagnosis of metastatic disease within four months after diagnosis of the primary tumor.
nous vs. synchronous). Patients with MSI-H CRCs were significantly younger than patients without MSI-H CRCs (59.5 vs. 64.6 years, p = 0.032).
Incidence of p53 overexpression and correlation between p53 overexpression and clinicopathological variables 211 out of 229 (92.1 %) tumor samples were successfully analyzed for p53 overexpression. In 51 of 211 (24.2 %) less than 1 % of nuclei were stained, in 22 (10.4 %) cases a nuclear staining of 1 – 10 % of tumor cells was observed. A staining of 10 – 50 % of the nuclei was found in 18 (8.5 %) tumor samples and 120 (56.9 %) samples displayed a p53 overexpression in more than 50 % of all nuclei. p53 overexpression defined as staining in more than 10 % of tumor cells was observed in 138 (65.4 %) samples. No correlation between p53 overexpression and age, sex, primary tumor localization, treatment (FUFOX vs. CAPOX) and type of metastatic disease (metachronous vs. synchronous) was observed.
Correlation between MSI and remission status No significant correlation was found between MSI-H-status and objective response rate (CR+PR) rate (p = 0.782). MSS/MSI-L patients harbored a trend to better disease control rate (defined as CR+PR+SD) compared with MSI-H patients (85.3 % vs. 64.3 %). However this result did not reach the predefined significance lev" Table 1). el (p = 0.055, ●
Correlation between p53 overexpression and remission status No correlation between p53 status and objective response rate or " Table 1). clinical disease control rate was detectable (●
Correlation between MSI and OS and PFS Patients with MSS/MSI-L tumors had a median PFS of 7.5 months (95 % CI, 6.7 – 8.4 months), whereas patients with MSI-H tumors had a median PFS of 4.5 months (95 % CI, 1.8 – 7.2 months, " Fig. 1a). The median OS for patients with MSI-H tup = 0.431) (● mors was 24 months (95 % CI, 19.1 – 29.5 months) and among patients with non-MSI-H tumors the OS was 17.5 months (95 % CI, " Fig. 1b). 15.5 – 19.7 months, p = 0.228, ●
Table 1
MSI and p53 status in relation to treatment response.
MSI-H
n = 14 CR
1
MSS/
P53 wild-
P53 over-
MSI-L
type
expression
n = 71
n = 135
n = 190 8
p 0.17
2
7
PR
6
99
32
75
SD
2
55
23
35
PD
5
28
14
18
p 0.321 1
objective response
Incidence of MSI-H and correlation between MSI and clinicopathological variables 204 out of 229 (89.1 %) tumor samples were successfully analyzed by PCR-based MSI testing and immunohistochemistry. We identified fourteen (7.4 %) MSI-H tumors. MSI-H was not correlated with sex, treatment (FUFOX vs. CAPOX) primary tumor localization (colon or rectum) and type of metastatic disease (metachro-
CR + PR
83
107
0.782 34
82
SD + PD
7
7
37
53
CR + PR + SD
9
162
0.055 57
117
PD
5
28
14
18
0.104 2
disease control 0.232 2
CR complete remission, PR partial remission, SD stable disease, PD progressive disease 1 Pearson’s Chi-square test 2 Fisher’s exact test
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Table 2 Overall survival (OS) and post-progression survival (PPS) in correlation to p53 overexpression in patients with or without subsequent therapies after oxaliplatin failure.
no p53 overexpression
p53 overexpression
all
(n = 73)
(n = 138)
(n = 211)
overall survival (months)
15.8
19.6
18.0
95 % CI
12.6 – 18.9
16.7 – 22.6
15.7 – 20.2
PPS in patients with 2 nd/3 rd line therapy (months)
11.4
15.4
13.5
95 % CI
10.3 – 12.5
13 – 17.9
12.1 – 14.8
PPS in patients without subsequent therapy (months)
3.2
3.2
3.2
95 % CI
0.6 – 5.8
1.0 – 5.5
1.3 – 5.1
Correlation between p53 overexpression and OS and PFS The median PFS was 6.8 months (95 % CI, 5.6 – 8.0 months) for patients without p53 overexpression and 7.8 months (95 % CI, 6.8 – 8.7 months) for patients with p53 overexpression (p = 0.907, ●" Fig. 2a). The median OS of patients with p53 overexpression was significantly longer in comparison to patients without p53 overexpression 19.6 months (95 % CI, 16.7 – 22.6 months) vs. 15.8 " Fig. 2b). This effect months (95 % CI, 12.7 – 18.8 months; p = 0.05, ● was more pronounced if tumors with complete negative staining were compared with tumors with a nuclear staining of 1 or more percent of nuclei: 14.2 months (95 % CI, 11.3 – 17.1 months) vs. " Fig. 2c). 19.7 months (95 % CI, 17.0 – 22.3; p = 0.022; ● Of note, survival curves began to separate after 12 months. Therefore we hypothesized that this difference may have been caused by subsequent therapies. The post-progression survival (PPS) in 121 patients treated with at least one second line chemotherapy was significantly better for patients with p53 overexpressed tumors compared to patients with tumors without p53 overexpression. PPS of p53 overexpressed patients was 15.4 months (95 % CI, 10.3 – 12.5 months) compared to tumors without p53 overexpression with 11.4 months (95 % CI, 13.0 – 17.9 " Fig. 2 d). Since almost all patients receiving months, p = 0.004; ● a second line chemotherapy were treated with irinotecan-based protocols (117 of 120) this effect may be contributed to irinotecan. Patients with p53 overexpressed and without p53 overexpression receiving irinotecan had a median OS of 15.8 and 11.9
p value
0.05 0.004 0.882
Table 3 Median post-progression survival (PPS) in months of patients receiving subsequent chemotherapies after oxaliplatin failure with or without Cetuximab in correlation to p53 overexpression.
