Application of Carbon Nanoparticles in Tracing Lymph Nodes and Locating Tumors in Colorectal Cancer: A Concise Review

Introduction

Colorectal cancer is one of the most common malignancies across all genders.¹ Headway in the treatment strategies, including improved surgical techniques, evolvements in chemotherapy, advances in radiotherapy, and the development of targeted therapies, are tremendously valuable in survival gains for these patients in recent decades.² However, complete surgical resection remains the cornerstone of curative treatment to manage resectable tumors.³ Lymphadenectomy, an important part of radical surgery, is one of the key factors influencing the prognosis, not only to eliminate possible lymphatic metastasis of cancer cells,^4,5 but also to assist in accurate lymph node (LN) staging.

Therefore, accurate evaluation of metastatic LNs constitutes an essential role in predicting the prognosis of CRC patients receiving radical resection.⁶ Inadequate retrieval of LNs would predispose the patients at risk of understaging the disease, causing lifesaving adjuvant treatment to be delayed. Multiple reports have illustrated that inadequate lymph node evaluation is positively correlated with shorter overall survival in CRC patients with or without metastatic LNs.^7–10 Adequate lymph nodes are not only critical for accurate LN staging, but also have significance in choosing an adjuvant treatment program after radical resection as well as in predicting the prognosis after disease management.^11–13 Even though different requirement of the minimum number of LNs harvested have been reported in previous studies, pathologists still should try their best to obtain as many LNs as they can.¹² Lymph nodes, however, were often not harvested sufficiently in many patients diagnosed with CRC.¹⁴ Therefore, tools or techniques reserved for harvesting adequate LNs and accurate LN staging were urgently demanded.

The rapidly emerging research of nanotechnology provides exciting new possibilities for developing more and more new nanomaterials for practical use.^15,16 CNPs, one of the most representative nanomaterials, has been widely used for drug and gene delivery,^17,18 biosensors,¹⁹ molecular imaging age,²⁰ and LN tracing in various surgeries.^21–23 CNPs can be absorbed selectively by lymphatics and stain the LNs black after being injected into the submucosal layer around the tumor, and the CNPs would not permeate into the blood capillaries due to different permeability between lymph and blood systems,²⁴ where CNPs are too large to enter blood circulation, thus leading to few toxic side effects.²⁵ Therefore, this method has been widely approved by some countries in clinical application.

Several clinical studies have demonstrated CNPs could be helpful in accurately staging LNs when performing lymphadenectomy,^23,26–36 resulting in reduced operation time^28,36 as well as less intraoperative blood loss.^24,28,36 Nevertheless, some contradictory studies, which showed that CNPs did not have much advantage either in retrieving LNs or locating tumors, were still reported.^24,37 Therefore, a systematic review was performed to comprehensively evaluate the safety and effectiveness of the CNPs in assisting in tracing of LNs and localization of CRC from currently available data.

Materials and Methods

This study systematically reviewed the current literature which had evaluated the safety and effectiveness of CNPs suspension in tracking LNs and tumor location of CRC. With respect to LN retrieval, total and mean number of LNs, percentages of metastatic lymph nodes and those patients with metastatic LNs, percentages of small LNs and those with insufficient dissected LNs (<12) were evaluated. Regarding tumor location, time to locate the primary tumor, total operation time and volume of bleeding were all evaluated. Postoperative complications and other outcomes related to tumor and LNs resection were assessed as well, including time to harvest LNs and percentages of stained LNs, metastatic LNs and small LNs. This systematic review was performed according to the Cochrane Handbook for Systematic Reviews of Intervention.³⁸ The protocol for this systematic review is available in PROSPERO (CRD42020192248).

Search Strategy

A search strategy was performed, including the terms below: (carbon nanoparticles OR activated carbon nanoparticles) AND (colon cancer OR rectal cancer OR colorectal cancer). Electronic databases PubMed, EMBASE, Cochrane and Ovid MEDLINE were searched from inception to May 30, 2020. A bibliographic review was performed to ensure capture of all relevant articles. There were no restrictions on study design, year, language, or country published.

