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Annals of Surgical Oncology 8:311-318 (2001)
© 2001 Society of Surgical Oncology

ORIGINAL ARTICLES

p27kip1 Expression Is Associated With Tumor Response to Preoperative Chemoradiotherapy in Rectal Cancer

Giovanni Esposito, MD, Salvatore Pucciarelli, MD, Rita Alaggio, MD, Luciano Giacomelli, PhD, Elisabetta Marchiori, MD, Gaetano Antonio Iaderosa, MD, Maria Luisa Friso, MD, Paola Toppan, MD, Luigi Chieco-Bianchi, MD and Mario Lise, MD

From the Sections of Oncology (GE, LC-B), II Clinical Surgery (SP, EM, PT, ML), and Pathology (RA, LG, GAI) of the Department of Oncology and Surgery of the University of Padova; and the Molecular Diagnostic Oncology Service (GE, LC-B), and Division of Radiology (MLF) of the City Hospital of Padova; Padova, Italy.

Correspondence: Address correspondence and reprint requests to: Giovanni Esposito, MD, Servizio Citodiagnostica Molecolare Oncologica, Via Gattamelata, 64, 35128 - Padova, Italy; Fax: 39-049-8072854; E-mail: cmaltese{at}ux1.unipd.it


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background: Our aim was to ascertain whether or not the response to preoperative chemoradiotherapy for rectal cancer is associated with p27kip1 and p53 protein expression.

Methods: Thirty-eight patients (27 male, 11 female) with a mean age of 59 years (age range 33–87) and stage II-III rectal cancer received preoperative chemoradiotherapy (45–50.4 Gy; 5-FU 350 mg/m2/day and leucovorin 10 mg/m2/day). Thirty-one underwent low anterior resection; seven underwent abdominoperineal excision. Endoscopic tumor biopsies, performed before adjuvant therapy, were evaluated for: histologic type, tumor differentiation, mitotic index, and p27kip1 and p53 protein expression which were immunohistochemically determined. p53 expression was graded as: a) absent or present in <=10% of tumor cells; b) present in 11–25%; c) present in 26–75%; and d) present in >75% of tumor cells. p27kip1 expression was assessed using both light microscopy (percent of stained cells x10 HPF) and cytometry with an image analysis workstation. Tumor response, ascertained with histology, was classified using a scale from 0 (no response) to 6 (complete pathologic response).

Results: The mitotic index for the endoscopic biopsies was low in 14 cases, moderate in 17 cases, and high in 7 cases. p53 protein expression was found in 21 (a), 3 (b), 3 (c), and 11 (d) cases. The mean percentage of cells expressing the p27kip1 protein was 34 (range 0–77.14%). A close correlation was found between cytometric and light microscopy findings for p27kip1 (r2 = 0.92, P = .0001). Tumor differentiation was good in 5 cases, poor in 2 cases, and moderate in the remaining 31 cases. While the response to adjuvant therapy was good/complete in 25 (65.78%) cases, it was absent/poor in 13 (34.21%) cases. Univariate analysis associated type of adjuvant therapy (chemoradiotherapy, P = .0428) and p27kip1 protein lower expression (P = .0148) with a poor response to adjuvant treatment. Stepwise linear regression found overexpression of p53 and p27kip1 and young age to be independent variables that were linked to a good response to adjuvant therapy.

Conclusions: Lack of p27kip1 and p53 protein expression in rectal cancer is associated with a poor response to preoperative adjuvant therapy.

Key Words: p27kip1 • p53 • Chemoradiotherapy • Rectal cancer


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Rectal carcinoma, one of the most frequent forms of cancer in Western countries, is not susceptible to radical surgery when diagnosed because of local or distant spread in about 20% of cases.1 After radical surgery, the five-year survival rate is 60% to 80% for stage II (T3-T4, N0) and 30% to 50% for stage III (any T, N+) patients.2

Over the last 50 years, no real improvement has been achieved in the survival percentages after resection. Because surgery alone cannot be considered curative for many stage II and III patients, adjuvant therapy is recommended in these cases. Several authors3–5 have observed a reduced local recurrence rate and an improved survival rate after radiotherapy.6 An improvement in survival rate has also been observed after radiotherapy and 5-fluorouracil-based chemotherapy.4,7,8

Further reduction in local and distant recurrences and an increase in survival rate may also be achieved by administering preoperative chemoradiotherapy.9–11 Neoadjuvant chemoradiotherapy allows tumor downstaging (size and local invasion reduction), with a consequently improved possibility for achieving curative sphincter-sparing surgery (low anterior resection rather than abdominoperineal resection) and an important effect on quality of life.12,13 The percentage for complete response to neoadjuvant therapy (i.e., no neoplasia on the surgical specimen) ranges from 5% to 14% in some studies, but is 35% in other studies.2,10,12,14–18 This discrepancy probably exists because of differences in the treatments given and in variations in intrinsic characteristics of each tumor. It is therefore important to identify biological parameters that will enable us to predict individually whether or not patients will respond to neoadjuvant chemotherapy, thus sparing potentially nonresponsive patients from unnecessary treatment.