Cetuxi-
p53
mab no (n = 81) yes (n = 42)
95 % CI
p value
(months) no overexpression
11.9
9.2 – 14.5
overexpression
12.5
7.4 – 17.6
no overexpression
13.4
7.6 – 19.2
overexpression total (n = 123)
PPS
23.0
18.9 – 26.9
13.5
12.1 – 14.8
0.31
0.031
" Table 2). PPS in patients who months respectively (p = 0.004, ● did not receive second line chemotherapy did not depend on the " Fig. 2e). p53 overexpression (● We also observed a survival benefit in cetuximab-treated patients with p53 overexpression as compared to patients without p53 overexpression. The median PPS among p53 overexpressed patients undergoing a cetuximab containing chemotherapy was significantly longer compared to tumors without p53 overex" Table 3). pression (22. 9 vs. 13.37 months, p = 0.031, ●
Nöpel-Dünnebacke S et al. Prognostic value of … Z Gastroenterol 2014; 52: 1394–1401
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Fig. 1 a Progression-free survival (PFS in months) for patients with MSI-H and MSS/MSI-L tumors. b Overall survival (OS in months) for patients with MSI-H and MSS/MSI-L tumors.
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Fig. 2 a Progression-free survival (PFS in months) for patients with p53 overexpression and without p53 overexpression. b Overall survival (OS in months) for patients with p53 overexpression (nuclei stained positively > 10 %) and without p53 overexpression. c Overall survival (in months) p53 overexpression (nuclei stained positively > 1 %) vs. without p53 overexpression (0 % stained positively). d Post-progression survival (PPS in months) for
patients with p53 overexpression and without p53 overexpression tumors after oxaliplatin failure. e Post-progression survival (PPS in months) for patients with p53 overexpression and without p53 overexpression tumors after oxaliplatin failure without second line therapy. f Post-progression survival (PPS in months) for patients with p53 overexpression and without p53 overexpression tumors after oxaliplatin failure treated with irinotecan.
Nöpel-Dünnebacke S et al. Prognostic value of … Z Gastroenterol 2014; 52: 1394–1401
Discussion !
The aim of the study was to determine the prognostic value of MSI-H and p53 in mCRC treated with a standard chemotherapy regimen of a fluoropyrimidine and oxaliplatin. We analyzed a subgroup of patients included in the Phase III trial of the German AIO group comparing FUFOX against CAPOX in mCRCs [24]. In our trial the incidence of MSI-H in mCRCs was 7.4 %. Our study confirms previous studies, reporting an incidence of MSI-H in mCRC of 4 – 5 % [14, 31] as compared to 10 to 15 % in stage III CRC and 18 to 25 % in stage II CRC [7, 32]. We observed p53 overexpression in 65.4 % of mCRC which is concordant to previous findings [18, 19]. MSI status is currently regarded as an important predictive marker for a lack of benefit from 5-FU-based adjuvant chemotherapy [4 – 6] although a recent study suggested that the lack of benefit may be restricted to cases with sporadic MSI whereas patients with Lynch syndrome may benefit from adjuvant 5-FUbased chemotherapy [7]. In addition Tejpar et al. reported data from the PETACC-3 trial which showed that patients with MSI-H CRC may benefit from adjuvant 5-FU-based chemotherapy [32]. Only three retrospective studies explored the clinical impact of MSI-H status in the adjuvant treatment with FOLFOX. Kim et al. detected no difference in the outcome between MSI-H CRC (n = 12) and MSS CRC (n = 122) treated with adjuvant FOLFOX [8]. In a recent study, 303 patients with stage III received adjuvant FOLFOX, among this population 34 patients (11.2 %) had a MSI-H CRC [10]. The 3 years-DFS was significantly higher in the MSI-H CRC group as compared to the MSS CRC group (90.5 % vs. 73.8 %, HR, 2.16; 95 % CI, 1.09 – 4.27; p = 0.027). Based on this data the MMR-status in CRCs was identified as an independent prognostic marker for DFS (HR, 4.90; 95 % CI, 1.46 – 16.39; p = 0.01). One small retrospective study compared the outcome of 32 MSI-H stage III CRC treated with FOLFOX (n = 20) vs. 5-FU (n = 12) only [23]. 3-year DFS was 100 % vs. 57 % for FOLFOX vs. 5-FU-based adjuvant chemotherapy resulting in a reduced Hazard ratio of 0.17 (95 % CI, 0.04 – 0.68; p = 0.01) [9]. Only few trials investigated the prognostic value of microsatellite instability in mCRC treated with palliative chemotherapy. Regarding 5-FU-based chemotherapy; Liang et al. reported a superior survival for MSI-H mCRCs patients when treated with 5-FUbased chemotherapy 169 patients were included in this study with a response rate of 65.7 % versus 35.1 % (p = 0.001) and median OS of 24 months versus 13 months (p = 0.0001) comparing MSI-H and non-MSI-H patients [33]. In addition Brueckl et al. reported similar results [34]. MSI-H patients (7 of 43) had a better response rate, which led to a significant better median survival (33 months vs. 19 months; p = 0.021) in comparison to MSS patients. Another study performed by Chen et al., confirmed these findings. MSI-H patients in Dukes D stage survived longer compared to MSS patients (37 months vs. 26 months), however the difference did not reach statistical significance [35]. A meta-analysis by Propat et al., demonstrated a significant better OS among MSI-H tumors compared to MSS metastatic tumors with a pooled HR of 0.64 (95 % CI, 0.46 to 0.89; [36]. Regarding a combination therapy with oxaliplatin and fluoropyrimidines, des Guetz et al. did not observe a significant difference between MSI-H and non-MSI mCRCs with respect to OS, PFS and RR [11]. In a systematic review by des Guetz et al. the authors analyzed least eight studies and did detect different outcomes [12]. Accordingly we previously reported no prognostic value of microsatellite instability in mCRC in first subset of tumor samples
