p21WAF1 expression is associated with improved survival after adjuvant chemoradiation for pancreatic cancer

p21WAF1 expression is associated with improved survival after adjuvant chemoradiation for pancreatic cancer Steven A. Ahrendt, MD, Heather M. Brown, MD, Richard A. Komorowski, MD, Yong-ran Zhu, MD, Stuart D. Wilson, MD, Beth A. Erickson, MD, Paul S. Ritch, MD, Henry A. Pitt, MD, and Michael J. Demeure, MD, Milwaukee, Wis

Background. Cell cycle arrest after DNA damage is partly mediated through the transcriptional activation of p21WAF1 by the p53 tumor suppressor gene. p21WAF1 and p53 are both critical in maintaining cell cycle control in response to DNA damage from radiation or chemotherapy. Therefore, we examined the role of p21WAF1 and p53 in the determination of outcome for patients who receive radiation and/or chemotherapy for pancreatic cancer. Methods. p21WAF1 and p53 protein expression were determined (with the use of immunohistochemistry) in specimens from 90 patients with pancreatic cancer. Forty-four patients underwent surgical resection, and 46 patients had either locally unresectable tumors (n = 9 patients) or distant metastases (n = 37 patients). Seventy-three percent of the patients who underwent resection and 63% of the patients who did not undergo resection received radiation and/or chemotherapy. Results. p21WAF1 expression was present in 48 of 86 tumors (56%) and was significantly (P < .05) associated with advanced tumor stage. Median survival among patients with resected pancreatic cancer who received adjuvant chemoradiation with p21WAF1-positive tumors was significantly longer than in patients with no p21WAF1 staining (25 vs 11 months; P = .01). Fifty of 89 tumors (56%) stained positive for p53 protein. p53 overexpression was associated with decreased survival in patients who did not undergo resection. Conclusions. Normal p21WAF1 expression may be necessary for a beneficial response to current adjuvant chemoradiation protocols for pancreatic cancer. Alternate strategies for adjuvant therapy should be explored for patients with pancreatic cancer who lack functional p21WAF1.(Surgery 2000;128:520-30.) From the Departments of Surgery, Pathology, Radiation Oncology, and Medicine, Medical College of Wisconsin, Milwaukee, Wis

PANCREATIC CANCER IS THE FIFTH leading cause of cancer deaths in the United States with an overall 5-year survival rate after diagnosis of 1% to 2%.1 Most patients with pancreatic cancer have metastases at the time of presentation, and current chemotherapeutic agents have had little impact on survival in these patients. For a few patients with localized pancreatic cancer, resection offers the only chance for long-term survival. However, 5-year survival rates for patients with pancreatic cancer who have undergone resection remain only 18% Presented at the 57th Annual Meeting of the Central Surgical Association, Chicago, Ill, March 2-4, 2000. Reprint requests: Michael J. Demeure, MD, Department of Surgery, The Medical College of Wisconsin, 9200 West Wisconsin Ave, Milwaukee, WI 53226. Copyright © 2000 by Mosby, Inc. 0039-6060/2000/$12.00 + 0 11/6/108052 doi:10.1067/msy.2000.108052

520 SURGERY

to 24%.2,3 A small randomized trial published in 1987 by the Gastrointestinal Tumor Study Group demonstrated improved median and long-term survival rates in patients who received adjuvant 5FU and external beam radiation.4 A larger nonrandomized series has confirmed these findings, with a significant increase in the median survival from 14 to 20 months in patients who received postoperative adjuvant therapy.2 More recently, the European Organization for Research and Treatment of Cancer has completed a prospective randomized trial of adjuvant chemoradiation using infusional 5-FU versus observation in 114 patients with pancreatic cancer.3 Although the median survival time was increased with chemoradiation from 13 to 17 months, this increase failed to reach statistical significance (P = .099).3 Inactivation of the p53 gene is common in most human cancers, including more than 50% of pancreatic cancers.5 Wild-type p53 plays an important

