|Ahead of print
|Is it possible that we can increase the lymph node number in cases with rectum tumor receiving neoadjuvant therapy?
Selma Şengiz Erhan1, Sevinç Hallaç Keser2, Gülçin Harman Kamalı1, Sibel Sensu3, Selvi Dinçer4, Fazıl Sağlam5
1 Departments of Pathology, Istanbul Prof. Dr. Cemil Taşcıoğlu City Hospital, Istanbul, Turkey
2 Department of Pathology, Kartal Dr. Lutfi Kirdar City Hospital, Istanbul, Turkey
3 Department of Pathology, Medical Faculty, University of Istinye, Istanbul, Turkey
4 Department of Radiation Oncology, Istanbul Prof. Dr. Cemil Taşcıoğlu City Hospital, Istanbul, Turkey
5 Department of Surgery, Istanbul Prof. Dr. Cemil Taşcıoğlu City Hospital, Istanbul, Turkey
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|Date of Submission||19-Dec-2021|
|Date of Decision||25-Apr-2022|
|Date of Acceptance||28-Apr-2022|
|Date of Web Publication||27-Jan-2023|
| Abstract|| |
Background: Though the recommended sampled lymph node number in colorectal carcinomas is at least 12, due to shrinkage after preoperative neoadjuvant chemoradiotherapy (NCRT), it can be difficult to attain that number. Aim: Our aim is to increase the lymph node number by applying alcohol fixation on the formalin-fixed resection materials of the patients that received or not received neoadjuvant therapy and to evaluate the changes in staging due to obtained lymph nodes. Settings and Design: Non-randomized controlled trial. Materials and Methods: Lymph node dissection was performed in the resection materials with rectum tumor which were formalin- and afterwards, alcohol-fixed. The number of lymph nodes obtained by both of the methods and status of metastasis were evaluated statistically. Results: Of the total 76 rectal tumors, 57 had and 19 had not received NCRT. The number of lymph node was adequate in 89.5% cases with no NCRT and in 63.2% cases with NCRT. While no change was observed after the alcohol fixation in the cases fulfilling adequacy criterion among those with no NCRT (p = 1.000), the adequacy rate increased from 63.2% to 87.7% in those with NCRT (p < 0.001). Although statistically insignificant, there was a change in pN stage in eight cases. In three of them, the stage varied from pN0 to pN1c, and in five cases, from pN1a to pN1b. Conclusion: Using solutions as alcohol during fixation might facilitate the identification of metastatic lymph nodes, might change the stage of the disease and therefore, might affect the patient-based therapy.
Keywords: Alcohol fixation, lymph node, neoadjuvant chemoradiotherapy, rectum
|How to cite this URL:|
Erhan S&, Keser SH, Kamalı GH, Sensu S, Dinçer S, Sağlam F. Is it possible that we can increase the lymph node number in cases with rectum tumor receiving neoadjuvant therapy?. Indian J Pathol Microbiol [Epub ahead of print] [cited 2023 Nov 30]. Available from: https://www.ijpmonline.org/preprintarticle.asp?id=368574
| Introduction|| |
Sampling adequate number of lymph node from the resection materials of colorectal tumors is important for the accurate staging and directing the treatment to be administered following the surgery. For an appropriate staging, dissection of at least 12 lymph nodes is recommended. Yet, different factors, such as age over 60 years, obesity, female gender, small tumor size, poorly differentiation, and neoadjuvant chemoradiotherapy (NCRT) might affect sampling., NCRT leads to shrinkage of the lymph nodes and difficulty to attain the minimum recommended number. The limited number of dissected lymph nodes might result in staging error (underdiagnosis) and inappropriate postoperative adjuvant chemotherapy which can affect the survival.