Several authors have demonstrated that apoptosis is one of the mechanisms underlying chemoradiotherapy-induced tumor regression,19–21 with the response to adjuvant therapy due mainly to neoplastic cells’ ability to trigger the apoptotic cascade.22 The present study focused on two of several proteins involved in the regulation of apoptosis: the p53 and the p27kip1 proteins. Alterations in the p53 gene are found frequently in different types of tumors.23–28 In particular, p53 mutations have been found in 45% to 70% of carcinomas of the large bowel.29–32 The p53 gene is involved in the regulation of the cell cycle, in the repair of DNA, in cellular death through apoptosis, and, therefore, in the maintenance of genomic integrity. As a response to DNA damage, the p53 protein accumulates in the cell and causes the cell cycle to arrest in the G1 phase, thus allowing repair mechanisms to intervene before the alteration is fixed in the genome and transmitted to daughter cells.33,34 If this does not occur, p53 induces a "suicidal" response in the cell by activating apoptosis.35,36

In 1994, p27kip1 was identified as a member of the universal cyclin-dependent kinase inhibitor family, like p21cip1/waf1 and p57kip2.37–42 More recently, it has been suggested that this protein is the product of a putative tumor suppressor gene,42 that it regulates cell differentiation43 and drug resistance in solid tumors,44 and that it also promotes apoptosis.45

The present study’s aim was to establish whether or not the expression of p27kip1 and p53 proteins in tumor representative preoperative biopsies is correlated with a response to neoadjuvant-combined chemoradiotherapy, classified as the histological degree of tumor regression assessed on surgical specimens. Both proteins were also evaluated on surgical specimens in order to identify any variations in their expression in resected tumors after neoadjuvant therapy.


   PATIENTS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patient Selection
Between March 1988 and March 1999 and after preoperative chemoradiotherapy, 79 consecutive patients with primary rectal adenocarcinoma of the middle and lower rectum underwent surgery at Clinica Chirurgica II, University of Padova. Criteria for giving neoadjuvant therapy were:

(1) histologically proven adenocarcinoma of the middle and lower rectum; (2) preoperative clinical TNM (tumor, node, metastasis) stage II-III; and (3) Eastern Cooperative Oncology Group (ECOG) performance status score of 0 to 2. Forty-one patients were excluded because preoperative endoscopic biopsies or radiotherapy had been performed elsewhere (n = 37) or because the biopsy was not sufficiently representative of the tumor (n = 4). The study group consisted of the remaining 38 patients (27 males, 11 females) with a mean age of 59 years (range, 33–87 years). Thirty-six underwent surgery between 1994 and 1999 and two between 1988 and 1993.

Preoperative Staging and Treatment
The preoperative clinical (pelvic CT scan, transrectal and liver ultrasonography, and chest x-ray) and postoperative pathological stages were defined using the TNM stage classification system.46 The clinical stage (cTNM) was II in 11 cases and III in 27 cases.

Thirty-three patients underwent preoperative combined chemoradiotherapy; five underwent radiotherapy alone. External beam radiotherapy was administered by a linear accelerator x-ray (10 MV). The four beam "box" technique was used, with the upper border extending to L-5/S-1. The total dose received was 45 to 50.4 Gy (1.8 Gy/day per fraction). Thirty-three patients received concomitant chemotherapy (5-fluorouracil 350 mg/m2/day and leucovorin 10 mg/m2/day), administered in bolus, on days 1 through 5 and 29 through 33 of radiotherapy.

The surgical procedures performed were: low anterior resection with colorectal or coloanal anastomosis (n = 31) and abdominoperineal resection (n = 7). The mean time interval between the end of neoadjuvant treatment and surgery was 38 days (range 10–66).

Histology
In each case, the preoperative biopsies (obtained before neoadjuvant therapy) and the surgical specimens were analyzed. Parameters considered were: histological type, tumor grading, and mitotic index. The mitotic index was considered low when mitotic figures were <=2/HPF, moderate when they were from >2 to <=5, and high when mitoses were >5/HPF.

