Originally published as JCO Early Release 10.1200/JCO.2003.07.077 on July 28 2003

This Article Abstract Full Text (PDF) Erratum (v22,p576) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Email this article to a colleague Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Save to my personal folders Download to citation manager Rights & Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Rau, B. Articles by Daniel, P. T. Search for Related Content PubMed PubMed Citation Articles by Rau, B. Articles by Daniel, P. T. Social Bookmarking                            What's this?

Dynamic Expression Profile of p21^WAF1/CIP1 and Ki-67 Predicts Survival in Rectal Carcinoma Treated With Preoperative Radiochemotherapy

Beate Rau, Isrid Sturm, Hermann Lage, Stefan Berger, Ulrike Schneider, Steffen Hauptmann, Peter Wust, Hanno Riess, Peter M. Schlag, Bernd Dörken, Peter T. Daniel

From the Charité Medical School, Humboldt University of Berlin; Campus Berlin-Buch, Robert-Rössle Klinik, Department of Surgery and Surgical Oncology; Campus Berlin-Buch, Robert-Rössle Klinik, Department of Hematology, Oncology and Tumor Immunology; Campus Mitte, Institute for Pathology; Campus Virchow-Klinikum, Department of Radiation Oncology; Campus Virchow-Klinikum, Department of Hematology and Oncology; and Max Delbrück Center for Molecular Medicine, Berlin, Germany.

Address reprint requests to Beate Rau, MD, Charité Medical School, Campus Berlin-Buch, Humboldt University, Department of Surgery and Surgical Oncology, Robert-Roessle Klinik, Lindenberger Weg 80, 13125 Berlin, Germany; e-mail: rau{at}rrk-berlin.de.

	ABSTRACT

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION AUTHORS’ DISCLOSURES OF... REFERENCES

 
Purpose: We investigated p53 and its downstream effectors p21^WAF1/CIP1, BAX, and hMSH2 as well as the proliferation marker Ki-67 (mki-67/MIB-1) in patients undergoing preoperative radiochemotherapy for rectal carcinoma to identify prognostic and predictive factors. The focus of this study was on the dynamics of these genetic markers in a longitudinal study—that is, before and after radiochemotherapy.

Patients and Methods: Expression of p53, BAX, p21^WAF1/CIP1, Ki-67, and hMSH2 was investigated by immunohistochemistry in pre- and posttherapeutic tumor samples in 66 patients. Tumor DNA was screened for p53 mutations by single-strand conformation polymorphism–polymerase chain reaction (SSCP-PCR). Paired tumor samples (pretherapy and posttherapy) were collected prospectively.

Results: Patients with a decrease in p21 expression following radiochemotherapy had better disease-free survival (P = .03). Similarly, patients with an increase in proliferative activity as measured by increased Ki-67 expression posttherapy had better disease-free survival (P < .005). In addition, we observed a significantly better prognosis for patients with high hMSH2 expression. In contrast, pretherapeutic levels of p53, BAX, or p21 expression and p53 mutation had no prognostic value, indicating that the combination of radiotherapy and chemotherapy might override defects in these genes.

Conclusion: These findings are novel and support the clinical relevance of p21 in the suppression of both proliferation and apoptosis. Thus, the dynamic induction of p21^WAF1/CIP1 was associated with a lower proliferative activity but an ultimately worse treatment outcome following neoadjuvant radiochemotherapy and tumor resection. Induction of p21, therefore, represents a novel resistance mechanism in rectal cancer undergoing preoperative radiochemotherapy.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION AUTHORS’ DISCLOSURES OF... REFERENCES

 
CANCER DEVELOPS in consequence of both disrupted cell cycle control and apoptosis.^1,2 In addition, the disruption of these regulatory networks, especially of those involved in cell death signaling, plays a key role in the acquired or constitutive resistance of malignant tumors to cytotoxic anticancer therapies. Thus, there is broad evidence that cytotoxic drugs or ionizing irradiation trigger tumor cell death via the induction of apoptosis and that the inactivation of key switches by loss of function of proapoptotic^3–5 or gain of function of antiapoptotic genes³ results in resistance to anticancer therapy.

In this line, we have shown previously that the disruption of components of the p53 signaling pathway is associated with a poor prognosis in gastrointestinal tumors.^6–10 The p53 tumor suppressor is a key regulator of cell death and cell cycle arrest on DNA damage.¹¹ Various downstream effectors of the p53 gene are involved in the p53-dependent cellular response to DNA damage (ie, cell cycle regulation, DNA repair, and apoptosis).

The aim of this study was, therefore, to determine the prognostic relevance of the p53 pathway components in patients with locally advanced rectal cancer treated with preoperative radiochemotherapy (RCT).

To this end, we analyzed the p53 gene and its downstream effectors p21^WAF1/CIP1 (cell cycle regulation),¹² BAX (pro-apoptotic Bcl-2 family member),¹³ and the DNA mismatch repair protein hMSH2,^14,15 in context with the proliferation marker Ki-67. Protein expression was determined in paired samples (pre- and posttherapy) from 66 patients treated for advanced rectal carcinoma with preoperative RCT.

In this longitudinal study, we identified a subset of tumors with an increase of p21^WAF1/CIP1 expression after cytotoxic therapy. Surprisingly, these patients had significantly reduced disease-free survival (DFS). The increase in expression of p21^WAF1/CIP1 was accompanied by a decrease in Ki-67 expression—that is, proliferative acivity. This is in line with the cell cycle arresting function of the cyclin-dependent kinase inhibitor p21^WAF1/CIP1. Thus, the decrease in Ki-67 expression after therapy, as compared with pretreatment values, was also found to be associated with a markedly reduced DFS.