from the Porschen trial [13]. However we did find that MSI-H was correlated with a lower disease control rate compared to non-MSI-H patients. We therefore decided to reevaluate the prognostic value in a larger number of patients. In the present study, we could not detect a prognostic value of MSI-H status in patients with mCRC treated with FUFOX or CAPOX regarding RR, PFS and OS. However, MSI-H tumors tended to have a lower disease control rate although this failed to reach statistical significance. We did not observe a difference with regard to RR, DCR and PFS between patients with p53 overexpressed and without p53 overexpressed tumors treated with FUFOX or CAPOX. We observed a n improved OS and PPS for patients with p53 overexpressed tumors compared to patients without p53 overexpressed tumors. PPS in patients who did not receive second line chemotherapy did not depend on p53 overexpression. In contrast PPS was significantly better for patients with p53 overexpressed tumors receiving second or third line chemotherapies. Since almost all patients receiving a second line chemotherapy were treated with irinotecan-based protocols this effect may be contributed to irinotecan. Patients with or without p53 overexpression receiving irinotecan had a median PPS of 15.8 and 11.9 months. Weekers et al. postulated a survival benefit of patients with p53 overexpression undergoing second line chemotherapy with an irinotecan containing regimen. Response to irinotecan was correlated with a longer median survival time (39 vs. 19 months; p = 0.008). In addition response rate was higher in patients with p53 overexpression (38 % vs. 25 %) [37]. Yu et al have shown that irinotecan is more effective in inducing apoptosis in CRC when the P53 ratio between tumor and normal tissue is high [38]. In that in vitro study the RNA expression of 24 irinotecan pathways genes was analyzed from 54 tumor tissues of Dukes C CRC patients. Another in vitro study showed higher cell apoptosis among p53 overexpressing CRC cell lines treated with irinotecan and 5-aza [39]. The results of these studies still have to be validated in clinical trials but may indicate that p53 has a potential to predict responsiveness to irinotecan-based chemotherapy in mCRC. Among the 120 patients with further treatment lines more than one third of patients received cetuximab. At time of the clinical trial cetuximab was approved only for chemotherapy of mCRC patients after experiencing irinotecan failure independent of K-RAS status. In the context of our study cohort the cetuximab patients therefore represent a group of patients with third line therapy. We observed a survival benefit in cetuximab-treated patients with p53 overexpression as compared to patients without p53 overexpression. In line with our finding Oden-Gangloff et al. previously reported 46 chemorefractary patients treated with cetuximab and demonstrated a significant increase of the median time to progression for patients with p53 overexpressed tumors compared to patients without (20 vs. 12 weeks, p = 0.004). In addition p53 overexpression was associated with better disease control (p = 0.037) [40]. However, we cannot rule out that the group of patients not receiving cetuximab may represent a group of patients with poorer performance status at the time of progress of second line therapy as compared to patients who did receive cetuximab and may not have been eligible for an additional treatment line. In conclusion, we observed in our study that mCRC p53 overexpressed patients undergoing an irinotecan containing second- or third-line chemotherapy have a significant benefit in comparison to patients without p53 overexpressed tumors. To validate the
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clinical impact of our findings trials with irinotecan- and/or cetuximab-based treatment regimens need to be explored regarding the effect of p53 alterations in a higher number of patients. MSI seems to have only a modest prognostic effect in patients with mCRC treated with a combination of oxaliplatin and 5-FU.
14
15
Affiliations 1
2 3
4 5 6
Department of Internal Medicine, Ruhr-University of Bochum, Knappschaftskrankenhaus, Bochum Departement of Oncology, Klinikum Arnsberg Departement of Hematology and Oncology, Ruhr-University of Bochum, St. Josef Hospital, Bochum Clinic of Internal Medicine, Hospital Bremen East, Bremen Institute of Pathology, Ruhr-University of Bochum Medizinische Klinik, Maria-Hilf-Krankenhaus, Mönchengladbach
16 17 18 19
20
Acknowledgement !
The authors wish to thank the AIO colorectal cancer study group for approval of the biomarker study, participating AIO study centers und their pathologists for providing tissue blocks, patients for providing written consent for biomarker studies, Petra Freitag for administrative regarding tissue acquisition and Sabine Geiger for DNA isolation.
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Nöpel-Dünnebacke S et al. Prognostic value of … Z Gastroenterol 2014; 52: 1394–1401
Originalarbeit
Prognostic value of microsatellite instability and p53 expression in metastatic colorectal cancer treated with oxaliplatin and fluoropyrimidine-based chemotherapy
Authors
S. Nöpel-Dünnebacke1, K. Schulmann2, A. Reinacher-Schick3, R. Porschen4, W. Schmiegel1, A. Tannapfel5, U. Graeven6
Affiliations
Affiliation addresses are listed at the end of the article.
Schlüsselwörter
Zusammenfassung
Abstract
"
!
!