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role in the cellular response to DNA damage from chemotherapeutic agents and irradiation.6 In response to DNA damage, p53 induces the synthesis of the cell-cycle inhibitor p21WAF1, which leads to the inhibition of the cyclin-cdk complexes that are required for the transition from G1 to S phase.7,8 Both p53 and p21WAF1 are also required for the maintenance of the G2/M checkpoint, which prevents cells from progressing into mitosis in the presence of unrepaired chromosomal alterations.7 Numerous studies have examined the effects of p53 and p21WAF1 inactivation on the response to chemotherapy and radiation.6,9,10 However, the results have been inconsistent; some studies have indicated increased sensitivity, and other studies have indicated decreased sensitivity to the same agents.6 Although adjuvant chemoradiation increases median and long-term survival and benefits some patients with pancreatic cancer, one third of patients clearly derive little benefit, dying within 1 year of diagnosis.2,3 To further distinguish which patients are most likely to benefit from adjuvant therapy, we examined the effect of p21WAF1 and p53 expression on the response to chemotherapy and radiation therapy in patients with pancreatic cancer. PATIENTS AND METHODS Patient characteristics. Formalin-fixed, paraffinembedded tumor tissue was available from 90 patients who were diagnosed with pancreatic ductal adenocarcinoma at Froedtert Memorial Lutheran Hospital/Medical College of Wisconsin between April 1994 and May 1999. Distal bile duct, ampullary, duodenal adenocarcinomas, and other pancreatic neoplasms were excluded from this study. The diagnosis of pancreatic cancer was confirmed in 85 patients at either open or laparoscopic exploratory operation and by percutaneous or endoscopic needle biopsy in 5 patients with metastatic disease. Fortyfour patients had resectable tumors (pancreatoduodenectomy, n = 37 patients; distal pancreatectomy, n = 5 patients; or total pancreatectomy, n = 2 patients). The remaining 46 patients had either locally unresectable tumors (n = 9 patients) or metastatic disease (n = 37 patients). All pathology reports were reviewed, and the TNM stage and grade were assigned with the use of the American Joint Committee on Cancer criteria.11 Surgical margins were considered positive if infiltrating adenocarcinoma was present at the uncinate process, retroperitoneal soft tissue, or final pancreatic neck margin. Clinical information was obtained from a review of hospital and physician charts or from the Froedtert Memorial Lutheran Hospital Tumor

Table I. Correlation between p53 overexpression and p21 expression in pancreatic cancer (P = .9) p53 Negative (%) p53 Positive (%)

p21 Negative (n)

p21 Positive (n)

17 (20) 20 (24)

20 (24) 28 (33)

Registry. Sixty-one patients (68%) received external beam radiation and/or chemotherapy. Twentynine of the 44 patients who underwent resection (66%) received postoperative adjuvant chemoradiation. Standard therapy included external beam irradiation to the pancreatic bed and regional lymphatics (5040-5400 cGy) with concurrent infusional 5-FU (before January 1998) or infusional 5-FU and weekly gemcitabine (after January 1998). Three patients with resected pancreatic cancer received chemotherapy or radiation therapy alone; 12 patients with resected pancreatic cancer declined further treatment. Twenty-seven patients with unresectable pancreatic cancer (59%) also received both combined radiation and chemotherapy (locally unresectable disease) or chemotherapy alone (distant metastases). Immunohistochemistry. Three-micron tissue sections were deparaffinized, rehydrated, and incubated in methanol/hydrogen peroxide. After antigen retrieval (DAKO Target Retrievel; DAKO Corp, Carpinteria, Calif) and blocking, sections were incubated with either anti-human p53 (dilution 1:2000, D07, code number M7001; DAKO) or p21WAF1 (dilution 1:100, SX118, code number M7202; DAKO) monoclonal antibody. Immunolocalization was performed with the Immunostaining System LSAB (number K0690; DAKO) and chromagen. Slides were counterstained with hematoxylin before dehydration and mounting. All sections were reviewed by 2 pathologists (H.M.B., R.A.K.) and graded on the basis of the percentage of tumor cells that demonstrated p53positive or p21-positive staining. Only nuclear staining was regarded as positive. Tumors were considered positive for p53 immunoreactivity when more than 33% of the nuclei exhibited staining and considered positive for p21 immunoreactivity when more than 5% of nuclei exhibited staining. Statistical analysis. Data are presented as mean ± SEM. Clinical and pathologic characteristics among groups were compared with chi-squared or Fisher’s exact test for categoric and the Student t test for continuous variables. Survival curves were generated by the Kaplan-Meier method. Survival among groups was compared with the use of the log-rank test. Pathologic and clinical variables and

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Table II. Clinical and pathologic characteristics of pancreatic cancer with normal and abnormal p21 and p53 expression p21

No. of patients (%) Age (y) Gender (n) Male (%) Female (%) Race (n) White (%) Black (%) Hispanic (%) Smoking history (n) None (%) Positive (%) Ethanol intake <1 Drink/d ≥1 Drink/d Tumor stage 1 2 3 4 Resection (n) Yes (%) No (%)

p53

Negative

Positive

Negative

Positive

38(44) 63 ± 1

48(56) 63 ± 1

39(44) 61 ± 1

50(56) 65 ± 1

25(53) 13(33)

22(47) 26(67)

23(53) 16(33)

24(47) 26(67)

36(45) 1(25) 1(50)

44(55) 3(75) 1(50)

35(45) 3(25) 1(50)

48(55) 1(75) 1(50)

17(49) 19(40)

18(51) 29(60)

12(33) 24(50)

24(67) 24(50)

11(46) 23(43)

13(54) 30(57)

12(50) 24(43)

12(50) 32(57)

10(71) 3(50) 11(55) 14(30)

4(29)* 3(50) 9(45) 32(70)†

6(43) 1(17) 11(50) 21(45)

8(57) 5(83) 11(50) 26(55)

25(60) 13(30)

17(40)‡ 31(70)

17(40) 22(48)

26(60) 24(52)

