Abstract
Background
To explore the impact of using nano-carbon suspension for lymph node tracing on the number of detected lymph nodes and short-term clinical outcomes in patients undergoing laparoscopic radical resection for rectal cancer following neoadjuvant therapy.
Methods
This study retrospectively analyzed clinical data from 109 patients who underwent neoadjuvant therapy and laparoscopic radical resection for rectal cancer at Weifang People's Hospital from January 2020 to December 2022. Of these, 43 patients received an endoscopic submucosal injection of nano-carbon suspension (experimental group), with 22 patients receiving the injection before neoadjuvant therapy and 21 patients receiving it 24 h before surgery. The remaining 66 patients did not receive the nano-carbon injection (control group). All patients received neoadjuvant therapy according to guidelines and were operated on by the same surgical team. By comparing the number of detected lymph nodes and short-term clinical outcomes among the three groups, the study aimed to investigate the impact of the endoscopic submucosal injection of nano-carbon and the timing of injection on the surgical quality for patients with rectal cancer undergoing neoadjuvant therapy.
Results
The number of detected lymph nodes in the groups injected with nano-carbon before neoadjuvant therapy and 24 h before surgery was significantly higher than that in the non-injected group (P = 0.000), with a significant increase in the proportion of detecting ≥ 12 lymph nodes (P = 0.016), showing statistical significance. There is no statistically significant difference in the number of detected lymph nodes between the group injected with nano-carbon before neoadjuvant therapy and the group injected 24 h before surgery (P = 0.141).
Conclusions
Endoscopic submucosal injection of nano-carbon suspension for lymph node tracing can increase the number of detected lymph nodes in rectal cancer surgery following neoadjuvant therapy, enabling more precise postoperative tumor staging. Although pre-neoadjuvant nano-carbon injection yielded a numerically higher lymph-node detection rate, this trend was not statistically significant (P = 0.141). Consequently, the optimal injection timing remains unconfirmed and should be validated in larger prospective studies.
Keywords: Rectal cancer, Neoadjuvant therapy, Nano-carbon, Lymph node dissection
Background
Colorectal cancer is one of the most prevalent malignancies globally, ranking third in incidence and second in mortality among all cancers [1]. Due to the rectum's unique anatomical position and the absence of obvious clinical symptoms, most patients are diagnosed at an advanced stage. The standard treatment for locally advanced rectal cancer is neoadjuvant chemoradiotherapy followed by total mesorectal excision [2]. Studies have demonstrated that neoadjuvant therapy, compared to adjuvant therapy after surgery, can enhance the rate of intraoperative tumor resection and the preservation of the anal sphincter, while also improving local tumor control and reducing adverse effects [3–5].
Lymph node dissection is a crucial component of radical surgery for rectal cancer. Standardized lymph node dissection and obtaining an adequate number of lymph nodes are vital for effective tumor eradication. Insufficient lymph node retrieval not only leads to inaccurate staging but also reduces postoperative survival [6]. After neoadjuvant therapy, regional lymph nodes undergo regressive changes, increasing the difficulty of detection [7]. Radiotherapy can cause lymphocyte depletion within the lymph nodes, along with interstitial proliferation and fibrosis, resulting in lymph node shrinkage. This makes distinguishing lymph nodes morphologically challenging, thereby complicating their detection through visual inspection and sampling [8]. Several international studies have shown that under the same surgical and pathological examination standards, neoadjuvant chemoradiotherapy significantly reduces the number of lymph nodes retrieved [9–11]. One study even found that up to 72% of patients had fewer than 12 lymph nodes retrieved in their surgical specimens, and 23% had fewer than 5 lymph nodes retrieved [8].
The use of effective lymph node tracers during surgery can increase the number of lymph nodes dissected and the detection of micrometastases. In laparoscopic surgery, effective lymph node tracers can compensate for the inability to palpate lymph nodes, further highlighting regional lymphatic tissue and avoiding damage to surrounding structures. However, for patients undergoing neoadjuvant therapy, chemoradiotherapy may cause fibrosis in lymphatic vessels and lymph nodes, potentially affecting the uptake of nano-carbon by lymph nodes [12]. Currently, most studies inject nano-carbon into the submucosa 24 h before surgery, which may result in some regional lymph nodes not being stained. Therefore, our center has conducted a study on the impact of nano-carbon submucosal injection on radical surgery for rectal cancer post-neoadjuvant therapy. This study aims to explore the effects of nano-carbon submucosal injection and its timing on the number of lymph nodes retrieved and postoperative complications.