Studie: Evaluation des prognostischen Wertes von MSI-H und p53-Überexpression bei metastasierten kolorektalen Karzinomen (mKRK), die mit oxaliplatin- und fluoropyrimidin-basierter Erstlinienchemotherapie behandelt wurden. Methodik: Retrospektiv wurden Tumorgewebeproben von 229 Patienten einer prospektiven randomisierten Phase-III-Studie der kolorektalen Studiengruppe der AIO, welche CAPOX gegen FUFOX bei mKRK verglich, analysiert. Ein immunhistochemischer Nachweis einer p53-Überexpression bzw. der MMR-Proteine sowie Mikrosatellitenanalysen wurden durchgeführt. Ergebnisse: Die Inzidenz von MSI-H und p53Überexpression lag bei 7,9 % bzw. 65,4 %. Der MSH-I-Status korrelierte nicht mit der ORR, dem PFS oder OS. Ein Trend unter MSI-H-Tumoren zu einer geringeren DCR war festzustellen (65 % vs. 85 %). Eine p53-Überexpression korrelierte nicht mit der DCR, ORR, oder dem PFS. Das mediane OS der Patienten mit p53-überexprimierten Tumoren war signifikant länger verglichen mit Tumoren ohne p53-Überexprimierung (19,6 vs. 15,8 Monate; p= 0,05). Das PFS der p53 überexprimierenden Patienten unter der Zweit- und Drittlinienchemotherapie mit Irinotecan und/oder Cetuximab war signifikant länger als bei Patienten ohne p53-Überexpression. Diskussion: MSI-H Tumoren zeigten unter einer Kombinationstherapie aus Oxaliplatin/Fluoropyrimidin tendenziell eine schlechtere DCR. p53 überexprimierte mKRK unter einer irinotecanhaltigen Zweit- bzw. Drittlinienchemotherapie nach Oxaliplatinversagen hatten ein signifikant längeres PFS im Vergleich zu nicht p53 überexprimierenden Tumoren. Um die klinische Bedeutung dieser Beobachtung zu valuieren, werden weitere Studien benötigt.
Purpose: The aim of this study was to evaluate the prognostic value of MSI-H and p53 overexpression in metastatic colorectal cancer (mCRC) treated with oxaliplatin and fluoropyrimidinebased first line chemotherapy. Methods: Tumour samples were retrospectively obtained from 229 patients from a prospective randomised phase III trial of the AIO colorectal study group, comparing CAPOX and FUFOX in mCRC. Immunohistochemistry of p53 and MMR proteins as well as microsatellite analysis were performed. Results: The incidence of MSI-H and p53 overexpression was 7.9 % and 65.4 %, respectively. MSI-H status was not correlated with ORR, PFS and OS. We observed a trend to lower DCR for MSI-H tumours (65 % vs. 85 %, p = 0.055). p53 overexpression was not correlated with DCR, ORR and PFS. The median OS of patients with tumors with p53 overexpression was significantly longer compared to tumors withhout p53 overexpression (19.6 vs. 15.8 months; p = 0.05). The post-progression survival (PPS) of p53-positive patients undergoing 2nd and/or 3rd line chemotherapy with irinotecan and/or cetuximab was significantly longer compared to p53-negative patients. Conclusion: MSI-H tumours tend to have lower disease control rates when treated with an oxaliplatin/fluoropyrmidin combination. mCRC patients with p53 overexpression undergoing an irinotecan containing second- or third-line chemotherapy after oxaliplatin failure have a significantly longer post-progression survival compared to patients without p53 overexpression. To validate the clinical impact of p53 in patients with mCRC treated with irinotecan- and/or cetuximab further studies are needed.
● metastasierters kolorektales ● ● ● ● " " " "
Karzinom Mikrosatelliteninstabilität p53 Oxaliplatin Prognose
Key words
● metastatic colorectal cancer ● microsatellite instability ● p53 ● oxaliplatin ● prognosis " " " " "
received accepted
14.11.2013 15.6.2014
Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1366781 Z Gastroenterol 2014; 52: 1394–1401 © Georg Thieme Verlag KG Stuttgart · New York · ISSN 0044-2771 Correspondence Dr. Stefanie NöpelDünnebacke Department of Internal Medicine, Ruhr-University of Bochum, Knappschaftskrankenhaus In der Schornau 23–25 44892 Bochum Germany [email protected]
Nöpel-Dünnebacke S et al. Prognostic value of … Z Gastroenterol 2014; 52: 1394–1401
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Prognostischer Einfluss der Mikrosatelliteninstabilität und p53-Überexpression bei metastasierten kolorektalen Karzinomen unter Oxaliplatin- und 5-FU-haltiger Chemotherapie
Originalarbeit
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Colorectal cancer (CRC) is the fourth common malignant tumor in the world and causes over 500 000 deaths each year [1]. At least three different molecular subtypes of CRC are currently known. The traditional type is characterized by chromosomal instability (CIN) (80 – 85 %). The second type is characterized by high levels of microsatellite instability (MSI-H) (10 – 23 %) and is caused by a deficient mismatch repair (MMR) system. The third type is characterized by widespread hypermethylation of CpG islands in the promoter region of tumor suppressor genes (CpG island methylator phenotype (CIMP)) which overlaps to some amount to CIN and MSI phenotypes [2, 3]. Tumors lacking MSI-H are usually classified as microsatellite stable (MSS). To improve outcomes, reduce toxicities and therefore tailor therapeutic concepts in metastatic CRC (mCRC) prognostic and predictive biomarkers are urgently needed. In addition to its prognostic value, the MSI-H phenotype is a predictive marker for a lack of benefit from 5-FU-based adjuvant chemotherapy in stage II and III [4 – 6], although a recent study suggested that the lack of benefit may be restricted to cases with sporadic MSI whereas patients with Lynch syndrome may benefit from adjuvant 5-FUbased chemotherapy [7]. Few small studies explored the clinical impact of MSI-H status in the adjuvant treatment with FOLFOX. Kim et al., reported no difference in the outcome between MSI CRC and MSS CRC when treated adjuvant with FOLFOX [8]. However, a more recent retrospective analysis of the MOSAIC trial by Flejou demonstrates a benefit from FOLFOX in stage III MSI CRC as compared to an adjuvant 5-FU-based chemotherapy [9]. Another study by Zaanan et al. observed a significant longer 3-year disease free survival (DFS) among MSI-H CRC compared to MSS CRC [10]. Data regarding the value of MSI-H in mCRC undergoing palliative 5-FU based chemotherapy are rare. Des Guetz et al. did not find significant differences in