| Abstract|| |
Context: There is a correlation between prognosis of the colorectal carcinomas and the number of retrieved and metastatic lymph nodes (LNs) from mesentery/mesorectal region. At least 12 LNs must be sampled for accurate evaluation of patients. A number of factors related to surgeon, pathologist, patient and disease could affect the total LN number. For maximizing LN yield, pathologist can use ancillary methods, as fat clearance and special solutions. Aims: This study investigates the effect of second evaluation after ethanol fixation on total and metastatic LN number and assesses factors that influence the dissected LN number. Materials and Methods: 177 colorectal resections were refixed with ethanol for a night, after standard LN sampling. Mesentery/mesorectal tissue was reevaluated for missed LNs. Results were statistically analyzed, P values <0.05 were considered significant. Results: Mean LN number increased from 26 to 30 (median: 20 to 25, P < 0.001) after ethanol fixation. Fourteen cases had additional metastatic LNs after reevaluation of the fat tissue and 5 of them upstaged. 22.5% (44/177) of the patients had <12 LNs before ethanol fixation and this decreased to 14.3% (26/177) after ethanol fixation. Resection type and length, tumor localization, size and histologic degree, pT and neoadjuvant therapy (P < 0.001) had an impact on the LN number (P = 0.034 for histologic degree, P = 0.02 for pT, P < 0.001 for others). Conclusions: Carrying out a second evaluation with ethanol fixation increased total and metastatic LN number and could lead upstage of pN. Ethanol fixation is cost-effective, easy accessible and applicable method; it may improve accuracy of LN assessment and staging, which are important for patients' outcome.
Keywords: Colorectal carcinoma, ethanol, fat clearance, lymph node, lymph node sampling
|How to cite this article:|
Cakir A, Turkmen &C, Akhan AU, Akkoc M, Korkmaz P. Second evaluation of the mesenteric tissue after ethanol fixation improved the total and metastatic number of lymph nodes in colorectal resections. Indian J Pathol Microbiol 2019;62:31-5
|How to cite this URL:|
Cakir A, Turkmen &C, Akhan AU, Akkoc M, Korkmaz P. Second evaluation of the mesenteric tissue after ethanol fixation improved the total and metastatic number of lymph nodes in colorectal resections. Indian J Pathol Microbiol [serial online] 2019 [cited 2019 Apr 24];62:31-5. Available from: http://www.ijpmonline.org/text.asp?2019/62/1/31/251234
| Introduction|| |
Colorectal carcinoma (CRC) is the most common tumor of the gastrointestinal tract. Survival and prognosis of CRC is improved when the number of harvested lymph nodes (LNs) identified in resection specimen increase. According to the current classifications; staging and therapy options depend on the involvement of LNs; and LN metastasis is one of the major prognostic factors. Many associations recommend 12 LNs as a minimum number for accurate and reliable staging.,,
A number of clinicopathological factors have influence on nodal count. These are patient age, gender, obesity, surgeons', pathologists' and pathology assistants' experience and skill, surgical technique, time spent for and ancillary dissection techniques used for retrieving LNs, tumor site, differentiation, stage and neoadjuvant therapy.
Regarding pathology related factors, sampling the LNs in the grossing room is critical. Pathologists', residents' or assistants' experience and time are important factors. The standard LN sampling method is serial sectioning of the mesenteric tissue, visual inspection and palpation. With this method, especially small LNs can be overlooked. For achieving not only sufficient but also maximum LN number in gross room, various ancillary techniques have been reported. These target to clear mesenteric fat for a straightforward sighting of LNs. Many different methods such as fat clearance, Schwartz solution, GEWF (glacial acetic acid, ethanol, water and formalin) solution, fat dissociation method, are defined in the literature. Although all methods have some disadvantages such as time consumption, toxicity, specialized preparation and cost, eventually they achieved to increase total and metastatic LN number.,,,,,,,
The purpose of this study was to evaluate ethanol fixation effect on the number of harvested and metastatic LNs in colorectal resections and to identify factors associated with the total LN number.