Altogether, our finding suggests a significant role for p21^WAF1/CIP1 in the development of resistance to therapy in rectal carcinoma. A similar effect connecting p21^WAF1/CIP1 and resistance to chemotherapy was previously demonstrated in the HCT116 cell line model, where the targeted knockout of the p21^WAF1/CIP1 gene results in sensitivity to DNA-damaging chemotherapy.¹⁶ Thus, the dynamic induction of p21^WAF1/CIP1 expression being associated with poor prognosis may provide a rational basis for the treatment of colorectal cancer (eg, by the pharmacological disruption of cell cycle arrest programs in conjunction with RCT). Our data therefore suggest that targeting cell cycle arrest programs may provide a suitable target for therapeutic intervention.

	PATIENTS AND METHODS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION AUTHORS’ DISCLOSURES OF... REFERENCES

 
Patients
In this study, 66 patients with rectal cancer, endosonographically classified as uT3 (n = 58) or uT4 (n = 8) without distant metastases, underwent preoperative RCT alone (n = 35) or combined with regional hyperthermia (hyperthermic radiochemotherapy [HRCT]; n = 31). The mean age was 59 years (range, 39 to 74 years) with a sex distribution of 25 women (38%) and 41 men (62%). Ten patients were treated in a phase II study, a detailed description of which is available,¹⁷ and 10 patients were treated outside the protocol. (One patient had a gastric cancer 13 years ago without any signs of recurrent disease, and nine patients rejected random assignment.) Forty-six patients were recruited from a consecutive phase III trial applying the same treatment protocol as the phase II study, and were randomly assigned either in the preoperative RCT or the HRCT arm. Patients were accrued from September 1993 to January 1999. Patient characteristics are presented in Table 1. All patients gave their informed consent for tissue banking and molecular analyses. The study was performed in accordance with the Declaration of Helsinki.

Patients were observed for a median of 39.3 months (range, 11.3 to 83.4 months). Follow-up was done in our outpatient clinic every 3 months for the first 2 years, and every 6 months thereafter.

Tissue Samples and Pathology Evaluation
Biopsies of the representative central tumor area of the rectal cancer were taken by two investigators, (B.R. and U.S.) 1 week before and 4 to 6 weeks after therapy. After therapy, the tumor biopsy was taken from the specimen together with the pathologist. The percentage of tumor regression was determined by the ratio of necrotic or fibrotic areas to the area of intact cells on histopathological examination of the resected material post-RCT. Furthermore, lymphatic and venous vessel infiltration by tumor cells was evaluated in the resected specimen after RCT.

R0 resection was defined as > 1 mm distance to the circumferential resection margin and was microscopically assessed by the pathologist.

Definition of Response
Response to preoperative treatment was classified according to the WHO criteria¹⁸ using all imaging modalities (computed tomography [CT], magnetic resonance imaging [MRI], and endosonography) and endoscopy. Complete remission (CR) was defined after successful surgical resection by histopathological examination of the resected specimen for the absence of vital tumor cells. Partial remission (PR) was defined by a decrease in the depth of tumor infiltration, as determined by pathological examination, compared with the pretherapeutic uT values (ie, a reduction of T category) or in cases in which the maximum tumor diameter (transverse or longitudinal) measured by endoscopy, or CT or MRI, showed a decrease of at least 50%. Responders in this investigation are those patients with a CR or PR, and nonresponders are all others.

Immunohistochemistry for p53, BAX, p21^WAF1/CIP1, hMSH2, and Ki-67
For immunohistochemistry, 4-µm slices from paraffin-embedded tissue were stained applying standard techniques.¹⁸ The primary antibody was a mouse monoclonal antibody for BAX (clone YTH-2D2; Trevigen, Gaithersburg, MD; dilution, 1:750), a mouse monoclonal antibody for p53 (clone DO-7; Dako, Glostrup, Denmark; dilution, 1:75), a mouse monoclonal antibody for p21^WAF1/CIP1 (clone 6B6; Pharmingen, San Diego, CA; dilution, 1:75), a mouse monoclonal antibody directed against Ki-67 (clone MIB-1; Dianova, Hamburg, Germany; dilution, 1:1,000), and a mouse monoclonal antibody specific for hMSH2 (clone FE11; Oncogene Research Products, San Diego, CA; dilution 1:50). Two observers, without knowledge of the clinicopathologic data, performed blinded analysis of slides. Four high-power fields (400x) were evaluated for percentage of positive cells (0% to 100% in 5% steps) and staining intensity (0 to +++). For further analysis, we used the staining index (SI; the product of the percentage of positive cells and staining intensity).