Data are presented as number (percentage) or as mean ± SEM. *P = .01 versus stage IV patients. †P = .008 versus stage I, II, and III patients. ‡P = .01 versus patients who did not undergo resection.

p53 and p21 status were evaluated in univariate and multivariate models to determine their impact on survival. Multivariate analysis was performed with the Cox proportional hazards model. RESULTS Patient and tumor characteristics. The mean age of the 90 patients with pancreatic cancer was 63 ± 1 years. There were 48 men and 42 women, with 84 white patients, 4 black patients, and 2 Hispanic patients. Fifteen patients had stage I tumors; 6 patients had stage II tumors; 22 patients had stage III tumors, and 47 patients had stage IV disease. Fifty-seven percent of the patients (50/87 patients) had a history of cigarette smoking; 69% of the patients had a history of alcohol consumption. The median diameter of the 44 resected cancers was 3.1 cm (range, 0.9-9.0 cm). Lymph node metastases were identified in 23 of the resected specimens (52%). Negative histologic margins were present in 34 of 44 resection specimens (77%). Twelve tumors were well differentiated; 23 tumors were moderately differentiated, and 8 tumors were poorly differentiated.

p21WAF1 and p53 immunohistochemistry. p21WAF1 and p53 immunohistochemistry was interpretable with adequate positive and negative controls in 86 and 89 patients, respectively. p21WAF1 protein expression was present in 48 of 86 pancreatic cancers (56%). Fifty of 89 (56%) tumors stained positive for p53 protein. No correlation was observed between p21WAF1 and p53 expression (Table I). The relationship between patient and tumor characteristics and both p21WAF1 protein expression and p53 overexpression is shown in Table II. p21WAF1 expression was present in 67% of women and 47% of men. In addition, p21WAF1 expression was also significantly (P = .02) associated with advanced tumor stage (stage IV) and unresectable tumors. The mean age of patients with tumors that demonstrated p53 overexpression was greater than of patients with p53negative tumors (65 ± 1 years vs 61 ± 1 years; P < .05). Neither p21WAF1 expression nor p53 overexpression was associated with cigarette smoking or alcohol consumption. The relationship between patient and tumor characteristics and both p21WAF1 protein expression and p53 overexpression among surgically

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Table III. Clinical and pathologic characteristics of resected pancreatic cancer with normal and abnormal p53 and p21 expression p21

No. of patients (%) Age (y) Gender (n) Male (%) Female (%) Tumor size (cm) T stage (n) T1 (%) T2 (%) T3 (%) T4 (%) N stage (n) N0 (%) N1 (%) Nx (%) Tumor stage (n) 1 (%) 2 (%) 3 (%) 4 (%) Tumor grade (n) Poor (%) Moderate (%) Well (%) Margin status (n) Negative (%) Positive (%) *P

p53

Negative

Positive

Negative

Positive

25(60) 64 ± 1

17(40) 64 ± 2

17(40) 61 ± 2

26(60) 65 ± 1

17(74) 8(40) 3.3±0.3

6(26)* 12(60) 3.6±0.5

10(44) 7(35) 3.2±0.2

13(56) 13(65) 3.5±0.4

3(60) 10(67) 11(55) 1(50)

2(40) 5(33) 9(45) 1(50)

1(20) 7(44) 8(40) 1(50)

4(80) 9(56) 12(60) 1(50)

13(65) 11(55) 1(50)

7(35) 9(45) 1(50)

7(35) 9(43) 1(50)

13(65) 12(57) 1(50)

10(71) 3(50) 11(58) 1(33)

4(29) 3(50) 8(42) 2(67)

6(43) 1(17) 9(45) 1(33)

8(57) 5(83) 11(55) 2(67)

5(62) 14(64) 8(62)

3(38) 8(36) 5(38)

2(25) 8(35) 6(55)

6(75) 15(65) 5(45)

17(53) 8(80)

15(47) 2(20)

13(39) 4(40)

20(61) 6(60)

= .05 versus female patients.

resected patients is shown in Table III. p21WAF1 expression was more common in women than men (60% vs 23%; P = .05). No relationship between tumor size, stage, or grade or in the incidence of positive resection margins and either p21WAF1 or p53 expression was observed. Survival analysis.The median follow-up time for the entire group of 90 patients was 10 months and was 8 months (range, 1-64 months) for those patients still alive at the time of the survival analysis. The median survival for all patients with cancer of the pancreatic head, body, tail, or uncinate process who had undergone resection was 18 months, with actuarial 1-, 2-, 3-, and 5-year survival rates of 68%, 25%, 18%, and 18%, respectively. Median and actuarial 5-year survival in patients with resected pancreatic cancer and who received chemo- and/or radiation therapy (20 months; 25%) were significantly (P = .04) longer than in patients with resected pancreatic cancer and who received no adjuvant therapy (16 months; 0%). The median survival for all unresectable pancreat-

ic cancer was 7 months, with actuarial 1-, 2-, 3-, and 5-year survival rates of 28%, 6%, 6%, and 0%, respectively. Clinical and pathologic factors that could potentially impact survival were examined in a univariate model (Table IV). Patients with early stage (I-III) or resectable tumors and those who received either chemotherapy or radiation therapy had a significantly longer survival than patients with stage IV or unresectable tumors or those patients who did not receive additional therapy. Neither p21WAF1 nor p53 expression was predictive of survival in the entire group of patients. A multivariate analysis was undertaken to further define factors associated with survival in the entire group of patients (Table V). Early tumor stage (I-III) and radiation therapy were both independently predictive of increased survival. A similar analysis was also completed to identify prognostic factors in the subgroup of patients with resected and unresectable pancreatic cancer (Table IV). Positive p21WAF1 expression was significantly

