K-ras Point Mutation in the Nerve Plexuses Around the Superior Mesenteric Artery in Resectable Adenocarcinoma of the Pancreatic Head: Distribution Pattern and Related Factors

Background  Adenocarcinoma of the pancreas is likely to spread into the nerve plexuses around the superior mesenteric artery (SMA) at a microscopic level. Since there has been no detailed report on how minute cancer invasion is distributed among the peri-SMA plexuses or which cases are more vulnerable to such an event, it has long been controversial how to treat this area when resecting the pancreatic head cancer.

Hypothesis  The K-ras mutation assay is more sensitive than the conventional histologic diagnosis in detecting minute cancer invasion around the SMA.

Design  Prospective consecutive series.

Setting  Cancer center hospital.

Patients and Methods  The entire circle of the peri-SMA tissues was obtained from 24 patients who had received an extended pancreatectomy for adenocarcinoma of the pancreatic head. They were divided into right and left hemicircular samples (48 samples), and each sample was used for both histologic and genetic diagnoses. Since all patients' primary tumors were positive for point mutation at codon 12 of the K-ras gene, the presence or absence of the mutation was determined for the peri-SMA plexuses using the mutant allele specific amplification method.

Results  Compared with results of the histologic examination, the K-ras mutation assay was more sensitive in detecting positive findings in the peri-SMA plexuses (12 samples from 9 patients). According to the distribution of the K-ras mutation into the right- and left-half samples, 24 patients were classified into the following 4 patterns (right/left): negative/negative in 15 patients; positive/negative in 6 patients; positive/positive in 3 patients; and negative/positive in 0 patients. In 3 patients who showed a positive/positive pattern in the genetic diagnosis, their right-half samples included more cancer cells that were detectable by routine microscopy. There was no relation between K-ras mutation and lymphatic invasion, while K-ras mutation was particularly related with the invasion of portal vein (P = .04) and posterior peripancreatic tissues (P = .002). All 3 patients with K-ras mutation in bilateral plexuses were classified by the TNM staging system as T4 using Union Internationale Contre le Cancer classification.

Conclusions  The K-ras mutation (at codon 12) assay indicated a simple and regular pattern of cancer extension into the nerve plexuses around the SMA from adenocarcinoma of the pancreatic head: (1) The left half of the plexus was unlikely to be involved by cancer in cases in which the right half was intact. (2) Cancer extension into the peri-SMA plexuses occurred after the posterior confine of the pancreas had been involved by direct invasion from the primary pancreatic tumor. (3) The left half was not involved in cancerous tumors classified as T1 to T3 but was occasionally involved in those classified as T4 tumors. These data seem to provide a useful indicator of some additional treatments (resection, irradiation, etc) for the peri-SMA region when a locally advanced pancreatic head cancer is treated with a curative intent.

THE INCIDENCE of adenocarcinoma of the pancreas has risen in many countries, and surgical resection has offered the only curative strategy in the treatment of pancreatic head cancer to date. However, the long-term outcome after pancreatoduodenectomy is still pessimistic, and the locoregional recurrence is one of the major causes of death from cancer. The pancreatic bed, particularly around the superior mesenteric artery (SMA), offers the common site of locoregional recurrence. Accounting for this fact, Willett et al¹ showed that postoperative microscopic studies of the resected specimens occasionally revealed cancer cells around the cut margin of the retropancreatic connective tissues, suggesting a possible cancer residual. Nagakawa et al² also showed that the nerve plexuses around the SMA were most likely to be involved by cancer with a fashion of perineural invasion.

Regarding the treatment toward the peri-SMA region, we have performed an extended pancreatectomy in which the entire circle of the SMA was stripped of the surrounding nerve plexuses.³ Although this procedure resulted in a modest improvement in both 5-year survival rates and locoregional control,⁴ a severe (usually watery) and persistent (1-2 postoperative years) diarrhea developed in almost all the patients.⁵ From the viewpoint of patients' quality of life, it is more desirable to preserve as much of the nerve plexuses as possible when we can correctly predict that the remaining nerve plexuses are quite intact. Since Nagakawa et al² suggested that diarrhea was mild enough that no special treatment was necessary when half of the plexuses were left behind, we need detailed knowledge on how the cancer cells distribute among the peri-SMA plexuses. On the other hand, instead of surgical denervation around the SMA, a prophylactic irradiation⁶ has been occasionally employed because it does not cause postoperative diarrhea. However, the peri-SMA area is quite close to the anastomotic sites such as pancreatojejunostomy and gastrojejunostomy. When adjuvant irradiation therapy is intended toward the peri-SMA area immediately after pancreatoduodenostomy, it raises a fear that the anastomotic sites might be irradiated concomitantly. Therefore, when determining which individuals require irradiation of this area, it is also necessary to know which are at the higher risk of cancer extension toward the peri-SMA plexuses. Thus, the present study is designed to clarify these 2 problems in resectable adenocarcinoma of the pancreatic head. To not overlook the minute cancer invasion in the peri-SMA plexuses, a genetic diagnosis of K-ras point mutation is employed in addition to the histologic examinations.

