Immunohistochemical Evaluation
of Regional Lymph Nodes in
Stage I and II Colorectal Cancer:

Comparison Between Patients
With and Without Recurrence

 

Juan C. Paramo, MD^*

Mark Landeros, MD^*

Christopher I. Wilson, MD^†

Amadeo Cabral, MD^*

Robert J. Poppiti, MD^†

Thomas W. Mesko, MD, FACS^*

 

^*Department of Surgery, and

^†Department of Pathology

Mount Sinai Medical Center

Miami Beach, FL 33140

 

 

KEY WORDS: colorectal cancer, immunohistochemistry, lymph nodes, recurrence

ABSTRACT

Background and Objectives: The implications of up-staging patients with stage I or II colorectal cancer based on the presence of nodal micrometastases are controversial. Therefore, a case-control study was performed to evaluate possible differences in the immunohistochemistry results of patients with recurrent versus nonrecurrent stage I or II carcinomas.

Methods: All cases of stage I and II colorectal cancer from 1980 through 1998 were reviewed. The study group included all cases of documented recurrence. A control group of patients without recurrence was included in the study for comparison. All patients had negative lymph nodes on initial hematoxylin and eosin examination. Deeper sections from the tissue blocks were made at 5 different levels and stained with low-molecular-weight cytokeratin (CAM 5.2).

Results: The study group included 7 patients and 120 lymph nodes. The control group included 14 patients and 157 nodes. Immunohistochemistry stains showed micrometastases in 6% of nodes in the recurrence group and in 17% in the control (P = 0.01). Overall, 57% of the patients with recurrence and 36% of the patients without recurrence were up-staged based on the immunohistochemistry stains (P = 0.3).

Conclusions: Immunohistochemical stains do not determine which patients with stage I or II colorectal cancer will experience recurrence, and the presence of micrometastases according to immunohistochemical stains does not imply a worse prognosis.

INTRODUCTION

Recent technological advances have improved the detection of malignant disease at the cellular level. Among these evolving techniques, immunohistochemistry (IH) has become a very sensitive method for detecting malignant cells. IH has been applied diversely, especially in the study of sentinel lymph nodes in melanoma and breast cancer patients. However, the use of IH stains in colorectal cancer has produced uncertain results. In an attempt to further clarify this issue, we performed the present study, which summarizes our experience with IH evaluation in patients with stage I or II colorectal carcinoma.

MATERIALS AND METHODS

We reviewed all cases of colorectal carcinoma listed in the tumor registry at our institution from January 1980 through December 1998. Patients whose cancer was staged as I or II per the American Joint Committee on Cancer Staging System1 were selected. A case-control study was performed as follows: the study group consisted of all patients with documented recurrent disease. The control group was made up of randomly selected patients from the tumor registry database who had colorectal cancer but who did not have evidence of recurrence. No patients in either group had received chemotherapy.

Once the groups were established, the paraffin tissue blocks containing all mesenteric lymph nodes from the surgical specimens were reviewed. Lymph nodes had been identified by gross dissection and all had been reported as negative for malignancy on initial hematoxylin and eosin (H&E) examination. Deeper sections were made from the tissue blocks at 5 different levels and stained with low-molecular-weight cytokeratin (CAM 5.2; Becton Dickinson, San Jose, CA). Positive micrometastasic involvement of a lymph node was defined as the presence of at least one cell composed of a clearly defined nucleus surrounded by positive IH staining of the cytoplasm.

In every case, the age, gender, site of the tumor, stage (I or II), total number of dissected mesenteric nodes, number of positive nodes, and total length of follow-up were documented. In the study group, the time interval until recurrence was recorded. The overall mortality was calculated for both groups, and statistical analysis was performed using a 2-sample t test for ordinal variables. A Fisher exact test and z test with Yates correction for continuity were used for nominal variables. A P value of less than 0.05 was considered statistically significant.

RESULTS

A total of 458 patients with stage I or II colorectal carcinoma were reviewed. There were 20 patients with documented recurrence. Of these 20 patients, paraffin blocks were available for 7 patients, which formed the study group. A total of 14 patients were selected as the control group. Characteristics of both groups are summarized in Table 1.

The study group consisted of 6 women and one man, aged 31 to 85 years old (average, 65 years). Tumors were located in the right colon in one case, left colon in 2 cases, sigmoid colon in 3 cases, and rectum in one case. All cases were stage II. Average tumor size was 4.3 cm (range 1.5 to 10 cm). A total of 120 regional lymph nodes were studied using IH, with an average of 17 nodes per patient (range 4 to 57 nodes). Seven nodes (6%) were positive according to IH (Figure 1). Overall, 4 (57%) of the patients in this group had evidence of malignant nodal involvement according to the IH stains (Table 2).