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Table IV. Univariate analysis of prognostic factors in patients with pancreatic cancer N

Median survival (mo)

All patients (n = 90) Age (y) <60 35 >60 55 Gender Male 48 Female 42 Stage I, II, or III 43 IV 47 Management Resection 44 No resection 46 p53 Positive 50 Negative 39 p21 Positive 48 Negative 38 Chemotherapy Yes 56 No 31 Radiation therapy Yes 40 No 45 Patients with resection (n = 44) Gender Male 24 Female 20 Nodal status Negative 20 Positive 23 Tumor size <3.0 cm 22 >3.0 cm 24

P value

11.2 10.5

.48

11.0 10.8

.26

16.7 8.3

.001

18.2 7.0

.0001

10.0 11.4

.82

11.7 10.5

.43

12.7 7.4

.001

16.7 6.8

.0001

15.7 19.7

.19

19.2 17.5

.35

16.2 19.3

.29

N

Median survival (mo)

p53 Positive 26 Negative 17 p21 Positive 17 Negative 25 Chemotherapy Yes 30 No 13 Radiation therapy Yes 31 No 12 Margin status Positive 10 Negative 34 Patients with no resection (n = 46) Metastases Present 36 Absent 10 p53 Positive 24 Negative 22 p21 Positive 31 Negative 13 Chemotherapy Yes 26 No 18 Radiation therapy Yes 9 No 33

P value

18.4 16.4

.46

22.2 12.5

.006

19.4 16.3

.03

20.0 15.8

.01

14.3 18.2

.86

5.9 14.2

.001

5.8 9.7

.02

6.9 6.9

.67

9.6 4.9

.03

12.7 5.3

.01

Table V. Multivariate analysis of prognostic factors in pancreatic cancer Hazard ratio

95% Confidence interval

.0001 .91 .03

3.46 1.03 0.52

0.17-0.50 0.60-1.80 0.29-0.93

.002 .07

0.35 0.6

0.18-0.67 0.33-1.06

.003 .04

4.41 2.02

1.63-11.9 1.01-4.06

P value All patients (n = 90) Stage IV Chemotherapy Radiation therapy Patients with resection (n = 44) p21WAF1 expression Chemoradiation Patients with no resection (n = 46) Metastases p53 overexpression

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Fig 1. Actuarial survival for patients with unresectable pancreatic cancer (n = 46 patients). Survival in patients with p53-negative tumors (n = 22 patients) was significantly (P = .02) longer than in patients with p53-positive staining.

Fig 2. Actuarial survival for patients with resected pancreatic cancer (n = 40 patients). Patients with p21positive tumors who underwent resection and who received chemoradiation therapy (n = 11 patients) survived significantly longer than patients with p21-negative tumors who received chemoradiation therapy (n = 18 patients; P = .02) or patients who were not treated with adjuvant therapy (n = 11 patients; P < .001). Three patients who received chemotherapy or radiation therapy alone were not included.

associated with increased survival (22.2 vs 12.5 months; P = .006) in the patients with resected pancreatic cancer. Both adjuvant chemotherapy and radiation survival were also associated with a significant increase in survival in the univariate analysis. In a multivariate analysis including p21WAF1 expression, chemotherapy, and radiation therapy, only p21WAF1 expression was independently associated (P = .003) with improved survival. Because the vast majority of patients received chemotherapy and radiation therapy together, we included this treatment as a single

variable (chemoradiation). In this second model, p21WAF1 expression remained a significant predictor of prolonged survival, although a trend towards improved survival was observed with chemoradiation (Table V). In patients with unresectable pancreatic cancer, the presence of metastases and p53-positive staining (Fig 1) were both associated with decreased survival in both the univariate and multivariate models (Tables IV and V). The influence of p21WAF1 expression and p53 overexpression on survival was further examined in

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Fig 3. Actuarial survival for patients with resected pancreatic cancer (n = 40 patients). Patients with p53positive tumors who underwent resection and who received chemoradiation therapy (n = 16 patients) survived significantly longer than patients who were not treated with adjuvant therapy (n = 11 patients; P = .02). Three patients who received chemotherapy or radiation therapy alone were not included.