At least six samples of the tumor, or the entire fibrotic area in which regression was suspected, were examined microscopically to evaluate the degree of regression. Histological determination of tumor response on surgical specimens was performed with a seven categories scoring model, which was a modified version of the system proposed by Dworak et al.47 (Table 1). For statistical purposes, the seven classes were dichotomized as good/complete response (grades 4–6) and absent/poor response (grades 0–3). Consequently, there was one group (0-3) with (apart from concomitant necrotic or fibrotic areas) a vital neoplastic component that could reach 70%, and another (4-6), with a reduction in the neoplasia, of at least 60%.


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  		TABLE 1. Histopathological classification of tumor response to chemoradiotherapy
 
Immunohistochemistry
In each case, one slide corresponding to the most representative part of the tumor was chosen to make an immunohistochemical evaluation of p27kip1 and p53 protein expression.

Formalin-fixed, paraffin-embedded sections were deparaffinized and rehydrated. p27kip1 expression was analyzed using an anti-p27 monoclonal antibody (clone 57: Transduction Laboratories, Lexington, KY) in a 1:600 dilution at room temperature. p53 was detected by the monoclonal antibody PAb 1801 (Ab-2: Oncogene Research Products, Cambridge, MA) in a 1:100 dilution at 4°C. For each antibody, a step of heat-induced antigen retrieval was performed at 750W in a microwave oven using three 5-minute cycles for PAb 1801 and four 5-minute cycles for the anti-p27 antibody; then, an overnight incubation was made. After incubation with the primary antibodies, immunostaining was performed using the avidin-biotin-peroxidase complex technique and 3–3' diaminobenzidine as chromogen (VECTASTAIN ABC kit and DAB kit: Vector Laboratories, Burlingame, CA). Finally, the sections were counterstained with Mayer’s hematoxylin.

p53 and p27kip1 Scoring
p53 protein expression was graded as: (1) absent or present in <=10% of tumor cells; (2) present in 11–25%; (3) present in 26–75%, or (4) present in >75% of tumor cells.

p27kip1 protein expression was assessed using two modalities: (1) light microscopy (by calculating the percentage of stained cells over the total cells x10 HPF) using an eye-piece integration grid (preoperative biopsies and surgical specimens); (2) blindly, with a light-microscope connected to an image analysis system, with the final determination (i.e., the percentage of p27kip1 labeled cells) made as an area fraction (i.e., the percentage of nuclei with a positive immunostaining area against the total nuclear area) on preoperative biopsies only.

In all cases, 5 to 10 fields were evaluated at 20 x magnification. Measurements were made using the CIRES workstation (Zeiss, Jena, Germany), consisting of a light microscope (Axioscope model, Zeiss, Jena, Germany) connected to a 3-CCD color video camera (model KY-F55BE, JVC, Japan). Köhler illumination was used to reduce stray light, and images were analyzed using a PC with a frame grabber (Kontron, Eching, Germany).

Statistical Analysis
Univariate analysis was performed using the one-way analysis of variance. Linear regression was used for multivariate analysis. The most significant subset of variables, first selected using a stepwise procedure, was analyzed to measure any correlation. A P value of <= .05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Downstaging
Pathologic TNM staging on surgical specimens showed a downstaging of tumors compared to clinical TNM: 9 cases were classified as stage I (none in clinical staging), 13 as stage II (11 in clinical staging), 7 as stage III (27 in clinical staging), and 3 as stage IV (none in clinical staging). Six cases (15.78%), in which no tumor was found on the surgical specimen, were considered completely responsive to chemoradiotherapy.

Histology
No differences were found between tumor differentiation assessed in preoperative biopsies and that in surgical specimens. In preoperative biopsies, tumors were graded as well differentiated in 5 cases, moderately differentiated in 31 cases, and poorly differentiated in 2 cases. The surgical specimens were well differentiated in 1 case, moderately differentiated in 30 cases, and poorly differentiated in 1 case. The mitotic index in preoperative biopsies was low in 14 cases, moderate in 17 cases, and high in 7 cases. In surgical specimens, the mitotic index was found to be low in 11 cases, moderate in 17 cases, and high in 4 cases.

p53 and p27kip1 Expression
In preoperative biopsies, p53 protein expression was not detected in 21 cases (55.26%), whereas it showed different degrees of positivity in 17 cases (44.73%): 11–25% of immunostained tumor cells in 3 cases, 26–75% in 3 cases, and >75% in 11 cases. p27kip1 protein expression was present in 34 cases and completely absent in 4 cases. The mean percentage of cancer cells expressing p27kip1 at the light-microscopy count was 34 (range 0–77.14%). A close correlation was found between cytometric and light-microscopy findings for p27kip1 (r2 = 0.92; P = .0001) (Fig. 1).