overall survival (OS), progression free survival (PFS) or remission rate (RR) according to MMR status [11]. In a meta-analysis including eight trials in mCRC des Guetz did not find evidence for MSI having impact on 5-FU-based chemotherapy [12]. In a previous report we found evidence that MSI-H mCRC may result in lower disease control rates as compared to MSS mCRC when treated with FUFOX or CAPOX [13]. However the numbers of cases with MSI-H have been small. The largest trial investigating the value of MSI-H in mCRC so far, the FOCUS trial was not able to demonstrate a predictive value of MSI-H in patients randomized to three different chemotherapy regimens [14]. Another putative prognostic marker is the tumor suppressor gene p53. The p53 protein plays a central role in the cell cycle control by transcription regulation [15]. Mutations of p53 are the most common genetic alteration in human cancers [16, 17]. p53 mutations can be identified in approximately 50 % of CRCs [18, 19]. Inactivating point mutations of p53 lead to nuclear accumulation of p53 that can be detected by immunohistochemistry (IHC). Therefore p53 overexpression in a surrogate marker for p53 inactivation and loss of p53 function. The prognostic impact of p53 in CRCs treated with surgery with or out adjuvant chemotherapy remains controversial [20, 21]. A recent meta-analysis failed to demonstrate that p53 alterations influence the prognosis of patients treated with 5-FU-based adjuvant chemotherapy [22]. In addition Munro et al. detected no significant distinction in the outcome between p53 mutated CRCs in different stages and wild type under 5-FU-based chemotherapy [14, 18]. Adding oxaliplatin to 5-FU/leucovorin in adjuvant treated stage
III CRCs resulted in a significantly better disease free survival (DFS) for patients with p53 overexpression [23]. The therapeutic impact of p53 mutation in mCRCs remains unclear. In this study we retrospectively evaluated the prognostic value of p53 overexpression and MSI-H in mCRCs treated with a palliative first line combination therapy with oxaliplatin and fluoropyrimidines within a prospective randomized phase III trial from the German AIO colorectal cancer group comparing the FUFOX (oxaliplatin and 5-FU) and CAPOX (capectabine and oxaliplatin). The clinical results of the trial have been reported previously [24].
Material and Methods !
Study design and patient’s characteristics Tumor tissue samples from patients with mCRC participating in a prospective randomized phase III first-line chemotherapy trial of the AIO Group (Arbeitsgemeinschaft Internistische Onkologie of the German Cancer Society) were collected retrospectively. 474 patients were randomized to be treated with either FUFOX (oxaliplatin, 50 mg/m2; FA, 500 mg/m2; continuous 5-FU, 2000 mg/m2/22h; on day 1, 8, 15, 22; q day 36) or CAPOX (oxaliplatin, 70 mg/m2 on day 1 and 8; capecitabine, 2 × 1000 mg/m2/ day consecutively for 2 weeks, q day 22). Primary and secondary endpoints, as well as inclusion and exclusion criteria and eligibility are reported elsewhere [24]. The ethical committee of the Central Hospital Bremen and of the Medical Faculty of the RuhrUniversity Bochum approved the study. This analysis included a subgroup of 229 patients (48 % of all patients) with available tumor tissue. Overall response rate (ORR) was defined as partial remission (PR) plus complete remission (CR), and disease control rate (DCR) included CR, PR and stable disease (SD).
Pathological processing Formalin-fixed paraffin embedded samples containing tumor tissue and from tumor free resection margins (as a negative control) were collected from primary pathologists at the study sites. 219 (95 %) blocks were derived from primary tumors and 10 (5 %) from metastases. Blocks were cut in 1 µm thick sections and stained with hematoxylin and eosin (H&E) to identify tumor tissue and estimate tumor cell cellularity.
p53 immunohistochemistry As primary monoclonal antibody the mouse Do-7-antibody (DAKO, Glostrup, DK, 1:50) was applied. The determination of positive stained nuclei was accomplished semi-quantitatively and classified as follows: < 1 % lack of p53 overexpression, 1 – 10 %, 10 – 50 % and > 50 % of nuclei stained positively. When more than 10 % of tumor cells showed a nuclear staining for p53, we classified them according to previous studies as p53 overexpression positive [18, 24]. p53 overexpression is a surrogate for loss of p53 function. Staining intensity was scored as low, intermediate and strong. However almost all slides with positive staining revealed a strong staining intensity.
Immunhistochemistry of MMR proteins MMR immunhistochemistry was performed as described preciously [26]. The determination of stained nuclei was assessed semiquantitatively: < 1 % represented a lack of expression of the MMR proteins; 1 – 10 % indicated a reduction of expression; > 10 % was classified as retained expression. These criteria were adopted from the German HNPCC Consortium studies [27, 28].
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Primary mouse monoclonal antibodies for MLH1, MLH2 and MSH 6 (BD Bioscience Heidelberg, Germany, 1:20) were applied. As positive internal control, staining was considered informative when a nuclear staining in adjacent normal tissue or tumor infiltrating lymphocytes was observed.
Microsatellite analysis Tumor and normal tissue was microdissected. Tumor cell cellularity was required to be at least 70 %. Genomic DNA was isolated with the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany). All samples were analyzed for instability of the microsatellite marker Bat-26 which was performed as described before [26]. In cases with instability of Bat-26, a testing with the complete National Institute of Health reference marker panel (Bat-25, D5S346, D17S250 and D2S123) via PCR was performed [29]. The application of Bat-26 alone has a sensitivity of 95 % to detect MSI-H [30]. If at least two of five markers exhibited instability tumor tissue was considered MSI-H, low microsatellite instability (MSI-L) was defined if only one marker was instable. Tumors without instabilities were classified as MSS. The combination of two different methods to detect MSI-H increased the sensitivity and specificity.