Twenty-four patients with resectable carcinoma of the pancreatic head were enrolled in the present study. None of the patients showed distant metastases or direct invasion to the neighboring major arteries by intraoperative macroscopic inspection, and none showed positive results for the cytologic examination by peritoneal lavage. All patients underwent pancreatoduodenectomy with a wide range of lymphatic and connective tissue clearance (extended pancreatectomy) at the Osaka Medical Center for Cancer and Cardiovascular Diseases, Osaka, Japan. When the portal vein and/or the superior mesenteric vein were involved by cancer, these infected veins were resected together with the pancreatic head and reconstructed by an end-to-end anastomosis. By postoperative histopathologic examination, all primary tumors were proven to be invasive ductal adenocarcinoma, excluding the low-grade malignant tumors such as cystadenocarcinoma, in situ carcinoma, or mucin-producing carcinoma. In all 24 patients, their primary pancreatic tumors were positive for K-ras point mutation (at codon 12) by the mutant allele–specific amplification (MASA) method.⁷

During pancreatectomy, the SMA was carefully stripped of the surrounding connective tissues from the branching point of the aorta to the branching point of the second jejunal artery (Figure 1), although they were free from cancer invasion by a macroscopic inspection. From the resected tissues, we obtained a slice 3- to 5-mm thick from the layer that had previously faced the entire circle of this artery (Figure 2). The sample was divided into right- and left-half segments according to the anterior and posterior middle lines on the SMA. Each of the 2 halves were washed with physiologic isotonic sodium chloride solution and divided into 2 subsegments.

Each single subsegment and a part of the primary pancreatic tumor were immediately frozen in nitrogen liquid and stored at −80°C for genetic diagnosis. The DNA samples were extracted from the primary tumors, and the mutation at codon 12 of the K-ras gene was examined by MASA. The 3′ ends of the 20 base pair oligonucleotides used as primers for polymerase chain reaction (PCR) corresponded to variants of the first or second nucleotide of K-ras codon 12. To look for the corresponding genetic alterations in the peri-SMA tissues where mutations had been identified in the primary tumors, we used MASA primers with 3′ ends corresponding to each variant and amplified the respective peri-SMA tissue DNA by PCR with these primers. The MASA was performed with 35 cycles for 0.5 minutes at 95°C, 1.5 minutes at 65°C or 66°C, and 1.5 minutes at 72°C using a programmable thermal cycler (Gene Amp PCR system 9600; Perkin Elmer, Norwalk, Conn), followed by the method described previously.⁷ The PCR products were analyzed by electrophoresis in a 2% agarose gel.

Another subsegment was extended on the flat board, fixed in 20% formaldehyde, and embedded in paraffin block. A pair of 4-µ slices was obtained from both the front and back aspects of the paraffin block, and each slice was stained with hematoxylin-eosin for microscopic diagnosis (the initial histologic analysis).

When a different result was obtained between the (initial) histologic analysis and genetic diagnosis, 100 to 200 additional pairs of serial sections were obtained from the front and back aspects of the remnant paraffin block. They were also stained with hematoxylin-eosin for the microscopic observation (additional histologic analysis). Similarly, 6 samples were selected from the samples that had shown negative results in both the initial histologic analysis and genetic diagnosis, and they were used for the additional histologic testing.

The differences between the groups are examined by unmatched t test or χ² test. Differences were considered significant at P<.05.

All 24 primary tumors were positive for point mutation at codon 12 of K-ras gene, and their nucleotide transitioning patterns were as follows: GGT→GAT in 11 cases; GGT→GTT in 9 cases; GGT→GCT in 2 cases; and GGT→CGT in 2 cases. No tumor showed multiple (2 or more) transitioning patterns. Using the corresponding primer to the peri-SMA samples, K-ras point mutation was detected (Figure 3) in 12 samples obtained from 9 patients (Table 1, patients 1-9); both right and left halves were positive in 3 patients (patients 1-3); and the right half alone was positive in 6 patients (patients 4-9). There were no patients who had a positive mutation in the left half alone.