| Materials and Methods|| |
Patients with colorectal carcinoma, underwent any type of colorectal resection over a 3-years period were routinely handled. Each specimen was left to formalin fixation, then, after sampling tumor, surrounding tissue and surgical margins, dissecting of the LNs was performed according to this procedure:First, parallel sections, as thin as possible, were performed to mesentery/mesorectal tissue. Lymph nodes that could be seen by naked eye were sampled entirely. For smaller ones, fatty tissue was palpated and small ones were sampled entirely, also. After the manual dissection, the mesentery fat tissue was put in a 96% ethylene alcohol (Vinprom Peshtera, Bulgaria) fixation until the next day. Then all the fatty tissue was reevaluated for missed LNs by the staff that made the first dissection [Figure 1]. All picked up LNs were evaluated on H and E stained slides; total and metastatic number of LNs harvested before (standart technique) and after ethanol fixation, the diameters of the LNs found after ethanol fixation were noted.
|Figure 1: Residual small lymph nodes can be seen easily after ethanol fixation. Inset: Closer view of the lymph node|
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Patients' demographic data, type of surgery, surgeon, and neoadjuvant therapy were collected from the hospital system. Tumor site and size, resection length, histologic grade, pT, pN, therapy effect (according to AJCC 7th edition) were recorded from pathology reports, retrospectively. Correlation between total LN number and patients age, gender, material length, tumor size, site and grade, neoadjuvant therapy was also investigated.
Statistical analyses were done with Wizard for mac (Version 1.8.16 (182)). One-way ANOVA and the Student's t-test, Mann-Whitney U test and the Kruskal–Wallis tests were used for statistical analysis. Differences with P value <0.05 were considered statistically significant.
| Results|| |
Total 177 colorectal specimens resected for adenocarcinoma were evaluated. All patients' demographic and pathologic data are summarized in [Table 1].
There was a relationship between dissected LN number and resection type, length, tumor site and size, pT and histologic degree. In anterior resections, less number of LNs was detected (P < 0.001), while right hemicolectomies and subtotal colectomies had more LNs (P < 0.001). Tumors located in ascending colon and caecum had more LN (P = 0.006 and P < 0.001, respectively) but rectum and sigmoid colon located tumors had less LN (P < 0.001 and P = 0.006, respectively). Lymph node number was correlated with pT; pT2 tumors had less LN than pT3 and pT4 tumors (P = 0.02). Low-grade tumors had less LN (P = 0.034). Neoadjuvant therapy had also effect on the LN number (P < 0.001). Besides, when there was any degree of response to the neoadjuvant therapy, LN number decreased, compared with the no therapy response (P = 0.03). There was not any correlation between total LN number and surgeons' subspecialty, patients' gender and age, and tumor type.
When LNs were sampled by traditional palpation method, mean number of dissected LN was 26 (range: 2-99, median: 20). Seventy (39.5%) of the cases had metastatic LN with a mean number of 2 (min-max: 1-17). After second evaluation with ethanol fixation, the mean LN number increased to 30 (range: 4-102, median: 25); and 74 (41.8%) of the cases had newly found metastatic LNs [Table 2]. The second evaluation with the use of ethanol fixation resulted in increase in LN number (P < 0.001). The diameter of the missed LNs was ranged between 0.1 cm to 1.6 cm with mean and median value of 0.3 cm. After second evaluation with ethanol fixation, a total of 698 missed LNs were found in 156 cases. In 14 cases, 15 newly found LNs had metastasis. The probability of finding missed and metastatic LN in each specimen by second evaluation using ethanol fixation was 88% and 7.9%, respectively [Table 2].
|Table 2: Second evaluation with ethanol effect on lymph node numbers and pN|
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Of these 14 cases with new metastatic LNs, 4 had neoadjuvant therapy. Also, in 3 of these 14 cases, pN was upstaged from pN0 to pN1a, pN0 to pN1b and pN1b to pN2a. Additionally, in 2 cases, tumor nodules were found and these patients upstaged from pN0 to pN1c [Table 2].