Statistical analyses For all test p values < 0.05 were considered as significant. We used only two sided tests. To calculate the relationship between dichotomous and continuous variables Student´s Test was used. Fisher´s exact test was performed to estimate the associations between MSI-H / p53 and other dichtotomous variables. KruskalWallis test exhibited significant differences between continuous variables. To analyze overall survival (OS) progression-free survival (PFS) and post-progression survival (PPS) the Kaplan-Meier method was applied. PPS was defined as the time interval from disease progression until the date of death or date of last followup (whatever occurred first). To detect differences between groups the log-rank test was applied.
Results !
Patient’s characteristics This analysis included a subgroup of 229 patients (48 % of all trial patients) with available tumor tissue. The median PFS among this cohort was 7.5 months (95 % CI, 6.7 – 8.3 months) and the median OS was 17.5 months (95 % CI, 15.3 – 19.7 months), which did not differ from the whole clinical trial population reported previously [23]. Patients´ characteristics were: 142 male (62 %), with a median age of 64.2 years (range 35 – 82). 77 of 229 (33.6 %) patients had their primary tumor located in the rectum, in 141 of 229 (61.6 %) the primary tumor was located in the colon, in eleven patients the localization was not known. 116 (50.7 %) patients had synchronous metastases and 91 (39.7 %) metachronous metastases, in 22 cases data was missing. Synchronous metastasis was defined as diagnosis of metastatic disease within four months after diagnosis of the primary tumor.
nous vs. synchronous). Patients with MSI-H CRCs were significantly younger than patients without MSI-H CRCs (59.5 vs. 64.6 years, p = 0.032).
Incidence of p53 overexpression and correlation between p53 overexpression and clinicopathological variables 211 out of 229 (92.1 %) tumor samples were successfully analyzed for p53 overexpression. In 51 of 211 (24.2 %) less than 1 % of nuclei were stained, in 22 (10.4 %) cases a nuclear staining of 1 – 10 % of tumor cells was observed. A staining of 10 – 50 % of the nuclei was found in 18 (8.5 %) tumor samples and 120 (56.9 %) samples displayed a p53 overexpression in more than 50 % of all nuclei. p53 overexpression defined as staining in more than 10 % of tumor cells was observed in 138 (65.4 %) samples. No correlation between p53 overexpression and age, sex, primary tumor localization, treatment (FUFOX vs. CAPOX) and type of metastatic disease (metachronous vs. synchronous) was observed.
Correlation between MSI and remission status No significant correlation was found between MSI-H-status and objective response rate (CR+PR) rate (p = 0.782). MSS/MSI-L patients harbored a trend to better disease control rate (defined as CR+PR+SD) compared with MSI-H patients (85.3 % vs. 64.3 %). However this result did not reach the predefined significance lev" Table 1). el (p = 0.055, ●
Correlation between p53 overexpression and remission status No correlation between p53 status and objective response rate or " Table 1). clinical disease control rate was detectable (●
Correlation between MSI and OS and PFS Patients with MSS/MSI-L tumors had a median PFS of 7.5 months (95 % CI, 6.7 – 8.4 months), whereas patients with MSI-H tumors had a median PFS of 4.5 months (95 % CI, 1.8 – 7.2 months, " Fig. 1a). The median OS for patients with MSI-H tup = 0.431) (● mors was 24 months (95 % CI, 19.1 – 29.5 months) and among patients with non-MSI-H tumors the OS was 17.5 months (95 % CI, " Fig. 1b). 15.5 – 19.7 months, p = 0.228, ●
Table 1
MSI and p53 status in relation to treatment response.
MSI-H
n = 14 CR
1
MSS/
P53 wild-
P53 over-
MSI-L
type
expression
n = 71
n = 135
n = 190 8
p 0.17
2
7
PR
6
99
32
75
SD
2
55
23
35
PD
5
28
14
18
p 0.321 1
objective response
Incidence of MSI-H and correlation between MSI and clinicopathological variables 204 out of 229 (89.1 %) tumor samples were successfully analyzed by PCR-based MSI testing and immunohistochemistry. We identified fourteen (7.4 %) MSI-H tumors. MSI-H was not correlated with sex, treatment (FUFOX vs. CAPOX) primary tumor localization (colon or rectum) and type of metastatic disease (metachro-
CR + PR
83
107
0.782 34
82
SD + PD
7
7
37
53
CR + PR + SD
9
162
0.055 57
117
PD
5
28
14
18
0.104 2
disease control 0.232 2
CR complete remission, PR partial remission, SD stable disease, PD progressive disease 1 Pearson’s Chi-square test 2 Fisher’s exact test
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Table 2 Overall survival (OS) and post-progression survival (PPS) in correlation to p53 overexpression in patients with or without subsequent therapies after oxaliplatin failure.