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  		FIG. 1. p27kip1 expression in preoperative biopsies: concordance between light microscopy and cytometry findings.

 
When comparing the preoperative and postoperative specimens, changes were found in the levels of expression of both p53 and p27kip1. For p53 expression, a shift from negativity to strong positivity was found upon immunostaining in three cases. For p27kip1 expression, a shift was found (i.e., a difference of at least 20% of immunostained neoplastic cells counted) toward a reduced expression in four cases and toward an increased expression in six cases (Table 2).


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  		TABLE 2. Variations between p27kip1 expression in preoperative biopsies and surgical specimens
 
The histological degree of response to neoadjuvant therapy, scored using a modified version of the Dworak tumor regression grade system, is shown in Fig. 2. The response was good/complete in 25 cases (65.78%) and absent/poor in 13 (34.21%). The mean (Standard Error) expression of p27kip1 was respectively 42.84% (3.74) in the former group and 25.08% (6.56) in the latter.



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  		FIG. 2. Tumor response and percent of p27kip1 expressing cells in pretreatment biopsies.

 
Univariate analysis found that type of neoadjuvant therapy (combined chemoradiotherapy vs. radiotherapy alone; P = .0428) and p27kip1 degree of expression (P = .0148) were associated with response to treatment. No other variables were associated with tumor response to the preoperative therapy (Table 3). Multivariate analysis found that p53 and p27kip1 expression and young age to be independent variables linked to a good response to neoadjuvant therapy (Table 4.).


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  		TABLE 3. Variables associated with tumor response to neoadjuvant therapy upon univariate analysis
 

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  		TABLE 4. Regression coefficients of variables associated with tumor response to neoadjuvant therapy upon multivariate regression analysis
 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Neoadjuvant therapy for carcinoma of the middle and lower rectum, with radiotherapy only or with combined chemoradiotherapy, can induce a tumor downstaging, thus allowing more sphincter-saving procedures to be performed, and also ensuring an encouraging oncologic outcome.48 However, in about 30% of the cases the response to therapy is poor or absent. The identification of factors prognosticating response to neoadjuvant therapy may allow a more accurate selection of patients—sparing those with nonresponsive tumors from neoadjuvant therapy, or allowing those patients to undergo alternative treatment with different drugs.

Therefore, with the aim of identifying any correlation between tumor response as histologically determined on surgical specimens and the features observable in preoperative biopsies, we analyzed the behavior of p53 and p27kip1. Both are involved in the inhibition of the cell cycle and the induction of apoptosis. For this purpose, a careful evaluation was made of the histological degree of tumor regression. Our grading system was based on the identification of fibrotic changes and necrosis in preoperative and surgical specimens in an effort to distinguish between the desmoplasia and necrosis characteristic of the neoplasia and the effect of therapy. Moreover, we found it important that the entire neoplastic area be embedded for regression in order to be considered complete. In six cases classified as grade 5, there were only microscopic foci of residual neoplasia that might have remained undetected, thus potentially leading to an erroneous diagnosis of complete regression.

For p27kip1 expression scoring on biopsies, light-microscope evaluation and the image analysis system, which were used blind, gave a good concordance of data. This suggested that the imaging analysis system, which is less time-consuming, could be used on a more widespread scale.

Both univariate and multivariate analysis found a significant correlation between the increase in the percentage of tumor cells expressing p27kip1 on biopsies and the increase in the histological degree of response to therapy (Fig. 3).



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  		FIG. 3. A-B: High expression of p27kip1 in a preoperative biopsy (A) with an almost histologically complete (grade 5) response to neoadjuvant therapy, as showed in the surgical specimen with only isolated neoplastic glands (arrowhead) enclosed in a fibrotic stroma (B). C-D: preoperative biopsy with diffuse negativity for p27kip1 (C); note the internal positive control represented by infiltrating lymphocytes (arrowhead), and absent response to neoadjuvant therapy in surgical specimen (D). A-C: immunohistochemical analysis - Mayer’s hematoxylin counterstaining - original magnification 20x. B-D: H&E staining - original magnification 20x.