Table VI. Effect of adjuvant chemoradiation and p21WAF1 or p53 expression on median survival (months) in patients with resected pancreatic cancer*

Immunohistochemistry p21WAF1 positive p21WAF1 negative p53 positive p53 negative

Chemotherapy No radiation and or radiation chemotherapy therapy (mo) (mo) P value 25.4 10.6 22.2 15.4

11.1 15.8 13.0 16.6

.001 .95 .02 .71

*Excludes

patients whose condition was managed with either chemotherapy or radiation therapy alone.

patients with resected pancreatic cancer who received adjuvant chemoradiation (Table VI). Adjuvant chemoradiation significantly (P < .001) increased survival in patients with p21WAF1-positive tumors. No survival difference was observed among treated and untreated patients with p21WAF1-negative tumors. Overall survival in patients who received adjuvant chemoradiation with p21WAF1-negative tumors was similar to patients with resected pancreatic cancer who did not receive adjuvant therapy (Fig 2). Adjuvant chemoradiation also improved survival in patients with p53-positive tumors (Fig 3) but not in patients with p53-negative tumors. DISCUSSION Current adjuvant therapy protocols for pancreatic cancer have produced limited increases in

patient survival. p21WAF1 and p53 play an integral role in the cellular response to DNA damage and may play a role in the determination of tumor cell sensitivity to adjuvant therapy. In the present study, adjuvant chemoradiation improved patient survival. p21WAF1 expression was associated with prolonged survival after chemoradiation, whereas patients with p21WAF1-negative tumors appeared to derive no survival benefit from adjuvant therapy. Patients with p53-positive tumors also survived longer after chemoradiation. In contrast, patients with metastatic pancreatic cancer, who received chemotherapy alone, survived longer if their tumors did not overexpress p53. The p53 protein plays a central role in the repair of genetically damaged cells. In pancreatic cancer, loss of functional p53 protein through gene mutation is common and leads to the failure of cell-cycle arrest and genomic instability, as evidenced by gene amplification, aneuploidy, and/or chromosome loss through DNA-strand breakage and rejoining.12,13 p21WAF1 is an important mediator of p53-induced cell-cycle arrest.7,8,14 The p21WAF1 gene is rarely mutated in human cancer, and p53 is the major transcriptional regulator of p21WAF-1 expression in mammalian cells.15 However, p21WAF1 is also induced through several p53-independent pathways (TGFβ, DPC4), which also play a significant role in the development of pancreatic cancer.16,17 The inability to delay cell division and allow adequate DNA repair to take place increases the probability that DNA damage will remain uncorrected during

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DNA replication, leading either to cell death or the proliferation of genetically damaged cells with a selective growth advantage.12 The effect of abnormal p53 and p21WAF1 function on the efficacy of chemotherapy and radiation therapy has been the focus of many studies.6 Recently, the inactivation of single genes through homologous recombination has permitted the role of individual genes in this response to be demonstrated both in in vitro and in vivo animal models. The most commonly used drug in adjuvant therapy protocols for pancreatic cancer, 5-FU, produces rapid apoptosis in cells with intact p53 independent of p21WAF1.6 p53-deficient cells are resistant to apoptosis induced by 5-FU. In contrast, DNA-damaging agents such as adriamycin or γ-irradiation produce opposite effects to 5-FU, which lead to apoptosis in both p53- and p21-deficient cells.6 However, extrapolating these in vitro data to in vivo models may not accurately predict the response of a heterogeneous tumor to a given treatment. For example, p53-deficient cells sensitive to irradiation in vitro were as resistant to radiation as cells with intact p53 when xenografted into a mouse model.6 Thus, the response of a tumor to either radiation or chemotherapy is likely to be more complex than that easily explained by any single genetic alteration.6 The p53 protein is overexpressed in 40% to 58% of pancreatic adenocarcinomas.5,18-22 Several studies have reported decreased survival in patients with p53-positive pancreatic cancer. However, in multivariate analyses including other significant prognostic factors such as tumor stage, p53 overexpression has not been an independent predictor of survival in resected pancreatic cancer.18-22 Thirty-eight percent to 57% of pancreatic cancers express p21WAF1.18-21 Despite the coupling of p53 and p21WAF1 expression in normal cells, the present series and other studies demonstrate that p21WAF1 expression often occurs independent of p53 expression in pancreatic cancer (Table I).18,23 Furthermore, p21WAF1 expression is often absent in cells that normally express p53. The effect of p21WAF1 on prognosis in pancreatic cancer remains unclear; several studies demonstrated no influence of p21WAF1 on survival,19-21 although 1 study has demonstrated increased survival in patients with resected p21-positive tumors in a multivariate analysis.18 Several studies have further examined the role of these 2 genes on the response to adjuvant therapy in pancreatic cancer. Dergham et al21 studied 75 patients with pancreatic cancer and demonstrated an increase in survival in p53-negative and p21-positive patients who were treated with radiation therapy and/or chemotherapy when compared with