no p53 overexpression
p53 overexpression
all
(n = 73)
(n = 138)
(n = 211)
overall survival (months)
15.8
19.6
18.0
95 % CI
12.6 – 18.9
16.7 – 22.6
15.7 – 20.2
PPS in patients with 2 nd/3 rd line therapy (months)
11.4
15.4
13.5
95 % CI
10.3 – 12.5
13 – 17.9
12.1 – 14.8
PPS in patients without subsequent therapy (months)
3.2
3.2
3.2
95 % CI
0.6 – 5.8
1.0 – 5.5
1.3 – 5.1
Correlation between p53 overexpression and OS and PFS The median PFS was 6.8 months (95 % CI, 5.6 – 8.0 months) for patients without p53 overexpression and 7.8 months (95 % CI, 6.8 – 8.7 months) for patients with p53 overexpression (p = 0.907, ●" Fig. 2a). The median OS of patients with p53 overexpression was significantly longer in comparison to patients without p53 overexpression 19.6 months (95 % CI, 16.7 – 22.6 months) vs. 15.8 " Fig. 2b). This effect months (95 % CI, 12.7 – 18.8 months; p = 0.05, ● was more pronounced if tumors with complete negative staining were compared with tumors with a nuclear staining of 1 or more percent of nuclei: 14.2 months (95 % CI, 11.3 – 17.1 months) vs. " Fig. 2c). 19.7 months (95 % CI, 17.0 – 22.3; p = 0.022; ● Of note, survival curves began to separate after 12 months. Therefore we hypothesized that this difference may have been caused by subsequent therapies. The post-progression survival (PPS) in 121 patients treated with at least one second line chemotherapy was significantly better for patients with p53 overexpressed tumors compared to patients with tumors without p53 overexpression. PPS of p53 overexpressed patients was 15.4 months (95 % CI, 10.3 – 12.5 months) compared to tumors without p53 overexpression with 11.4 months (95 % CI, 13.0 – 17.9 " Fig. 2 d). Since almost all patients receiving months, p = 0.004; ● a second line chemotherapy were treated with irinotecan-based protocols (117 of 120) this effect may be contributed to irinotecan. Patients with p53 overexpressed and without p53 overexpression receiving irinotecan had a median OS of 15.8 and 11.9
p value
0.05 0.004 0.882
Table 3 Median post-progression survival (PPS) in months of patients receiving subsequent chemotherapies after oxaliplatin failure with or without Cetuximab in correlation to p53 overexpression.
Cetuxi-
p53
mab no (n = 81) yes (n = 42)
95 % CI
p value
(months) no overexpression
11.9
9.2 – 14.5
overexpression
12.5
7.4 – 17.6
no overexpression
13.4
7.6 – 19.2
overexpression total (n = 123)
PPS
23.0
18.9 – 26.9
13.5
12.1 – 14.8
0.31
0.031
" Table 2). PPS in patients who months respectively (p = 0.004, ● did not receive second line chemotherapy did not depend on the " Fig. 2e). p53 overexpression (● We also observed a survival benefit in cetuximab-treated patients with p53 overexpression as compared to patients without p53 overexpression. The median PPS among p53 overexpressed patients undergoing a cetuximab containing chemotherapy was significantly longer compared to tumors without p53 overex" Table 3). pression (22. 9 vs. 13.37 months, p = 0.031, ●
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Fig. 1 a Progression-free survival (PFS in months) for patients with MSI-H and MSS/MSI-L tumors. b Overall survival (OS in months) for patients with MSI-H and MSS/MSI-L tumors.
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Fig. 2 a Progression-free survival (PFS in months) for patients with p53 overexpression and without p53 overexpression. b Overall survival (OS in months) for patients with p53 overexpression (nuclei stained positively > 10 %) and without p53 overexpression. c Overall survival (in months) p53 overexpression (nuclei stained positively > 1 %) vs. without p53 overexpression (0 % stained positively). d Post-progression survival (PPS in months) for
patients with p53 overexpression and without p53 overexpression tumors after oxaliplatin failure. e Post-progression survival (PPS in months) for patients with p53 overexpression and without p53 overexpression tumors after oxaliplatin failure without second line therapy. f Post-progression survival (PPS in months) for patients with p53 overexpression and without p53 overexpression tumors after oxaliplatin failure treated with irinotecan.
Nöpel-Dünnebacke S et al. Prognostic value of … Z Gastroenterol 2014; 52: 1394–1401
Discussion !
The aim of the study was to determine the prognostic value of MSI-H and p53 in mCRC treated with a standard chemotherapy regimen of a fluoropyrimidine and oxaliplatin. We analyzed a subgroup of patients included in the Phase III trial of the German AIO group comparing FUFOX against CAPOX in mCRCs [24]. In our trial the incidence of MSI-H in mCRCs was 7.4 %. Our study confirms previous studies, reporting an incidence of MSI-H in mCRC of 4 – 5 % [14, 31] as compared to 10 to 15 % in stage III CRC and 18 to 25 % in stage II CRC [7, 32]. We observed p53 overexpression in 65.4 % of mCRC which is concordant to previous findings [18, 19]. MSI status is currently regarded as an important predictive marker for a lack of benefit from 5-FU-based adjuvant chemotherapy [4 – 6] although a recent study suggested that the lack of benefit may be restricted to cases with sporadic MSI whereas patients with Lynch syndrome may benefit from adjuvant 5-FUbased chemotherapy [7]. In addition Tejpar et al. reported data from the PETACC-3 trial which showed that patients with MSI-H CRC may benefit from adjuvant 5-FU-based chemotherapy [32]. Only three retrospective studies explored the clinical impact of MSI-H status in the adjuvant treatment with FOLFOX. Kim et al. detected no difference in the outcome between MSI-H CRC (n = 12) and MSS CRC (n = 122) treated with adjuvant FOLFOX [8]. In a recent study, 303 patients with stage III received adjuvant FOLFOX, among this population 34 patients (11.2 %) had a MSI-H CRC [10]. The 3 years-DFS was significantly higher in the MSI-H CRC group as compared to the MSS CRC group (90.5 % vs. 73.8 %, HR, 2.16; 95 % CI, 1.09 – 4.27; p = 0.027). Based on this data the MMR-status in CRCs was identified as an independent prognostic marker for DFS (HR, 4.90; 95 % CI, 1.46 – 16.39; p = 0.01). One small retrospective study compared the outcome of 32 MSI-H stage III CRC treated with FOLFOX (n = 20) vs. 5-FU (n = 12) only [23]. 