Clinical and molecular prognostic factors in sphincter-preserving surgery for rectal cancer

Clinical and Molecular Prognostic Factors in Sphincter-Preserving Surgery for Rectal Cancer j. Milburn Jessup, Massimo Loda, and Ronald Bleday As many as a third of patients with rectal cancers may be candidates for sphincter preservation surgery. The goal of the conservative management of adenocarcinoma of the distal rectum is to preserve rectal sphincter function without sacrificing local tumor control. To achieve this goal, a combined modality approach is necessary because multimodality therapy for more advanced disease has improved both local control and survival. Candidates for local excision are those with adenocarcinomas with a maximal diameter of less than 4 cm, mobile, and not poorly differentiated or mucinous and within 10 cm of the anal verge--usually within 6 cm. These criteria should be defined objectively by biopsy combined with state-of-the-art endorectal imaging. Newer molecular markers that are associated with prognosis and response to therapy may also be important for assessing prognosis, probability of local recurrence, and whether conservative treatment is appropriate. Patients with T0-3 NO lesions meeting these

standard clinicopathologic criteria have been treated successfully with wide local excision combined with chemotherapy and radiotherapy, Patients with larger or more advanced lesions may undergo low anterior resection with coloanal anastomosis. After resection, radiotherapy to at least 45 to 50 Gy is delivered to the pelvis and tumor bed often with concomitant chemotherapy. The overall rate of local failure in prospective singleinstitution trials in which local excision is performed with postoperative chemoradiotherapy has been 5% for T1 lesions, 7% for T2 lesions and 24% for T3 lesions. Although single-institution studies have supported the concept of conservative therapy, the safety and efficacy of this approach must still be confirmed in a multicenter, prospective trial, such as that underway in several of the cooperative oncology groups, before it may be considered a standard of practice.

denocarcinoma of the rectum afflicted about 37,100 people with approximately 1 in 4, or 8,300 people, dying of the disease in 1997.1 Not onlyis this a lethal cancer, but also its treatment has a major impact on the lifestyle of a patient because the majority of distal cancers are still treated with abdominoperineal resection and colostomy as described by Miles in 1908. 2The loss of a rectal sphincter from this type of radical resection may be curative but carries with it a high incidence of bladder and sexual dysfunction as well as the social problems caused by a permanent colostomy. Because conservative surgical procedures combined with chemotherapy and radiotherapy have achieved survival results similar to that for radical resection in both breast cancer and skeletal and soft tissue sarcoma with improved quality of life, considerable enthusiasm has been generated for procedures that spare the rectal sphincter, avoid a

permanent colostomy, and yet achieve a local control rate similar to that of radical resection. In a review of colorectal carcinomas by the National Cancer Data Base, a cancer management and outcomes database for health care organizations, 37% of rectal adenocarcinomas were either stage 0 or I lesionsP Thus, nearly a third of patients with rectal cancer may be candidates for a conservative approach. Candidates for rectal sphincter preservation, however, must be chosen carefully because if the operation fails to provide local control, the patient may die from pelvic recurFence. The identification of appropriate candidates for sphincter-preserving surgery depends on the careful analysis of the characteristics of rectal carcinomas and their propensity for local invasion and recurrence as well as the sensitivity of the cancer to radiation and chemotherapy. Although the abilitr of cancers to invade and metastasize is great, standard clinicopathologic features do define behavior within broad categories. The difference in behavior of individual lesions based on histological grade, depth of rectal wall invasion, and status of the draining lymph nodes is quite large, with the probability of disease progression for stage II or III rectal carcinomas varying by as much as 30% for each stage. 4,5 This article defines the clinicopathologic factors that are used to identify patients with rectal carcinoma who are candidates for sphincter preservation.

A

From the Departments of Surgery Jess'up,Bleday) and Pathology (Loda), Beth Israel DeaconessMedical Center,Boston, MA. This work was supported in part by grants CA 44704 and CA 42857 from the Department ofHealth and Human Services, Washington, DC. The views are those of the authors and do not necessarilyreJIectthe views of the Federalgovernment. Address reprint requefts to J. Milburn Jessup, MD, Department of Surge~, Beth Israel Deaconess"Medical Center, West Campus, One DeaconessRd, Boston, MA 02215. Copyright 9 1998 by W..B. Saunders Company 1053-4296/98/0801-000958.00/0

54

Copyright9 1998by W.B. Saunders Company

Seminars in Radiation Oncology, Vol 8, No 1 (January), 1998:pp 54-69

Clinical and MolecularPrognostic Factors in Sphincter-PreservingSurgery

The focus is on standard prognostic variables that may be measured objectively on biopsy or by endorectal imaging. The utility of molecular markers expressed within primary rectal carcinomas is also described briefly, although these markers have not yet been incorporated into clinical practice. These molecular markers have usually been shown to be significant covariates within multivariate analyses from single-institution studies and require validation either in multiple-institution series or as companion studies to large cooperative oncology group therapy trials. Because few of these molecular markers have yet to be validated by such confirmatory studies, they are not yet ready to be incorporated into daily practice, and our review should not be considered to endorse any particular marker. The clinicopathologic and molecular markers that we consider are listed in Table 1. This review focuses on local excision of low Table 1. Clinicopathologic Variables and Intratumoral Markers That May Affect Sphincter Preservation in Distal Rectal Carcinoma* Variable or Marker

Clinicopathologic variable T stage N stage Size Grade Degree of mobility Mucin production Lymphatic, vascular, or neural invasion Radial and mucosal margin Ulceration Molecular prognostic markers Mucin genes and related epitopes (MUC 1, Muc2, Sialosyl-Tn, Sialosyl-Lex, and Sialosyl-LeA) DCC Sucrase-isomaltase DNA ploidy and proliferation p27VdP1

Angiogenesis (VEGF, PD-ECGF) Thymidylate synthase

Therapeutic Prognosis Respome

+++ +++ _+ +++ +++ _+

+++ +++ • + +++ •

_+ _+ +

• • •

+++ +++ ++ _

~ _+

+ + +

•

++ ++

+++

*Marker utility for establishing prognosis at the time of definitive therapy or predicting therapeutic response either in the adjuvant setting (for stage IMII rectal carcinoma or stage III colon carcinoma as determined by survival benefit compared with marker negative carcinomas) or in advanced disease: + + + , marker shown to be significant covariate in multivariate analysis in trials with more than 100 patients; + +, marker significant covariate in multivariate analysis in trials of less than 100 patients; +, marker is significant in univariate Kaplan-Meier analyses only; _+, marker utility is equivocal, but data are of interest and may be useful in future studies; ?, no data concerning use of marker but biological rationale exists for evaluation.

55

rectal adenocarcinomas (0 to 6 cm from anal verge) and describes both the selection of patients for a local surgical approach and the current results. This review focuses on local control because both surgery and radiation therapy are local treatments that ablate tumor at the primary or regional site but do not affect tumor deposits outside the surgical field or radiation port. Therefore, prevention of locoregional failure is the principal measure of the usefulness of rectal sphincter preservation by conservative methods.

Standard Clinicopathologic Factors for Distal Rectal Cancer T Stage The most important single factor for predicting the success of rectal sphincter preservation is the T stage or depth of penetration by the carcinoma into or through the bowel wall. Cohen et al6 showed that in tumors that were contained within the bowel wall, well or moderately differentiated, less than 3 cm in diameter, and exophytic, the probability of metastasis in regional lymph nodes was only 10%. Similar results were reported by Morson. 7 Further, Jass et al 8 showed that only a 5-mm invasion of the perirectal tissue by the primary carcinoma significantly increased the recurrence of rectal carcinoma. Sixty percent to 70% of carcinomas that penetrate through the bowel wall (T3) metastasize to regional pararectal lymph nodes compared with 10% to 15% of carcinomas that invade the submucosa (T1) or 15% to 25% that penetrate into the muscularis propria (T2). 6-1~The importance of using these prognostic factors to identify the risk of recurrence is borne out by Pilipshen et al, 11 who reported that 96% of patients who developed a pelvic recurrence died of their disease compared with only 23% of patients who did not develop a local or regional failure. Even the performance of a radical resection does not necessarily prevent local or regional recurrence of rectal carcinoma. Local recurrences occur after radical resections in direct proportion to the stage of disease (0 to 27% for stage I [T1-2NOM0], 12% to 83% for stage II [T3NOM0], and 27% to 71% for stage III [T1-3N1M0]. 12-17)These locoregional recurrences may develop from either retained nodal micrometastases or contiguous spread from the primary cancer. Recurrence does not depend on the proximal and distal margins as much as it does on the lateral margin of the surgical specimen in the mesorectum or pararectal tissues, which is a function of the depth