Patients and methods
General data
A retrospective analysis was conducted on the clinical and pathological data of 109 rectal cancer patients who underwent surgical treatment at the Department of Colorectal and Anal Surgery, Weifang People's Hospital, from January 2020 to December 2022. All patients underwent laparoscopic radical resection for rectal cancer following neoadjuvant therapy. Any patient scheduled to receive neoadjuvant therapy was routinely recommended to undergo submucosal nano-carbon injection; final assignment to the “injection” versus “non-injection” group depended solely on patient preference after informed consent. Among those who accepted injection, the surgical statistician prepared a pre-generated random-number table (odd numbers: pre-neoadjuvant injection; even numbers: pre-operative injection). Patients were enrolled in strict chronological order, with the next unused number on the list determining group assignment. Among the patients, 43 received a nano-carbon injection; 22 of these patients received an endoscopic submucosal injection of nano-carbon before neoadjuvant therapy, and 21 received a nano-carbon injection 24 h before surgery. The remaining 66 patients did not receive a nano-carbon submucosal injection for lymph node tracing. The inclusion and exclusion criteria were as follows:
Inclusion criteria:
Preoperative pathological diagnosis of adenocarcinoma
Preoperative pelvic MRI or CT assessment indicating tumor T stage as T3 or T4, or suggesting lymph node metastasis
No evidence of distant metastasis, such as in the lungs or liver, as determined by chest and abdominal CT; radical surgery is feasible
Exclusion criteria:
Distant metastasis to the liver, lungs, or other organs during neoadjuvant therapy
Symptoms such as intestinal obstruction or bleeding during neoadjuvant therapy requiring emergency surgery
Patients unable to tolerate chemoradiotherapy and who did not complete the full course of neoadjuvant therapy
Patients with severe underlying diseases who could not tolerate surgery
Patients considered to have complete clinical response (cCR) after neoadjuvant therapy and requested watch-and-wait (W&W) management
Patients with other malignant tumors or a history of malignant tumors
This study obtained approval from the Ethics Committee of Weifang people’s Hospital, and due to its retrospective nature, the need for obtaining written informed consent was waived.
Methods
We confirmed that all methods were carried out in accordance with relevant guidelines and regulations. Patients undergoing lymph node tracing received an endoscopic submucosal injection of nano-carbon suspension under intravenous general anesthesia either before neoadjuvant therapy or 24 h before surgery. This procedure was performed by experienced gastroenterologists.
The injection sites varied depending on the location of the tumor. Typically, nano-carbon was injected submucosally at three sites on both the posterior and lateral walls of the rectum. The anterior rectal wall was not used as an injection site. The injection sites were approximately 0.5 cm away from the tumor, starting with the intestinal wall at the same plane as the tumor, followed by the proximal end of the tumor. For tumors that could not be accessed by colonoscopy, the distal end was chosen for injection. Prior to the nano-carbon injection, normal saline was injected into the submucosal layer to create a transparent submucosal bulge. Subsequently, the nano-carbon suspension was drawn and injected at each site. This is shown in Fig. 1.
Fig. 1.
Endoscopically injected carbon nanosuspension submucosa
All patients received preoperative neoadjuvant therapy according to the following regimen: a total radiotherapy dose of 45–50 Gy, administered at 1.8–2.0 Gy per fraction, five fractions per week, completed within five weeks; concurrent chemotherapy with capecitabine at a dose of 1250 mg/m2, taken orally twice daily. Preoperative assessment was performed eight weeks after the completion of neoadjuvant radiotherapy, followed by laparoscopic radical resection for rectal cancer. All surgeries adhered to the principles of TME (total mesorectal excision) and mesorectal dissection. As shown in Fig. 2, lymph nodes in non-surgical areas without suspected metastases were not routinely dissected. For the inferior mesenteric artery (IMA), the surgeon decided whether root-level resection was necessary on a case-by-case basis, weighing factors such as diabetes, vascular calcification, advanced age, sigmoid-colon length, and mesenteric length. Whenever feasible without adding operative complexity or prolonging the procedure, the left colic artery was preserved in principle. Post-surgery, the specimens were sent to the pathology department, where pathologists retrieved the lymph nodes and processed the tumor specimens.