patients who received neither therapy. However, the expression of p53 and p21WAF1 were not independently associated with survival after adjustments were made for stage and treatment. Nio et al18 reported 58 patients with resected pancreatic cancer, including 30 patients whose conditions were managed with adjuvant chemotherapy.18 None of these patients received radiation therapy, and most of these patients received either the combination of ftorafur/uracil or cyclophosphamide. As in the current study, chemotherapy increased median survival in patients who were p53 positive (15 vs 8 months) but not in patients who were p53 negative (13 vs 12 months).18 Chemotherapy also increased survival in patients who were p21 positive (15 vs 10 months) but not in patients who were p21 negative (10 vs 10 months). In a multivariate analysis, p21WAF1 expression (P = .009) and not adjuvant therapy (P = .129) was independently associated with survival.18 Several pathologic factors (tumor size less than 3 cm, negative lymph nodes, negative margins) previously associated with improved survival after pancreatic cancer resection were not independent predictors of survival in our study.2 The relatively small number of patients with resected pancreatic cancer in this series likely contributed to this (type II error). Furthermore, the percentage of node-negative patients in our series is higher than in other series. However, most of these patients harbor occult lymph node micrometastases.24 In conclusion, p21WAF1 and p53 expression may help select those patients with pancreatic cancer who are likely to benefit from chemo- or radiation therapy. p53 overexpression is associated with decreased survival in patients with metastatic pancreatic cancer, although normal p21WAF1 expression may be necessary for a significant response to current adjuvant chemoradiation protocols. Alternate strategies for adjuvant therapy should be explored for patients with pancreatic cancer who are lacking a functional p21WAF1. REFERENCES 1. Landis SH, Murray T, Bolden S, Wingo PA. Cancer statistics, 1999. CA Cancer J Clin 1999;49:8-31. 2. Yeo CJ, Abrams RA, Grochow LB, Sohn TA, Ord SE, Hruban RH, et al. Pancreaticoduodenectomy for pancreatic adenocarcinoma: postoperative adjuvant chemoradiation improves survival. Ann Surg 1997;225:621-36. 3. Kinkenbijl JH, Jeekel J, Sahmoud T, van Pel R, Couveur ML, Veenhof CH, et al. Adjuvant radiotherapy and 5-fluorouracil after curative resection of cancer of the pancreas and periampullary region. Ann Surg 1999;230:776-84. 4. Gastrointestinal Tumor Study Group. Further evidence of effective adjuvant combined radiation and chemotherapy

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following curative resection of pancreatic cancer. Cancer 1987;59:2006-10. Ruggeri BA, Huang L, Berger D, Chang H, Klein-Szanto AJP, Goodrow T, et al. Molecular pathology of primary and metastatic ductal pancreatic lesions. Cancer 1997;79:700-16. Bunz F, Hwang PM, Torrance C, Waldman T, Zhang Y, Dillehay L, et al. Disruption of p53 in human cancer cells alters the responses to therapeutic agents. J Clin Invest 1999;104:263-9. Bunz F, Dutriaux A, Lengauer C, Waldman T, Zhoud S, Brown JP, et al. Requirement for p53 and p21 to sustain G2 arrest after DNA damage. Science 1998;282:1497-501. Waldman T, Kinzler KW, Vogelstein B. p21 is necessary for the p53-mediated G1 arrest in human cancer cells. Cancer Res 1995;55:5187-90. Koch WM, Brennan JA, Zahurak M, Goodman SN, Westra WH, Schwab D, et al. p53 mutation and locoregional treatment failure in head and neck squamous cell carcinoma. J Natl Cancer Inst 1996;88:1580-6. Wahl AF, Donaldson KL, Fairchild C, Lee F, Foster SA, Demers GW, et al. Loss of normal p53 function confers sensitization to taxol by increasing G2/M arrest and apoptosis. Nature Med 1996;2:72-9. Fleming ID, Cooper JS, Henson DE, Hutter RVP, Kennedy BJ, Murphy G, et al, editors. American Joint Committee on cancer: exocrine pancreas: AJCC cancer staging manual. 5th ed. Philadelphia: Lippincott-Raven; 1997. p. 121-6. Sidransky D, Hollstein M. Clinical implications of the p53 gene. Annu Rev Med 1996;47:285-301. Hartwell LH, Kastan MB. Cell cycle control and cancer. Science 1994;266:1821-8. El-Deiry WA, Tokino T, Velculescu VE, Levy DB, Parsons R, Trent JM, et al. WAF1, a potential mediator of p53 tumor suppression. Cell 1993;75:817-25. Shiohara M, El-Deiry WS, Wada M, Nakamaki T, Takeuchi S, Yang R, et al. Absence of WAF1 mutations in a variety of human malignancies. Blood 1994;84:3781-4. Grau AM, Zhang L, Wang W, Ruan S, Evans DB, Abbruzzese JL, et al. Induction of p21WAF1 expression and growth inhibition by transforming growth factor β involve the tumor suppressor gene DPC4 in human pancreatic adenocarcinoma cells. Cancer Res 1997;57:3929-34. Hahn SA, Schutte M, Shamsul Hoque ATM, Moskaluk CA, DaCosta LT, Rozenblum E, et al. DPC4 a candidate tumor suppressor gene at human chromosome 18q21.1 Science 1996;271:350-3. Nio Y, Dong M, Uegaki K, Hirahara N, Minaru Y, Sasaki S, et al. Comparative significance of p53 and WAF/1-p21 expression on the efficacy of adjuvant chemotherapy for resectable invasive ductal carcinoma of the pancreas. Pancreas 1999;18:117-26. Harada N, Gansauge S, Gansauge F, Gause H, Shimoyama S, Imaizumi T, et al. Nuclear accumulation of p53 correlates significantly with clinical features and inversely with expression of the cyclin-dependent kinase inhibitor p21WAF1/CIP1 in pancreatic cancer. Br J Cancer 1997;76:299-305. Coppola D, Lu L, Fruehauf JP, Kyshtoobayeva A, Karl RC, Nicosia SV, et al. Analysis of p53, p21WAF1, and TGF-β1 in human ductal adenocarcinoma of the pancreas. Am J Clin Pathol 1998;110:16-23.