There exist different methods such as the prolonging of fixation period and administration of different solutions that could reveal the lymph nodes otherwise could be missed by palpation.,,, Most studies reveal that there is an increase in the number of lymph nodes by administration of different solutions compared to the manual lymph node dissection. Yet, in very few studies, the changes in the N stage have been concluded as significant.,
In this study by applying alcohol fixation to formalin-fixed rectum resection materials of the patients that had or had not taken NCRT, we aimed to increase the number of identified lymph nodes and explore the changes in stage.
| Material and Methods|| |
The cases that were operated between 2018 and 2020 due to localized tumor in the rectum were included in the study. The study consisted of two groups. The first group included cases that received preoperative NCRT due to advanced stage local rectal tumor (T3-T4 and/or N1-N2), while the second group, with rectal tumor that did not receive NCRT. Low anterior resection surgery including the total mesorectal excision line was applied to all of the cases. The demographic features (age, gender) for both of the groups were obtained via the hospital archive system. The ethical approval for this study was obtained from the Ethics Committee with the number 12.01.2021/9.
Macroscopic sampling of the resection materials
The resection materials of both groups were fixed at least for two days in 10% formaldehyde solution. Tumor and surgical borders with surrounding mucosa were sampled according to the macroscopic sampling protocol by one pathologist. In the thin slices performed on mesentery and mesorectal fatty tissue, the lymph nodes and structures which were detected by observation and palpation and suspected of being tumor deposits were dissected. Afterwards, to identify the lymph nodes that were not detected by observation and palpation, alcohol fixation as an additional method applied to the same resection materials. After formaldehyde fixation (FF), all resection materials were fixed in 96% ethyl alcohol (AF) (Alkomed, Turkey) for three days including the weekends., Then, the tissues were reevaluated and sampled by the same pathologist with a second coding.
In the study, the materials in both groups were dissected after FF and, afterwards, were taken into AF. Thus, there was no group of materials dissected only following an AF.
Microscopic examination of the resection materials and obtaining of the data
The tumor size, histological type, grade, pathological T stage and effect of therapy on the cases that received NCRT were microscopically evaluated. The treatment effect for the cases that received NCRT was determined based on the modified Ryan scoring. Not detecting live tumor tissue was score 0 (complete response), tracking single or small groups of tumor tissue along with prevalent fibrosis, score 1 (near complete response), tracking more than single or small groups of tumor along with fibrosis, score 2 (partial response), the presence of prevalent residual tumor, score 3 (poor response). The treatment effect was split into two groups as score 0-1 (good response) and score 2-3 (poor response) following the evaluation. The lymph nodes, metastatic lymph nodes, and tumor deposits dissected after both methods were determined separately and in total. The lymph nodes were also evaluated as per the changes depending on the treatment impact for the cases that received NCRT.
Statistical Package for Social Sciences (SPSS), Windows version 23.0 (SPSS Inc. Chicago, USA) software package program was used for the statistical analyses of the research data. In the descriptive statistics aspect, the categorical variables were presented as number and percentage while the continuous variables were presented as mean, ± standard deviation and median (the lowest-highest value). The fit of the continuous variables to the normal distribution was evaluated visually (histogram and probability graphs) along with employing analytical methods (i.e. Kolmogorov-Smirnov/Shapiro-Wilk tests). The result of the normalization analyses showed that the data belonging to the continuous variables had normal distribution. Student t-test was used for the comparative analyses between the two independent groups concerning the data that fit normal distribution. Pearson's Chi-square test, Fisher Chi-square test and McNemar test were used for the comparative analysis conducted with respect to categorical variables between the groups. P < 0.05 was accepted to indicate significance.
| Results|| |
General properties of the cases
76 cases (51 (67.1%) male and 25 (32.9%) female) were included in the study. The average age was 61.88 ± 10.19. The number of cases that received NCRT (first group) was 57 (75%) and the number of cases with no NCRT (second group) was 19 (25%). One case (5.3%) that did not receive NCRT was diagnosed with villous adenoma following the histopathological examination. All the other cases in both of the groups were invasive tumor (adenocarcinoma (78.9% vs 89.5%)) and mucinous carcinoma (5.3% vs 5.3%). A significant relationship was detected between the first group and second group in terms of tumor size with student t-test (p = 0.010). The tumor size in the second group was larger than first group.
