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Prognostic Significance of Cytokeratin-20 Reverse Transcriptase Polymerase Chain Reaction in Lymph Nodes of Node-Negative Colorectal Cancer Patients

From the Chirurgische Klinik und Poliklinik and Institut für Pathologie und Pathologische Anatomie, Klinikum rechts der Isar, Technische Universität München, Munich, Germany.

Address reprint requests to Hjalmar Nekarda, MD, Chirurgische Klinik und Poliklinik, Klinikum rechts der Isar der Technischen Universität München, Ismaningerstr. 22, 81675 Munich, Germany; email: nekarda{at}nt1.chir.med.tu-muenchen.de

	ABSTRACT

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
PURPOSE: Approximately 20% to 30% of patients with curatively resected, node-negative (pN0) colorectal cancer die of tumor recurrence, which can be caused by minimal residual disease. To identify patients with an increased risk of tumor recurrence and evaluate the prognostic value of cytokeratin-20 (CK-20), we detected CK-20–positive cells in histopathologically tumor-free lymph nodes (pN0) of patients with colorectal cancer.

PATIENTS AND METHODS: Two peritumoral lymph nodes each from 85 patients with completely resected (R0) colorectal cancer without lymph node metastases (pN0) by routine examination were analyzed using a CK-20–specific reverse transcriptase polymerase chain reaction (RT-PCR) and compared with CK-20–specific immunohistochemistry (IHC). The results were correlated with histopathologic findings and with survival.

RESULTS: CK-20 RT-PCR was positive in 44 patients (52%) and detected 83% of cancer-related death. Positive RT-PCR was significantly correlated with poorer overall survival (P < .009). Comparing RT-PCR with IHC, 13 patients with positive RT-PCR were identified, where the CK-20 expression was caused by tumor cell contamination located exclusively outside the lymph node capsule and had no prognostic impact. Defining these 13 patients as RT-PCR negative improved specificity of the RT-PCR assay from 57% to 75%. The 5-year overall survival of the 31 RT-PCR–positive patients was 71%, compared with 96% in the 54 negative patients (P < .001). Multivariate analysis showed expression of CK-20 mRNA to be an independent prognostic factor with a relative risk of cancer-related death of 6.1.

CONCLUSION: CK-20 RT-PCR in peritumoral histopathologic tumor-free (pN0) lymph nodes of colorectal cancer is an independent prognostic factor for overall survival. Additional CK-20 IHC improves the specificity and prognostic value of RT-PCR for cancer-related death.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
DESPITE RECENT PROGRESS in the diagnosis of and therapy for colorectal carcinoma over the past few decades, its overall prognosis has not markedly improved. Lymph node involvement is one of the most important predictors,¹ but even in completely resected International Union Against Cancer (UICC) criteria of tumor response stage I and II cases (pN0) in which a good long-term patient survival might be expected, recurrent disease occurs in approximately 20% to 30% of patients.² To more accurately identify patients who will later develop recurrent disease, additional methods have been evaluated to identify minimal residual disease in regional lymph nodes, peripheral blood, and bone marrow.³ Previous studies have shown that using immunohistochemistry (IHC)^4-9 or polymerase chain reaction (PCR) amplification,^10,11 tumor cells can be identified in 20% to 60% of patients with histopathologically negative lymph nodes of colorectal carcinoma by routine hematoxylin and eosin (H&E) staining. However, the prognostic impact of these newly identified tumor cells is uncertain^12,13 because of the significant differences in the interpretation of IHC¹⁴ or reverse transcriptase (RT)-PCR analyses,^15,16 because the markers used vary in their tumor specificity,^17,18 and because uniform protocols for the analyses of tissues do not exist.

In a previous study, we showed that RT-PCR detection of cytokeratin-20 (CK-20) mRNA in lymph nodes of patients with stage I and II colorectal cancer can be used to identify patients who develop tumor recurrence.¹⁹ In agreement with other studies based on RT-PCR,^11,20 we detected tumor cells in a high proportion of patients (61%) but identified 56% of the patients who did not develop tumor recurrence, indicating that the method was not prognostically relevant because of a high rate of false-positive detection. The aim of the present study was to additionally evaluate the use of CK-20 RT-PCR as a predictor of cancer-related death in colorectal carcinoma by increasing the number of patients and using additional CK-20 IHC to study the phenomenon of positive RT-PCR in patients who do not develop tumor recurrence (false-positive detection).