56

Jess@, Loda, andBleday

of penetration by the tumor. In a prospective study of 190 patients, Adam et al~a observed that 78% of patients who underwent a potentially curative operation for low rectal carcinoma but had a positive lateral margin developed a local recurrence compared with 10% of patients with a negative margin. Chan et all9 reported that the lateral margin was often less than 6 mm for both Dukes B and C carcinomas that had been resected for cure in a series of 50 patients. Because of the anatomy of the pelvis, the close lateral margin may cause local recurrences in pelvic viscera and around the sacrum. Heald et al2~ have popularized total excision of the mesorectum because they found microscopic deposits of carcinoma several centimeters beyond the edge of the rectal cancer in the mesorectum, which would have been left in situ if an extended dissection of the mesorectum was not performed. There were no local recurrences in 50 patients whose mesorectum did not demonstrate any micrometastases. More recently, Heald and Karanjia 21 have updated their results and found that overall recurrence in 152 curatively resected patients was less than 4% even though a large proportion of patients had negative lateral margins that were less than 1 cm. More recent analysis of distal margin of rectal carcinomas further underscores the importance of the T stage. Shirouzu et al ~2 performed a detailed study of both the intramural and extramural extent of disease distal to rectal carcinomas and found that none of 150 T1-T2N0 lesions extended beyond the obvious gross margin, whereas only 1.2% of T3N0 lesions extended 1 cm from the gross edge of the cancer. For cancers with positive nodes, the intramural or extramural extension was more than 1 cm in 4.7% of 195 cases. As a result, in patients who are chosen appropriately with T1-T2 depth of penetration, the gross margin of resection on the rectal mucosa can be approximately 1 cm. The radial or deep margin, however, must still be negative if the risk of local recurrence is to be minimal. Thus, the amount of tissue removed by an abdominoperineal resection does not guarantee an adequate radial or deep margin because the narrow confines of the pelvis may force the surgeon to compromise on a wide resection. As a result, local failure often results from contiguous spread of rectal carcinoma into tissues that are not removed by the surgeon who fears that removal of such tissues may cause morbidity or an alteration of lifestyle that is not acceptable to the patient. Gunderson and Sosin 17demonstrated that local recurrence in the perineum or midpelvis

was a component of 92% of all recurrences in rectal carcinoma and was the only site of failure in approximately 50% of patients who failed. Because these sites are encompassed within a standard pelvic radiation port, the addition of treatment modalities such as radiation therapy may sterilize micrometastases in the mesorectum and regional nodes. This is borne out in Table 2, in which the results of several, prospective institutional trials are presented and demonstrate that locoregional failure after local excision with or without radiation therapy and a radiosensitizing dose of chemotherapy is proportional to the depth of bowel wall penetration by a primary cancer: 5% for T1 lesions, 7% for T2 lesions, and 24% for T3 lesions. When a multivariate analysis is performed of patients with stage I or II rectal carcinoma who have undergone curative radical procedures at the Beth Israel Deaconess Medical Center, West Campus that includes grade and other clinicopathologic variables and molecular markers, the depth of bowel wall penetration is still the most significant marker (Table 3).

N Stage The status of the draining nodes is the next most important clinical variable for determining the outcome of rectal carcinoma. This variable is often the least reliable or available because detection of nodes in patients who are candidates for local excision depends on imaging studies that have at best an accuracy of 80% for detecting metastasis. 23 Clearly, if positive nodes are identified by endorectal ultrasound (ERUS) with biopsy, ~4 local excision is not appropriate because the probability of residual nodes containing tumor is high.

State of Differentiation Poorly differentiated carcinomas have long been considered more likely to recur than well-differentiated to moderately differentiated tumors. This concept may have originated with the early investigations of Spratt and Spjut. l~ When large data sets are considered, the state of differentiation is associated with advanced stage of disease in colon25 and rectal (unpublished results of the National Cancer Data Base) carcinoma. A multivariate analysis demonstrated that poorly differentiated colorectal carcinomas have a worse survival than well-differentiated or moderately differentiated carcinomas. 26Thus, poorly differentiated carcinomas may be more likely to spread to distant organs or regional nodes. Faivre et a127 found that poorly differentiated rectal carcino-

Clinical and Molecular PrognosticFactors in Sphincter-PreservingSurgery

57

T a b l e 2. The Effect of Local Excision With or Without Radiotherapy or Chemotherapy on the Incidence of Local Failure in Patients With Adenocarcinoma of the Rectum

Stage ofPrima~ Series Tubingcn 2a NEDH 3~ MSKCC a2 FCCC 137 UTMDACC m M G H 139 U F 14~

Radiation Therapy

5-Fluorouracil*

T1

None Bolus None None CI (T3 only) Bolus None

2/48 (4)

QO (0)

!/7 (14)

2/21t

0/21 (0)

2/5 (40)

0/4 (0) 1/2 (50) 0/17 (0) 0/5 (0) 1/13 (8)

2/12 (17) 2/15 (13) 1/14 (7) 0/11 (0) 1/4 (25)

2/6 (33) 1/4 (25) 3/15 (20) 0/1 (0)

6/110 (5)

6/86 (7)

9/38 (24)

None 50 50 50 53 45 55

Totals

T2

T3

*5-Fluorouracil was given as radiosensitizer in several of the trials either as a bolus the first and last 3 days of the radiation therapy (Bolus) or as a continuous infusion throughout (CI). tThese patients did not receive radiation therapy; please see text for details. m a s t e n d e d to r e c u r m o r e o f t e n t h a n b e t t e r d i f f c r c n t i a t e d cancers. M e n t g e s e t al 2a i n c l u d e d poor s t a t e o f d i f f e r e n t i a t i o n as a c r i t e r i o n for t h e i r h i g h - r i s k t r a n s a n a l excisions, b u t n o n e o f t h e s e p a t i e n t s r e c u r r e d locally d u r i n g t h e p e r i o d o f follow-up. Volk et a F 9 i n t h e i r s t u d y f r o m t h e C l e v e l a n d Clinic o f endoscopic p o l y p e c t o m y f o u n d t h a t poorly d i f f e r e n t i a t e d carcinom a s w e r e m o r e likely to c o n t a i n r e s i d u a l c a r c i n o m a

at o p e r a t i v e l c-ic~cctio~l o~ to d c , c l o p d i s t a i n m c ~ , ~ tases. I n o u r r e t r o s p e c t i v e series o f s t a g e I to II r e c t a l c a n c e r p a t i e n t s w h o w e r e o p e r a t e d o n b e t w e e n 1965 a n d 1977 a n d s t u d i e d for m o l e c u l a r p r o g n o s t i c f a c t o r e x p r e s s i o n ( T a b l e 3), t h e only p a t i e n t w i t h a poorly differentiated carcinoma developed metastases and died o f disease. T h i s a n e c d o t a l e v i d e n c e of m o r e aggressive b e h a v i o r m a y n o t p r e c l u d e a s p h i n c t e r

T a b l e 3. Univariate and Multivariate Analysis of Clinicopathologic Variables and Selected Molecular Markers in Patients with Stage I-II Adenocarcinoma of the Rectum Operated on Between 1965 and 1977 at the Beth Israel Deaconess Medical Center, West Campus*

Cox ProportionalHazards

Univariate Factor T stage Grade Sex Vascular invasion Sucrase-isomaltase

p27

DCC

Category

N

%NED

Log Rank P

CategoV

P

T1-T2 T3 W-M Poor Female Male Present Absent Absent Low High Absent Low High Absent Present

17 24 39 1 15 26 19 22 17 16 8 3 5 12 9 4

76 33 51 0 23 50 42 59 59 62 13 0 40 50 33 75

.007

T1-T2 T3 W-M Poor

.030

Absent Low High Absent Low High

.036

.001

Relative Risk

95% C.L

.061 --

.01-.77

.133 .103 -5.51 5.96 --

.02-.94 .02-.63

NS

.74 .36 .004

.032

.109

.73-42 .50-70

.25

"41 patients underwent radical resection between 1965 and 1977 who had stage I or II rectal adenocarcinoma and were studied as part of our original molecular marker study, n2 These patients would have been candidates for conservative surgery now but received radical resection with full pathological evaluation, which was standard therapy, and, as a result, are used to assess the impact of clinicopathologic variables and molecular markers on outcome. W-M is well and/or moderately differentiated. Poor is poorly differentiated. NS, not significant. VascularInvasion includes lymphatic and neural as well as vascular invasion by treatment. Absent, < 1% of tumor cells stain for marker on indirect immunoperoxidase staining. Present,-->1% of tumor cells stain for marker on indirect immunoperoxidase staining. Low, <50% of tumor cells display marker on indirect immunoperoxidase staining. High, -->50% of tumor cells display marker on indirect immunoperoxidase staining. See text for actual description of markers. % NED, % no evidence of disease. Univariate analysis was performed using a Kaplan-Meier Life-Table method. Variables that have a Log RankP value --<.10were included in a Cox proportional hazards analysis and P values, relative risks, and 95% confidence intervals (95% C.I.) calculated.

Jessup, Loda, and Bleday

58

preservation approach if the cancer is small enough, negative margins are achieved, and adjuvant chemoradiation therapy is given. Poorly differentiated carcinomas, however, generally have other characteristics that make them less likely to be appropriate for sphincter preservation. In the prospective series of local excisions performed at the Beth Israel Deaconess Medical Center and reported by Bleday et at, 3~ there were only four locoregional failures in 48 patients, but one of these was in a poorly differentiated cancer that was also a T3 lesion, had a positive radial margin, produced mucin, and invaded lymphatics. Thus, poorly differentiated cancers may be more likely to recur, but their behavior may be due to other attributes that are more likely to make the cancers unfavorable candidates for conservative therapy.

Ulceration One of those attributes that is characteristic of an aggressive carcinoma is ulceration. Cohen et al 6 stressed that an important poor prognostic factor was ulceration that made the cancer appear to be fiat with rolled edges. More recently, 4 of 5 (80%) rectal cancers that were ERUS staged as T1-T2 lesions but were ulcerated recurred after 75 to 120 Gy of endocavitary radiation compared with only 4 of 27 (15%) of exophytic lesions that were treated to the same dose. 31 In a multivariate analysis of the factors associated with failure after sphincter preservation for distal rectal cancers, Minsky et al32 found that ulceration increased locoregional failure in addition to T stage.

Mucin Production The production of significant amounts of extracellular mucin has long been considered a poor prognostic sign for colon and rectal cancer. GrinnelP 3 may have been the first to realize that a subset of eolorectal carcinomas produces excess extracellular (mucinous or colloid carcinomas) or intracellular (signet-ring cell carcinomas) mucin and created the pathological criteria that define these two types of mucinous carcinomas. Although several studies 343a supported the concept that mucinous carcinomas have a worse prognosis than nonmucinous carcinomas, other studies 39-41 have not demonstrated that extracellular mucinous carcinomas have a worse prognosis than carcinomas of the same histological state of differentiation. In a local excision series, Faivre et al27 suggested that mucinous carcinomas have a significantly greater propensity for locoregional failure. Heys et al42 found that in a multivariate analysis of 92

Scottish patients less than 45 years old with colorectal cancer, mucinous carcinomas were 4.9 times more likely to develop local recurrence than nonmucinous carcinomas. The prognosis of mucinous carcinomas may reflect the biological effects of the histological grade because mucinous carcinomas are often poorly differentiated. The exclusion of mucinous rectal carcinomas from conservative sphincter-preserving surgery requires a closer examination in future trials and consideration of the state of differentiation of the cancer as a whole.