Sixty five (36.7%) patients had neoadjuvant therapy, in our study group. With traditional LN sampling method, mean total LN number was 15 (median: 12, ranging 2-66), after second evaluation with ethanol fixation; mean total LN number was 17 (median: 14, ranging 4-68). This increase was statistically significant (P < 0.001). Missed metastatic LNs were found in 4 cases; 1 LN in 2 cases, 2 LNs in 1 case and 3 LNs in 1 case.
With traditional sampling method, 41 (22.5%) of the cases had total number of LN less than 12, but after second evaluation with ethanol fixation, it decreased to 26 (14.3%) cases. Ten of these 26 cases had no additional LNs despite second evaluation and ethanol fixation.
| Discussion|| |
Correct staging of CRC is crucial for planning therapy and determining the prognosis. pT and pN, depending on the depth of tumor invasion and metastatic LN numbers, are critical for staging.,, Not only the metastatic LNs but also the total number of dissected LNs is important. Studies showed that approximately 20-25% of the node negative patients relapse and especially stage II patients with more number of LNs have better prognosis.,, The possible explanation is when more LNs are evaluated, the chance of finding positive node increases, and by lymphadenectomy, metastatic spread and tumor clearance could be achieved more successfully., Also, entity of lymphadenectomy can be used as a quality parameter for pathology laboratories.,
Some authors believe that there should be no limit for the number of evaluated LNs., But for correct staging, the minimum/accurate LN number '12' was first established at the World Congress of Gastroenterology in 1990 and after that many guidelines, such as Collage of American Pathologists, refereed this number.,
In this study, retrieved LN number ranged between 4 and 102 with a mean of 30. We achieved to dissect 12 and more LNs in 85.7% (156/177) of the cases. Before second evaluation with ethanol fixation, the ratio was 77.5% (141/177). Compared with the reported series that ranged from 26.5% to 93.6%, our finding is one of the highest values.,, We can explain this by our dissection method. Because, in our routine practice, we aim to find as much as LNs we can, so that we perform second evaluation to mesenteric/mesorectal fat tissue after 1 night ethanol fixation. Ethanol fixation makes LNs easily visible in second evaluation.
Different variables can affect the harvested LN number. These can be divided into two categories as modifiable and nonmodifiable factors. Modifiable factors are surgeon's and pathologist's experience, surgical technique, ancillary dissection techniques and time spend for the retrieval by pathologists; and nonmodifiables are patients age, gender, obesity and tumor site, stage and neoadjuvant therapy.,
Most of the studies indicated that advanced age and rectum located tumor can affect LN number negatively while right colon location of tumor, grade and pathologic T stage can effect positively.,,,,,, There are conflict data on the effect of gender and obesity.,,, In our study, age and gender of the patients, tumor type and pT did not correlate with the LN number. Similarly with the publications, there were less LN in anterior resection specimens and the tumors located at rectum and sigmoid colon (P < 0.001). There was positive correlation between length of the resection and tumor size with total LN number (P < 0.001, for both). Since rectum and sigmoid colon location and neoadjuvant therapy had negative effect on total LN number, we advise to seek LNs with additional techniques such as second evaluation, ethanol fixation for these patients.
Although surgeons' experience reported as an independent factor for number of removed LNs by some publications, in 3 studies comparing surgeons, there was no exact difference between dissected LN numbers and the experienced/inexperienced surgeon (eg. 13 vs 11 LN, 11 vs 9 LN and achieving >12 LN in the 86% and 83% of the patients).,,,, In our study, there were 2 surgeons performing the operations; one of them was a colorectal surgeon. Although case number of the noncolorectal surgeon was less (n = 16), there was no statistically significant difference between two of them (P = 0.9). Also, we couldn't find any difference between two surgeons when LN cutoff value was taken as 12 (P = 0.2).