	PATIENTS AND METHODS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Patients and Specimens
Eighty-five consecutive patients with stage I or II colorectal carcinoma (UICC classification)²¹ were operated on in the Department of Surgery at the Klinikum rechts der Isar of the Technical University in Munich between February 1995 and August 1988. Patients were retrospectively studied, and only completely resected patients (UICC R0-tumor resection) with histopathologically negative lymph nodes who did not receive preoperative chemotherapy or radiation therapy were included in the study. All patients entered on the study were consecutive patients treated at our institution between August 1988 and February 1995 and were selected if lymph node sampling was performed and fresh frozen lymph nodes were available from our tumor bank. No other exclusion criteria were used in this study. All patients entered retrospectively into the study. No histopathologically node-positive patients were included in the study population. The site, tumor-node-metastasis system classification, and stage of the tumor are shown in Table 1. Two peritumoral lymph nodes, which were located close to the primary tumor and appeared macroscopically enlarged, were taken from the fresh specimen in each case and divided in half. Lymph nodes were collected by a surgeon with 3-year pathology training. For practical reasons, the lymph nodes were dissected with the same set of instruments that was used for lymph node harvesting and preparation of the primary tumor. One half was examined by routine histopathology and the other was frozen at -80°C within 1 hour of removal. The median number of lymph nodes identified in the specimens was 25, with a range of six to 72.

Eighteen patients (21%) developed recurrent disease and died during follow-up period, 11 (61%) with distant metastases alone (liver in nine patients, lung in one patient, and liver and lung in one patient), five with local recurrence and distant metastases (liver in one patient, lung in two patients, liver and lung in one patient, and skin in one patient), and two with local recurrence alone. Ten patients had recurrent tumor by 36 months, 15 had recurrent tumor by 60 months, and all 18 recurrent tumor by 72 months. The 18 patients who developed recurrent or metastatic disease were treated surgically with resection or palliative chemotherapy or radiotherapy according to the decision of our multidisciplinary oncological board. Seven of the 11 patients with distant metastases alone received a palliative chemotherapy consisting of fluorouracil and folic acid (according to the Ardalan scheme), one patient underwent liver resection and postoperative chemotherapy, and three patients were treated only symptomatically because of progressive tumor cachexia. Three of the five patients with local recurrence and distant metastases underwent local tumor resection with postoperative radiochemotherapy (50 Gy and fluorouracil/folic acid). One patient was treated only by radiochemotherapy, and another patient was treated only symptomatically. One of the two patients with local recurrence alone underwent radiochemotherapy, and the other patient underwent only symptomatic therapy. Ten (15%) of the 67 patients who did not develop tumor recurrence died of causes unrelated to cancer during the follow-up period and were treated as censored by statistical survival analysis.

RT-PCR for CK-20
Thirty 15-µm-thick cryostat sections were used for RNA extraction. The total RNA was extracted using the silica gel–based membrane system RNeasy (Quiagen, Hilden, Germany). The extracted RNA was quantified by spectrophotometry at 260- and 280-nm wavelengths, demonstrating that 40 to 60 µg of total RNA was extracted. RNA quality was tested by identification of 18s and 28s rRNA bands on a 2% agarose gel. Intact RNA was extracted from all 170 lymph node samples.

Using the set of CK-20–specific primers described by Funaki et al,²² RT-PCR amplification was performed in a Landgraf Varius-V thermocycler with the Titan One Tube RT-PCR kit (Roche Diagnostics, Mannheim, Germany), as described in a previous study.¹⁹ The reactions were carried out in a reaction volume of 50 µL with 2 µg of total RNA per sample. The reactions steps consisted of reverse transcription (30 minutes at 50°C), template denaturation (5 minutes at 93°C), 10 cycles of denaturation (1 second at 94°C), annealing (20 seconds at 63°C), elongation (10 seconds at 72°C), and 35 additional cycles with the same conditions, with a prolongation of 5 seconds per cycle at 72°C and a final step at 72°C for 5 minutes. Gel electrophoresis was performed on 2% agarose gels. The amplified CK-20 cDNA fragments were 370 bp in length. Sequencing of the PCR products confirmed that the selected portion of the target mRNA had been amplified (data not shown). Efficient amplification was consistently obtained from all samples and was monitored with a control ß₂-microglobulin PCR using the primers M1, 5'-CCT GAA TTG CTA TGT GTC TGG GTT TCA TCC A-3', and M2, 5'-GGA GCA ACC TGC TCA GAT ACA TCA AAC ATG G-3'. Dilution series of the colorectal cancer cell line CaCo-2 and HT-29 were used for sensitivity testing and revealed that the CK-20 RT-PCR was able to detect one tumor cell in 10⁶ mononuclear cells.