3-year DFS was 100 % vs. 57 % for FOLFOX vs. 5-FU-based adjuvant chemotherapy resulting in a reduced Hazard ratio of 0.17 (95 % CI, 0.04 – 0.68; p = 0.01) [9]. Only few trials investigated the prognostic value of microsatellite instability in mCRC treated with palliative chemotherapy. Regarding 5-FU-based chemotherapy; Liang et al. reported a superior survival for MSI-H mCRCs patients when treated with 5-FUbased chemotherapy 169 patients were included in this study with a response rate of 65.7 % versus 35.1 % (p = 0.001) and median OS of 24 months versus 13 months (p = 0.0001) comparing MSI-H and non-MSI-H patients [33]. In addition Brueckl et al. reported similar results [34]. MSI-H patients (7 of 43) had a better response rate, which led to a significant better median survival (33 months vs. 19 months; p = 0.021) in comparison to MSS patients. Another study performed by Chen et al., confirmed these findings. MSI-H patients in Dukes D stage survived longer compared to MSS patients (37 months vs. 26 months), however the difference did not reach statistical significance [35]. A meta-analysis by Propat et al., demonstrated a significant better OS among MSI-H tumors compared to MSS metastatic tumors with a pooled HR of 0.64 (95 % CI, 0.46 to 0.89; [36]. Regarding a combination therapy with oxaliplatin and fluoropyrimidines, des Guetz et al. did not observe a significant difference between MSI-H and non-MSI mCRCs with respect to OS, PFS and RR [11]. In a systematic review by des Guetz et al. the authors analyzed least eight studies and did detect different outcomes [12]. Accordingly we previously reported no prognostic value of microsatellite instability in mCRC in first subset of tumor samples
from the Porschen trial [13]. However we did find that MSI-H was correlated with a lower disease control rate compared to non-MSI-H patients. We therefore decided to reevaluate the prognostic value in a larger number of patients. In the present study, we could not detect a prognostic value of MSI-H status in patients with mCRC treated with FUFOX or CAPOX regarding RR, PFS and OS. However, MSI-H tumors tended to have a lower disease control rate although this failed to reach statistical significance. We did not observe a difference with regard to RR, DCR and PFS between patients with p53 overexpressed and without p53 overexpressed tumors treated with FUFOX or CAPOX. We observed a n improved OS and PPS for patients with p53 overexpressed tumors compared to patients without p53 overexpressed tumors. PPS in patients who did not receive second line chemotherapy did not depend on p53 overexpression. In contrast PPS was significantly better for patients with p53 overexpressed tumors receiving second or third line chemotherapies. Since almost all patients receiving a second line chemotherapy were treated with irinotecan-based protocols this effect may be contributed to irinotecan. Patients with or without p53 overexpression receiving irinotecan had a median PPS of 15.8 and 11.9 months. Weekers et al. postulated a survival benefit of patients with p53 overexpression undergoing second line chemotherapy with an irinotecan containing regimen. Response to irinotecan was correlated with a longer median survival time (39 vs. 19 months; p = 0.008). In addition response rate was higher in patients with p53 overexpression (38 % vs. 25 %) [37]. Yu et al have shown that irinotecan is more effective in inducing apoptosis in CRC when the P53 ratio between tumor and normal tissue is high [38]. In that in vitro study the RNA expression of 24 irinotecan pathways genes was analyzed from 54 tumor tissues of Dukes C CRC patients. Another in vitro study showed higher cell apoptosis among p53 overexpressing CRC cell lines treated with irinotecan and 5-aza [39]. The results of these studies still have to be validated in clinical trials but may indicate that p53 has a potential to predict responsiveness to irinotecan-based chemotherapy in mCRC. Among the 120 patients with further treatment lines more than one third of patients received cetuximab. At time of the clinical trial cetuximab was approved only for chemotherapy of mCRC patients after experiencing irinotecan failure independent of K-RAS status. In the context of our study cohort the cetuximab patients therefore represent a group of patients with third line therapy. We observed a survival benefit in cetuximab-treated patients with p53 overexpression as compared to patients without p53 overexpression. In line with our finding Oden-Gangloff et al. previously reported 46 chemorefractary patients treated with cetuximab and demonstrated a significant increase of the median time to progression for patients with p53 overexpressed tumors compared to patients without (20 vs. 12 weeks, p = 0.004). In addition p53 overexpression was associated with better disease control (p = 0.037) [40]. However, we cannot rule out that the group of patients not receiving cetuximab may represent a group of patients with poorer performance status at the time of progress of second line therapy as compared to patients who did receive cetuximab and may not have been eligible for an additional treatment line. In conclusion, we observed in our study that mCRC p53 overexpressed patients undergoing an irinotecan containing second- or third-line chemotherapy have a significant benefit in comparison to patients without p53 overexpressed tumors. To validate the
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clinical impact of our findings trials with irinotecan- and/or cetuximab-based treatment regimens need to be explored regarding the effect of p53 alterations in a higher number of patients. MSI seems to have only a modest prognostic effect in patients with mCRC treated with a combination of oxaliplatin and 5-FU.
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Affiliations 1
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Department of Internal Medicine, Ruhr-University of Bochum, Knappschaftskrankenhaus, Bochum Departement of Oncology, Klinikum Arnsberg Departement of Hematology and Oncology, Ruhr-University of Bochum, St. Josef Hospital, Bochum Clinic of Internal Medicine, Hospital Bremen East, Bremen Institute of Pathology, Ruhr-University of Bochum Medizinische Klinik, Maria-Hilf-Krankenhaus, Mönchengladbach
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Acknowledgement !
The authors wish to thank the AIO colorectal cancer study group for approval of the biomarker study, participating AIO study centers und their pathologists for providing tissue blocks, patients for providing written consent for biomarker studies, Petra Freitag for administrative regarding tissue acquisition and Sabine Geiger for DNA isolation.
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