Histologically, the peri-SMA samples consisted of nerve fibers that formed bundle structures (nerve plexuses) of varying sizes (Figure 4), together with many collagen fibers and adipose tissues. It was not difficult to detect the cancer cells among these background components because cancer cells were characterized by nuclear irregularity, far larger sizes of cytoplasm and nuclei, and higher ratios of nuclei to cytoplasm (Figure 4B). In addition, cancer cells, when present, were likely to be located just adjacent to or at the periphery of the nerve plexuses.

The initial histologic study revealed cancer cells in 5 samples obtained from 3 patients (Table 1, patients 1-3). Additional histologic tests were performed on 7 samples (left half in patient 3 and right half in patients 4-9), the findings for which had once been judged negative by initial histologic analysis but positive by the following K-ras mutation assay, and cancer cells were newly detected in 3 (43%) of the samples (left half in patient 3 and right half in patients 4 and 5). Also, 6 samples were randomly selected for the additional histologic examinations from the samples that were negative in both the initial histologic analysis and K-ras mutation assay (patients 10-24), and no cancer cells were detected.

The background factors of the 9 patients with K-ras mutation in at least the right half (positive group; patients 1-9) were compared with those of the other 15 patients (negative group; patients 10-24) to determine which patients are at a high risk of cancer cell invasion into the peri-SMA plexuses (Table 2). Between the 2 groups, no significant differences were seen regarding sex, age, initial symptoms, histologic differentiation, and status of nodal involvement or invasion into the lymphatic channels. The size of the primary tumor in the positive group was significantly larger than that of the negative group, and all patients (100%) in the former group had the tumors extending beyond the confines of the pancreas (Union Internationale Contre le Cancer [UICC] TNM staging system classifications of T3 or T4 tumors).

According to the distribution of K-ras mutation among the peri-SMA plexuses, the 24 patients were classified into 3 groups (Table 3), and the background factors associated with locoregional extension of the tumor were compared. As a result, the distributing pattern of K-ras mutation was well-associated with T factor in the UICC classification (P = .002). In the T1 to T2 group, none of the patients showed the K-ras mutation in the peri-SMA plexuses. On the other hand, among 3 patients in whom bilateral segments were positive, all of their primary tumors were categorized as T4. Since T factor in the UICC classification consists of many subfactors based on macroscopic inspection, each subfactor was determined by the histopathologic findings. As a result, the invasion toward the portal vein (P = .041) and the posterior confine (P = .002) was more associated with the distributing pattern of the K-ras mutation than the invasion toward other directions (anterior confine [P = .32], duodenum, or bile duct [P>.99]). In the 3 patients who showed the K-ras mutation for both right and left halves of the plexuses, all of their primary tumors were categorized as T4 (UICC) by invading both the portal vein and posterior confine of the pancreas.

It is our great concern to know how and when the peri-SMA plexuses should be treated when resecting the pancreatic head. However, the data in the previous reports hardly answered our question because most of them were based on the light-microscopic observation alone using a small number of histologic sections. Therefore, first in the present study, the peri-SMA plexuses were classified into the right and left halves for clinical practice. Second, a genetic technique was employed because it offered more accurate assessment of minute cancer invasion in the peri-SMA tissues than did conventional histologic analysis. The positive rate of K-ras point mutation occurring at codon 12 was as high as 80% to 95% in the invasive ductal adenocarcinoma of the pancreas,^8,9 and the MASA method was so sensitive that even one cancer cell with K-ras point mutation was detectable in a background of thousands of mutation-negative cells.⁷ Third, our samples were limited to the extrapancreatic parenchymal tissues so that there was no fear of false-positive results by a contamination with nonmalignant pancreatic duct cells, which sometimes show the K-ras mutation.¹⁰ As a result, we had no samples that showed negative results for the K-ras mutation but positive results on histologic analysis. Also, when the specimens that had been positive in K-ras mutation while negative in the initial histologic analysis were serially sliced again and examined, we detected cancer cells newly in 43% of the samples (3 of 7). Recently, Demeure et al¹¹ examined regional lymph nodes for mutated K-ras in 22 patients with pathologic stage I (T1-T2, N0, M0) pancreatic cancer and reported that mutated K-ras was found in at least 1 regional lymph node in 16 patients (73%). Thus, our results obtained by genetic diagnosis seem to be suited for the following discussion on the detailed mode of cancer extension into the peri-SMA plexuses.