Fig. 2.
Lymph nodes in non-operative areas without suspected metastasis were not routinely dissected
Observation indicators
The primary observation indicators included the number of lymph nodes retrieved and the proportion of patients from whom ≥ 12 lymph nodes were retrieved.
The secondary indicators recorded were operative time, blood loss, tumor regression grade, postoperative complication rate, postoperative length of stay, and hospitalization costs.
Statistical analysis
Data were analyzed using SPSS 24.0 statistical software. Categorical data are presented as frequencies (n) and were compared using the χ2 test, the corrected χ2 test, or Fisher's exact test. For continuous variables, the normality is tested using the Shapiro–Wilk test. Normally distributed continuous data are presented as mean ± standard deviation (x̄ ± s) and were compared using one-way analysis of variance (ANOVA) or the Brown-Forsythe/Welch test. Non-normally distributed continuous data are presented as median (interquartile range, IQR) and were compared using the Kruskal–Wallis test (a non-parametric test for independent samples). Using the number of harvested lymph nodes as the dependent variable, univariate analysis was conducted on the relevant clinical and pathological data. Indicators with a P-value less than 0.1 were selected for multivariate analysis using a generalized linear model. A P-value < 0.05 was considered statistically significant.
Results
Clinical and pathological data
Based on the inclusion and exclusion criteria, a total of 109 patients were included in this study, with 66 patients in the Non-injection group, 22 patients in the Pre-neoadjuvant injection group, and 21 patients in the Preoperative injection group. The general clinical and pathological data for these three groups are presented in Table 1. No significant differences were observed in gender (P = 0.563), age (P = 0.578), BMI (P = 0.791), comorbidity (P = 0.755), pre-treatment cT stage (P = 0.255), pre-treatment cN stage (P = 0.108), tumor distance from anal verge (P = 0.533), surgical approach (P = 0.075), procedure (P = 0.741), inferior mesenteric artery ligation level (P = 0.547) or ASA classification (P = 0.551).
Table 1.
Clinical and pathological data
| Non-injection group n = 66 | Pre-neoadjuvant injection group n = 22 | Preoperative injection group n = 21 | P value | |
|---|---|---|---|---|
| Gende, n(%) | 0.563* | |||
| Male | 43 (65.2%) | 16 (72.7%) | 12 (57.1%) | |
| Female | 23 (34.8%) | 6 (27.3%) | 9 (42.9%) | |
| Age (year),M(IQR) | 61.5 (16) | 62.0 (20) | 58.0 (15) | 0.578** |
| BMI(kg/m2),M(IQR) | 23.10 (3.20) | 22.73 (6.23) | 23.44 (4.14) | 0.791** |
| Comorbidity, n(%) | 0.755* | |||
| Yes | 23 (34.8%) | 9 (40.9%) | 9 (42.9%) | |
| No | 43 (65.2%) | 13 (59.1%) | 12 (57.1%) | |
| Pre-treatment cT stage, n(%) | 0.255*** | |||
| T2 | 1 (1.5%) | 1 (4.5%) | 1 (4.8%) | |
| T3 | 50 (75.8%) | 12 (54.5%) | 15 (71.4%) | |
| T4 | 15 (22.7%) | 9 (40.9%) | 5 (23.8%) | |
| Pre-treatment cN stage, n(%) | 0.108*** | |||
| N0 | 1 (1.5%) | 1 (4.5%) | 3 (14.3%) | |
| N1 | 25 (37.9%) | 4 (18.2%) | 7 (33.3%) | |
| N2 | 40 (60.6%) | 17 (77.3%) | 11 (52.4%) | |
| Tumor distance from anal verge (cm),M(IQR) | 5.0 (4.0) | 4.0 (4.0) | 5.0 (3.5) | 0.533** |