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21. Dergham ST, Dugan MC, Joshi US, Chen YC, Du W, Smith DW, et al. The clinical significance of p21WAF1/CIP-1 and p53 expression in pancreatic adenocarcinoma. Cancer 1997;80:372-81. 22. DiGiuseppe JA, Hruban RH, Goodman SN, Polak M, Van Den Berg FM, Allison DC, et al. Overexpression of p53 protein in adenocarcinoma of the pancreas. Am J Clin Path 1994;101:684-8. 23. DiGiuseppe JA, Redston MS, Yeo CJ, Kern SE, Hruban RH. p53-independent expression of the cyclin-dependent kinase inhibitor p21 in pancreatic carcinoma. Am J Pathol 1995;147:884-8. 24. Demeure MJ, Doffek KA, Komorowski RA, Redlich PN, Zhu Y, Erickson B, et al. Molecular metastases in stage I pancreatic cancer: improved survival with adjuvant chemoradiation. Surgery 1998;124:663-9.

DISCUSSION Dr Fabrizio Michelassi (Chicago, Ill). In recent years there has been an increasing understanding of the biologic basis of tumor behavior. Our better understanding of genetic events not only has provided clues about tumorigenesis but has also formed the basis of translational investigations similar to the present study. These studies hold the promise of contributing to improvements in the treatment of this lethal disease by not only affording a more accurate risk stratification but also by identifying potential patient subgroups for novel treatment modalities with the use of gene therapy technology. The results of this present study are intriguing. p21 and p53 expression as measured by immunohistochemistry may aid in the selection of patients likely to benefit from adjuvant therapy. The end result would potentially translate, clinically, into a welcome refinement in treatment planning and modalities. Immunohistochemistry offers the simplest analysis for p53 molecular integrity but does not directly detect p53 mutations. In pancreatic cancer, some studies have demonstrated a concordance between genetic and immunohistochemical analysis in only approximately 50% of cases. Could you please comment on this? Additionally, immunohistochemical quantitation is often difficult and fraught with both false positives and negatives, depending on the tissue studied. How did you derive the threshold for what constituted a positive nuclear staining pattern of 33% for p53 and 5% for p21? Finally, what specifically did you use for positive and negative controls in your assays? If wild-type p53 regulates expression of cell-cycle inhibitor p21, which inhibits the transition from G1 to S phase and maintains the G2/M checkpoint, how do you explain your data that demonstrate that adjuvant chemotherapy significantly improves survival in both the p53 positive tumors (which presumably represents an accumulation of abnormal mutated p53 product) and in tumors that express normal p21 protein, which presumably require a normal wild-type p53 gene product? Are

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there other pathways that would explain this apparent contradiction? Dr Thomas A. Stellato (Cleveland, Ohio). This is really an intriguing paper. What was the chemotherapy and radiation, and how was it administered? More importantly, the article raises the question as to the role of adjuvant and neoadjuvant treatment. I suggest it also raises the role of resection in that the patients who were unresected and who were p21 positive had no different survival versus those who were resected and who were p21 negative; that is, their median 1-, 2-, and 3year survival rates were essentially identical. Can you comment on how you will use this in the future and what you think the significance of this is in defining a patient’s resectability? Dr Richard A. Prinz (Chicago, Ill). Pancreas cancer is a devastating and frustrating disease. Increased understanding of its molecular biologic features has promised to improve our ability to diagnose it at an earlier stage, select the most appropriate treatment for individual patients, and better predict prognosis. Nevertheless, there is little tangible evidence to indicate that genetic markers have favorably impacted any of these areas. First, although the authors found that positive p21 staining was associated with longer survival in their patients with resected pancreatic cancer who were undergoing postoperative chemoradiation, I am unconvinced of the clinical utility of this finding. I would like them to discuss their definition of positive staining (which was different for p53 and p21) and why such a low level of p21 staining is positive in their study. Second, p53 induces synthesis of the cell-cycle inhibitor p21. However, your data show uncoupling of p53 and p21. Can you explain this to us? Can you also tell us whether any of the various combinations of positive and negative p53 and p21 had any demonstrable effect on survival? Finally, you had 12 patients with resected pancreatic cancer and 19 patients who did not undergo resection who did not receive postoperative adjuvant therapy. What was the p53 and p21 status in these patients? Did it correlate in any way with their survival? I understand that the survival in this group was very short. But you should share with us the staining results and whether there was any difference. You should also tell us why they did not receive adjuvant therapy. Dr Arthur W. Boddie, Jr. (Chicago, Ill). My perception of p21 is that it is a schizophrenic gene because in damaged tumor cells it can either allow the tumor cells to go into cell-cycle arrest and repair themselves or push them into programmed cell death. If you are talking about apoptosis as a mechanism for killing in these particular treatments, you should consider looking at the ratio of bax (a pro-apoptolic gene)and bcl-2 (an anti-apoptolic gene), upregulation of bax may be 1 way that intact p53 induces apoptosis in response to cellular injury.