Average time until recurrence was 14 months (range, 2 to 33 months), and mean follow-up time was 30 months (range, 2 to 67 months) in the study group. Patients with evidence of micrometastases were followed up on average for 17 months. Only one patient (14%) in this group died, at 14 months postoperatively due to recurrent disease.

The control group consisted of 14 patients, 7 men and 7 women. Five tumors were located in the right colon, one in the left colon, 6 in the sigmoid colon and 2 in the rectum. Three cases were stage I, and 11 cases were stage II. Average age was 73 years (range, 56 to 86 years). Mean tumor size was 4.5 cm (range, 2.5 to 10 cm) in this group. A total of 157 nodes underwent IH staining, with an average of 11 nodes per patient (range, 2 to 23 nodes). Twenty-six nodes (17%) were positive according to IH. Overall, 5 (36%) of the patients in this control group had evidence of micrometastases using IH staining (Table 2).

The average follow-up interval in the control group was 39 months (range, 16 to 79). Mean follow-up time in patients with micrometastases was 42 months. Two patients (14%) in this group died, at 16 and 79 months, respectively, due to medical reasons not related to their colon cancer.

Comparison of the results from the 2 groups showed no statistically significant differences with regards to age (P = 0.2), mean tumor size (P > 0.5), average number of nodes per patient (P = 0.5), percentage of patients with micrometastases (P = 0.3) or average follow-up time (P = 0.5). There was a statistically significant difference with regards to the percentage of positive nodes, which was higher in the control group (P = 0.01).

DISCUSSION

Colorectal cancer is the most common cancer of the gastrointestinal tract, with a worldwide population-adjusted incidence of approximately 45 to 50 cases per 100,000.2 Early recognition of this disease is critical for successful therapy. However, even after an adequate surgical resection, the cancer recurs in 10% to 40% of patients. Therefore, identifying patients at risk continues to be a challenge.3,4

Risk factors associated with increased incidence of recurrence include the site of origin of the carcinoma, the depth of penetration of the tumor into the intestinal wall, degree of differentiation of the carcinoma, presence and number of lymph nodes with metastasic disease, vascular invasion, and overall stage.2,4–7

Previous studies have demonstrated the benefits of adjuvant therapy in advanced colon cancer, with the use of chemotherapy in stage III carcinomas considered the standard of care.2 There are no established guidelines for systemic therapy for stage II colon carcinoma, although several clinical trials including subsets of these patients are ongoing. Stage II rectal cancer patients receive adjuvant chemoradiation therapy.

The use of IH has had a wide acceptance because of its various diagnostic applications. IH is a sensitive technique that is used as a complementary diagnostic modality. Because IH is a more sensitive technique, micrometastases can be found with IH in nodes determined to be negative by H&E. However, IH is more expensive and may not be cost-effective for widespread use. In colorectal carcinoma, some studies have shown important benefits of performing IH in mesenteric nodes while other experts believe that it should not be used routinely.

The prognostic implications of micrometastasic nodal involvement are debatable. Previous reports have shown that between 25% and 54% of patients with stage I or II colorectal cancer can have evidence of micrometastases on IH only.8–11 Some studies have shown IH results to be a prognostic factor,10 although others have shown contradictory results.9 In our study, the rate of micrometastasic involvement shown by IH was 57% in patients with recurrence and 36% in patients without recurrence (P = 0.3). These results suggest that positive IH cells in previously negative H&E lymph nodes may not be of clinical significance. Other studies have shown that even though occult tumor cells might increase the risk for local recurrence, they do not influence the patients’ prognosis.12 Our study confirms the limited benefit of IH as a predictor of tumor recurrence.

However, if the presence of nodal micrometastases implies up-staging of stage I or II colon cancer to stage III, IH results would have therapeutic implications because adjuvant chemotherapy would then be indicated.2,13 In cases of rectal cancer, up-staging stage I tumors would likewise indicate adjuvant therapy.14 Therefore, these issues would suggest justification for the use of IH staining. Some studies, however, show that overall survival and outcomes are not altered by the presence of micrometastases.8,10,15–17 Our study corroborates this finding because the evidence of nodal micrometastases in the group without recurrent disease was not statistically different from that in the group with recurrent disease.

The lack of clinical significance of IH micrometastases may have 2 possible explanations. First, not all cytokeratin-positive cells are tumor cells; they may be mesothelial cells. Second, even if they are tumor cells, clinically significant metastases may need more than just their presence in the lymph node to develop. That is, some other unknown factors may be responsible for initiating and sustaining the growth of these cells. 