| Surgical approach,n(%) | 0.075*** | |||
| Laparotomy, | 2 (3.0%) | 0 (0) | 3 (14.3%) | |
| Laparoscopy | 64 (97.0%) | 22 (100%) | 18 (85.7%) | |
| Surgical procedure, n(%) | 0.741*** | |||
| Dixon | 7 (10.6%) | 0 (0) | 1 (4.8%) | |
| Dixon + colostomy | 44 (66.7%) | 16 (72.7%) | 14 (66.7%) | |
| Harrtmann | 1 (1.5%) | 0 (0) | 0 (0) | |
| Miles | 14 (21.2%) | 6 (27.3%) | 6 (28.6%) | |
| inferior mesenteric artery ligation level, n(%) | 0.547* | |||
| high | 32 (48.5%) | 13 (59.1%) | 9 (42.9%) | |
| low | 34 (51.5%) | 9 (40.9%) | 12 (57.1%) | |
| ASA classification, n(%) | 0.551*** | |||
| II | 55 (83.3%) | 20 (90.9%) | 19 (90.5%) | |
| III | 11 (16.7%) | 2 (9.1%) | 2 (9.5%) | |
*χ2 test
** Kruskal–Wallis test
*** Fisher's exact test
Intraoperative data and short-term clinical outcomes
The intraoperative conditions and short-term clinical outcomes for patients in each group are shown in Table 2. No significant differences were observed in pT stage (P = 0.527), pN stage (P = 0.964), yPTNM stage (P = 0.752), tumor differentiation (P = 0.953), or tumor regression grade (P = 0.876) among the groups. The number of dissected lymph nodes (P = 0.000) and the proportion of patients with ≥ 12 lymph nodes (P = 0.016) in the pre-neoadjuvant injection group and the preoperative 24-h injection group were significantly higher than those in the non-injection group. There is no statistically significant difference in the number of detected lymph nodes between the group injected with nano-carbon before neoadjuvant therapy and the group injected 24 h before surgery (P = 0.141). However, there were no significant differences in tumor size (P = 0.135), distal margin (P = 0.721), surgery duration (P = 0.776), blood loss (P = 0.637), postoperative hospital stay (P = 0.076), hospital costs (P = 0.170), or overall complication rates (P = 0.623) among the groups. Specifically, intra-abdominal infection (P = 0.844), anastomotic leakage (P = 0.458), urinary tract infection (P = 1.000), and hemorrhage rates (P = 0.636) did not differ significantly between the groups. Moreover, no induration or ecchymosis was observed at the nano-carbon injection site during surgery, and there was no additional bleeding attributable to the tracer. Nano-carbon injection did not affect tumor-related indicators such as tumor staging and tumor regression grade after chemoradiotherapy, nor did it increase postoperative complications.
Table 2.
Intraoperative data and short-term clinical outcomes
| Non-injection group n = 66 | Pre-neoadjuvant injection group n = 22 | Preoperative injection group n = 21 | P value | |
|---|---|---|---|---|
| pT stage, n(%) | 0.527*** | |||
| T0 | 8 (12.1%) | 2 (9.1%) | 2 (9.5%) | |
| T1 | 3 (4.5%) | 0 (0) | 2 (9.5%) | |
| T2 | 15 (22.7%) | 5 (22.7%) | 1 (4.8%) | |
| T3 | 38 (57.6%) | 15 (68.2%) | 15 (71.4%) | |
| T4 | 2 (3.0%) | 0 (0) | 1 (4.8) | |
| pN stage, n(%) | 0.964*** | |||
| N0 | 43 (65.2%) | 16 (72.7%) | 14 (66.7%) | |
| N1 | 18 (27.3%) | 5 (22.7%) | 5 (23.8%) | |
| N2 | 5 (7.6%) | 1 (4.5%) | 2 (9.5%) | |
| yPTNM stage, n(%) | 0.752*** | |||
| 0 | 7 (10.6%) | 2 (9.1%) | 2 (9.5%) | |
| I | 16 (24.2%) | 3 (13.6%) | 3 (14.3%) | |