The histopathological findings are presented in [Table 1].
|Table 1: Clinicopathological findings of the cases that received NCRT and that did not receive NCRT|
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Lymph nodes and tumor deposits
In the first group, following FF, 36 cases had ≥ 12 lymph nodes (63.2%). In 21 of the cases (36.8%), the number of lymph nodes was < 12. The number of cases with adequate number of lymph nodes after AF was 50 (87.7%), yet it was not possible to attain an adequate level of lymph node for seven cases (12.3%). Thus, there was a 24.5% increase in the number of cases that fulfilled the adequacy criterion with AF. Treatment-related changes in the lymph nodes were observed in 24 of the cases (42.1%). These changes were detected in 56 of 424 lymph nodes (13.2%). Chi-square test for the lymph nodes with treatment-related changes revealed no statistically significant difference between score 0-1 group and score 2-3 group (p = 0.335).
In the second group, following FF, 17 cases had ≥ 12 lymph nodes (89.5%) and in 2 cases, the number of lymph nodes was below 12 (10.5%). There was an increase in the lymph node number after AF, yet the dissected lymph nodes were detected in the cases that fulfilled the adequacy criterion. As a result, there was no change in the number of cases that met adequacy criterion in this group. The result of the Chi-square test yielded a significant relationship between the first group and second group in terms of lymph node number dissected following FF (p = 0.031). Based on this, the adequacy rate for the lymph node dissected after FF in the cases with no NCRT was significantly higher compared to those that received NCRT. Student t-test also yielded a statistically significant difference between the first and the second group in terms of total lymph node number dissected following FF (p = 0.001) and AF (p = 0.005). Based on this, the number of total lymph node number dissected after both of the solutions in the cases with no NCRT was significantly higher than those that received NCRT. McNemar test did not identify a statistically significant difference in terms of lymph node numbers between the two sampling methods in the cases with no NCRT (p = 1.000). In contrast, the difference was significant for those that received NCRT (p < 0.001). Thus, the adequacy rate with the lymph nodes obtained after AF among the ones that received NCRT was significantly higher compared with the first sampling.
No significant relationship was detected between the lymph node number and age, gender, tumor size and differentiation in either of the groups.
No change was found after AF in the number of cases that had metastatic lymph nodes following FF in both of the groups; however, there was an increase in the number of metastatic lymph nodes after AF (range 0-4 vs 0-1). In 29 of the cases, the number of metastatic lymph nodes detected after FF was 80. The largest lymph node was 1.5 cm. In 10 cases, the number of metastatic lymph nodes detected at ≤0.5 cm was 12 (15%). Following AF, 14 metastatic lymph node was detected in 10 of the cases and the largest metastatic lymph node was 0.4 cm.
Tumor deposit was observed in six of the cases after FF (10.5%) and in four of the cases after AF (7%) in the first group. Tumor deposit was detected one in four cases by both of the methods, and the detection was observed in only three of them after AF. In the second group, tumor deposit was observed in five of the cases after FF (26.3%) and no additional tumor deposit was detected after AF.
The data belonging to the lymph node, metastatic lymph node, and tumor deposits obtained by both of the methods are presented in [Table 2].
|Table 2: Changes related to lymph node, metastatic lymph node, and tumor deposit after the fat clearing technique in cases that received NCRT and that did not receive NCRT|
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Changes in pN stage
In the study, there was a change in N stage in eight cases. Only one of those cases had not received NCRT, while seven of the cases were in the first group that had received NCRT. In addition, in one case that received NCRT and in one case with no NCRT, the lymph node number was below the adequacy criterion. In three of the cases, the stage changed from pN0 to pN1c upon tumor deposit detection after AF and from pN1a to pN1b in five cases with the metastatic lymph node number increase.