Immunohistochemistry
Three 6-µm cryostat sections for IHC and H&E staining were prepared from the lymph node samples before and after the 30 sections for RT-PCR (sandwich technique). Two of the six sections were stained with H&E, and none revealed any identifiable tumor cells in any of the 85 patients. The remaining four sections for IHC were placed onto glass slides treated with aminosilane (Menzel, Braunschweig, Germany) and were stained by IHC using the monoclonal antibody CK-20 (Dako Diagnostika, Hamburg, Germany). After a 30-minute incubation with the primary antibody at a concentration of 1:100 at room temperature, the reaction was developed using the alkaline phosphatase-antialkaline phosphatase technique.²³ A frozen section of each of the 85 primary colorectal tumors was also stained with CK-20 IHC. Positive staining for CK-20 was seen in every tumor.

Controls
Fourteen primary colorectal carcinoma specimens and five lymph nodes with metastatic carcinoma were used as positive controls for both CK-20 RT-PCR and IHC and were consistently positive using both methods. As negative controls, mRNA from 20 lymph nodes from six patients with ulcerative colitis and four patients with Crohn disease were used. RT-PCR studies on histopathologically positive lymph nodes and colon cancer cell lines (CaCo-2 and HT-29) were included as positive controls. The RT-PCR reactions from the negative control tissues were consistently negative throughout the experiments.

Statistical Analysis
The primary end points for the study were tumor recurrence and overall survival. The ² test was used for group comparisons, and survival was calculated according to the Kaplan-Meier method. Survival differences between groups were analyzed using the log-rank test. Cox regression analysis was used for multivariate analysis. All tests were two-sided, and P < .05 was considered to be of statistical significance.

	RESULTS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
CK-20 RT-PCR and Immunohistochemistry
CK-20 RT-PCR was positive in 44 (52%) of the 85 patients (Fig 1 and Table 2). Tumor cells were detected by CK-20 IHC in the lymph node samples of 47 (55%) of the 85 patients. However, in 23 of these patients, the tumor cells were located exclusively outside of the lymph node in the surrounding tissue, so that only 24 (28%) of the 85 patients were judged by CK-20 IHC to have true lymph node involvement by disseminated tumor cells.

View larger version (50K): [in this window] [in a new window]   Fig 1. RT-PCR screening in lymph nodes of patients with colorectal carcinoma stage I/II. (A) CK-20 expression (370 bp). (B) ß2-microglobulin expression (441 bp). Patients no. 2, 3, 5-7, 10-12 = CK-20 detection. Patients no. 1, 4, 8-9 = no CK-20 detection. N, negative control; M, molecular weight marker; P1/P2, positive control.

CK-20 RT-PCR and Survival and Prognosis
Of the 44 positive RT-PCR patients, 15 (34%) developed recurrence and died, compared with three cancer-related deaths in the 41 (7%) negative RT-PCR patients. Among the remaining 29 CK-20 RT-PCR–positive patients, 25 patients were still alive at the end of follow-up period and four died of causes unrelated to cancer. Thirty-two of the 38 negative RT-PCR patients were still alive at the end of the study, and six patients died of causes unrelated to cancer. The 5-year overall survival rates for the patients with a positive CK-20 RT-PCR (77.9% ± 6.5%) were significantly worse than those of the patients with a negative CK-20 RT-PCR (94.7% ± 3.7%) (P < .009) (data not shown). Multivariate analysis revealed CK-20 RT-PCR to be an independent prognostic factor, predicting overall survival (including age, sex, tumor site, grading, pT category, UICC stage, and lymphatic vessel involvement) with a relative risk of 3.9 (CI, 1.1 to 13.7). Other independent prognostic factors were the tumor site (colon v rectum) and the presence of lymphatic vessel involvement.