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Dr Ahrendt. Several of the discussants asked similar questions, and I will try to answer these collectively. One major criticism of p53 immunohistochemistry has always been the rather low correlation with p53 mutation analysis. One may assume that p53 mutation analysis should be a better prognostic marker. However, mutation analysis is difficult in pancreatic cancers because the extensive desmoplastic reaction makes it difficult to get tumor DNA for analysis that is not mixed with a large amount of normal DNA. Several laboratories have used xenografting to obtain pure tumor tissue from pancreatic cancer for genetic analysis. Let me first point out briefly that genetic analysis also has its pitfalls. We recently compared the oligonucleotide p53 gene chip, which remains in the investigational stage, with dideosynucleotide sequencing of exons 5 through 9 of the p53 gene in 100 lung cancers. Both of these mutation detection techniques had a sensitivity of about 80% at detecting p53 mutations. So, genetic analysis is not 100% accurate either. Immunohistochemical staining also has its drawbacks. One is the inconsistent cut-off between negative and positive staining. In addition, there are no standards among different laboratories that perform immunohistochemistry, and different monoclonal antibodies that react with different parts of the protein are used. However, immunohistochemistry does have the advantage of being simple, inexpensive, and quick; and often it seems to correlate with clinical outcome. We selected our cut-off between negative and positive in part on the basis of what we observed with our staining and what other studies on pancreatic cancer have reported. A number of tumors exhibited weak staining of a small percentage of tumor cells with the p53 antibody, so we used a higher cut-off with p53. In contrast, diffuse staining of nearly all the cells within a tumor was much more common with the p53 antibody than with p21WAF1 antibody. We did not observe this diffuse p21WAF1 staining to the same degree as we did the p53, and most samples that scored positive with the p21WAF1 antibody had staining of about 10% to 33% of the tumor. Several questions related to the discordant results between p21WAF1 and p53 staining. Our study and several others have shown no relationship among p21WAF1 and p53 expression in pancreatic cancer. p53 is certainly the major regulator of the p21WAF1 gene in normal cells. However, other p53-independent pathways exist for the induction of p21WAF1, including TGF-β. One gene involved in that pathway is DPC-4, which is inactivated in over 50% of pancreatic cancers. We did not look for DPC-4 gene mutations in these patients, and this pathway and others may influence p21WAF1 expression in pancreatic cancer independent of p53. Several other questions related to how p21WAF1 and p53 may influence the response to both radiation and

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chemotherapy. Our standard regimen for adjuvant therapy included external beam irradiation of 54 Gy to the pancreatic bed and regional lymphatics with concurrent infusional 5-FU or infusional 5-FU and weekly gemcitabine after January 1998. Most patients were offered adjuvant therapy. Like other series, approximately 25% of patients declined adjuvant therapy. Although median survival among patients with resected pancreatic cancer who received adjuvant therapy was longest in patients with p21WAF1-positive and p53-positive tumors, this difference was not statistically significant, and only p21WAF1 staining was independently predictive of survival in the multivariate analysis. Cancers are heterogeneous and contain multiple inactivated or activated genes, and it is unlikely that a single gene will predict response to therapy in all tumors. In

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addition, radiation and different chemotherapeutic agents have different effects on tumor cells in vitro; that is, certain drugs such as 5-FU require an intact p53 gene to cause apoptotic cell death, whereas, radiation and other agents (such as taxol and doxorubicin [Adriamycin]) are effective in p53-deficient cells. Similarly, radiation and Adriamycin also lead to apoptotic cell death in p21WAF1-deficient cells. Because most patients received both radiation and 5-FU and/or gemcitabine, it is hard to dissect out which agent is having a significant effect on a given tumor and correlate this with p21WAF1 or p53 status. At present, we are not making treatment decisions on the basis of either the p21WAF1 or p53 status. If these results are confirmed in a prospective analysis, p21WAF1 status may help select patients who should be included in studies of new adjuvant chemotherapeutic regimens.

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