| II | 20 (30.3%) | 11 (50.0%) | 9 (42.9%) | |
| III | 23 (34.8%) | 6 (27.3%) | 7 (33.3%) | |
| Differentiation, n(%) | 0.953*** | |||
| Low | 12 (18.2%) | 3 (13.6%) | 3 (14.3%) | |
| Middle | 38 (57.6%) | 15 (68.2%) | 13 (61.9%) | |
| High | 0 (0) | 0 (0) | 0 (0) | |
| Unknown | 16 (24.2%) | 4 (18.2%) | 5 (23.8%) | |
| Tumor regression grade, n(%) | 0.876*** | |||
| 0 | 7 (10.6%) | 2 (9.1%) | 2 (9.5%) | |
| 1 | 22 (33.3%) | 7 (31.8%) | 5 (23.8%) | |
| 2 | 29 (43.9%) | 11 (50.0%) | 9 (42.9%) | |
| 3 | 8 (12.1%) | 2 (9.1%) | 5 (23.8%) | |
| Tumor size (cm),M(IQR) | 2.0 (2.0) | 3.0 (1.9) | 2.5 (2.0) | 0.135** |
| Distal margin (cm),M(IQR) | 1.5 (1.6) | 1.5 (2.1) | 1.5 (1.9) | 0.721** |
| Total lymph nodes harvested,M(IQR) | 14.0 (5.0) | 20.0 (7.0) a | 16.0 (5.0) a | 0.000** |
| ≥ 12 lymph nodes harvested, n(%) | 0.016*** | |||
| Yes | 55 (83.3%) | 22 (100%) | 21 (100%) | |
| No | 11 (16.7%) | 0 (0) | 0 (0) | |
| Surgery duration (min),M(IQR) | 221.0 (79.0) | 230.5 (115.0) | 225.0 (117.0) | 0.776** |
| Blood loss (ml),M(IQR) | 100.0 (103) | 100.0 (113) | 100.0 (150) | 0.637** |
| Postoperative hospital stay (day),M(IQR) | 11.0 (5) | 8.5 (4) | 10.0 (3) | 0.076** |
| Hospital costs(RMB),M(IQR) | 61,311.29 (12,116.71) | 55,391.29 (14,543.13) | 62,335.23 (9603.36) | 0.170** |
| Complication, n(%) | 0.623*** | |||
| Yes | 16 (24.2%) | 6 (27.3%) | 3 (14.3%) | |
| No | 50 (75.8%) | 16 (72.7%) | 18 (85.7%) | |
| Intra-abdominal infection, n(%) | 8 (12.1%) | 2 (9.1%) | 3 (14.3%) | 0.844*** |
| Anastomotic leakage, n(%) | 6 (9.1%) | 2 (9.1%) | 0 (0) | 0.458*** |
| Urinary tract infection, n(%) | 2 (3.0%) | 1 (4.5%) | 0 (0) | 1.000*** |
| Hemorrhage, n(%) | 1 (1.5%) | 1 (4.5%) | 0 (0) | 0.636*** |
** Kruskal–Wallis test
*** Fisher's exact test
"a"indicates a statistically significant difference compared to the non-injected group. All comparisons were corrected using the Bonferroni method
Univariate and multivariate analysis of lymph node harvested
Table 3 presents the univariate analysis of factors influencing the total number of lymph nodes harvested. The group factor showed a significant difference, with the Pre-neoadjuvant injection group having the highest median lymph node count (20.0) compared to the Non-lymph node injection group (14.0) and Preoperative injection group (16.0) (P = 0.000). Additionally, tumor size was found to be significantly associated with the total number of lymph nodes harvested (P = 0.049), with tumors ≤ 2 cm having a lower median lymph node count than those > 2 cm. Other variables such as gender (P = 0.637), age (P = 0.656), BMI (P = 0.875), comorbidity (P = 0.751), pre-treatment cT stage (P = 0.687), pre-treatment cN stage (P = 0.243), tumor distance from anal verge (P = 0.342), surgical approach (P = 0.744), surgical procedure (P = 0.455), inferior mesenteric artery ligation level (P = 0.195), ASA classification (P = 0.656), pT stage (P = 0.451), pN stage (P = 0.826), ypTNM stage (P = 0.514), differentiation (P = 0.266), tumor regression grade (P = 0.863), distal margin (P = 0.064), surgery duration (P = 0.752), and blood loss (P = 0.337) did not show significant associations with the total number of lymph nodes harvested.
Table 3.