Lymph node dissecting pathology staff, such as pathology assistants, residents and pathologists, could affect the number of sampled LN and some studies indicate that it is an independent factor as surgeons' experience.,,, Time, educational training and skills are important factors intervening with this finding. Bomboat et al. and Kuijpers et al. showed that pathology assistants and first year pathology residents can achieve dissecting more LNs, especially in rectal resections, than experienced pathologists because they have more time, less distractions and may use ancillary techniques such as fat clearing solutions and intra-arterial methylene blue injection.,,
American Collage of Pathologists recommend to use ancillary techniques while or after manual sampling of the LNs especially if the LN number is less than 12, because the remaining mesentery tissue could contain additional LNs., There are several methods for this purpose, such as fat clearance, Schwartz solution, GEWF solution, fat dissociation method, transluminating the mesentery, entirely sampling the mesenteric tissue, extending the fixation time and dye injection. Solutions contain various proportions and combinations of some chemical agents, such as acetone, acetic acid, diethyl ether, ethanol, formalin, hydrochloric acid and xylene.,,,,,,, With these methods, additional mean LN numbers varied from 1 to 48.,,,,, But in some studies no statistically significant improvement was found when compared to manual dissection., Increase in metastatic LN number and even upstaging of pN, ranging 1% to 33% of the cases, was also reported by authors.,,, Size of the missed LNs ranged from 1 to 15 mm, usually 1-5 mm.,,,,,,, However, there are some limitations for each procedure. These are time consumption (1-9 days), special preparation need, toxicity and additional cost.,,,,,, There is no consensus on which method is more efficient but all these methods aim to clear fat and visualize the LNs distinctly and easily without damaging the tissue. Among all the solutions and methods, GEWF is one of the most studied solutions in the literature. It is reported as a safe and efficient lymph node revealing solution, and also preparing is quick, cheap and easy.,,
Not only for achieving the minimum required number, but also finding the missing LNs and reaching the maximum LN number, we performed additional second evaluation with ethanol fixation to our colorectal resection specimens after standard LN sampling. Ethanol was easily accessible, cost-effective for laboratory, and was no need to prepare new solutions. In our study, mean and median number of LNs with traditional sampling was 26 and 20 (ranging 2-99), while after second evaluation with ethanol, mean and median number were 30 and 25 (ranging 4-102). This increase in LN number was statistically significant (P < 0.001). Usually, small LNs were found after second evaluation, with a mean size of 0.3 cm. Ethanol fixation made these remaining small LNs readily visible. Metastatic LN number also increased in 14 cases, in our study. Moreover, new tumor nodules were found in 2 cases. Eventually 3 cases (1.7% of all cases) upstaged by additional metastatic LNs and 2 cases (1.1% of all cases) upstaged pN0 to pN1c.
Neoadjuvant therapy also affects the LN sampling by posttherapy inflammation and fibrosis.,, Reported mean LN numbers ranged from 7 to 9.8 for patients treated with neoadjuvant therapy, and Marks et al. concluded that limit of 12 LN cannot be used for these patients.,,, This decrease is statistically significant and more distinct when radiotherapy and chemotherapy are used in combination., This finding can also be interpreted as the positive response to the therapy. In our study, 35.7% (65/177) of the patients received neoadjuvant therapy. It was not surprising for us that 84.6% of the cases, which had less than 12 LNs, received neoadjuvant therapy. Mean LN numbers were less for patients who had neoadjuvant therapy than the patients that did not have neoadjuvant therapy but after second evaluation with ethanol fixation mean LN number increased from 15 to 17. This increase was statistically significant. In 4 cases having neoadjuvant therapy, we found additional metastatic LNs after second evaluation with ethanol fixation.
| Conclusion|| |
In conclusion, we highly recommend second evaluation of LNs with using additional visual enhancement techniques, such as ethanol fixation in routine practice for all case but especially in these conditions: i. anterior resection specimens, ii. patients taking neoadjuvant therapy, iii. when total LN number is <12. With ethanol fixation, LNs, even the small ones, can be visualized easily. Second evaluation and ethanol fixation are effective, simple, cheap and accessible methods for all pathology laboratories.