Lymphatic, Vascular, or Neural Invasion Considerable evidence suggests that the presence of

invasion of vessels, lymphatics, or nerves by the carcinoma is an important poor prognostic variable. In the Beth Israel Deaconess Medical Center series, 3~ both of the locoregional failures in T1 cancers occurred in patients with lymphatic invasion. Faivre et al27 also found that this was a significant indicator of increased risk of recurrence in their local excision series. In addition, Heys et a142 found that vascular invasion by carcinomas was a significant risk factor that increased local recurrence 9.4-fold compared with tumors that lacked vascular invasion. The importance of neural invasion has been underscored by a French study43 that demonstrated in 339 rectal carcinomas that the presence of neural invasion was a significant covariate in a multivariate Cox proportional hazards even in the presence of stage and grade variables, whereas vascular invasion was not an independent factor. Because neural invasion decreased survival by a relative risk of 1.6-fold, it may not be a strong enough factor for the small singleinstitution studies seen to date. It would be of interest to determine in larger multicenter trials of sphincter preservation if neural invasion was a significant indicator of poor prognosis. Minsky et al $2 found that vascular invasion, similar to ulceration, of the primary cancer tended to increase locoregional failure. Thus, practical experience suggests that the identification of invasion of lymphatics, vessels, or nerves warrants at least external-beam radiotherapy with chemotherapy for radiosensitization for T1 lesions and possibly more radical resection of T2 or T3 primary cancers, especially if the margins are positive.

Margins, Tumor Fixation, and Digital Rectal Examination It is an axiom of oncologic surgery that the margins surrounding a cancer must be negative to achieve

Clinical and Molecular PrognosticFactors in Sphincter-PreservingSurgery

local control. If there is disease in the bed of the resection, the local recurrence rate is high unless the residual cancer is controlled by such other modalities as radiation therapy or chemotherapy. For local excision of rectal carcinoma to succeed, the amount of microscopic residual carcinoma must be minimal. This means that the margins of resection are microscopically negative and the probability of microscopic lymph node metastasis is low enough that chemoradiation therapy can control disease. Herrera and Brown44 have found that the majority of lymph node metastases are less than 5 m m in rectal carcinoma, especially in T1 and T2 carcinomas. This size is amenable to control by external-beam radiotherapy with radiosensitization. The identification of residual microscopic disease in the operative margins, however, may not necessarily be controllable by radiation therapy. Rich et al45 demonstrated that in 15 rectal carcinomas treated with local excision control of positive microscopic disease was achieved when the tumor dose was greater than 45 Gy delivered in conventional 1.8-Gy fractions. In the Beth Israel Deaconess series, 3~ two of the four recurrences are associated with positive margins. As a result, our policy is to resect until margins are clear, even if that requires a second procedure. It is important to stress that the most important margin may be the hardest to clear: the deep or radial margin. Adam et al la have found that a microscopically involved positive radial margin after radical resection is associated with a positive predictive value of 85% for local recurrence, de Haas-Kock et al.46 have performed the clearest study yet of the relative contribution of the radial margin to outcome. In a series of 325 patients treated with radical surgery, they observed that a radial margin of less than 1 m m (a positive margin) was associated in a multivariate analysis with an increased risk of local recurrence and that this association was stronger than either the depth of penetration into the rectal wall (T89 versus T35) or the status of the regional nodes (NO versus N+). Although local recurrence was significantly associated with the status of the radial margin, the association between the radial margin and survival was more complex. Only in node-positive patients was the status of the radial margin significant for the development of distant metastases. Overall survival in this series was associated with the status of the lymph nodes and not with either the T stage or the status of the radial margin. These results suggest that salvage of patients with local recurrence may be feasible. Thus, local recur-

59

rence because of near or positive radial margins may be similar in its biological properties to local recurrence after breast-conserving surgery. Because the consequences of locoregional failure that is not controlled by salvage therapy are often more dire in the pelvis than on the chest wall, however, it is imperative that local control be achieved with as high a frequency as possible. Tumor fixation is also related to margins because tethering to the perirectal tissues either denotes direct tumor invasion or inflammatory changes that cause the fibrosis that tethers the tumor. Either situation suggests that the cancer is likely to have penetrated the rectal wall to extend into the perirectal fat (at least a T3 lesion). Although ERUS is likely to identify or confirm these deep lesions, the question of tumor fixation to the pelvic structures results from a subjective impression formed during a digital rectal examination (DRE). Because DRE is the mainstay for many of the guidelines of screening for colorectal cancer as well as for the staging and therapeutic decision making for rectal cancer, it is important to define the accuracy of DRE. Studies have compared DRE with ERUS and other imaging modalities. Stark et aP 7 compared DRE with ERUS in a series of 34 rectal cancers prospectively. DRE detected only 68% of the lesions that were later confirmed at surgery. In contrast, 88% of the lesions were detected by ERUS. The sensitivity, specificity, and positive predictive value of DRE were 75%, 86%, and 90%, whereas the sensitivity, specificity, and positive predictive value of ERUS were 91%, 91%, and 95%. Similar results were reported by Rafaelsen et al,48 who studied 107 patients with ERUS and DRE. Both techniques were quite similar in their ability to detect rectal wall penetration by tumor (T3 or T4 disease), but DRE overestimated the depth of penetration in rectal cancers that were limited to the rectal wall (T1 or T2 disease). In 33 patients with T1-T2 rectal cancers, ERUS identified the correct T stage of disease in 26 patients, but DRE was correct in only 13 patients. Thus, DRE seems to detect rectal penetration that may be associated with tumor fixation or loss of mobility. It may not be that accurate, however, for detecting and evaluating correctly more limited extents of rectal cancer. In this series, 19 patients had lymph node metastases. ERUS detected metastases in 11 of these patients, whereas DRE did not detect metastases in any of the patients. As a result, it is important that patients who are candidates for sphincter-preserving therapy undergo ERUS to image and stage the patient as carefully as possible.

Jessup, Loda, and Bleday

60

Molecular Markers as an Aid to Determining Prognosis in Rectal Cancer Conservative therapy of rectal cancer may be enhanced if there are other prognostic markers that help assess the likelihood of either locoregional failure or disease progression. Our group and others have been interested in the use of the expression of certain molecules associated with differentiation, neoplastic transformation, or the cell cycle within primary carcinomas as indicators of the biological behavior of a cancer. We have three markers that may help assess prognosis and contribute to the determination of whether a T2 or T3 lesion may need chemoradiation therapy because it has a low probability of recurrence compared with a T1 rectal cancer that may require a radical resection with adjuvant chemotherapy and radiation therapy because of a high likelihood of recurrence. Each of these markers has provided significant prognostic information in multivariate analyses of a large data set of colon and rectal carcinomas. Therefore, each marker may provide information that may be available from a biopsy that could enhance the estimation of outcome over that provided by just T stage and state of differentiation. The markers that we discuss briefly are a putative tumor suppressor gene (Deleted in Colorectal Carcinoma [DCC]), a differentiation-associated marker (sucrase-isomaltase [S-I]), and p27 Kipl, a cyclin-dependent kinase inhibitor. In addition, other promising intratumoral markers for rectal cancer are thymidylate synthase and molecules associated with angiogenesis. For a more complete review of prognostic markers in rectal carcinoma, please see Jessup and Loda. 49 All of these intratumoral markers are detectable by immunohistochemistry of formalinfixed tissue. Therefore, if the expression of these molecules is validated as a useful and independent prognostic marker, these molecules should be able to be used in community hospitals. DCC

DCC is a tumor-suppressor gene (TSG) that may be an important prognostic marker. The primary function of DCC may be cell adhesion because its nucleotide sequence is homologous to neural cell adhesion molecule (NCAM), an important intercellular adhesion molecule for neural cells. 5~This gene was shown in early studies of loss of heterozygosity (LOH) by Kern et a151 as well as O'Connell et a152 to be a significant adverse prognostic factor when L O H for 18q was present. Laurent-Puig et al,53 however, did

not confirm that LOH for 18q was significant prognostically. Zetter 54observed that patients who developed liver metastases were more likely to lose expression of DCC than patients who retained expression.Jen et a155 then examined LOH for 18q using microsatellite markers that are near the DCC locus. Microsatellites are short stretches of mononucleotide, dinucleotide, trinucleotide, and tetranucleotide repeats that are scattered throughout the genome and display significant heterozygosity within the population. When the polymerase chain reaction (PCR) is used to amplify microsatellite DNA, each allele produces slightly different size DNAs, which can be detected on a gel after electrophoretic separation. If an allele is missing because of LOH, only one allele is detected. By comparing the PCR results from the tumor samples with those obtained from normal tissues adjacent to the tumor, Jen et a155 found that allelic loss of chromosome 18q was associated with a 2.46-fold increase in death from colorectal carcinoma after correction for TNM stage, tumor differentiation, and vascular invasion. Allelic loss of DCC was a significant independent prognostic variable in stage II colorectal cancer in multivariate analysis. Because small numbers of patients were studied who had stage II disease, site of the primary was not a significant factor, and the data suggested that L O H for 18q had similar interactions with both rectum and colon primary carcinomas. Retention of both alleles of 18q21 in stage II colorectal cancer had a 93% 5-year overall survival compared with a 54% survival in stage II patients with allelic loss. Although this seemed to be strong evidence for the role of DCC in stage II colorectal cancer, the subsequent identification of another TSG that also mapped to 18q21, the Deleted in Pancreas Cancer 4 (DPC4) gene, 56 raised questions about the relevance of DCC because the microsatellite marker used to map the allelic loss was as close to DPC4 as DCC. Subsequent work also suggested that another TSG was located in this 18q21 region, the MADR2 gene, 57 which similar to DPC4 has homology to the MAD genes that are involved in the transforming growth factor-[3 (TGF-[3) signal transduction pathway. MADR2 was found to be mutated in colorectal carcinoma, 57 but mutations in DPC4 appear to be uncommon in colorectal carcinoma. 5a As a result, there was considerable confusion about whether DCC, MADR2, or DPC4 was the TSG for colorectal carcinoma. Shibata et a159have provided an answer by demonstrating that expression of DCC detected by immunohistochemistry of formalin-fixed tissues was a signifi-

Clinical and MolecularPrognosticFactors in Sphincter-PreservingSurgery

cant predictor of outcome in both stage II and III colorectal cancer and was a significant covariate in a multivariate analysis that included stage and tumor differentiation. These data suggested that loss of expression of DCC was associated with a 30% to 40% decrease in survival in both stage II and III colorectal carcinoma. Site of primary did not appear to be important because rectal primaries behaved similarly to colon primary cancers in regards to expression of DCC and to DCC-related outcomes. By measuring the specific gene product, Shibata et al may have clarified whether DCC acts as the primary TSG for colorectal carcinoma on 18q21.