Mutation Analysis of p53
DNA was extracted from biopsy probes snap frozen in liquid nitrogen or collected in a guanidinium thiocyanate buffer and stored at -80°C until analysis. Extraction of genomic DNA was done using the Invisorb Spin Tissue Kit (Invitek, Berlin, Germany). For storage, the DNA was eluted in 10 mmol/L Tris HCl and 0.1 mmol/L EDTA buffer (pH 8.7). p53 mutations in the DNA binding region were detected by single-strand conformation polymorphism–polymerase chain reaction (SSCP-PCR) analysis. Precise description and primer sequences of the method are given elsewhere.^7,19 Briefly, exons 5 to 8 of the DNA binding domain of the p53 gene were amplified, and for SSCP analysis, 5 µL of the amplified fragments were diluted in 7 µL loading buffer (82% formamide, 10 mmol/L NaOH, 50 mmol/L EDTA, bromophenol blue, xylene xyanole dye). The samples were denatured at 95°C for 5 minutes and cooled on ice. The denatured fragments were analyzed on a 10% nondenaturing polyacrylamide gel at 500 V and at 50 mA for 2 hours at 10°C or 22°C, respectively, in a Multiphor electrophoresis chamber (Pharmacia, Freiburg, Germany), and were subsequently visualized by silver staining.

Statistical Analysis
DFS was estimated by the Kaplan-Meier method. Patients were observed for a median of 39.3 months (range, 11.3 to 83.4 months). Recurrent disease was defined as either local relapse or recurrence with distant metastases. Events were defined as documented recurrence. Disease recurrence was diagnosed for local relapse by endosonographic ultrasound, CT, or MRI scan. Distant metastases were identified by routinely performed abdominal transcutaneous sonography and chest x-ray. Additional diagnostic procedures were performed when clinical signs were present. The survival curves were compared using the log-rank and Cox-Mantel test. Univariate analyses were performed using the Cox proportional hazards model. For the immunhistochemical analyses, the median of the protein expression SI was used as cutoff value (ie, median SI or greater was considered as "high expressing," and less than median SI was considered as "low expressing"). For the longitudinal expression studies, the difference of posttherapy and pretherapy SI was calculated for paired samples. A positive value (>0) indicated an increase in expression posttherapy as compared with pretherapy, and a negative value (<0) was defined as decrease after therapy.

For comparison of pre- and posttherapy samples, the Wilcoxon test for paired samples was used. For the comparison of categorical variables, the ² test was applied. All tests were two-sided, and P < .05 was considered statistically significant. The analyses were done using SPSS version 9.0 (SPSS Inc, Chicago, IL).

	RESULTS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION AUTHORS’ DISCLOSURES OF... REFERENCES

 
Pathological Response Parameters
All tumors were resectable, and in 91% of the patients, an R0 resection was achieved. In six patients (9%) the RCT resulted in CR as determined by pathology review (pCR). All six patients with a pCR were R0 resected. The infiltration depth of the tumors after the preoperative RCT is shown in Table 1. Twenty-four patients (36%) presented with locoregional lymph node metastases at the time of surgery. The mean ± SD number of excised lymph nodes and lymph node metastases was 18 ± 12 and 7 ± 11 nodes, respectively. Tumor infiltration of lymph vessels (L1) and venous vessels (V1) was observed in 12 and nine specimens after RCT, respectively. The analysis of tumor grading showed a mainly moderately differentiated tumor in 68% of cases (Table 1). Tumor necrosis of less than 90% of the tumor was seen in 38 specimens, whereas 23 patients showed a tumor necrosis 90% after preoperative RCT. (For five patients, this information was not available.) As expected, the extent of tumor necrosis posttherapy correlated well with the clinical response (P < .001).

Clear prognostic relevance of the pathological characteristics was seen for complete resection, the infiltration depth into the muscularis layer (ypT0 to T2), the absence of lymph node metastases, and the grading of the tumor, as presented in Table 2. In contrast, sex, venous vessel infiltration, and tumor necrosis were not associated with DFS (Table 2).

Regarding correlations of the pretherapy expression profiles and clinicopathological data, we observed a significant correlation between p53 protein expression and the presence of lymph node metastases — patients with lymph node metastases (N1, N2) showed a significantly lower expression of p53 protein. Such a difference was not apparent when p53 mutation was analyzed, indicating that this difference relates to expression of wild-type p53 protein (Table 4). A similar correlation was observed for hMSH2 that also showed higher expression levels in the N0 group (Table 4). This, together with the p53 expression data, suggests that N0 status is associated with an intact p53/hMSH2–signaling pathway. Nevertheless, such a correlation was not found for p21 and BAX. Similarly, hMSH2 levels were associated with tumor grading and tumors with a G0 to G2 grading showed significantly higher hMSH2 levels (Table 4). The only significant correlation concerning p21 was found in the case of the response status where nonresponders tended to have lower p21 expression in the pretherapy tumor samples (Table 4).

View larger version (12K): [in this window] [in a new window]   Fig 1. Impact of pretherapeutic p53 expression level or p53 mutation on disease-free survival (DFS). (A) p53 protein expression, (B) p53 mutation. Solid white line, high expression, dashed line; low expression. Censored patients are indicated. Log-rank, all P > .05.

View larger version (12K): [in this window] [in a new window]   Fig 2. Impact of pretherapy expression of p53 effector genes (A) BAX, (B) hMSH2, and (C) p21 on tumor free survival. Solid white line, high expression; dashed line, low expression. Censored patients are indicated. Log-rank, P = .04 for hMSH2; for all others, P > .05. DFS, disease-free survival.

View larger version (16K): [in this window] [in a new window]   Fig 3. Dynamic expression of p53, BAX, hMSH2, and p21 in paired samples (posttherapy-pretherapy staining index and disease-free survival [DFS]) in longitudinal study. (A) p53, (B) BAX, (C) hMSH2, and (D) p21. Solid white line, longitudinal decrease; dashed line, longitudinal increase of expression. Censored patients are indicated. Log-rank for p21, P = .03; for all others, P > .05.