Eligibility Criteria

The following criteria were used to select studies: (1) full paper or conference abstract available; (2) studies that compared carbon nanoparticles with control groups in locating tumors and (or) tracing LNs in colorectal cancer; (3) studies sorted as RCT, cohort study, or retrospective study. Studies with duplicate data or data not relevant to CNPs were excluded.

Data Extraction

Two Investigators independently extracted data according to the predefined form, including the following information: author, year published, country, study type, sample size, age, gender, tumor location, nanoparticle size, injection time, injection dose and site, tumor diameter, operation time, time to locate the primary tumor, amount of bleeding, total and mean numbers of the dissected LNs, numbers of harvested metastatic LNs, patients with positive LNs, dissected LNs <12 and percentage of LNs smaller than 5 mm in diameter, dyed LNs, dyed metastatic LNs, dyed small metastatic LNs and rate of postoperation complications.

Results

Studies Retrieved and Characteristics

By searching, 268 potential relevant records were identified. After screening the titles and abstracts,140 records were originally included, and 21 records were subsequently acquired after full texts being read, 14 papers eventually met the inclusion criteria and were included for final analysis (Figure 1). These studies include eight randomized controlled trials, three case–control studies, and three retrospective studies, involving a total of 1618 participants (887 patients in CNPs group and 731 in control groups). The baseline information for the CNPs group and the control group were comparable in these studies (Table 1). All the patients were confirmed pre-operationally by endoscopic biopsy or other methods. In this review, we included all these patients diagnosed as carcinoma of colon or rectum, and tumor location in these studies were listed in Table 1.

Table 1 The Basic Information for the Two Groups in All These Studies

Figure 1 Study selection flowchart.

After analysis, most of the studies in the CNPs group validated that primary tumors and LNs could be tattooed by injecting 0.5–1mL of CNPs suspension into the submucosal layer around the lesions 10 min to seven days before the operation (Table 2). Two studies underwent submucosal injection surrounding the tumor intraoperatively.^34,39 In another study assessing CNPs’ feasibility in advanced CRC, the CNPs detection efficacy was evaluated 8 weeks after chemoradiotherapy (about 14 weeks before surgery).³⁶ Moreover, there was also study indicated that the time of injection had no significantly relationship with the efficacy of the method.²⁸

Table 2 Injection Information

Whether these patients, with or without preoperation adjuvant chemotherapy, should receive laparoscopic or open radical operation was based on the clinical guidelines and surgeons’ preference. No significant differences in tumor diameters, T-stage, degree of differentiation and other baseline characteristics were noted between CNPs and control groups across all the studies.

LN Retrieval

Thirteen studies had reported total and (or) average number of dissected LNs in the CNPs group and the control group, respectively (Table 3). Eleven of 13 studies showed that the total (and /or) mean number of detected LNs per patient was significantly higher in the CNPs group than in the control group,^23,26–36 illuminating the superiority of CNPs in tracing LNs. Seven studies recorded the numbers of harvested metastatic LNs,^{26–28,30–34} wherein two studies found more metastatic LNs in CNPs group, while four studies showed no significant difference between the two groups. Seven studies recorded the patients with metastatic LNs,^{23,26,28,31–34} three studies detected more LN-positive patients in the CNPs group than in the control group, while four studies showed no significant difference. Six studies had recorded and compared the ratio of patients whose detected LNs were less than 12,^{23,26,28,30,32,35} where four of these studies reported a lower ratio of <12 LNs in the CNPs group,^23,26,32,35 while another two studies showed no significant difference.^28,30

Table 3 Information About the Lymph Node Retrieval

The mean number of LNs with their greatest diameters less than 5 mm were also evaluated in six studies,^{26,27,29,30,32,34} all the studies showed that CNPs assisted lymph node retrievals were beneficial to harvest more small LNs (less than 5 mm in diameter). Two studies had calculated the percentages of stained LNs in the total lymph (84.8% and 56.8%) and the stained metastatic LNs in total metastatic LNs (77.6% and 65.9%),^23,27 where one study had reported the stained small LNs (75.9%). Moreover, two studies found the numbers of metastatic LNs with their greatest dimension less than 5 mm were different (31.6% and 63.8%), of note, most of these metastatic small LNs were stained black (89.2% and 75.9%).^23,32