DNA Content, Ploidy, and Proliferation Abnormal DNA content (aneuploidy) and high fraction of cells synthesizing DNA (S phase fraction [SPE]) have been considered to be hallmarks of poor outcome in colorectal carcinoma. The guidelines of both the College of American Pathologists6~and the American Society of Clinical Oncology,61 however, indicate that the measurement of either ploidy or proliferation using flow cytometry is inconclusive as a prognostic factor that contributes to predicting outcome when added to the standard clinicopathologic variables of stage, nodal status, and tumor differentiation. Wolley et al62 observed in a retrospective study that DNA diploid colorectal carcinomas had a 65% survival compared with a 7.5% survival in aneuploid tumors. When ploidy was examined in a multivariate analysis, stage and differentiation were more important, and ploidy was not a significant covariate. Schutte et al63 found that ploidy was associated with survival but only in stage III patients. Kokal et a164did demonstrate that ploidy was a significant covariate in a retrospective study. Prospective trials by Jones et al~5 or retrospective analysis of a prospective rectal cancer adjuvant trial by Fisher et al,6G however, demonstrated that aneuploidy was associated in univariate but not multivariate analysis with outcome. Albe et a167 have performed static cytometry on smears of 211 colorectal carcinomas and found that ploidy was a significant independent covariate in multivariate analysis. Measurements of proliferation by flow cytometry are also inconclusive. Bauer et al6~and Harlow et a169 have performed careful analyses of both ploidy and proliferation and found that nodal status was a stronger covariate than proliferation, although high proliferative rate was a significant variable in stage III patients with nodal metastases. In contrast, Witzig et al 7~ observed in a large retrospective study of

61

694 colorectal carcinoma patients that both ploidy and proliferation were important and that patients whose cancers were diploid but with high SPF had a worse outcome than patients with diploid cancers and low SPF. Cytometry is technically challenging because the sample preparation for flow cytometry must be exquisitely controlled to provide reproducible data, and static cytometry may yield information similar to flow cytometry but is so labor intensive as to be prohibitively expensive. As a result, investigators have more recently begun to explore alternative molecular markers to assess proliferative capacity in carcinomas. The cell cycle regulatory proteins may be important surrogate markers because they are involved in the regulation of proliferation by controlling how cells progress through G1 to S phase and then through G2 and mitosis. Two major participants are the @ins--proliferating cell nuclear antigen (PCNA), which is essential for passage through S-phase, and MIB-1 antigen, which is a related molecule that also interacts with PCNA during Sphase. Immunohistochemistry of fixed tissues with monoclonal antibodies to PCNA or MIB-1 (also known as Ki-67) may provide surrogates for assessing SPF. Lazaris et a171 found that PCNA staining in 35 adenomas and 60 colorectal carcinomas correlated with SPF determined by flow cytometry, but that neither were independent prognostic factors. Linden et al, 72however, did not confirm that PCNA or MIB- 1 staining correlated with flow cytometry in a retrospective study. Finally, Neoptolemos et a173observed that PCNA staining was not a significant prognostic indicator for patients with rectal cancer in a study that compared PCNA staining with standard clinicopathologic staging, whereas Sun et al TM had similar data in colorectal cancer patients. Thus, neither PCNA nor MIB-1 may be significant prognostic factors because proliferation is not associated with outcome in colorectal carcinoma. Although PCNA staining may not be a prognostic factor for outcome in rectal cancer, it may be important for predicting the response to preoperative radiation therapy. Willett et a175,76at the Massachusetts General Hospital have shown that small rectal cancers with high PCNA staining in preoperative biopsy specimens respond well to 45 to 50 Gy radiation therapy. In contrast, PCNA staining in the postirradiation biopsy specimen was often significantly decreased compared with that in the preoperative biopsy specimen and that high PCNA staining of the postirradiation tissue was associated with better

Jessup, Loda, andBleday

62

local control than MIB-1 staining. 77 Low PCNA staining in T3/T4 rectal cancers, either preirradiation or postirradiation, was associated with a higher rate of disease progression. Desai et al78 presented somewhat contrary data because they found that high PCNA staining in the preirradiation biopsy specimen was associated with locoregional failure in 23 patients with T3 or T4 rectal cancers. In their study, the postirradiation tissues could not be evaluated reliably because the radiation appeared to decrease expression of the antigens. They did observe a subset of tumors with low PCNA staining and lacking expression of p53 that appeared to respond well to radiation. Thus, the presumed presence of wild-type p53 in this study may be associated with a better response to radiation perhaps because tumor cells retained the elements to undergo apoptosis. Obviously, more work is necessary to define better the role of PCNA, MIB-1, and other proliferation markers in predicting both outcome and response to radiation therapy.

Cell Cycle Regulatory Proteins A number of other cell cycle regulatory proteins are currently being evaluated for possible use as prognostic factors and as therapeutic indicators. Cyclins D 1 and E are involved in progression through the G1 phase of the cell cycle. Overexpression of both have been reported in colorectal carcinoma, 79a2 whereas overexpression of p34 cdc2, a kinase that activates cyclins in the G2 phase of the cell cycle, has also been described in colorectal carcinoma. 8~ The prognostic or therapeutic significance of these alterations in molecules that drive cells through the cell cycle has not yet been defined. Of more interest may be overexpression of molecules that inhibit cell cycle progression, p21 wAF1 is one such cell cyclin-dependent kinase inhibitor (CKI) that mediates the G1 arrest that occurs when DNA damage is induced in cells that contain a wild-type p53. DNA damage from radiation or chemotherapy induces cells to arrest in G1 in an attempt to repair their DNA. 84 If p53 is mutated, such repair does not occur, and the cell proceeds through S phase into G2AVI.85Although this mutation in p53 may allow for terminal proliferation if lethal DNA damage is not repaired, it may also allow a cell that has not received a lethal dose of either radiation or chemotherapy to survive, p21 wAFl does not appear to be mutated in colorectal cancer, 86 but it is not clear that the expression of p21 wAF1 is a prognostic factor. Because p21 wAFI is induced when wild-type p53 is induced, it may be important only in

the small subset of colorectal carcinomas that contain wild-type or nonmutated p53. Although most research has thus far centered on p53's control of the G1/S checkpoint, evidence points to a possible control by p53/p21 of the G2/M checkpoint as well, further underscoring the central role of p53 in cancer. 85 p21 waF1 may also be up-regulated by TGF-[387 as well as by the cell adhesion molecule cx6134 integrin. 88 Further work is necessary to evaluate the role of p21 waF1 in colorectal carcinoma prognosis and response to therapy.

p27~ 1 A CKI that may be a more important prognostic factor is p27 raP1. Related by sequence homology to p21 w~l, p27 ram inhibits cell cycle progression from G1 into S phase by binding to and inhibiting cyclin E/CDK2 (for review see Steeg and Abrahmsag). Its expression is regulated by ubiquitin-mediated proteolysis. 9~Loda et aP 1 have shown that patients whose tumors express p27 ram have a significantly better outcome than patients whose tumors do not express p27 ram. This finding also was true for stage II colorectal cancer patients in whom the risk of dying from disease was 12-fold greater in patients whose cancers either did not express any p27 ~ 1 or had low expression of p27 vael (<50% of tumor cells displayed p27 FaP1 on IHC [immunohistoehemistry]) compared with high expression of p27 ~ 1 (->50% of the tumor cells stained for p27K1el). Similar associations between expression of p27 Krm and outcome have been reported for breast cancer. 92-94Thus, p27 ~rm appears to be a potent prognostic marker whose expression is associated with a good outcome and may have as strong an association with positive outcome in multivariate analyses as DCC. Rectal carcinomas were as likely to express p27 rael as colon primary cancers. 91 Furthermore, analysis of the subset of patients with stage I to HI rectal cancer reveals that p27 ~ 1 expression was a significant positive prognostic factor in univariate analysis (Table 3). Finally, p27 ram has been implicated as a regulator of resistance to chemotherapy. 95 Another set of CKIs (cyclin-dependent kinase inhibitors) are members of the INK family and are important TSGs in such cancers as melanomaP 6 There are few data, however, that these molecules are involved in colorectal carcinoma. As a result, one of the most promising molecular markers for prognosis in colorectal carcinoma is p27 ~ 1 . It is important that this marker be evaluated in confirmatory trials soon.