Pattern of Recurrence and Dynamic Expression of Protein Expression of p53, hMSH2, BAX, and p21^WAF1/CIP1
In 4 of 66 patients, we observed a local tumor recurrence, and in 22 of 66 patients, relapse with distant metastases (3 of whom had local tumor recurrence at the same time). Thus, the statistical power was too low to analyze the impact of the p53 pathway components or Ki-67 on local relapse. Nevertheless, we observed a trend for the dynamic expression of p21^WAF1/CIP1 and Ki-67 being associated with the occurrence of distant metastases (dynamic p21^WAF1/CIP1 increase and dynamic Ki-67 decrease were more frequent in the group with distant metastases, P = .09 and P = .005, respectively).

Proliferative Activity (Ki-67) in Relation to p21 Expression
Tumor cell proliferation, as assessed by Ki-67 expression in the pretherapy tumor samples, had no prognostic relevance per se (Table 5; Fig 4A). In contrast, a clear prognostic relevance for the dynamic expression of Ki-67 was seen — an increase of Ki-67 posttherapy was associated with a 5-year DFS rate of 91%, while a decrease of Ki-67 after therapy was associated with a 5-year DFS rate of only 34% (P = .005; Table 5 and Fig 4B). Ki-67 expression showed, however, no significant association with the p53 status (ie, p53 protein overexpression or p53 mutation; ² test, all P > .05). Nevertheless, we observed an inverse relationship between the longitudinal dynamic profile of p21^WAF1/CIP1 and Ki-67, which failed however, to reach statistical significance (² test, P = .10); in 9 of 10 cases with an increase of p21^WAF1/CIP1 posttherapy, the Ki-67 decreased posttherapy.

View larger version (13K): [in this window] [in a new window]   Fig 4. Ki-67 expression and disease-free survival (DFS). (A) pretherapy Ki-67 expression, (B) longitudinal study of Ki-67 expression (posttherapy-pretherapy staining index). (A) Solid white line, low expression; dashed line, high expression. (B) Solid white line, longitudinal increase; dashed line, longitudinal decrease of expression. Censored patients are indicated. Log-rank for dynamic Ki-67 expression, P < .005; pretherapy Ki-67, P > .05.

	DISCUSSION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION AUTHORS’ DISCLOSURES OF... REFERENCES

 
Anticancer therapies aim for the complete erradication of tumor cells. Nevertheless, it is well known that although some tumors respond to cytotoxic therapies, such as irradiation and chemotherapy, as expected, other tumors persist or relapse early after therapy. Until now, little has been known about the mechanisms responsible for persistence or recurrence of tumor cells after cytotoxic therapies. One difficulty is the rare availability of longitudinal tumor material (ie, tumor samples before and after cytotoxic therapy). Here, we present a study of 66 patients with advanced rectal carcinoma who were uniformly treated with a multimodal regimen consisting of RCT with or without hyperthermia.

It is widely accepted that antitumor therapies (here, FU/folinic acid, irradiation, and hyperthermia) induce apoptosis. A central regulator of the minute balance of cell death and cell proliferation is the p53 gene.¹¹ The p53 tumor suppressor can be activated by endogenous as well as exogenous stimuli, and it is involved in DNA repair, cell cycle regulation and induction of apoptosis. We previously observed that a pathway analysis of p53 and its downstream pro-apoptotic effector BAX identifies patients with poor or good prognosis in colorectal^7,8 and in esophageal carcinoma.⁶ Furthermore, we found a high proliferative activity to be associated with reduced survival in metastatic colorectal cancer.²¹ We therefore analyzed p53 mutation and overexpression in combination with expression of key downstream effectors of p53 (ie, the proapoptotic BAX protein, the cell cycle inhibitor p21^WAF1/CIP1, and the DNA mismatch repair enzyme hMSH2) in paired tumor samples obtained before and after RCT.

In this setting, we observed a central role for p21 and its dynamic expression under RCT. While pretherapy p21 expression did not have a clear prognostic impact, the posttherapy value is relevant: A reduction of p21 expression in the tumor cells surviving after therapy is associated with improved DFS. In contrast, patients with tumors showing an increase in post-RCT p21 levels had a significantly shorter DFS. Of note, the dynamic expression profiles could be investigated only in those patients in whom vital tumor cells were still present in the resected specimen after RCT (ie, a prognostically unfavorable cohort). Thus, the induction of p21 expression (or selection of p21 expressing tumor cells) seems to be a major factor in the development of resistance to RCT.

Previous in vitro experiments depicted the role of p21 in cancer therapy. p21 is a cyclin dependent kinase inhibitor capable of inducing cell cycle arrest at checkpoints in the G1, S, and G2 phases of the cell cycle. More importantly, experiments in a cell line model showed that p21 expression is not only linked to cell cycle arrest but also with chemoresistance — HCT116 cells with a targeted p21^WAF1/CIP1 gene knockout failed to undergo cell cycle arrest, showed DNA endoreplication, and underwent apoptosis on DNA damaging chemotherapy.¹⁶ Similarly, intact cell cycle checkpoint control (ie, G1 or G2/M arrest after ionizing irradiation) have been connected in vitro to apoptosis resistance and tumor cell survival resulting in regrowth of tumor cells.^22,23 In line with these findings, downregulation of p21 by antisense treatment impaired G2 checkpoint control and radiosensitized cancer cells.²⁴ In our series, we observed a concomitant increase of wild-type p53 and p21^WAF1/CIP1 protein expression. Since p53 is a well established transcriptional activator of p21,¹² this implies that the induction of p21 expression we observed in a subset of the rectal carcinomas occurs via p53-dependent transcription. Nevertheless, the increase of p21^WAF1/CIP1 expression may occur by selection of tumor cells with a priori high p21^WAF1/CIP1 expression. Unlike the situation with B-cell chronic lymphocytic leukemia, where prolonged treatment with alkylating anticancer drugs is related to the occurrence of p53 mutation and development of resistant disease,²⁵ we did not observe an increased rate of p53 mutations after preoperative RCT.