Univariate analysis of lymph node harvested
| Variable | N(%) | Total lymph nodes harvested, M (IQR) | P value |
|---|---|---|---|
| Group | 0.000** | ||
| Non-injection | 66(60.6%) | 14.0(5) | |
| Pre-neoadjuvant Injection | 22(20.2%) | 20.0(7) | |
| Preoperative injection | 21(19.3%) | 16.0(5) | |
| Gender | 0.637# | ||
| Female | 38(34.9%) | 15.5(6) | |
| Male | 71(65.1%) | 15.0(6) | |
| Age | 0.656# | ||
| ≤ 50 | 28(25.7%) | 16.0(7) | |
| > 50 | 81(74.3%) | 15.0(7) | |
| BMI | 0.875** | ||
| < 18.5 kg/m2 | 2(1.8%) | 18.0(-) | |
| 18.5–24.9 kg/m2 | 81(74.3%) | 15.0(7) | |
| 25.0–29.9 kg/m2 | 23(21.1%) | 15.0(4) | |
| ≥ 30 kg/m2 | 3(2.8%) | 18.0(-) | |
| Comorbidity | 0.751# | ||
| No | 68(62.4%) | 15.0(6) | |
| Yes | 41(37.6%) | 16.0(7) | |
| Pre-treatment cT stage | 0.687** | ||
| T2 | 3(2.8%) | 14.0(-) | |
| T3 | 77(70.6%) | 15.0(6) | |
| T4 | 29(26.6%) | 15.0(7) | |
| Pre-treatment cN stage | 0.243** | ||
| N0 | 5(4.6%) | 16.0(9) | |
| N1 | 36(33.0%) | 14.0(7) | |
| N2 | 68(62.4%) | 15.5(6) | |
| Tumor distance from anal verge | 0.342# | ||
| ≤ 5 cm | 74(67.9%) | 15.0(5) | |
| > 5 cm | 35(32.1%) | 18.0(8) | |
| Surgical approach | 0.744# | ||
| Laparoscopy | 104(95.4%) | 15.0(6) | |
| Laparotomy | 5(4.6%) | 14.0(17) | |
| Surgical procedure | 0.455** | ||
| Dixon | 8(7.3%) | 15.0(7) | |
| Dixon + colostomy | 74(67.9%) | 15.5(7) | |
| Harrtmann | 1(0.9%) | - | |
| Miles | 26(23.9%) | 15.0(5) | |
| inferior mesenteric artery ligation level | 0.195# | ||
| high | 54(49.5%) | 15.0(5) | |
| low | 55(50.5%) | 18.0(7) | |
| ASA classification | 0.656# | ||
| II | 94(86.2%) | 15.0(7) | |
| III | 15(13.8%) | 14.0(5) | |
| pT stage | 0.451** | ||
| T0 | 12(11.0%) | 15.5(9) | |
| T1 | 5(4.6%) | 14.0(5) | |
| T2 | 21(19.3%) | 15.0(8) | |
| T3 | 68(62.4%) | 15.0(6) | |
| T4 | 3(2.8%) | 19.0(-) | |
| pN stage | 0.826** | ||
| N0 | 73(67.0%) | 15.0(7) | |
| N1 | 28(25.7%) | 15.0(6) | |
| N2 | 8(7.3%) | 15.0(6) | |
| yp TNM stage | 0.514** | ||
| 0 | 11(10.1%) | 14.0(9) | |
| I | 22(20.2%) | 14.5(6) | |
| II | 40(36.7%) | 16.0(7) | |
| III | 36(33.0%) | 15.0(6) | |
| Differentiation | 0.266** | ||
| Low | 18(16.5%) | 14.0(2) | |
| Middle | 66(60.6%) | 16.0(6) | |
| Unknown | 25(22.9%) | 17.0(8) | |
| Tumor regression grade | 0.863** | ||
| 0 | 11(10.1%) | 14.0(9) | |
| 1 | 34(31.2%) | 15.5(9) | |
| 2 | 49(45.0%) | 15.0(6) | |
| 3 | 15(13.8%) | 15.0(6) | |
| Tumor size | 0.049# | ||
| ≤ 2 cm | 50(45.9%) | 14.0(6) | |
| > 2 cm | 59(54.1%) | 17.0(7) | |
| Distal margin | 0.064# | ||
| ≤ 1.5 cm | 63(57.8%) | 15.0(7) | |
| > 1.5 cm | 46(42.2%) | 17.0(5) | |
| Surgery duration | 0.752# | ||
| ≤ 225 min | 55(50.5%) | 15.0(6) | |
| > 225 min | 54(49.5%) | 16.0(7) | |
| Blood loss | 0.337# | ||
| < 100 ml | 36(33.0%) | 15.5(5) | |
| ≥ 100 ml | 73(67.0%) | 15.0(7) | |
**Kruskal–Wallis test
# Mann–Whitney U test
Table 4 presents the multivariate analysis of factors influencing the total number of lymph nodes harvested. The group factor showed significant differences, with the Non-injection group having a coefficient of −2.913 (P = 0.013) and the Pre-neoadjuvant injection group having a coefficient of 3.837 (P = 0.007), indicating a lower and higher number of lymph nodes harvested compared to the Preoperative injection group, respectively. Other variables such as tumor size (≤ 2 cm) (P = 0.452) and distal margin (≤ 1.5 cm) (P = 0.052) were not found to be significant predictors in the multivariate model.