 
Data reported in literature on the relation between p27kip1 and apoptosis, and response to therapy, are contradictory. Some authors45,49,50 found in vitro that high p27kip1 levels, induced by adenovirus vectors, led to an arrest in growth as well as an increase in apoptosis in various cell lines from different species and tissues of various origin. In vitro studies by other authors44,51 found p27kip1 overexpression to induce a resistance to some drugs through the inhibition of apoptosis. On the other hand, there is a growing body of in vivo evidence showing a close correlation between p27kip1 expression and apoptosis. A strong correlation has been found between p27kip1 expression and the apoptotic index by Fujieda et al.52 in 60 oral and oropharyngeal squamous cell carcinoma patients, by Othani et al.54 in 225 non-early stage gastric carcinoma patients,53 and by Wu et al in 181 breast cancer patients. In the last study, p27kip1 was also found to be predictive of chemotherapy benefit. Our finding of an association between p27kip1 and response to preoperative chemoradiotherapy may therefore be explained by the pro-apoptotic activity of p27kip1.

However, from a clinical viewpoint, it would be useful to establish a reliable p27kip1 cut-off value for the prediction of the response to neoadjuvant therapy. Interestingly, when arbitrarily stratifying p27kip1 expression as absent, present in <50%, and present in more than 50% of neoplastic cells, we still found a correlation between expression and response. Moreover, upon histological evaluation, all four tumors that were negative for p27kip1 expression presented a regression that was classified as absent/poor. These findings should, however, be confirmed in a larger patient population.

In several different tumor types, gene mutations have been found for p53 and other molecular prognostic factors, whereas it has been observed that p27 gene mutations are not present in a variety of human tumors.54 The levels of p27kip1 expression seem to be regulated mainly at a posttranscriptional level, via a ubiquitin-proteasome mediated proteolysis mechanism.55–57 These observations give a reliable meaning to the immunohistochemical data for p27kip1.

A more vexing issue is the interpretation of data for p53. Although univariate analysis found no association between p53 expression and tumor response to neoadjuvant therapy, multivariate analysis found that p53 expression on preoperative biopsies (together with the p27kip1 percentage of expression and young age) was predictive of a better response to therapy. Data reported in the literature are contradictory: whereas some authors report an association between p53 overexpression and a lower response rate to treatment,58,59 others do not.22

The antibodies normally used recognize stabilized forms of the p53 molecule and are not specific for mutant forms. It is well-known that mutated forms of p53 are generally stabilized with a longer half-life than the wild-type protein; this makes them detectable by immunohistochemistry. The immunohistochemical detection of protein is, therefore, considered synonymous with protein mutation; however, this is not necessarily the case (i.e., stabilization can depend on an interaction with other cellular oncogene products, or viral molecules).60,61 Clearly, an analysis of the p53 gene is required for an adequate understanding of the meaning of p53 protein expression.

Two hypotheses may be put forward to explain our findings: (1) the presence of a mutated protein that, however, maintains its pro-apoptotic activity,62–64 or (2) the presence of a stabilized wild-type protein responding to DNA damage.

After comparing p53 and p27kip1 expression in preoperative and surgical specimens, we found that there was a shift in three cases from p53 negativity in preoperative biopsies to strong positivity in posttherapy samples. This was probably related to p53 gene activation subsequent to DNA damage induced by therapy, or it may have been due to new gene mutations. For p27kip1, differences were found in 10 cases: the posttherapy specimens showed a significantly reduced expression in four cases and an increase in six cases with respect to corresponding preoperative biopsies. As with p53, therapy-induced damage may affect p27kip1 expression. However, our data confirm the need to exclude patients undergoing preoperative therapy from studies performed on surgical samples.

In conclusion, we found a close correlation between the level of p27kip1 expression and the degree of tumor regression upon histological evaluation of surgical specimens. This relationship, if confirmed in larger series, may become a useful tool for identifying subgroups of patients more likely to benefit from neoadjuvant therapy.

The finding for the p53 protein is controversial, calling above all for a knowledge of the type of protein expressed.


   Acknowledgments
 
The authors thank Dr. Carlo Schievano for the statistical analysis, Mrs. Dina Pozza for technical assistance, Mr. Pierantonio Gallo for artwork, and Mrs. Sara Pearcey for assistance with English. This study was supported in part by a grant from Associazione Italiana per la Ricerca sul Cancro (AIRC), Milano, Italy.

Received for publication July 7, 2000. Accepted for publication December 4, 2000.


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 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
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