Our finding that induction of p21 is linked to poor prognosis is supported by the analysis of tumor cell proliferation. As expected, the patients with higher p21 expression are mainly the same as those with a reduced Ki-67 expression. Thus, induction of p21 was linked to a decrease in proliferative activity. This, in turn, shows that the increase in p21 is functionally relevant and is a further argument for the functional integrity of the p21 detected posttherapy. Similar to p21, the reduction of Ki-67 expression after RCT was associated with a worse DFS.

The pretherapeutic p21 expression level was previously suggested to affect disease prognosis in colon cancer.²⁶ Nevertheless, we did not observe such a prognostic impact of p21 in this and in previous studies.⁷ This is well in line with a recent report that clearly confirmed that the constitutive levels of p21 expression are not predictive for the response to adjuvant chemotherapy in colon cancer.²⁷ Thus, the dynamic expression and the propensity of carcinoma cells to upregulate p21 levels appears to be important rather than constitutive p21 expression.

There is not much mechanistic evidence available as to how p21 can antagonize chemosensitivity. The majority of reports suggest that p21 inhibits cell death indirectly, ie, through its cell cycle arresting function. Inhibition of the cell cycle at the the G1/S boundary through interaction with the cyclin-dependent kinases cdk2, cdk4, and cdk6 prevents entry into the S phase, where cells are most sensitive to DNA damage.²⁸ Thus, G1 arrest by p21 can counteract sensitivity to DNA damaging therapies by preventing DNA synthesis and accumulation of replication errors. Inhibition of G2/M progression prevents the uncoordinated entry into mitosis and is involved in DNA-damage activated checkpoint control. The best evidence for loss of G2 checkpoint control as the major mechanism underlying sensitization for apoptosis on inactivation of p21 comes again from the HCT116 system. There, exposure to DNA damaging treatment results in G2 arrest in p21 proficient cells while HCT116 p21 knockout cells undergo DNA endoreplication followed by apoptosis.¹⁶ p21-sensitive checkpoint control occurs in concert with p53 that maintains G2 checkpoint control not only via a p21, but also by a 14-3-3–dependent pathway.^29,30 Nevertheless, there is also evidence that p21 can directly inhibit signaling for apoptosis. Evidence for such a mechanism comes from the physical interaction of p21 with CARB (Cip-1–associated regulator of cyclin B), a death promoting protein that induces apoptosis in the absence of p21.³¹

Another interesting finding of our study is that p53 expression and mutation and BAX expression levels do not correlate with disease prognosis. This is in contrast to our previous investigations in gastrointestinal cancer in which inactivation of the p53/BAX pathway was associated with a shorter overall survival.^6–8 We favor the interpretation that RCT (and hyperthermia in a subset of the patients) may override defects in the p53/BAX pathway. This is especially relevant since the patients in our previous studies were uniformly treated with surgery and additional cytotoxic treatment was not applied in general.

Evidence has accumulated that members of the DNA-mismatch repair system, including hMSH2, serve as a detection system for DNA damage that activates apoptotic signal-transduction pathways and cell cycle arrest.³² In this study, we demonstrate for the first time that a high pretherapeutic hMSH2 expression correlates with a significantly better 5-year DFS. This is of considerable interest in view of the impact of mismatch repair deficiency on disease prognosis in colon cancer in which patients with tumors arising in consequence of mismatch repair deficiency (which may have lost hMSH2 expression³³) tend to have a better disease prognosis. We also see a significantly higher hMSH2 expression in tumors without lymph node involvement and in low-grade tumors. One possible explanation is that high hMSH2 expression is a characteristic of a more global "early stage disease." According to the entrance criteria in this study (locally advanced rectal carcinoma), we could not determine whether there is also an association between low infiltration depth (ie, T stage) and hMSH2 expression. Nevertheless, our observation is well in line with in vitro data that demonstrated a high activity of the DNA-mismatch repair system to be correlated with sensitivity of cancer cells to DNA damaging therapies.^32,34 Thus, loss of hMSH2 may impair the activation of apoptosis. Another study described downregulation of hMSH2 in ovarian carcinoma in therapy with cisplatinum.³⁵ This was not the case in our study, and RCT did not affect the hMSH2 expression status in our patient cohort. Finally, loss of hMSH2 may impede, like loss of p21, the G2/M arrest program.^36,37 Of note, inactivation of hMSH2-signaling may be involved in resistance to FU-based anticancer therapy,³⁸ while the tumor cell survival after ionizing irradiation does not seem to depend on hMSH2 pathway.³⁹ Thus, our data on hMSH2 clearly delineate the relevance of DNA repair pathways and cell cycle arrest programs in the response to cytotoxic chemotherapies. This study does not include results from multivariate analysis. Such data would be interesting with respect to the interaction of the variables with each other and with clinicopathological data. However, the number of patients in this study did not allow us to perform such an analysis reliably. This question should therefore be clarified in a prospective study.