Tumor Location

Four studies had reported the operation time,³⁹ however, only one study delineated CNPs could significantly decrease the operation time³⁶ (Table 4). There were also studies that showed that CNPs could be helpful in rapid detection of lesions^28,36 and harvest lymph nodes^23,31 during surgery, thus could substantially diminishing the time cost for surgeons and pathologists. In addition, five studies had recorded the bleeding volume during the surgery,^{28,33,36,37,39} three of them demonstrated the blood loss during operation was less in the CNPs group than in the control group.^28,36,39 Besides, one study had described a longer distance between the tumor and the circumferential resection margin in the CNPs group.²⁹ One study illustrated the protective ileostomy rates were lower in the CNPs group, however, the differences reached no statistical significance.³⁷

Table 4 Surgical Outcomes

Complications

Among all reported research, no patients in the CNPs group experienced adverse effects by injection of carbon nanoparticle suspension. While six studies documented some surgically relevant complications (Table 5).^{26,28,33,34,37,39} No significant differences in blood loss, postoperative hospital length of stay, rehospitalization, reoperation and motility were found between the two groups. Furthermore, no obvious difference was reported regarding postoperative complications, including anastomotic leakage, anastomotic hemorrhage, ileus, lymphorrhagia, perianal abscess, pneumonia, infection, intestinal obstruction and some other special complications.

Table 5 Information About the Postoperative Complications

Discussion

Theoretically, an optimal size of lymph node tracer should range between 50 and 200 nm, which would be small enough to enter into the lymphatic capillaries (diameter of 150–500 nm) and travel rapidly to the LNs, while simultaneously being large enough to remain in the sentinel nodes long enough for staining (by macrophage phagocytosis).⁴⁰ Carbon nanoparticles, with an average diameter of 150 nm, were the most commonly used agent when tracing lymph nodes and locating tumors in CRC surgery, and these nanoparticles could be accumulated in the LNs long enough for subsequent identification during surgery. Besides, due to the means of administration (injection around tumors), together with their strong affinity to lymphatic systems, CNPs could mainly be deposited in LNs, while not entering into the cardiovascular system, thus, to some extent, exempting the potential toxic effect to cardiovascular, respiratory, and other systems (or organs).^41,42 And this could explain why there were no significant differences in the prevalence of postoperative complications, suggesting CNPs are a safe agent in CRC surgery.

Another critical issue in LN identification is injection dose.⁴³ Low dose of CNPs may be insufficient to dye the LNs, yet an overdose may cause other concerns. Previous clinical experiences have demonstrated that 0.5 or 1 mL (25 or 50 mg) of CNPs suspension, with or without being dissolved in appropriate physiological saline, was sufficient and recommended for identification of LNs. The dose of CNPs varies as application situations change,⁴⁴ and timing of the application differs as well. In most studies, CNPs were injected one day before surgery (listed in Table 2). Some studies evidenced that more LNs were stained black as time passed,⁴³ however, the best time to administrate CNPs needs to be further investigated.

Over past years, several techniques such as Indian ink and indocyanine green (ICG) have been utilized for tattooing tumors and LNs, unfortunately, they all have drawbacks, like inevitable adverse event, short imaging time, and even having certain interference with the visual field of operation.³⁵ Intriguingly, as the development and evolvement of nanotechnology in recent years,^45,46 biological application of nanoparticles has increased rapidly, thus making CNPs suspension used for lymph node tracing feasible.²⁸

In clinic, neoadjuvant chemoradiotherapy could compellingly reduce detected LNs,⁴⁷ a number of other patient- and tumor-related factors also play some roles in LNs yielding and assessment, such as the patient’s gender, age, tumor grade, surgical type, body mass index, and so on.^48–50 Thereby, it would be regarded to have high risk factor if the number of LNs harvested per specimen were fewer than 12, according to the guideline of National Comprehensive Cancer Network (NCCN).^14,51,52 Other studies also claimed that more LNs are needed for staging, decision making, and prognosis predicting.^53,54 Besides, the proportion of the number of involved LNs upon the examined LNs—defined as metastatic LN ratio (mLNR)—has also been considered as a prognostic parameter in CRC, and high mLNR was significantly correlated with worse survival.^55–58 Despite different minimum required numbers of LNs being reported in previous studies, pathologists still should try their best to obtain as many LNs as they can. Unluckily, it is often possible that adequate LNs are not adequately harvested in many patients diagnosed with CRC.⁵⁹ In this review, we found that most of the studies (84.6%) had proven that CNPs can assist in harvesting more LNs than traditional visual and tactile methods. CNPs are significantly beneficial to detect more LNs (especially small LNs) and for accurate LN staging. Strikingly, four of six studies reported a notably lower ratio of patients whose detected LNs were less than 12 in the CNPs group.