As a result of the Mann–Whitney U test, no statistically significant difference was detected between the first and second group as regards the metastatic lymph node number dissected by the two sampling methods; and based on the Chi-square result, no statistically significant difference was detected either between the tumor deposits (p > 0.05).
The data related to the changes in the lymph node stage are presented in [Table 3].
|Table 3: Changes detected in the pN stage after formaldehyde and alcohol fixation|
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| Discussion|| |
It is possible to attain an adequate number of lymph nodes in 25% to 86% of the cases in the studies which included cases with rectal tumor that received NCRT.,,,,,, In our study, which includes cases with NCRT, the rate we have detected has been above the data provided in the previous studies of the literature. While the factors that are related to the patient and tumor have unalterable impact on the lymph node number, surgical procedure and the pathologist's work have alterable impact.,,, Age over 60 years, obesity, female gender, right sided tumor, small size, poorly differentiation, and NCRT can be stated as the unalterable factors. The number of lymph node dissected in females and right sided tumors could be high while age over 60 years, small tumor size and receiving NCRT leads to fewer number of dissected lymph nodes., No significant relationship is detected in our study between the lymph node number and age, gender, tumor size, and differentiation.
NCRT could lead to necrosis with fibrosis in tumor as well as loss of lymphocytes in the lymph nodes and stromal atrophy. As a result, shrinking in the lymph node size is observed. Especially, as the period between the operation and NCRT gets longer, more regression and fibrosis develop in the tissues. Consequently, the total lymph node number will further decrease along with the metastatic lymph node number., In concordance with the literature, it has been revealed in our study that the tumor size was smaller in the cases that received NCRT compared to those that did not receive NCRT. It has also been observed that the total lymph node number dissected in the cases that received NCRT was lower than the ones that did not receive NCRT.
Tumor regression score shows the malignant cell rate that remains live after NCRT which was demonstrated as a potentially important prognostic factor in different studies. In some of the studies, it was stated that low lymph node number did not show inadequate resection or understaging in the cases that received NCRT, rather it manifested increased sensitivity towards treatment., On the other hand, some studies reported that it could not be related to good tumor regression and survival. In our study, no significant relationship between total lymph node and total metastatic lymph node numbers and good response (score 0-1) and poor response (score 2-3) has been observed. Besides this, no significant relationship has been identified either between the changes depending on the treatment impact detected in the lymph nodes and good and poor response.
The surgical procedure which is an alterable factor affecting the lymph node number can be modified. Besides the experience, skill and patience of the pathologist, different macroscopic techniques and use of solutions revealing lymph nodes could be among the factors that can lead to increase in the lymph node number.,,,, More than half of the metastatic nodes are of small size, being below 5 mm. Solutions that reveal lymph node could be of particular assistance in detecting the lymph nodes of such a size that could be easily missed. We have obtained results that verify this data. Only in 15% of the cases that were pN + after FF had lymph node dimension at 5 mm and below 5 mm. However, all of the metastatic lymph nodes dissected after AF had dimensions below 5 mm.
Xylene, acetone, alcohol and GWEF (glacial acetic acid, ethanol, water, formalin) as well as methylene blue injection are among the solutions used.,,,,,, The ones that are more preferable are GWEF and methylene blue injection, indicated to be advantageous in terms of time.,, The drawbacks of these solutions are cost, need for extra time, work burden, and exposure to chemical solution like xylene whose carcinogenic potential is known., The solution we preferred to use in our study was alcohol. The solutions that show the lymph nodes are at least two times expensive than formaldehyde solution. The most affordable of these solutions were alcohol and xylene. We preferred alcohol in our study since it was safer than xylene regarding toxicity.