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| References|| |
Edge SB, Compton CC. The American joint committee on cancer: The 7th
edition of the AJCC cancer staging manual and the future of TNM. Ann Surg Oncol 2010;17:1471-4.
Nelson H, Petrelli N, Carlin A, Couture J, Fleshman J, Guillem J, et al.
Guidelines 2000 for colon and rectal cancer surgery. J Natl Cancer Inst 2001;93:583-96.
Washington MK, Berlin J, Branton P, Burgart LJ, Carter DK, Fitzgibbons PL, et al.
Protocol for the examination of specimens from patients with primary carcinoma of the colon and rectum. Arch Pathol Lab Med 2009;133:1539-51.
Li Destri G, Di Carlo I, Scilletta R, Scilletta B, Puleo S. Colorectal cancer and lymph nodes: The obsession with the number 12. World J Gastroenterol 2014;20:1951-60.
Morikawa E, Yasutomi M, Shindou K, Matsuda T, Mori N, Hida J, et al.
Distribution of metastatic lymph nodes in colorectal cancer by the modified clearing method. Dis Colon Rectum 1994;37:219-23.
Chapman B, Paquette C, Tooke C, Schwartz M, Osler T, Weaver D, et al.
Impact of schwartz enhanced visualization solution on staging colorectal cancer and clinicopathological features associated with lymph node count. Dis Colon Rectum 2013;56:1028-35.
Gregurek SF, Wu HH. Can GEWF solution improve the retrieval of lymph nodes from colorectal cancer resections? Arch Pathol Lab Med 2009;133:83-6.
Hernanz F, García-Somacarrera E, Fernández F. The assessment of lymph nodes missed in mesenteric tissue after standard dissection of colorectal cancer specimens. Colorectal Dis 2010;12:e57-60.
Newell KJ, Sawka BW, Rudrick BF, Driman DK. GEWF solution. Arch Pathol Lab Med 2001;125:642-5.
Fujino S, Miyoshi N, Ohue M, Noura S, Tomita Y, Yano M, et al.
New enhanced and effective method for staging cancer to detect lymph nodes after fat-dissociation. Oncol Rep 2014;32:922-6.
Vogel C, Kirtil T, Oellig F, Stolte M. Lymph node preparation in resected colorectal carcinoma specimens employing the acetone clearing method. Pathol Res Pract 2008;204:11-5.
Horne J, Bateman AC, Carr NJ, Ryder I. Lymph node revealing solutions in colorectal cancer: Should they be used routinely? J Clin Pathol 2014;67:383-8.
Norwood MG, Sutton AJ, West K, Sharpe DP, Hemingway D, Kelly MJ, et al.
Lymph node retrieval in colorectal cancer resection specimens: National standards are achievable, and low numbers are associated with reduced survival. Colorectal Dis 2010;12:304-9.
Le Voyer TE, Sigurdson ER, Hanlon AL, Mayer RJ, Macdonald JS, Catalano PJ, et al.
Colon cancer survival is associated with increasing number of lymph nodes analyzed: A secondary survey of intergroup trial INT-0089. J Clin Oncol 2003;21:2912-9.
Stocchi L, Fazio VW, Lavery I, Hammel J. Individual surgeon, pathologist, and other factors affecting lymph node harvest in stage II colon carcinoma. Is a minimum of 12 examined lymph nodes sufficient? Ann Surg Oncol 2011;18:405-12.
Shia J, Wang H, Nash GM, Klimstra DS. Lymph node staging in colorectal cancer: Revisiting the benchmark of at least 12 lymph nodes in R0 resection. J Am Coll Surg 2012;214:348-55.
McDonald JR, Renehan AG, O'Dwyer ST, Haboubi NY. Lymph node harvest in colon and rectal cancer: Current considerations. World J Gastrointest Surg 2012;4:9-19.