Altogether, we show in this report that the dynamic induction of genes acting in DNA damage response pathways may play a key role in treatment outcome following RCT in rectal carcinoma. The induction of p21^WAF1/CIP1 resulted not only in shorter DFS but was also associated with a decrease in tumor cell proliferation. Apart from delineating a novel, p21^WAF1/CIP1-mediated, resistance mechanism, these data therefore suggest that targeting cell cycle checkpoint signaling and arrest programs may provide a suitable target for therapeutic intervention. Evidence for the usefulness of such a strategy comes again from cell line data for which disruption of G2/M checkpoint control (eg, by the use of flavopiridol, an inhibitor of cyclin dependent and other kinases) sensitizes carcinoma cells for chemotherapeutics.^40–43 Such strategies therefore warrant evaluation in a clinical setting to overcome acquired resistance to therapy in consequence of dynamic induction of p21^WAF1/CIP1.

	AUTHORS’ DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION AUTHORS’ DISCLOSURES OF... REFERENCES

 
The authors indicated no potential conflicts of interest.

	ACKNOWLEDGMENTS

 
We thank Jana Rossius and Sylvia Scheele for expert technical assistance.

	NOTES

 
Supported by the Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 273 and 506, and grant No. Da238/4.

	REFERENCES

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION AUTHORS’ DISCLOSURES OF... REFERENCES

 
1. Evan GI, Vousden KH: Proliferation, cell cycle, and apoptosis in cancer. Nature 411:342–348, 2001[CrossRef][Medline]

2. Daniel PT: Dissecting the pathways to death. Leukemia 14:2035–2044, 2000[CrossRef][Medline]

3. Radetzki S, Köhne CH, von Haefen C, et al: The apoptosis promoting Bcl-2 homologues Bak and Nbk/Bik overcome drug resistance in Mdr-1-negative and Mdr-1 overexpressing breast cancer cell lines. Oncogene 21:227–238, 2002[CrossRef][Medline]

4. Friedrich K, Wieder T, von Haefen C, et al: Overexpression of caspase-3 restores sensitivity for drug-induced apoptosis in breast cancer cells with acquired drug resistance. Oncogene 20:2460–2749, 2001

5. Bosanquet AG, Sturm I, Wieder T, et al: BAX expression correlates with cellular drug sensitivity to doxorubicin, cyclophosphamide and chlorambucil but not fludarabine, cladribine, or corticosteroids in B cell chronic lymphocytic leukemia. Leukemia 16:1035–1044, 2002[CrossRef][Medline]

6. Sturm I, Petrowsky H, Volz R, et al: Analysis of p53/BAX/p16ink4a/CDKN2 in esophageal squamous cell carcinoma: High BAX and p16ink4a/CDKN2 identifies patients with good prognosis. J Clin Oncol 19:2272–2281, 2001[Abstract/Free Full Text]

7. Sturm I, Kohne CH, Wolff G, et al: Analysis of the p53/BAX pathway in colorectal cancer: Low BAX is a negative prognostic factor in patients with resected liver metastases. J Clin Oncol 17:1364–1374, 1999[Abstract/Free Full Text]

8. Schelwies K, Sturm I, Grabowski P, et al: Analysis of p53/BAX in primary colorectal carcinoma: Low BAX protein expression is a negative prognostic factor in UICC stage III tumors. Int J Cancer 99:589–596, 2002[CrossRef][Medline]

9. Mrozek A, Petrowsky H, Sturm I, et al: Combined p53/BAX mutation results in extremely poor prognosis in gastric carcinoma with low microsatellite instability. Cell Death Differ 10:461–467, 2003[CrossRef][Medline]

10. Güner D, Sturm I, Hemmati PG, et al: Multigene analysis of Rb-pathway and apoptosis-control in esophageal squamous cell carcinoma identifies patients with good prognosis. Int J Cancer 103:445–454, 2003[CrossRef][Medline]

11. May P, May E: Twenty years of p53 research: Structural and functional aspects of the p53 protein. Oncogene 18:7621–7636, 1999[CrossRef][Medline]

12. El-Deiry W, Harper JW, O’Connor PM, et al: WAF1/CIP1 is induced in p53-mediated G1 arrest and apoptosis. Cancer Res 54:1169–1174, 1994[Abstract/Free Full Text]

13. Miyashita T, Reed JC: Tumor suppressor p53 is a direct transcriptional activator of the human BAX gene. Cell 80:293–299, 1995[CrossRef][Medline]

14. Warnick CT, Dabbas B, Ford CD, et al: Identification of a p53 response element in the promoter region of the hMSH2 gene required for expression in A2780 ovarian cancer cells. J Biol Chem 276:27363–27370, 2001[Abstract/Free Full Text]

15. Scherer SJ, Maier SM, Seifert M, et al: p53 and c-Jun functionally synergize in the regulation of the DNA repair gene hMSH2 in response to UV. J Biol Chem 275:37469–37473, 2000[Abstract/Free Full Text]

16. Waldman T, Lengauer C, Kinzler KW, et al: Uncoupling of S phase and mitosis induced by anticancer agents in cells lacking p21. Nature 381:713–716, 1996[CrossRef][Medline]

17. Rau B, Wust P, Hohenberger P, et al: Preoperative hyperthermia combined with radiochemotherapy in locally advanced rectal cancer: A phase II clinical trial. Ann Surg 227:380–389, 1998[CrossRef][Medline]