In very few studies, like in the study of ours, solution that reveals the lymph node in the same resection material (applied before and after) has been used., The lymph node stage was observed to change between 0.96% and 27% in these studies and it is reported that the solutions used could cause an increase in the lymph node number but do not have a significant effect on the stage.,,, In a study in which the cases that received and did not receive NCRT were compared, unlike the cases that did not receive NCRT, the cases that received NCRT had increase in the lymph node number. Besides this, a significant change was observed in the metastatic lymph nodes and N stage as well. Like in our study, alcohol was used in that study as the solution revealing the lymph node. Through the alcohol we used in our study, the cases that had 12 and more number of lymph nodes have increased by 18.42%. A significant increase in lymph node number has been detected particularly in the cases that received NCRT. In five cases, though insignificant, stage rose from pN1a to pN1b due to increased number of metastatic lymph nodes.
Tumor deposits are defined as the tumor focus in the lymph drainage area in the pericolic or perirectal fatty tissue without belonging to lymph node or vascular structure. Regardless of the pT stage in cases which do not have metastatic lymph node, the classification thereof is made as pN1c. Tumor deposits detected in some parts of the cases with colorectal tumor without metastatic lymph node in the literature have been found to be correlated with poor prognosis., In three of the cases in our study, N stage changed with tumor deposit we detected after AF, rising from pN0 to pN1c.
There are some limitations in our study. The fact that similar number of pT stage cases did not exist in the group that received and did not receive neoadjuvant therapy could have prevented us from achieving statistical significance. Due to only six cases having died during the follow-up of the cases, the relationship between survival and prognostic parameters have not been evaluated, which can be stated as another limitation of our study.
| Conclusion|| |
Since a more precise N staging in colorectal cancers is required especially in cases who received preoperative NCRT, we have increased the rate of the cases with the adequacy criterion by 18.42% through the alcohol fixation and pN stage has changed in 10.5% of the cases. Although not statistically significant, as in our study, increase in metastatic lymph node number and tumor deposits detected by FF and following AF might change the stage and accordingly, patient-based treatment protocols (especially in patients receiving NCRT). Therefore, to identify lymph nodes in cases with minute lymph node numbers, FF followed by AF can be a method used in daily practice.
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Conflicts of interest
There are no conflicts of interest.
| References|| |
Wang H, Safar B, Wexner S, Zhao R, Cruz-Correa M, Berho M. Lymph node harvest after proctectomy for invasive rectal adenocarcinoma following neoadjuvant therapy: Does the same standard apply? Dis Colon Rectum 2009;52:549-57.
Jessup JM, Goldberg RM, Asare EA, Benson AB, Brierley JD, Chang GJ, et al
. Colon and rectum. In: Amin MB, editor. AJCC Cancer Staging Manuel. 8th
ed. Switzerland: Springer; 2017. p. 251-71.
Lindboe CF. Lymph node harvest in colorectal adenocarcinoma specimens: The impact of improved fixation and examination procedures. APMIS 2011;119:347-55.
Madbouly KM, Abbas KS, Hussein AM. Metastatic lymph node ratio in stage III rectal carcinoma is a valuable prognostic factor even with less than 12 lymph nodes retrieved: A prospective study. Am J Surg 2014;207:824-31.
O'Neil M, Damjanov I. Histopatology of colorectal cancer after neoadjuvant chemoradiation therapy. Open Pathol J 2009;3:91-8.
Doll D, Gertler R, Maak M, Friederichs J, Becker K, Geinitz H, et al
. Reduced lymph node yield in rectal carcinoma specimen after neoadjuvant radiochemotherapy has no prognostic relevance. World J Surg 2009;33:340-7.
Markl B, Schaller T, Krammer I, Cacchi C, Arnholdt HM, Schenkirsch G, et al
. Methylen blue-assisted lymph node dissection technique is not associated with an increased detection of lymph node metastases in colorectal cancer. Mod Pathol 2013;26:1246-54.
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.
Yeh CC, Pan CF, Liu HW, Lin JC, Fang LH, Lee HS, et al
. Using the fa-clearing technique to improve lymph node retrieval in colorectal cancer. Int J Surg Pathol 2021;29:385-91.
Abbassi-Ghadi N, Boshier PR, Goldin R, Hanna GB. Techniques to increase lymph node harvest from gastrointestinal cancer specimens: A systematic review and meta-analysis. Histopathology 2012;61:531-42.