RT-PCR Versus Immunohistochemistry and Prognosis
Comparing the CK-20 RT-PCR results with CK-20 IHC, we evaluated the survival data of the different subgroups (Table 3). The subgroup of 13 patients (8%) with positive RT-PCR but evidence of exclusively extranodal tumor cells detected by IHC had the same 5-year overall survival as both RT-PCR–negative groups. Defining these 13 patients, who were found to have positive RT-PCR and solely extracapsular tumor cells by IHC, there were 21 positive RT-PCR patients with confirmation by IHC and 10 positive patients that were negative by IHC for a total of 31 (36%). Fourteen of the 31 patients developed tumor recurrence and died, compared with only four of 54 patients with a negative RT-PCR or a positive RT-PCR shown by IHC to be attributable to extranodal tumor. The overall 5-year survival rates of the IHC-controlled RT-PCR positive group were again significantly worse than the negative group (P < .001), and the differences were greater than with RT-PCR alone (95.9% ± 2.9% for the negative patient group v 70.6% ± 8.3% for the positive patient group). Theq! Kaplan-Meier curves for overall survival for IHC-controlled RT-PCR evaluation are shown in Fig 2.

View larger version (14K): [in this window] [in a new window]   Fig 2. Ten-year overall survival curves of the 85 patients with colorectal carcinoma stage I or II according to the immunohistochemically controlled CK-20 mRNA expression in histopathologic tumor-free lymph nodes.

Comparison With Clinicopathologic Variables and Multivariate Analysis
As shown in Table 1, positivity by IHC-controlled CK-20 RT-PCR was more likely to be found in female patients (P < .01) and in younger patients (average age of 58 years) (P < .04). No correlation was seen with tumor site, grading, pT category, stage, lymphatic vessel involvement, or serum carcinoembryonic antigen (CEA) levels. Multivariate analysis (Table 4) revealed that RT-PCR combined with IHC was the strongest prognostic factor, predicting overall survival with a relative risk of cancer-related death of 6.1 (CI, 1.9 to 19.4). Other independent prognostic factors were the tumor site (colon v rectum), with a relative risk of 3.4 (CI, 1.3 to 9.1), and the presence of lymphatic vessel involvement (RR, 4.4; CI, 1.3 to 15.1).

	DISCUSSION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

To date, the only other study that has shown an independent prognostic impact for RT-PCR–based tumor detection in colorectal carcinoma was reported by Liefers et al²⁵ using the CEA as the mRNA target. This study included 192 histopathologically tumor cell–free lymph nodes from 26 patients with stage II colorectal cancer. CEA mRNA expression in lymph nodes was found in 54% of the patients. Compared with a 5-year survival rate of 91% in the group without CEA-expressing tumor cells, the survival of the CEA mRNA positive group was significantly worse at 54%.

The RT-PCR target in our study, cytokeratin-20, is an intermediate filament that is expressed in both normal and malignant gastrointestinal epithelium.²⁷ In contrast to other cytokeratin filaments, no CK-20 pseudogenes have been identified, so it should be a highly sensitive and specific target for both IHC and RT-PCR.^17,18,28 Our preliminary RT-PCR study for regional lymph node tumor detection in colorectal carcinoma compared CEA and CK-20 for use as RT-PCR targets. Whereas CK-20 positivity showed a statistical trend toward recurrence-free survival, CEA was not associated with prognosis, although the same target was used as in the study by Liefers et al.²⁵

A novel aspect of our study is the fact that only two peritumoral lymph nodes were examined by RT-PCR in each case, meaning that this procedure could be used in a routine clinical setting. This strategy simulates the concept of sentinel lymph node mapping, in which the status of the first lymph node draining the tumor, which should have the most prognostic impact, is used to provide information about potential tumor involvement of more distant regional lymph nodes.^29-31

A second important aspect is the combination of RT-PCR with IHC for CK-20 to identify reasons for CK-20 expression in lymph nodes of patients who did not develop tumor recurrence (false-positive detection). Several previous studies have also highlighted the underappreciated problem of tumor cell contamination of tissues for use with PCR amplification.^15,32 Even when standard sampling procedures are used, contamination in the course of sampling of fresh tissues for PCR can often occur.³² Because the extranodal tumor cells had no contact to the lymph node capsule and patients with exclusively extranodal tumor cells had an equally good prognosis as patients with negative CK-20 RT-PCR results, it is important to be able to remove these cases from the positive-defined cases using IHC. In addition, we did not find a correlation with the IHC-detected extranodal tumor cells with the presence of lymphangiosis (data not shown). Therefore, these tumor cells do not correspond to tumor cells that were released from open lymph vessels during lymph node sampling but reflect possible tumor contamination that must occur during lymph node sampling. We suggest preparing the resected tumor specimen with two different sets of instruments in additional studies. One set should be used only for lymph node dissection, and the other for primary tumor preparation. The primary tumor and the clamped bowel ends have to stay untouched by the pathologist until the lymph nodes or, in future studies, the sentinel lymph nodes are dissected. After identification and resection, the lymph nodes should be washed with NaCl solution to prevent any epithelial contamination. Then the lymph nodes should be separated from mesenterial fat tissue, if no macroscopic tumor involvement is present, and should be separated from the specimen. Subsequently, the preparation of the primary tumor, including antimesenterial opening of the bowel, should be performed.