18. Miller AB, Hoogstraten B, Staquet M, et al: Reporting results of cancer treatment. Cancer 47:207–214, 1981[CrossRef][Medline]

19. Sturm I, Papadopoulos S, Hillebrand T, et al: Impaired BAX protein expression in breast cancer: Mutational analysis of the BAX and the p53 gene. Int J Cancer 87:517–521, 2000[CrossRef][Medline]

20. Hermann S, Sturm I, Mrozek A, et al: BAX expression in benign and malignant thyroid tumors: Dysregulation of wild-type p53 is associated with a high BAX and p21 expression in thyroid carcinoma. Int J Cancer 92:805–811, 2001[CrossRef][Medline]

21. Petrowsky H, Sturm I, Graubitz O, et al: Relevance of Ki-67 antigen expression and K-ras mutation in colorectal liver metastases. Eur J Surg Oncol 27:80–87, 2001[CrossRef][Medline]

22. Wouters BG, Giaccia AJ, Denko NC, et al: Loss of p21WAF1/CIP1 sensitizes tumors to radiation by an apoptosis-independent mechanism. Cancer Res 57:4703–4706, 1997[Abstract/Free Full Text]

23. Wang YA, Elson A, Leder P: Loss of p21 increases sensitivity to ionizing radiation and delays the onset of lymphoma in atm-deficient mice. Proc Natl Acad Sci U S A 94:14590–14595, 1997[Abstract/Free Full Text]

24. Tian H, Wittmack EK, Jorgensen TJ: p21WAF1/CIP1 antisense therapy radiosensitizes human colon cancer by converting growth arrest to apoptosis. Cancer Res 60:679–684, 2000[Abstract/Free Full Text]

25. Sturm I, Bosanquet AG, Hermann S, et al: Mutation of p53 and consecutive selective drug resistance in B-CLL occurs as a consequence of prior DNA damaging chemotherapy. Cell Death Differ 10:477–484, 2003[CrossRef][Medline]

26. Tsihlias J, Kapusta L, Slingerland J: The prognostic significance of altered cyclin-dependent kinase inhibitors in human cancer. Annu Rev Med 50:401–423, 1999[CrossRef][Medline]

27. Watanabe T, Wu TT, Catalano PJ, et al: Molecular predictors of survival after adjuvant chemotherapy for colon cancer. N Engl J Med 344:1196–1206, 2001[Abstract/Free Full Text]

28. Loeb LA, Kunkel TA: Fidelity of DNA synthesis. Annu Rev Biochem 51:429–457, 1982[CrossRef][Medline]

29. Chan TA, Hwang PM, Hermeking H, et al: Cooperative effects of genes controlling the G(2)/M checkpoint. Genes Dev 14:1584–1588, 2000[Abstract/Free Full Text]

30. Bunz F, Dutriaux A, Lengauer C, et al: Requirement for p53 and p21 to sustain G2 arrest after DNA damage. Science 282:1497–1501, 1998[Abstract/Free Full Text]

31. McShea A, Samuel T, Eppel JT, et al: Identification of CIP-1-associated regulator of cyclin B (CARB), a novel p21-binding protein acting in the G2 phase of the cell cycle. J Biol Chem 275:23181–23186, 2000[Abstract/Free Full Text]

32. Lage H, Dietel M: Involvement of the DNA mismatch repair system in antineoplastic drug resistance. J Cancer Res Clin Oncol 125:156–165, 1999[CrossRef][Medline]

33. Dietmaier W, Wallinger S, Bocker T, et al: Diagnostic microsatellite instability: Definition and correlation with mismatch repair protein expression. Cancer Res 57:4749–4756, 1997[Abstract/Free Full Text]

34. Fujieda S, Tanaka N, Sunaga H, et al: Expression of hMSH2 correlates with in vitro chemosensitivity to CDDP cytotoxicity in oral and oropharyngeal carcinoma. Cancer Lett 132:37–44, 1998[CrossRef][Medline]

35. Samimi G, Fink D, Varki NM, et al: Analysis of MLH1 and MSH2 expression in ovarian cancer before and after platinum drug-based chemotherapy. Clin Cancer Res 6:1415–1421, 2000[Abstract/Free Full Text]

36. Strathdee G, Sansom OJ, Sim A, et al: A role for mismatch repair in control of DNA ploidy following DNA damage. Oncogene 20:1923–1927, 2001[CrossRef][Medline]

37. Hawn MT, Umar A, Carethers JM, et al: Evidence for a connection between the mismatch repair system and the G2 cell cycle checkpoint. Cancer Res 55:3721–3725, 1995[Abstract/Free Full Text]

38. Carethers JM, Chauhan DP, Fink D, et al: Mismatch repair proficiency and in vitro response to 5-fluorouracil. Gastroenterology 117:123–131, 1999[CrossRef][Medline]

39. Yan T, Schupp JE, Hwang HS, et al: Loss of DNA mismatch repair imparts defective cdc2 signaling and G(2) arrest responses without altering survival after ionizing radiation. Cancer Res 61:8290–8297, 2001[Abstract/Free Full Text]

40. Li W, Fan J, Bertino JR: Selective sensitization of retinoblastoma protein-deficient sarcoma cells to doxorubicin by flavopiridol-mediated inhibition of cyclin- dependent kinase 2 kinase activity. Cancer Res 61:2579–2582, 2001[Abstract/Free Full Text]