Kim IK, Lim BJ, Kang J, Kim SA, Kang D, Sohn SK, et al
. Clinical impact of fat clearing technique in nodal staging of rectal cancer after preoperative chemoradiotherapy. J Korean Surg Soc 2013;85:30-4.
Burgart LJ, Chopp WV, Jain D. Protocol for the examination of resection specimens from patients with primary carcinoma of the colon and rectum. In: CAP Cancer and CAP Pathology Electronic Reporting Committees; 2021. p. 1-28.
Wang Y, Zhou M, Yang J, Sun X, Zou W, Zhang Z, et al
. Increased lymph node yield indicates improved survival in locally advanced rectal cancer treated with neoadjuvant chemoradiotherapy. Cancer Med 2019;8:4615-25.
Tsai HL, Huang CW, Yeh YS, Ma CJ, Chen CW, Lu CY, et al
. Factors affecting number of lymph nodes harvested and the impact of examining a minimum of 12 lymph nodes in stage I-III colorectal cancer patients: A retrospective single institution cohort study of 1167 consecutive patients. BMC Surg 2016;16:17.
Persiani R, Biondi A, Gambacorta MA, Zoccali MB, Vecchio FM, Tufo A, et al
. Prognostic implications of the lymph node count after neoadjuvant treatment for rectal cancer. Br J Surg 2014;101:133-42.
Gurawalia J, Dev K, Nayak SP, Kurpad V, Pandey. Less than 12 lymph nodes in the surgical specimen after neoadjuvant chemo-radiotherapy: An indicator of tumor regression in locally advanced rectal cancer? J Gastrointest Oncol 2016;7:946-57.
Orsenigo E, Gasparini G, Carlucci M. Clinicopathological factors influencing lymph node yield in colorectal cancer: A retrospective study. Gastroenterol Res Pract 2019;2019:
Ong MLH, Schofield JB. Assessment of lymph node involvement in colorectal cancer. World J Gastrointest Surg 2016;8:179-92.
Betge J, Harbaum L, Pollheimer MJ, Lindtner RA, Kornprat P, Ebert MP, et al
. Lymph node retrieval in colorectal cancer: Determining factors and prognostic significance. Int J Colorectal Dis 2017;32:991-8.
Dawson H, Kirsch R, Messenger D, Driman D. A review of current challenges in colorectal cancer reporting. Arch Pathol Lab Med 2019;143:869-82.
Ryan R, Gibbsons D, Hyland JMP, Treanor D, White A, Mulchay HE, et al
. Pathological response following long-course neoadjuvant chemoradiotherapy for locally advanced rectal cancer. Histopathology 2005;47:141-6.
Pheby DFH, Levine DF, Pitcher RW, Shepherd NA. Lymph node harvests directly influence the staging of colorectal cancer: Evidence from a regional audit. J Clin Pathol 2004;57:43-7.
Ludeman L, Shepherd NA. Macroscopic assesment and dissection of colorectal cancer resection specimens. Curr Diagn Pathol 2006;12:220-30.
Rodriguez-Bigas MM, Maamoun S, Weber TK, Penetrante RB, Blumenson LE, Petrelli NJ. Clinical significance of colorectal cancer: Metastasis in lymph nodes <5 mm in size. Ann Surg Oncol 1996;3:124-30.
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.
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.
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.
Wang Y, Zhang J, Zhou M, Yang L, Wan J, Shen L, et al
. Poor prognostic and staging value of tumor deposit in locally advanced rectal cancer with neoadjuvant chemoradiotherapy. Cancer Med 2019;8:1508-20.
Selma Şengiz Erhan,
Istanbul Prof. Dr. Cemil Taşcıoğlu City Hospital, Department of Pathology, Kaptanpasa Mah. Darulaceze Cad. No: 27 Okmeydani-Sisli/Istanbul
Source of Support: None, Conflict of Interest: None
[Table 1], [Table 2], [Table 3]
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