Clinical and MolecularPrognosticFactorsin Sphincter-PreseroingSurge~

Mucins Considerable interest has been expressed in the role of mucin and associated oligosaccharides as markers. 97 Bresalier et al 9s have reported that metastases expressed more MUC2 sialomucin than did the corresponding primary colorectal carcinomas. This mucin is the predominant mucin expressed in the gastrointestinal tract, is expressed normally in small and large intestine, contains variable numbers of tandem repeats of 23 amino acids, and expresses the O-linked oligosaccharides T and Tn. Bresalier et a198 demonstrated that the amount of sialic acid conjugated to T and Tn was increased in metastases and that the expression of sialyl-T and Tn was associated with increased adherence to cytokine-activated hepatic endothelial cells. Because sialyl-T and Tn are ligands for E-selectin, a cell adhesion receptor expressed on endothelial cells, 99 any increase in sialyl-T or Tn may increase the adherence of circulating metastatic precursor cells to endothelial cells within organs as an initial step in metastasis. Bresalier et a198 found that inhibition of E-selectin by specific monoclonal antibody inhibited the ability of human colorectal carcinoma cells to bind to activated hepatic endothelial cells. Earlier, Itzkowitz et al ~~176 had reported that T, Tn, and sialyl-Tn were expressed during fetal development and re-expressed in malignant bowel carcinomas, especially in poorly differentiated and mucinous tumors. Then Itzkowitz et al 1~ observed that expression of sialyl-Tn as detected by IHC with monoclonal antibody TKH2 was a determinant of poor outcome in colorectal carcinomas whose effects were independent of DNA ploidy, primary tumor location, stage, and grade. Similar increases in the degree of sialylation of blood group substances, oligosaccharides attached to extended carbohydrate chains on mucins, glycoproteins, and glycolipids, have been reported for Lewis ~ (Le a) and Lewisx (Lex). Nakagoe et al 1~ observed that expression of sialylLe x was a significant poor prognostic factor. The data suggest that examination of the primary for this marker may not be useful if the marker is upregulated in nodal or visceral metastases. Evaluation of the metastases, however, mayyield either therapeutic response or prognostic data that may guide treatment of patients with advanced colorectal carcinoma. Nakarnori et al 1~ examined 132 colorectal cancer patients with 114 undergoing curative resection. Expression in rectal carcinomas was similar to that in colon. Those primary carcinomas that did not express sialyl-Lex had a 93% 5-year survival compared with a 58% 5-year survival for those carcino-

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mas that did express sialyl-Lex. This was a significant factor in both univariate analysis and a Cox proportional hazards analysis. Thus, the expression of sialylLe x and possibly sialyl-Tn is an important prognostic marker that should be further evaluated. Another mucin whose expression is often upregulated during neoplastic transformation and progression is MUC 1 mucin. This mucin is composed of variable numbers of tandem repeats of 20 amino acids, and expression is often increased in dysplastic polyps 104 as well as in invasive carcinomas. Lise et al I~ found that expression of MUC1 mucin by immunohistochemistry with the monoclonal antibody DF3 trended with an inverse association with outcome but was not a significant covariate for outcome when stage-related and grade-related variables were included in the model. These different results with the expression of MUC1, MUC2, and related oligosaccharides suggest that these molecules may be useful but require more evaluation before they can be recommended for use. Sucrase-Isomaltase Our group has been interested in the expression of the disaccharidase sucrase-isomaltase (S-I) in eolorectal carcinoma, which is not involved in cell adhesion but is expressed on the cell surface. S-I expression is increased in severely dysplastic adenomatous polyps and in primary invasive carcinomas. 1~176 In contrast, normal adult colon adjacent to these neoplasms is negative for S-I by enzyme activity, protein, or gene transcript. ~~ Other investigators have confirmed that premalignant lesions and carcinomas in the esophagus are also associated with increased expression of S-I.l~176 The function of S-I in human colorectal carcinoma is not clear, but it may be involved with glucose metabolism and energy production.ll~ m S-I expression, however, was a significant predictor of disease recurrence in 183 stage I to III patients with colorectal carcinoma who had undergone potentially curative resection by either univariate or multivariate analysis. 11~S-I expression was similar in patients with either rectal or colon cancer. 112 When patients with stage I to ill rectal carcinoma were analyzed separately for the interaction between outcome and expression of S-I, there was a 10% better survival in patients whose tumors did not express S-I compared with those whose tumors did. Furthermore, in the subset of 41 patients that would have been candidates for conservative rectal surgery, expression of S-I in more than 50% of the tumor cells was a significant poor prognostic factor in both univariate

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Jessup, Loda, and Bleday

and multivariate analyses (Table 3). Thus, S-I expression should be pursued further as a marker of poor outcome, whereas the expression of p27 rdpi and DCC are markers associated with good outcomes.

ThymidylateSynthase Thymidylate synthase (TS) is the critical enzyme for 5-fluorouracil (5-FU) inhibition of cell proliferation. TS catalyzes the conversion of dUMP into dTMP for subsequent incorporation into DNA. Without TS, mammalian cells can salvage thymidine from the extracellular milieu only through the thymidine kinase pathway. TS is inhibited by a primary metabolite of 5-FU, FdUMP, and this inhibition is enhanced by 5,10-methylene-tetrahydrofolate (CH2-THF), which is a metabolite of leucovorin and reduced folates (for reviews, see Chu and Allegra 113). FdUMP and CH2-THF form a ternary complex with TS that physically prevents its catalysis of the conversion of dUMP to dTI'P. Cellular TS protein levels and activity within the cell, however, are more important than the ternary complex because they are prognostic and predict response to therapy. High TS protein levels lead to increased production of dTMP and DNA synthesis and are associated with resistance to 5-FU in v i t r o 114,u5 as well as in patients with advanced metastatic colorectal cancer.i16,ii7 Prior treatment with 5-FU is associated with increased levels of TS either during the acute response H6 or after therapy. 117The addition of leucovorin to 5-FU treatment of advanced metastatic cancers clearly reduces TS levels and activity.116 Thymidylate synthase expression in rectal cancer appears to be associated with prognosis in rectal cancer and may also predict response to adjuvant chemotherapy. Sanguedolce et a1118published a small series of colorectal cancer patients in which stage I cancers contained the highest levels of TS measured in a FdUMP-binding assay and higher levels of FdUMP-binding activity were associated with a better disease-free and overall survival. The prognostic association of TS-binding activity was independent of site of primary cancer (rectum versus right or left colon) and other standard clinicopathologic factors. In contrast,Johnston et a1119performed a large study in which formalin-fixed sections from 294 tumors of patients with rectal cancer who participated in the R-01 stage II to III rectal cancer adjuvant therapy trial of the National Surgical Adjuvant Breast and Bowel Project (NSABP) were analyzed for TS levels by immunohistochemistry using monoclonal antibody TS 106. Patients were randomized in this study

to either surgery alone, surgery plus radiation therapy, or surgery plus methylCCNU, 5-FU, and vincristine (MOF). Patients whose tumors displayed low levels of TS had a significantly better disease-free (49%) and overall (60%) survival compared with patients whose tumors displayed high levels of TS and who had 27% and 40% survival rates (both were significant at P < .01). Patients whose tumors displayed high levels of TS and received MOF chemotherapy enjoyed better survival than did patients who had high levels of TS and received only surgery. Patients with low levels of TS did not benefit further from chemotherapy compared with patients with low TS levels who had only surgery. Lenz et al I2~ have reported that TS expression as detected by IHC was also prognostic in a series of 25 patients with stage II colon cancer. This finding was interesting because it also observed that nuclear overexpression of p53 (presumably by mutation of p53) was associated with high levels of TS and that 91% of recurrences occurred in patients whose tumors had mutated p53 and high levels of TS. An interaction between TS and p53 occurs at the translational level because TS can bind to the mRNAs of both p53 and c-myc 121,122 and inhibit production of the protein product. The findings of Lenz et a112~ are important because they suggest that mutation in p53 may increase TS levels and cause resistance to chemotherapy. Further work is necessary to determine the usefulness of TS as a prognostic marker as well as an indicator of responsiveness to 5-FU-based regimens. At least suggested is an interesting interaction between the tumor suppressor gene p53, the proto-oncogene c-myc, and TS.

Angiogenesis Tumors must establish an adequate blood supply when they are 2 to 3 m m in diameter to increase in size, invade, and metastasize. 12STherefore, angiogenesis (ie, assessment of the number of microvessels adjacent to a tumor by IHC using antibodies that decorate endothelial-associated antigens on fixed sections of tumors) has been an important prognostic factor in several human cancers.124,125 Colorectal carcinoma may also demonstrate a similar relationship between angiogenesis and disease progression. In a study of rectal cancers, Saclarides et a1126found that there was an inverse association between a high concentration of microvessels and decreased survival. This group 12y also demonstrated an association between angiogenesis and survival in patients with stage II colon carcinoma. Takebayashi et a1128 found

Clinical and Molecular Prognostic Factors in Sphincter-PreservingSurgery

that microvessel density in a series of 166 colorectal carcinomas was a strong independent predictor of poor outcome even in a Cox proportional hazards analysis with stage variables included in the model. Engel et a1129 analyzed microvessel counts by IHC using a monoclonal antibody to CD31 in 35 patients with colorectal carcinoma: 13 had recurred and 22 had not. In this small sample, when microvessel counts were performed in a coded fashion, high microvessel counts (-->65 near the primary tumor) were associated with recurrence but were not an independent covariate of outcome in a multivariate analysis that included stage variables (P = . 12 with a relative risk of death of 2.97 for high microvessel count when stage h a d P = .003 and a relative risk of cancer-specific death of 9.09 in a proportional hazards model). There are several studies, however, that have not found a direct association between microvessel density and outcome. Bossi et all30 performed a larger study of 36 adenomas and 178 bowel carcinomas again using monoclonal antibody to CD31 to perform microvessel counts by IHC. Although Bossi et a113~demonstrated an increase in the density of microvessels around primary tumors that was greater than that in either normal mucosa or adenomas, the microvessel density was not associated with metastasis, stage of disease, or the survival of patients. Mooteri et all31 also found that angiogenesis was not associated with outcome in primary colorectal carcinomas, but this was a small series, and rectal carcinomas were not separated from colon cancers. In this study, angiogenesis in metastases was higher than in primary cancers, and higher microvessel counts in a metastasis were associated with a better survival. Microvessel counts may yield conflicting results because they involve selection of areas near or at the edge of a tumor to identify the highest areas of activity. Because this may introduce selection bias, Takebayashi et a1132 found that a surrogate for microvessel counts is the expression of thymidine phosphorylase, which is directly associated with microvessel counts. This association is supported by the fact that thymidine phosphorylase, or plateletderived endothelial cell growth factor, is a potent angiogenic factor. Thyrnidine phosphorylase expression as detected by IHC was significantly associated with worse outcome in 163 primary colorectal carcinomas and was a significant covariate in a Cox proportional hazards analysis. Takahashi et all33 also found that thymidine phosphorylase could be produced by host infiltrating cells and proposed that this is a novel way in which the tumor induces the host to provide