41. Hirose Y, Berger MS, Pieper RO: Abrogation of the Chk1-mediated G(2) checkpoint pathway potentiates temozolomide-induced toxicity in a p53-independent manner in human glioblastoma cells. Cancer Res 61:5843–5849, 2001[Abstract/Free Full Text]

42. Motwani M, Delohery TM, Schwartz GK: Sequential dependent enhancement of caspase activation and apoptosis by flavopiridol on paclitaxel-treated human gastric and breast cancer cells. Clin Cancer Res 5:1876–1883, 1999[Abstract/Free Full Text]

43. Schwartz GK, O’Reilly E, Ilson D, et al: Phase I study of the cyclin-dependent kinase inhibitor flavopiridol in combination with paclitaxel in patients with advanced solid tumors. J Clin Oncol 20:2157–2170, 2002[Abstract/Free Full Text]

Submitted July 15, 2002; accepted February 26, 2003.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Facebook    Reddit    Technorati    Twitter    What's this?

This article has been cited by other articles:

				A. Debucquoy, K. Haustermans, A. Daemen, S. Aydin, L. Libbrecht, O. Gevaert, B. De Moor, S. Tejpar, W. H. McBride, F. Penninckx, et al. Molecular Response to Cetuximab and Efficacy of Preoperative Cetuximab-Based Chemoradiation in Rectal Cancer J. Clin. Oncol., June 10, 2009; 27(17): 2751 - 2757. [Abstract] [Full Text] [PDF]

				P. Grabowski, J. Schrader, J. Wagner, D. Horsch, R. Arnold, C. N. Arnold, I. Georgieva, H. Stein, M. Zeitz, P. T. Daniel, et al. Loss of Nuclear p27 Expression and Its Prognostic Role in Relation to Cyclin E and p53 Mutation in Gastroenteropancreatic Neuroendocrine Tumors Clin. Cancer Res., November 15, 2008; 14(22): 7378 - 7384. [Abstract] [Full Text] [PDF]

				G. L. Cascini, A. Avallone, P. Delrio, C. Guida, F. Tatangelo, P. Marone, L. Aloj, F. De Martinis, P. Comella, V. Parisi, et al. 18F-FDG PET Is an Early Predictor of Pathologic Tumor Response to Preoperative Radiochemotherapy in Locally Advanced Rectal Cancer J. Nucl. Med., August 1, 2006; 47(8): 1241 - 1248. [Abstract] [Full Text] [PDF]

				A. L. Gartel Is p21 an oncogene? Mol. Cancer Ther., June 1, 2006; 5(6): 1385 - 1386. [Full Text] [PDF]

				H.-C. Kwon, C. H. Moon, S.-H. Kim, H.-J. Choi, H.-S. Lee, M. S. Roh, T.-H. Hwang, J.-S. Kim, and H.-J. Kim Expression of Double-stranded RNA-activated Protein kinase (PKR) and its Prognostic Significance in Lymph Node Negative Rectal Cancer Jpn. J. Clin. Oncol., September 1, 2005; 35(9): 545 - 550. [Abstract] [Full Text] [PDF]

				G. K. Schwartz Development of Cell Cycle Active Drugs for the Treatment of Gastrointestinal Cancers: A New Approach to Cancer Therapy J. Clin. Oncol., July 10, 2005; 23(20): 4499 - 4508. [Abstract] [Full Text] [PDF]

				B. M. Ghadimi, M. Grade, M. J. Difilippantonio, S. Varma, R. Simon, C. Montagna, L. Fuzesi, C. Langer, H. Becker, T. Liersch, et al. Effectiveness of Gene Expression Profiling for Response Prediction of Rectal Adenocarcinomas to Preoperative Chemoradiotherapy J. Clin. Oncol., March 20, 2005; 23(9): 1826 - 1838. [Abstract] [Full Text] [PDF]

				S. A. Waldman Markers of Cell Cycle-Mediated Drug Resistance And Prognosis Of Patients Receiving Preoperative Combined Modality Therapy For Rectal Cancer Ann. Surg. Oncol., November 1, 2004; 11(11): 949 - 950. [Full Text] [PDF]

				H. G. Moore, J. Shia, D. S. Klimstra, L. Ruo, M. Mazumdar, G. K. Schwartz, B. D. Minsky, L. Saltz, and J. G. Guillem Expression of p27 in Residual Rectal Cancer after Preoperative Chemoradiation Predicts Long-term Outcome Ann. Surg. Oncol., November 1, 2004; 11(11): 955 - 961. [Abstract] [Full Text] [PDF]

				J. I. Geller, K. Szekely-Szucs, I. Petak, B. Doyle, and J. A. Houghton P21Cip1 Is a Critical Mediator of the Cytotoxic Action of Thymidylate Synthase Inhibitors in Colorectal Carcinoma Cells Cancer Res., September 1, 2004; 64(17): 6296 - 6303. [Abstract] [Full Text] [PDF]

				C. H. Crane, H. D. Thames, and S. R. Hamilton Will Identifying or Targeting Altered Marker Expression in Response to Cytotoxic Therapy Be of Prognostic or Therapeutic Value? J. Clin. Oncol., September 15, 2003; 21(18): 3381 - 3382. [Full Text] [PDF]

This Article Abstract Full Text (PDF) Erratum (v22,p576) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Email this article to a colleague Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Save to my personal folders Download to citation manager Rights & Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Rau, B. Articles by Daniel, P. T. Search for Related Content PubMed PubMed Citation Articles by Rau, B. Articles by Daniel, P. T. Social Bookmarking                            What's this?