Table 4.
Multivariate analysis of lymph node harvested
| Variable | Coefficient | Standard Error | 95% confidence interval | P value | |
|---|---|---|---|---|---|
| Lower Limit | Upper Limit | ||||
| Intercept | 18.651 | 1.1618 | 16.374 | 20.929 | 0.000 |
| Group | |||||
| Non-injection | −2.913 | 1.1687 | −5.203 | −0.622 | 0.013 |
| Pre-neoadjuvant injection | 3.837 | 1.4123 | 1.069 | 6.605 | 0.007 |
| Tumor size | |||||
| ≤ 2 cm | −0.681 | 0.9046 | −2.454 | 1.092 | 0.452 |
| Distal margin | |||||
| ≤ 1.5 cm | −1.749 | 0.8997 | −3.512 | 0.015 | 0.052 |
| Scale | 21.390 | 2.8974 | 16.402 | 27.894 | |
Discussion
Neoadjuvant chemoradiotherapy combined with total mesorectal excision is widely recognized as the standard treatment for locally advanced rectal cancer due to its ability to reduce local tumor recurrence [2]. However, while neoadjuvant chemoradiotherapy is effective at killing tumor cells, it also leads to lymphocyte depletion in lymph nodes and causes stromal proliferation and fibrosis within them. This results in the shrinkage of lymph nodes, complicating their detection and reducing the number of detectable lymph nodes [8]. Nano-carbon has been utilized as a tumor localization marker and lymph node tracer in colorectal cancer surgeries that do not involve chemoradiotherapy, and studies have confirmed its efficacy in increasing the number of detectable lymph nodes [13, 14]. Nevertheless, there is a scarcity of research on its application in patients undergoing neoadjuvant therapy for rectal cancer. Research indicates that injecting nano-carbon suspension 24 h before surgery in patients receiving neoadjuvant therapy can enhance both the number of detectable lymph nodes and the proportion of patients with ≥ 12 detectable lymph nodes, aligning with our study results [15–17]. At present, there is a lack of studies comparing the effect of injection before neoadjuvant treatment and 24 h before surgery of nano-carbon suspension. Our study found that the number of lymph nodes detected after nano-carbon injection before neoadjuvant therapy, and 24 h before surgery, was significantly higher than in the group that did not receive nano-carbon injection. However, there is no statistically significant difference in the number of detected lymph nodes between the group injected with nano-carbon before neoadjuvant therapy and the group injected 24 h before surgery (20 vs.16, P = 0.141). Nevertheless, while we observed a trend towards higher lymph node detection in the pre-neoadjuvant injection group, we acknowledge that this finding is not definitive and requires further investigation with a larger sample size. Multivariate analysis confirmed that nano-carbon injection independently enhanced lymph node detection, with the pre-neoadjuvant injection group showing the highest lymph node harvested (coefficient = 3.837, P = 0.007). Tumor size (≤ 2 cm) was marginally significant in univariate analysis (P = 0.049) but not in multivariate models (P = 0.452), suggesting its association might be confounded by injection strategies rather than reflecting independent biological behavior.
A separate study on gastric cancer patients who underwent surgery after neoadjuvant therapy showed that lymph node detection was higher when tracing was performed before neoadjuvant therapy than 24 h before surgery [18]. Therefore, regarding the timing of nano-carbon injection, we prefer injection before neoadjuvant therapy. First, neoadjuvant therapy can cause lymphatic vessel fibrosis, obstructing normal lymphatic drainage. If nano-carbon is injected before surgery, the obstructed lymph nodes may not be adequately stained, increasing the risk of missed lymph nodes, particularly in patients with suspected lymph node metastasis prior to surgery. Second, injecting nano-carbon before neoadjuvant therapy can provide sufficient time for dispersion, allowing the lymph nodes in the drainage area to be stained as much as possible, which may help improve the accuracy of lymph node detection. Additionally, our study lacked pathological confirmation, such as data on the staining rate of lymph nodes and evidence of lymphatic vessel obstruction, which are critical for fully evaluating the effectiveness of the injection timing. Further studies with larger sample sizes, more detailed analyses, and comprehensive pathological assessments are needed to confirm the optimal timing of nano-carbon injection.