65

nutrients. Ellis et al134 demonstrated that inhibition of vascular endothelial growth factor (VEGF) by antisense DNA decreased the ability of human colorectal carcinoma cells to induce angiogenesis when implanted in nude mice. These results firmly link VEGF with angiogenesis in colorectal carcinoma. Vermeulen et al135 found that mutation in p53 as evidenced by nuclear protein overexpression in eolorectal carcinomas was associated with increased microvessel density in primary colorectal carcinomas. These authors 1~5 postulate that mutant p53 may stimulate either production of the angiogenesis inhibitor thrombospondin-1, which then indirectly enhances angiogenesis, or mutation in p53, which leads to a direct effect on tumor angiogenesis. Another stimulus for angiogenesis is hypoxia. Mattern et a1136 compared the oxygen tension within a series of rectal carcinomas with microvessel density and the expression of VEGF growth factor and such other molecules as GST-'rr (glutathione s-transferase -rr) and TS. Hypoxic tumors had fewer microvessels and higher expression of VEGF, GST--rr and the nuclear transcription factor c-jun than well-oxygenated tumors. As a result, hypoxia appears to be a stimulus for angiogenesis that is as strong as nutrient depletion. Thus, angiogenesis needs to be analyzed further in colorectal carcinoma before it can be recommended as a prognostic marker as part of standard practice.

Summary Adenocarcinomas of the distal rectum may be treated by conservative surgery and chemotherapy and radiotherapy, if they are selected appropriately. The criteria for candidates for local excision have been put forth as well as some of the newer prognostic factors that are still investigational. With better tools for evaluating the T and N stages and the biological aggressiveness of a cancer, it may be possible to tailor therapy so that radical resections are avoided. Another trend that has not been described here but is discussed elsewhere in this issue is the use of low anterior resection with coloanal anastomosis. This surgical approach may be used for those cancers that do not involve the sphincters and can be removed with negative margins. As a result, specific clinical or pathological factors may not necessarily be applied to those cancers other than the ability to achieve negative margins. Although local excision and chemoradiation therapy for rectal cancer appears to be a viable option for the care of patients with T1-T2 rectal cancer, the follow-up is still relatively short, and this

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treatment should not yet be considered standard of care until the phase II multicenter trial performed by the cooperative oncology groups matures and is published.

References 1. Parker SL, Tong T, Bolden S, et al: Cancer statistics, 1997. CancerJ Clin 47:5-27, 1997 2. Miles WE: A method of performing abdominoperineal excision for carcinoma of the rectum and of the terminal portion of the pelvic colon. Lancet 2:1812-1813, 1908 3. Steele GDJr,JessupJM: Cotorectat cancer, in Steele GDJr, JessupJM, Winchester DP, et al (eds): National Cancer Data Base: Annual Review of Patient Care 1995. Atlanta, GA, American Cancer Society, 1995, pp 66-83 4. Fisher ER, Robinsky B, Sass R, et al: Relative prognostic value of the Dukes and the Jass systems in rectal cancer: Findings from the National Surgical Adjuvant Breast and Bowel Projects (Protocol R-01). Dis Colon Rectum 32:944949, 1989 5. Jass JR, Atkin WS, Cuzick J, et al: The grading of rectal cancer: Historical perspectives and a multivariate analysis of 447 cases. Histopathology I0:437-459, 1986 6. Cohen AM, Wood WC, Gunderson LL, et ah Pathological studies in rectal cancer. Cancer 45:2965-2968, 1980 7. Morson BC: Factors influencing the prognosis &early cancer of the rectum. Proc R Soc Med 59:607-608, 1966 8. Jass JR, Atkin WS, Cuzick J, et al: The grading of rectal cancer: Historical perspectives and a multivariate analysis of 447 cases. Histopathology 10:437-459, 1986 9. Schmitz-Mormann P, Himmelmann GW, Baum U, et ah Morphological predictors of survival in colorectal carcinoma: Univariate and multivariate analysis. J Cancer Res Clin Oncol 113:586-592, 1987 10. SprattJSJr, Spjut HJ: Prevalance and prognosis of individual clinical and pathologic variables associated with colorectal carcinoma. Cancer 20:1976-i985, 1967 11. Pilipshen SJ, Heilweil M, Quan SH, et ah Patterns of pelvic recurrence following definitive resections of rectal cancer. Cancer 53:1354-1362, 1984 12. Whittaker M, Goligher JC: The prognosis after surgical treatment for carcinoma of the rectum. BrJ Surg 63:384-388, 1976 13. Williams NS, Dixon MF, Johnston D: Reappraisal of the 5 centimetre rule of distal excision for carcinoma of the rectum: A study of distal intramural spread and of patients' survival. BrJ Surg 70:150-155, 1983 14. Freedman LS, Macaskill P, Smith AN: Multivariate analysis of prognostic factors for operable rectal cancer. Lancet 2:733-736, 1984 15. Moossa AR, Ree PC, Marks JE, et ah Factors influencing local recurrence after abdominoperineal resection for cancer of the rectum and rectosigmoid. BrJ Surg 62:727-730, 1975 16. McDermott F1~, Hughes ES, Pihl E, et al: Local recurrence after potentially curative resection for rectal cancer in a series of 1008 patients. BrJ Surg 72:34-37, 1985 17. Gunderson LL, Sosin H: Areas of failure found at reoperation (second or symptomatic look) following "curative surgery" for adenocarcinoma of the rectum. Cancer 34:1278-1292, 1974

18. Adam IJ, Mohamdee MO, Martin IG, et al: Role of circumferential margin involvement in the local recurrence of rectal cancer. Lancet 344:707-711, 1994 19. Chan KW, Bey J, Wong SK: A method of reporting radial invasion and surgical clearance of rectal carcinoma. Histopathology 9:1319-1327, 1985 20. Heald RJ, Husband EM, Ryall RD: Tile mesorectum in rectal cancer surgery: The clue to pelvis recurrence? Br J Surg 69:613-616, 1982 21. Heald RJ, Karanjia ND: Results of radical surgery for rectal cancer. WorldJ Surg 16:848-857, 1992 22. Shirouzu K, Isomoto H, Kakegawa T: Distal spread of rectal cancer and optimal distal margin of resection for sphincterpreserving surgery. Cancer 76:388-392, 1995 23. Hitdebrandt U, Schuder G, Feifel G: Preoperative staging of rectal and colonic cancer. Endoscopy 26:810-812, 1994 24. MilsomJW, Czyrko C, Hull TL, et al: Preoperative biopsy of pararectal lymph nodes in rectal cancer using endoluminal ultrasonography. Dis Colon Rectum 37:364-368, 1994 25. JessupJM, McGinnis LS, Steele GDJr, et al: National Cancer Data Base: Report on colon cancer. Cancer 78:918-926, 1996 26. D'Eredita G, Serio G, Nrei V, et ah A survival regression analysis of prognostic factors in colorectal cancer. Aust N ZJ Surg 66:445-451, 1996 27. FaivreJ, ChaumeJ, Pigot F, et al: Transanal electroresection of small rectal cancer: A sole treatment? Dis Colon Rectum 39:270-278, 1996 28. Mentges B, Buess G, Schafer D, et al: Local therapy of rectal tumors. Dis Colon Rectum 39:886-892, 1996 29. Volk EE, Goldblum JR, Petras RE, et al: Management and outcome of patients with invasive carcinoma arising in colorectal polyps. Gastroenterology 109:1801- 1807, 1995 30. BledayR, Breen E, JessupJM, et ah Prospective evaluation of local excision for small rectal cancers. Dis Colon Rectum 40:388-392, 1997 31. Reed WP, Cataldo PA, GarbJL, et al: The influence of local tumor ulceration on the effectiveness of endocavitary radiation for patients with early rectal carcinoma. Cancer 76:967971, 1995 32. Minsky BD, Enker WE, Cohen AM, et al: Clinicopathologic features in rectal cancer treated by local excision and postoperative radiation therapy. Radiat Med 13:235-241, 1995 33. Grinnelt RS: The grading and prognosis of carcinoma of the colon and rectum. Ann Surg 109:500-503, 1939 34. Wolfman EF, Astler VB, Coller FA: Mucoid adenocarcinoma of the colon and rectum. Surgery 42:847-852, 1957 35. Eker R: Some prognostic factors for carcinomas of the colon and rectum. Acta Chir Scand 126:636-656, 1963 36. Symonds DA, Vicery AL: Mucinous carcinoma of the colon and rectum. Cancer 37:1891-1900, 1979 37. Pihl E, Nairn RC, Hughes ESR, et al: Mucinous colorectal carcinoma: Immunopathology and prognosis. Pathology I2: 439-447, 1980 38. Umpleby HC, Ranson DL, Williamson RCN: Peculiarities of mucinous colorectal carcinoma. BrJ Surg 72:715-718, 1985 39. Halvorsen TB, Seim E: Influence of mucinous components on survival in colorectal adenocarcinomas: A multivariate analysis.J Clin Patho141:1068-1072, 1988 40. Wiggers T, Arends JW, Schutte B, et al: A multivariate analysis of pathologic prognostic indicators in large bowel cancer. Cancer 61:386-395, 1988