Tekkis PP, Smith JJ, Heriot AG, Darzi AW, Thompson MR, Stamatakis JD, et al.
Anational study on lymph node retrieval in resectional surgery for colorectal cancer. Dis Colon Rectum 2006;49:1673-83.
Lindboe CF. Lymph node harvest in colorectal adenocarcinoma specimens: The impact of improved fixation and examination procedures. APMIS 2011;119:347-55.
Mekenkamp LJ, van Krieken JH, Marijnen CA, van de Velde CJ, Nagtegaal ID, Pathology Review Committee and the Co-operative Clinical Investigators. et al.
Lymph node retrieval in rectal cancer is dependent on many factors – The role of the tumor, the patient, the surgeon, the radiotherapist, and the pathologist. Am J Surg Pathol 2009;33:1547-53.
Deodhar KK, Budukh A, Ramadwar M, Bal MM, Shrikhande SV. Are we achieving the benchmark of retrieving 12 lymph nodes in colorectal carcinoma specimens? Experience from a tertiary referral center in India and review of literature. Indian J Pathol Microbiol 2012;55:38-42. [Full text]
Valsecchi ME, Leighton J Jr., Tester W. Modifiable factors that influence colon cancer lymph node sampling and examination. Clin Colorectal Cancer 2010;9:162-7.
Evans MD, Barton K, Rees A, Stamatakis JD, Karandikar SS. The impact of surgeon and pathologist on lymph node retrieval in colorectal cancer and its impact on survival for patients with Dukes' stage B disease. Colorectal Dis 2008;10:157-64.
Gelos M, Gelhaus J, Mehnert P, Bonhag G, Sand M, Philippou S, et al.
Factors influencing lymph node harvest in colorectal surgery. Int J Colorectal Dis 2008;23:53-9.
Barbas A, Turley R, Mantyh C, Migaly J. Advanced fellowship training is associated with improved lymph node retrieval in colon cancer resections. J Surg Res 2011;170:e41-6.
Khatib WM, Jagtap SV, Demde RB, Pate PM. Study of lymph node involvement in colorectal cancer – A 5 year study in a tertiary care rural hospital. Indian J Basic Appl Med Res 2016;5:11-9.
Shaw A, Collins EE, Fakis A, Patel P, Semeraro D, Lund JN, et al.
Colorectal surgeons and biomedical scientists improve lymph node harvest in colorectal cancer. Tech Coloproctol 2008;12:295-8.
Leung KL, Kwok SP, Lam SC, Lee JF, Yiu RY, Ng SS, et al.
Laparoscopic resection of rectosigmoid carcinoma: Prospective randomised trial. Lancet 2004;363:1187-92.
Scott KW, Grace RH. Detection of lymph node metastases in colorectal carcinoma before and after fat clearance. Br J Surg 1989;76:1165-7.
Kim YM, Suh JH, Cha HJ, Jang SJ, Kim MJ, Yoon S, et al.
Additional lymph node examination from entire submission of residual mesenteric tissue in colorectal cancer specimens may not add clinical and pathologic relevance. Hum Pathol 2007;38:762-7.
Brown HG, Luckasevic TM, Medich DS, Celebrezze JP, Jones SM. Efficacy of manual dissection of lymph nodes in colon cancer resections. Mod Pathol 2004;17:402-6.
Ustün MO, Onal B, Tuğyan N, Rezanko T. Lymph node revealing solution: Is it effective on detecting minute lymph nodes? Adv Clin Path 1999;3:135-8.
Marks JH, Valsdottir EB, Rather AA, Nweze IC, Newman DA, Chernick MR, et al.
Fewer than 12 lymph nodes can be expected in a surgical specimen after high-dose chemoradiation therapy for rectal cancer. Dis Colon Rectum 2010;53:1023-9.
Department of Pathology, Istanbul Medipol University Hospital, Bagcilar, Istanbul
Source of Support: None, Conflict of Interest: None
[Table 1], [Table 2]