Previous studies based on immunohistochemistry alone for tumor cell detection in regional lymph nodes of colorectal carcinoma have failed to show any significant prognostic impact.^6-7,9,12-13 In our series, 28% of patients had intranodal CK-20–positive cells, and this correlated significantly with recurrent disease for a high specificity of 79%. However, because of the fact that RT-PCR detected five additional patients who developed recurrent disease, IHC had a much lower sensitivity than RT-PCR (27% v 56%) according cancer-related death. The combination of both techniques optimized the specificity and sensitivity of the techniques as a whole.

A discrepancy between our study and that of Liefers et al²⁵ is the fact that Liefers et al did not identify tumor cells by IHC for cytokeratins or CEA in any of 36 lymph nodes that were positive for RT-PCR. This difference is hard to explain but might be attributable to the different targets that were used, differing case selection criteria, or sampling methods. All of these factors vary widely among the many studies in this area, making comparison of any two studies extremely difficult.

A few of our patients had positive RT-PCR but did not develop tumor recurrence. There are a few methodical explanations, including contamination that was not detected by IHC. It is also possible that the detected CK-20 mRNA may represent biologically inactive tumor cells (dormant cells) that do not have the capacity to develop into metastases. Another explanation is that all RT-PCR–detected tumor cells were removed by the lymphadenectomy, which was performed with a median number of 25 resected lymph nodes. Although the RT-PCR seems to be sensitive for tumor cell detection, we saw three patients with positive tumor cells detected by IHC that were not confirmed by RT-PCR. Sampling errors have to be discussed in these cases, because single tumor cells that were located only in the cryostat sections examined by IHC cannot be found inside the sections that underwent RNA extraction for RT-PCR. These three patients did not develop recurrent disease, although the immunohistochemically detected tumor cells should reflect potential metastatic impact or may be attributable to biologically inactive tumor cells (dormant cells). When including the three exclusively IHC intranodal-positive patients with the 31 positive IHC-controlled RT-PCR patients, the prognostic impact decreased nonsignificantly (data not shown). The three patients that were negative by RT-PCR but positive for IHC were defined in the present IHC-controlled RT-PCR evaluation (Table 3) as negative because of the aim to examine the biologic valence of RT-PCR.

The evaluation of our complete colorectal cancer patient data confirms that the occurrence of lymph node metastases is significantly associated with raising pT category (data not shown). The finding that CK-20 RT-PCR detection in histopathologically node-negative lymph nodes did not increase with raising pT category underscores the independent prognostic value of CK-20 for identification of patients with increased risk of recurrent tumor. The phenomenon that 53% of the T2 patients were RT-PCR positive compared with 33% and 37% of patients with T3 and T4 tumors has a direct correlation with the proportion of recurrences in the respective groups. Six (40%) of the 15 patients with T2 tumors developed and died of tumor disease, whereas only seven (13%) of the 55 and three (38%) of the eight patients with T4 tumors experienced tumor recurrence. This additionally supports the clinical relevance of the used methodology for micrometastatic cell detection.

These theoretical possibilities reflect the complexity of the metastatic process that complicates the interpretation of RT-PCR for prognostic prediction. Improved prognostic prediction through PCR-based methods will increasingly have to take these factors into account, but for the short term, more standardized intraoperative lymph node sampling methods, uniform mRNA targets, and standardized patient selection and clinical follow-up criteria would be significant improvements to allow the comparison of the prognostic impact of different studies.

In summary, the detection of CK-20 mRNA in pN0 lymph nodes of colorectal cancer (UICC stage I and II) represents an independent prognostic factor. A subgroup of patients was identified that had a worse prognosis and would probably benefit from adjuvant therapy protocols. Additional immunohistochemistry allowed detection of extracapsular located tumor cells and improved the specificity and prognostic value of CK-20 RT-PCR.

	ACKNOWLEDGMENTS

 
We thank C. Marthen and D. Poehlmann for their expert technical assistance.

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Submitted May 15, 2001; accepted September 25, 2001.

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