Another issue is the routine use of serial sectioning as a method to increase the detection of lymph node metastases. In breast cancer, using step and serial sectioning with H&E staining alone identified “occult” metastases in 22% of patients.18 Another study concluded that although lymph node micrometastases may be more easily detected using IH, careful screening of H&E-stained sections by a pathologist appeared to be equally sensitive.19 Although we did not perform H&E staining on the serial sections in our study, we believe that IH detected metastases that would likely not be found on H&E, because several metastases were single cells or groups of only a few cells (Figure 1).

CONCLUSIONS

The immunohistochemical study of regional lymph nodes in patients with colorectal cancer did not provide prognostic or therapeutic implications. Immunohistochemical stains did not determine which patients with stage I or II colorectal cancer will experience recurrence. This needs to be further elucidated with larger studies.

 

REFERENCES

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6. Chung CK, Stryker JA, Demuth WE Jr: Patterns of failure following surgery alone for colorectal carcinoma. J Surg Oncol 22: 65–70, 1983.

7. Rich T, Gunderson LL, Lew R, et al: Patterns of recurrence of rectal cancer after potentially curative surgery. Cancer 52:1317–1329, 1984.

8. Adell G, Boeryd B, Franlund B, Hakansson L: Occurrence and prognostic importance of micrometastases in regional lymph nodes in Dukes’ B colorectal carcinoma: An immunohistochemical study. Eur J Surg 162:637–642, 1996.

9. Oberg A, Stenling R, Tavelin B, Lindmark G. Are lymph node micrometastases of any clinical significance in Dukes stages A and B colorectal cancer? Dis Colon Rectum 41:1244–1249, 1998.

10. Greenson JK, Isenhart CE, Rice R, et al: Identification of occult micrometastases in pericolic lymph nodes of Dukes’ B colorectal cancer patients using monoclonal antibodies against cytokeratin and CC49: Correlation with long term survival. Cancer 73:563–569, 1994.

11. Futamura M, Takagi Y, Koumura H, et al: Spread of colorectal cancer micrometastases in regional lymph nodes by reverse transcriptase-polymerase chain reactions for carcinoembryonic antigen and cytokeratin 20. J Surg Oncol 68:34–40, 1998.

12. Broll R, Schauer V, Schimmelpenning H, et al: Prognostic relevance of occult tumor cells in lymph nodes of colorectal carcinomas: An immunohistochemical study. Dis Colon Rectum 40:1465–1471, 1997.

13. Macdonald JS. Adjuvant therapy of colon cancer. Ca Cancer J Clin 49:202–219, 1999.

14. Engstrom PF, Benson AB III, Cohen A, et al: NCCN colorectal cancer practice guidelines. Oncology 10(suppl):140–75, 1996.

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16. Jeffers MD, O’Dowd GM, Mulcahy H, et al: The prognostic significance of immunohistochemically detected lymph node micrometastases in colorectal carcinoma. J Pathol 172:183–187, 1994.

17. Cutait R, Alves VAF, Lopes LC, et al: Restaging of colorectal cancer based on the identification of lymph node micrometastases through immunoperoxidase staining of CEA and cytokeratins. Dis Colon Rectum 34:917–20, 1991.

18. Pickren JW. Significance of occult metastases: A study of breast cancer. Cancer 14:1266–1271, 1961.

19. O’Brien MJ, Zamcheck N, Burke B, et al: Immunocytochemical localization of carcinoembryonic antigen in benign and malignant colorectal tissues. Am J Clin Pathol 75:283–290, 1981.

 

 

 

 

Table 1. Characteristics of patients with and without recurrence

 

               Study       Control      

               Group        Group       

            (recurrence) (no recurrence)

 

Age                        65 years               73 years           0.2 (NS)

                          (31–85 years)      (56–86 years)

 

Male:Female                1:6                        7:7                    NA

 

Tumor size               4.3 cm                   4.5 cm            > 0.5 (NS)

                            (1.5–10 cm)          (2.5–10 cm)

 

Follow-up time          30 mo                    39 mo              0.5 (NS)

                              (2–67 mo)           (16–79 mo)

 

NS = not significant ; NA = not applicable.

 

 

Figure 1. Mesenteric lymph node with evidence of micrometastases after immunohistochemical staining is seen. Notice the single darker tumor cell that had been undetected on initial hematoxylin and eosin examination.

 

Table 2. Lymph nodes results in patients with and
without recurrence

 

               Study       Control      

               Group        Group       

            (recurrence) (no recurrence)

 

Total lymph nodes      120                       157                    NA

 

Lymph nodes              17                         11                 0.5 (NS)
   per patient            (4–57)                   (2–23)

 

Positive nodes by IH    7                          26                    0.01

                                  (6%)                    (17%)

Patients with                4                           5                  0.3 (NS)
positive nodes by IH (57%)                  (36%)

 

NS = not significant ; NA = not applicable.