One of the problems with nano-carbon injection is the staining of the surrounding tissues and organs of the rectum. To minimize the adverse effects of nano-carbon on the surrounding tissues and organs of the rectum caused by staining, we strive to optimize the injection site and injection method in addition to having the procedure performed by experienced and professional endoscopists. Injecting saline into the submucosal layer separates the mucosa from the muscular layer, forming a natural cavity that completely encloses the nano-carbon suspension between the intestinal walls. This approach helps to avoid the injection needle from penetrating the intestinal wall to the greatest extent, thereby reducing the staining of surrounding tissues. This is shown in Fig. 3. Furthermore, due to the relatively weak mesorectum of the anterior rectal wall, the likelihood of nano-carbon staining surrounding organs (such as the prostate and vaginal wall) is significantly increased, which may complicate the dissection of the anterior rectal wall and increase surgical risk. Therefore, we chose the injection sites on the posterior and lateral walls of the rectum, where the mesorectum is relatively thick. Even if nano-carbon leaks, it will be contained within the mesorectum, minimizing the impact on the surgical plane and enabling better completion of total mesorectal excision. Staining near the proximal end of the tumor can further minimize the impact of nano-carbon on the assessment of the safe surgical margin for tumor resection. In our study, no induration or ecchymosis was observed at the nano-carbon injection site during surgery; the dissection planes remained clear, and there was no additional bleeding attributable to the tracer. The distal margin was determined primarily by intra-operative digital rectal examination; nano-carbon injection did not compromise the ability to achieve an adequate distal margin. Median distal margin length in the Preoperative injection group was comparable to that in the other groups (P = 0.721); all distal and circumferential margins were tumor-free.
Fig. 3.
The nano-carbon was wrapped in the mesorectum and did not affect the judgment of tissue space
This study has its limitations. First, the study lacks data on the actual rate of nano-carbon uptake within individual lymph nodes. Because our pathology workflow does not routinely record the staining status of each node, we cannot verify that all retrieved nodes contained nano-carbon or exclude the possibility that some unstained nodes were overlooked. This limitation means that the observed increase in nodal counts could, in part, reflect improved surgical technique rather than tracer efficacy alone, and it tempers the strength of any causal claims about nano-carbon’s impact. To address this limitation, future prospective trials should systematically document and confirm the staining status of every harvested node, thereby providing direct evidence of tracer efficacy. Second, While the present study demonstrates that nano-carbon can enhance nodal harvest after neoadjuvant therapy, we did not collect recurrence or survival data. Consequently, we cannot conclude that higher nodal counts translate into better disease-free or overall survival. Additionally, this retrospective study had a relatively small sample size, which may affect the accuracy of the results. Therefore, the findings need to be validated in a well-designed, large-scale randomized controlled trial with adequate follow-up to determine whether the observed increase in nodal retrieval ultimately improves long-term oncological outcomes.
Conclusion
Endoscopic submucosal injection of nano-carbon suspension for lymph node tracing can increase the number of lymph nodes detected during rectal cancer surgery after neoadjuvant therapy, thereby allowing for more accurate postoperative tumor staging. Although pre-neoadjuvant nano-carbon injection yielded a numerically higher lymph-node detection rate, this trend was not statistically significant (P = 0.141), and therefore requires further validation through larger studies. Future research should focus on optimizing the timing of nano-carbon injection and exploring its long-term impact on patient outcomes.
Author contribution
Shuai Shen: Research design, data collection and analysis, and drafting the initial manuscript Hui Gao: Research design, data collection and analysis, and drafting the initial manuscript Zhongzheng Cao: Assisted in research design, data collection and analysis, and manuscript revision Wenlong Xia: Assisted in data collection and analysis, manuscript revision Chengren Li: Research design and guidance, manuscript review and revision.
Funding
There was no funding for this study.
Data availability
No datasets were generated or analysed during the current study.
Declarations
Competing interests
The authors declare no competing interests.
Footnotes
Publisher's Note
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
No datasets were generated or analysed during the current study.