A study showed that the maximum diameter (less than or more than 5 mm in diameter) of the positive LNs was an independent indicator of prognosis, while there was no significant difference in overall survival rate and disease-free survival rate between them.⁵⁹ Therefore, increased number of the dissected small LNs (<5mm in diameter) might make some achievements in accurate staging and prognosis prediction. Of note, LNs are easy to be overlooked by traditional methods if their diameters are less than 5 mm, particularly when some of these small LNs are metastatic and tumors are at high risk of understaging. In this review, all six studies included have proved that CNPs had merits in assisting in harvesting significantly more small LNs than the control group,^{26,27,29,30,32,34} suggesting that the preoperative SLN detection with CNPs in CRC patients can evaluate the LN's status, guide the treatment and improve the prognosis.³² Although there is one study that demonstrated no significant differences in survival or recurrence between the two groups after a three-year follow-up,³⁹ more multicenter randomized controlled trails (such as NCT03550001) are required to explore the long-term clinical results.

Accurate tumor localization is of vital importance in CRC management as well, especially for patients with early-stage cancer or having previous resected tumors.⁶⁰ The demand for correctly localizing a lesion has increased as endoscopic operations soar in recent years, however, obstacles in precisely palpating the pathological colon during surgical intervention makes locating the tumor troublesome, increasing the difficulty of resection. Therefore, endoscopically tattooing adjacent tissue of the tumor can promote precise tumor recognition to ensure a more precise, minimally invasive, colorectal resection. Due to these merits, endoscopic tattooing is considered to be the gold standard for tumor localization prior to undertaking minimally invasive surgery.^61,62 More accurate localization may be associated with measurably shorter operation time,^28,36 less blood loss,^28,36,39 more accurate resection,^36,37 simpler method, and greater practicality in the CNPs group compared to the control group.²⁸

Interestingly, some studies illustrated that the overall time expenditure in the CNPs group was not greatly increased, or even reduced, when more LNs needed to be dissected.^31,39 Furthermore, the mean operation time in the CNPs group was also reduced compared to the control group as reported in some studies.^28,36 Thereby, these findings have validated the superiority of CNP staining in examining LNs (Table 6). Meanwhile, some challenges also emerge when using CNPs. Firstly, technical difficulties do exist when CNPs are injected into the submucosal layer around the tumor endoscopically. Given this, saline injection to construct a submucosal uplift prior to administering CNPs might be a feasible solution.³⁵ Secondly, leakage during injection could also perturb the resection of tumors, even causing other undesirable adverse effects. Thirdly, CNPs are a nonspecific lymph node tracer, which is incapable of discriminating the metastatic LNs from negative LNs. Besides, more harvested LNs denote more pathologic work, which would be a heavy burden for pathologists.

Table 6 The Merits of CNPs Compared with Control Group

Some limitations exist in this work. First, the included studies are not all randomized controlled trials, in which cohort studies and retrospective studies were all collectively included; second, most of the studies have very limited samples; third, the patients in this review are those with colon cancers and rectal cancers; fourth, the long-term observations of the clinical effects of CNPs in CRC patients are not confirmed in majority of the studies. However, our review also has some strengths, we have extensively incorporated and analyzed all relevant studies with respect to the CNPs application and their results, which indicate our review is a comprehensive study.

Conclusion

In conclusion, tattooing with CNPs in CRC surgery is safe and beneficial in both tumor localization and LN tracing, which shows the importance of CNPs in reducing the difficulty and operation time in CRC surgery should be emphasized. Therefore, the value of CNPs in surgical treatment of cancer should be recognized and its application might be promoted and expanded in the future. However, the exact merits and mechanisms with respect to prolonging survival are not illuminated and more randomized controlled trials are still needed to clarify and elucidate its underlying mechanism.