Clinical and Molecular Prognostic Factors in Sphincter-PreservingSurgery

4t. Sasaki O, Atkin WS, Jass JR: Mucinous carcinoma of the rectum. Histopathology 1t:259-272, 1987 42. Heys SD, Sherif A, BagleyJS, et al: Prognostic factors and survival of patients aged less than 45 years with colorectal cancer. BrJ Surg 81:685-688, 1994 43. Bognel C, Rekacewicz C, Mankarios H, et al: Prognostic value of neural invasion in rectal carcinoma: A multivariate analysis on 339 patients with curative resection. EurJ Cancer 31A:894-898, 1995 44. Herrera L, Brown MT: Prognostic profile in rectal cancer. Dis Colon Rectum 37:S1-5, 1994 45. Rich T, Weiss DR, Mies C, et al: Sphincter preservation in patients with low rectal cancer treated with radiation therapy with local excision or fulguration. Radiology 156:527-531, 1985 46. de Haas-Kock DFM, Baeten CGMI,JagerJJ, et al: Prognostic significance of radial margins of clearance in rectal cancer. BrJ Surg 83:781-785, 1996 47. Stark M, Bohe M, Fork F-T, et al: Endoluminal ultrasound and low-field magnetic resonance imaging are superior to clinical examination in the preoperative staging of rectal cancer. EurJ Surg 161:841-845, 1995 48. Rafaelsen SR, Kronborg O, Fenger C: Digital rectal examination and transrectal ultrasonography in staging of rectal cancer: A prospective, blind study. Acta Radiol 35:300-304, 1994 49. JessupJM, Loda M: Prognostic markers in rectal carcinoma. Semin Surg Oncol (in press) 50. Fearon ER, Cho KR, Nigro J'M, et at: Identification of a chromosome 18q gene which is altered in colorectal cancers. Science 247:49-56, 1990 51. Kern SE, Fearon ER, Tersmette KW, et al: Clinical and pathological associations with allelic loss in colorectal carcinoma.JAMA 261:3099-3103, 1989 52. O'Connell MJ, Schaid DJ, Ganju V, et al: Current status of adjuvant chemotherapy for colorectal cancer: Can molecular markers play a role in predicting prognosis? Cancer 70 (suppl):1732-1739, 1992 53. Laurent-Puig P, Olschwang S, Delattre O, et al: Survival and acquired genetic alterations in colorectal cancer. Gastroenterology 102:1136-1141, 1992 54. Zetter BR: Adhesion molecules in tumor metastasis. Semin Cancer Biol 4:219-229, 1993 55. JenJ, Kim H, Piantadosi S, et al: Allelic loss of chromosome 18q and prognosis in colorectal cancer. N Engl J Med 331:213-221, 1994 56. Hahn SA, Schutte M, Hoque AT, et al: DPC4, a candidate tumor suppressor gene at human chromosome 18q21.1. Science 271:350-353, 1996 57. Eppert K, Scherer SW, Ozcelik H, et al: MADR2 maps to 18q21 and encodes a TGFbeta-regulated MAD-related protein that is functionally mutated in colorectal carcinoma. Cell 86:543-552, 1996 58. Takagi Y, Kohmura H, Futamura M, et al: Somatic alterations of the DPC4 gene in human colorectal cancers in vivo. Gastroenterology 1t 1:1369-1372, 1996 59. Shibata D, Reale MA, Lavin P, et al: The DCC protein and prognosis in colorectal cancer. N EnglJ Med 335:1727-1732, 1996 60. Henson DE, Fielding P, Grignon DJ, et al: College of American Pathologists Conference XXVI on clinical rel-

67

evance of prognostic markers in solid tumors: Summary. Arch Pathol Lab Med 119:1109-1 t 14, 1995 61. Bast RC, Desch CE, Ravdin P, et al, and ASCO Expert Panel. Clinical practice guidelines for the use of tumor markers in breast and colorectal cancer: Report of the American Society of Clinical Oncology Expert Panel. J Clin Oncol 14:28432877, 1996 62. Wolley RC, Schreiber K, Koss LG, et al: DNA distribution in human colon carcinomas and its relationship to clinical behavior.J Natl Cancer Inst 69:15-22, 1982 63. Schutte B, Reynders MJ, Wiggers TH, et al: Retrospective analysis of the prognostic significance of DNA content and proliferative activity in large bowel carcinoma. Cancer Res 47:5494-5496, 1987 64. Kokal WA, Gardine RC, Sheibani K, et al: Tumor DNA content in resectable primary colorectal carcinoma. Ann Surg 209:188-193, 1989 65. Jones DJ, Moore M, Schofield PF, et al: Prognostic significance of DNA ploidy in colorectal cancer: A prospective flow cytometric study. BrJ Surg 75:28-33, 1988 66. Fisher ER, Siderits RH, Sass R: Value of assessment ofploidy in rectal cancers. Arch Pathol Lab Med 113:525-528, t989 67. Albe X, Vassilakos P, Helfer-Guarnori K, et al: Independent prognostic value of ploidy in colorectal cancer: A prospective study using image cytometry. Cancer 66:1168-1175, 1990 68. Bauer KD, Lincoln ST, Vera-Roman JM, et ai: Prognostic implications of proliferative activity and DNA aneuploidy in colonic adenocarcinomas. Lab Invest 57:329-335, 1987 69. Harlow SP, Eriksen BL, Poggensee L, et al: Prognostic implications of proliferative activity and DNA aneuploidy in Astler-Colter Dukes stage C colonic adenocarcinomas. Cancer Res 51:2403-2409, 1991 70. Witzig TE, Loprinzi CL, GonchoroffNJ: DNA ploidy and cell kinetic measurements as predictors of recurrence and survival on stages B2 and C colorectal carcinoma. Cancer 68:879-888, 1991 71. Lazaris AC, Davaris P, Nakopoutou L, et al: Correlation between immunohistochemical expression of proliferating cell nuclear antigen and flow cytometry parameters in colorectal neoptasia. Dis Colon Rectum 37:1083-1089, 1994 72. Linden MD, el-Naggar AK, Nathanson SD, et ai: Lack of correlation between flow cytometric and immunohistologic proliferation measurements of tumors. Mod Pathol 9:682689, 1996 73. Neoptolemos JP, Oates GD, Newbold KM, et al: Cyclin/ proliferation cell nuclear antigen immunohistochemistry does not improve the prognostic power of Dukes' or Jass' classifications for colorectal cancer. B r J Surg 82:184-187, 1995 74. Sun XF, CarstensenJM, St-al O, et al: c-erbB-2 oncoprotein in relation to DNA ploidy and prognosis in colorectal adenocarcinoma. APMIS 103:309-315, t 995 75. Willett CG, Warland G, Cheek R, et al: Proliferating cell nuclear antigen in rectal cancer: Predictor of response to preoperative irradiation.J Clin Oncol 12:679-682, 1994 76. Willett CG, Warland G, Coen J, et al: Rectal cancer: The influence of tumor proliferation on response to pre-operative irradiation. IntJ Radiat Oncol Biol Phys 32:57-61, 1995 77. Willett CG, Warland G, Hagan MP, et al: Tumor proliferation in rectal cancer following preoperative irradiation.J Clin Oncot 13:1417-1424, 1995 78. Desai GR, Myerson RJ, Higashkubo R, et al: Carcinoma of

68

79.

80.

81.

82.

83.

84.

85.

86.

Jessup, Loda, and Bleday

the rectum: Possible cellular predictors of metastatic potential and response to radiation therapy. Dis Colon Rectum 39:1090-1096, 1996 Arber N, Hibshoosh H, Moss SF, et al: Increased expression of cyclin D1 is an early event in multistage colorectal carcinogenesis. Gastroenterology 110:669-674, 1996 BartkovaJ, LukasJ, Strauss M, et al: The PRAD-1/cyclin D1 oncogene product accumulates aberrantly in a subset of colorectal carcinomas. IntJ Cancer 58:568-573, 1994 Yasui W, Kuniyashi H, Yokozaki H, et al: Expression of cyclin E in colorectal adenomas and adenocarcinomas: Correlation with expression of Ki-67 antigen and p53 protein. Virchows Arch 429:13-19, 1996 Kitahara K, Yasui W, Kuniyasu H, et al: Concurrent amplification of cyclin E and CDK2 genes in colorectal carcinomas. IntJ Cancer 62:25-28, 1995 Yasui W, Ayhan A, Kitadai Y, et al: Increased expression of p34cdc2 and its kinase activity in human gastric and colonic carcinomas. IntJ Cancer 53:36-41, 1993 el-Deiry WS, Tokino T, Velculescu VE, et al: WAF1, a potential mediator of p53 tumor suppression. Cell 75:817825, 1993 Waldman T, Lengauer C, Kinzler KW, et ah Uncoupling of S phase and mitosis induced by anticancer agents in cells lacking p21. Nature 381:713-716, 1996 Li Y-J, Laurent-Puig P, Salmon RJ, et al: Polymorphisms and probable lack of mutation ha the WAF1-CIP1gene in colorectal cancer. Oncogene 10:599-601, 1995

87. Li C-Y, Suardet L, Little JB: Potential role of WAF1/Cip l/p21 as a mediator of TGF-[3 cytoinhibitory effect. J Biol Chem 270:4971-4974, 1995 88. Clarke AS, Lotz IVlM, Chao C, et al: Activation of the p21 pathway of growth arrest and apoptosis by the [34 integrin cytoplasmic domain.J Biol Chem 270:22673-22676, 1995 89. Steeg PS, AbrahmsJS: Cancer prognostics: Past, present and p27. Nat Med 3:152-154, 1997 90. Pagano M, Tam SW, Theodoras AM, et al: Role of the ubiquitin-proteasome pathway in regulating abundance of the cyclin-dependent kinase inhibitor p27. Science 269:682685, 1995 91. Loda M, Cukor B, Tam SW, et al: Increased proteasomedependent degradation of the cyclin-dependent kinase inhibitor p27 in aggressive colorectal carcinomas. Nat Med 3:231234, 1997 92. Porter PL, Malone KE, Heagerty PJ, et al: Expression of cell-cycle regulators p27 mpl and cycfin E, alone and in combination, correlate with survival in young breast cancer patients. Nat Med 3:222-225, 1997 93. Catzavelos C, Bhattacharya N, Ung YC, et al: Decreased levels of the cell-cycle inhibitor p27Kipl protein: Prognostic implications in primary breast cancer. Nat Med 3:227-230, 1997 94. Tan P, Cady B, Wanner M, et al: The cell cycle inhibitor p27 is an independent prognostic marker in small (Tla,b) invasive breast carcinomas. Cancer Res 57:1259-1263, 1997 95. St. Croix B, Florenes VA, Rak JW, et al: Impact of the cyclin-dependent kinase inhibitor p27Kipl on resistance of tumor cells to anticancer agents. Nat Med 2:1204-1210, 1996 96. Kamb A: Cell-cycle regulators and cancer. Trends Genet 11:136-140, 1995 97. JessupJM, Gallick GE: The biology of colorectal carcinoma. Curr Prob Cancer 16:261-328, 1992