Since the SMA is encircled closely by many nerve fibers (plexuses), we had once postulated that they might form a complicated network communicating with each other and thereby facilitating the spread of cancer cells toward the various and random directions after one of the nerve fibers had become involved by cancer. However, in contrast to this speculation, our genetic diagnosis revealed a very simple result: carcinoma of the pancreatic head hardly extended into the left half of peri-SMA plexuses before reaching into the right half. In addition, cancer extension into the peri-SMA plexuses was associated well with the local growth of the tumor, particularly with the degree of tumor growth toward the retropancreatic direction (Table 2). Takahashi et al¹² also suggested that peripancreatic plexus invasion became more frequent after the retropancreatic invasion occurred. Although Urich¹³ reported that perineural spread might be a pathway for lymphatic spread of cancer cells, our results showed no relation between peri-SMA plexuses spread and the status of nodal involvement or the cancer invasion in the lymphatic channels. From these findings, the cancer extension into the peri-SMA plexuses seems to be within the range of locoregional therapy, differing from the hematogenous metastasis and peritoneal seeding.

The indication of locoregional therapy toward the peri-SMA plexuses should be determined carefully in consideration of other prognostic factors such as nodal involvement, histologic type, etc. However, our results indicated no need of additional therapy for this area if the posterior confine of the pancreas was quite intact. On the other hand, if the pancreatic tumor extended beyond the posterior confine of the pancreas, there was a higher possibility (9 [64%] of 14) that cancer cells may have extended in at least the right half of the peri-SMA plexuses. Likewise, according to T factor in the UICC-classification, the left half of the plexus was not involved at all in T1 to T3 cancers but was occasionally involved in T4 cancers. For T1 to T3 (UICC) cases, one reasonable strategy is to resect the right half of the plexus alone because there is smaller possibility that the left half of the plexus might be involved by cancer even after the right half has been affected (Table 3). It was reported previously that no severe diarrhea occurred after surgical resection in which the left half had been preserved.² It is necessary to develop a rapid and reliable technique to detect a microinvasion into the nerve plexuses during laparotomy. Intraoperative cytological analysis of the touch smear of the portal vein revealed a minute cancer invasion at an incidence of 30% in patients whose portal vein appeared macroscopically intact.¹⁴ Thus, this technique may be useful for analysis of the nerve plexuses and may have sensitivity comparable to that of genetic diagnosis.

Instead of surgical resection, prophylactic irradiation is another strategy because it does not cause severe diarrhea. Although postoperative irradiation therapy includes a risk that anastomotic sites might be irradiated, this problem could be solved by performing preoperative¹⁵ or intraoperative irradiation.¹⁶ Finally, future studies are needed to determine which treatment is most effective among the locoregional treatments (surgical resection of the peri-SMA plexuses, irradiation with or without chemotherapy, etc) to eradicate the possible cancer residual in this region.

Corresponding author and reprints: Hiroaki Ohigashi, MD, Department of Surgery, Osaka Medical Center for Cancer and Cardiovascular Diseases, 3-Nakamichi, 1-chome, Higashinari-ku, Osaka, 537-8511, Japan.

Some writers seem to have forgotten that there is more to surgery than what happens in an operating room. Surgery is not synonymous with operation.

In a manuscript I edited recently the author wrote "The patient is seen several weeks before the proposed surgery . . . " What is "seeing" the patient if not the provision of surgical care? This writer had focused on the operation he would perform rather than on the person seeking treatment.

When I edit a manuscript, I use the following guidelines: (1) Surgery is surgical care, surgical treatment, or surgical therapy; ie, the care provided by a surgeon with the help of nurses and other personnel from the first consultation and examination, through the hospital stay, operation, and postoperative care, until the last follow-up visit is complete. Surgery can last for years. (2) An operation is what happens between induction of and emergence from anesthesia—incision, dissection, excision, and closure—the surgical procedure. Surgical technique is the details of cutting and sewing. An operation rarely lasts more than several hours.

Used in sentences: The patient who needed cardiac surgery underwent more than one operation. Although the surgical technique was appropriate, the bypass graft failed, so transplantation was performed.

Surgery is what a surgeon practices. An operation is what a surgeon performs. In this context, there is no such word as surgeries. In Great Britain, surgeries are treatment rooms.

Neither an operation nor a patient is a case, but that's another commentary.—Catherine Judge Allen, MA, ELS, DeMotte, Ind

Reprinted from Arch Surg. 1996;131:128.