98. Bresalier RS, Ho SB, Schoeppner HL, et al: Enhanced sialylation of mucin-associated carbohydrate structures in human colon cancer metastasis. Gastroenterology 110:13541367, 1996 99. Varki A: Selectins and other mammalian sialic acid-binding lectins. Curr Opin Cell BM 4:257-266, 1992 i00. Itzkowitz SH, Yuan M, Montgomery CK, et al: Expression of Tn, Sialosyl-Tn, and T antigens in human colon cancer. Cancer Res 49:197-204, 1989 101. Itzkowitz SH, Bloom EJ, Kokal WA, et al: Sialosyl-Tn: A novel mcin antigen associated with prognosis in colorectal cancer patients. Cancer 66:1960-1966, 1990 102. Nakague T, Fukushima K, Hirota M, et ah Immunohistochemical expression of sialyl Lex antigen in relation to survival of patients with colorectal carcinoma. Cancer 72:23232330, 1993 103. Nakamori S, Kameyama M, Imaoka S, et al: Increased expression of sialyl Lewisx antigen correlates with poor survival in patients with colorectal carcinoma: Clinicopathological and immunohistochemical study. Cancer Res 53:56325637, 1993 104. Andrews CW Jr, Jessup JM, Goldman H: Localization of tumor-associated glycoprotein DF3 in normal, inflammatory, and neoplastic lesions of the colon. Cancer 72:3185-3190, 1993 105. Lise M, Loda M, Fiorentino M, et al: Association between sucrase-isomaltase (SI) and p53 expression in colorectal cancer. Ann Surg Oncol 4:176-183, 1997 106. Wiltz O, O'Hara CJ, Steele GD, et al: Sucrase-isomaltase: A marker associated with the progression of adenomatous polyps to adenocarcinomas. Surgery 108:269-276, 1990 107. Wiltz O, O'Hara CJ, Steele GD, et al: Expression of enzymatically active sucrase-isomaltase is a ubiquitous property of colon adenocarcinomas. Gastroenterology 200:1266-1278, 1991 108. Wu GD, Beer DG, MooreJH, et al: Sucrase-isomaltase gene expression in Barrett's esophagus and adenocarcinoma. Gastroenterology 105:837-844, 1993 109. Nikulasson S, Andrews CWJr, Goldman H, et ah Sucraseisomaltase expression in dysptasia associated with Barrett's esophagus and chronic gastritis. IntJ Surg Pathol (in press) 110. Chantret I, Rodolosse A, Barbat A, et ah Differential expression of sucrase-isomaltase in clones isolated from early and late passages of the cell line Caco-2: Evidence for glucosedependent negative regulation.J Cell Sci 107:213-225, 1994 111. Darmonl D, Baricault L, Sapin C, et al: Decrease of mRNA levels and biosynthesis of sucrase-isomaltase but not dipeptidylpeptidase IV in forskolin or monensin-treated Caco-2 cells. Experientia 47:1211-1215, 1991 112. Jessup JM, Lavin PT, Andrews CW Jr, et al: Sucraseisomaltase is an independent prognostic marker for colorectal carcinoma. Dis Colon Rectum 38:1257-1264, 1995 113. Chu E, Allegra CJ: The role of thymidylate synthase in cellular regulation. Adv Enzyme Regu136:143-163, 1996 114. Copur S, Aiba K, DrakeJC, et al: Thymidylate synthase gene amplification in human colon cancer cell lines resistant to 5-fluorouracil. Biochem Pharmaco149:1419-1426, 1995 115. Mulder NH, Timmer-Bosscha H, Meersma GJ, et ah Thymidylate synthase levels in tumor biopsies from patients with colorectal cancer. Anticancer Res 14:2677-2680, 1994 116. Peters GJ, van der Wilt CL, van Groeningen CJ, et ah Thymidylate synthase inhibition after administration offluo-

Clinical and Molecular Prognostic Factors in Sphincter-PreservingSurgery

rouracil with or without leucovorin in colon cancer patients: Implications for treatment with fluorouracil. J Ctin Oncol 12:2035-2042, 1994 117. Leichman L, Lenz HJ, Leichman CG, et al: Quantitation of intratumoral thymidylate synthase expression predicts for resistance to protracted infusion of 5-fluorouracil and weekly leucovorin in disseminated colorectal cancers: Preliminary report from an ongoing trial. EurJ Cancer 31A:t306-1310, 1995 1t8. Sanguedolce R, Brumarescu I, Dardanoni G, et al: Thymidylate synthase level and DNA-ploidy pattern as possible prognostic factors in human colorectat cancer: A preliminary study. Anticancer Res 15:901-906, 1995 119. Johnston PG, Fisher ER, Rockette I-IE, et al: The role of thymidylate synthase expression in prognosis and outcome of adjuvant chemotherapy in patients with rectal cancer.J Clin Oncol 12:2640-2647, 1994 120. Lenz HJ, Danenberg KD, Danenberg P, et al: p53 status and thymidylate synthase (TS) expression are associated and predict for recurrence in patients with stage II colon cancer (CC). Proc Am Soc Clin Oncol 15:215, 1996 121. Chu E, Voeller DM, Jones KL, et al: Identification of a thymidylate synthase ribonueleoproteincomplex in human colon cancer cells. MOl Cell Biol 14:207-213, 1994 122. Chu E, Takechi T, Jones KL, et al: Thymidylate synthase binds to c-myc RNA in human colon cancer cells and in vitro. MOl Cell Biol 15:179-185, 1995 123. FolkmanJ, Watson K, Ingber D, et al: Induction ofangiogenesis during the transition from hyperplasia to neoplasia. Nature 339:58-61, 1989 i24. Ribatti D, Vacea A, Palmer W, et al: Angiogensis during tumor progression in human malignant melanoma, In Steiner R, Weisz PB, Langer SR (eds): Angiogenisis: Key PrinciplesScience-Technology-Medicine.Basel, Birkhauser Verag, 1992, pp 415-419 125. Weidner N, Semple JP, Welch WR, et ah Tumor angiogenesis and metastasis---correlation in invasive breast cancer. N EnglJ Med 324:1-8, 1991 126. Saclarides TJ, Speziale NJ, Drab E, et ah Tumor angiogenesis and rectal carcinoma. Dis Colon Rectum 37:921-926, 1994 127. Frank RE, Sadarides TJ, Leurgans S: Tumor angiogenesis as a predictor of recurrence and survival in node-negative colon cancer. Ann Surg 222:695-699, 1995 128. Takebayashi Y, Aklyama S, Yamada K, et al: Angiogenesis as an unfavorable prognostic factor in human colorecta[ carcinoma. Cancer 78:226-23 I, 1996

69

129. Engel CJ, Bennett ST, Chambers AF, et al: Tumor angiogenesis predicts recurrence in invasive colorectal cancer when controlled for Dttkes staging. A m J SuN Patho120:1260-1265, 1996 130. Bossi P, Viale G, Lee AK, et al: Angiogenesis in colorectal tumors: Microvessel quantitation in adenomas and carcinomas with clinicopathological correlations. Cancer Res 55:50495053, 1995 131. Mooteri S, Rubin D, Leurgens S, et al: Tumor angiogenesis in primary and metastatic coloreetal cancers. Dis Colon Rectum 39:1073-1080, 1996 132. Takebayashi Y, Akiyama S, Akiba S, et al: Clinicopathologic and prognostic significance of an angiogenic factor, thymidine phosphorylase, in human colorectal carcinoma. J Natl Cancer Inst 88:1110-1117, 1996 133. Takahashi Y, Bucana CD, Liu W, et al: Platelet-derived endothelial cell growth factor in human colon cancer angiogenesis: Role of infiltrating cells.J Natl Cancer Inst 88:11461151, 1996 134. Ellis LM, Liu W, Wilson M: Down-regulation of vascular endothelial growth factor in hmnan colon carcinoma cell lines by antisense transfection decreases endothelial cell proliferation. Surgery 120:871-87821996 135. Vermeulen PB, Roland L, Mertens V, et al: Correlation of intratumoral microvessel density and p53 protein overexpression in human colorectal adenocarcinoma. Microvasc Res 51:164-174, 1996 136. Mattern J, Kallinowski F, Herfarth C, et al: Association of resistance-related protein expression with poor vascularization and low levels of oxygen in human rectal cancer. Int J Cancer 67:20-23, 1996 137. Fortunato L, Ahmad NR, Yeung RS, et ah Long-term follow-up of local excision and radiation therapy tor invasive rectal cancer. Dis Colon Rectum 38:1193-1 t99, 1995 138. Ota DM, SkibberJ, Rich TA: M.D. Anderson Cancer Center experience with local excision and multimodality therapy for rectal cancer. Surg Oncol Clin North Am 1:147-152, 1992 139. Wood WC, Willett CG: Update of the Massachusetts Genera[ Hospital experience of combined local excision and radiotherapy for rectal cancer. Surg Oncol Clin North Am t:131-136, 1992 140. Summers G, Mendenhall W, Copeland EM III: Update on the University of Florida experience with local excision and postoperative radiation therapy for the treatment of early rectal carcinoma. Surg Oncol Clin North Am 1:125-130, 1992