Factors affecting false-negative rates on ex vivo sentinel lymph node mapping in colorectal cancer

doi:10.1016/j.ejso.2009.06.007

European Journal of Surgical Oncology (EJSO)

Volume 36, Issue 2, February 2010, Pages 130-134

https://doi.org/10.1016/j.ejso.2009.06.007 Get rights and content

Abstract

Purpose

Despite the increasing use of sentinel lymph node (SLN) mapping after colorectal cancer resection, reported node identification and false-negative rates vary considerably. The main aim of this prospective study was to quantify the false-negative rates on SLN mapping after resection and to evaluate factors influencing them.

Methods

Sixty-nine patients with biopsy-proven cancer of the colon and rectum underwent SLN mapping according to a protocol involving the ex vivo submucosal and peritumoral injection of 2–4 ml of Patent Blue V dye. All lymph nodes visualized were marked as SLN and totally embedded, then two 4 μm sections were cut for hematoxylin and eosin staining, and cytokeratin (AE1/AE3) immunostaining. A standard examination of the whole specimen and of the regional non-sentinel lymph nodes was also performed.

Results

SLNs were identified in 97.3% of the evaluable cases. A mean of 5.0 SLNs were removed per patient (SD ± 4.2). Nine false negatives were identified. Rectal cancer, tumor size > 60 mm, number of metastatic non-sentinel lymph nodes, and mucinous tumors were associated with false-negative SLNs. At multivariate analysis, a rectal location and mucinous differentiation were independently associated with false-negative SLNs.

Conclusions

Ex vivo SLN mapping after colorectal cancer surgery is technically feasible with a high identification rate. Tumor size and stage, rectal involvement and a mucinous histology seem to interfere with the reliability of SLN staging. It is mandatory to standardize the procedure and selection criteria in order to deal with the question of the reliability of SLN mapping in colorectal cancer.

Introduction

Lymph node metastases are the most important predictors of survival in patients with potentially curable colorectal cancer (CRC). Adjuvant chemotherapy is given only to patients with positive nodes, whereas node-negative patients are considered to be cured by surgery.1, 2 Up to 30% of patients with negative nodes will recur, however, in the course of their lives.³ This is at least partially due to the understaging of the regional lymph nodes: patients with metastatic nodal disease that goes unnoticed are not given adjuvant chemotherapy and are at higher risk of recurrence.

There are two possible reasons why regional nodes go understaged:

1.
Not all metastatic nodes are identified due to an inadequate extension of the surgical resection, or some are overlooked during standard pathological dissection; 70% of metastatic lymph nodes are <5 mm in size and may escape from notice because of their small size.⁴ Cawthorn et al.⁵ recommended using a xylene alcohol clearance technique to increase the number of nodes identified, but the procedure is time-consuming and has not been widely adopted;
2.
Some metastatic deposits in harvested nodes are too small to be detected in 1–2 sections stained with hematoxylin and eosin (H&E) at standard pathological analysis. Stepwise sectioning of the nodes and the extensive use of immunohistochemistry and molecular analysis (reverse transcription polymerase chain reaction, RT-PCR) on the lymph nodes may improve the detection threshold for such metastases but, here again, such methods are labor-intensive and expensive, and they cannot be used routinely on all lymph nodes detected.⁶

Sentinel lymph node mapping (SLNM) has been recommended in cases of CRC to help overcome these problems and improve disease staging.7, 8, 9 Unlike the situation in breast cancer and melanoma, where the SLN concept has been used to lower the morbidity of regional lymphadenectomy,10, 11 SLNM for CRC enables the pathologist to focus on the node(s) at greatest risk of spread and examine these nodes with multiple slices and immunohistochemistry, and/or RT-PCR, and thereby detect metastatic tumor cells that would otherwise go unrecognized.

The results achieved with SLNM in CRC vary, however, in terms of the identification, false-negative and upstaging rates12, 13 and several questions remain to be addressed. It is still not clear which patients benefit most from this procedure (case selection), the best timing of the injection, the best tracer, the ultrastaging protocol and the prognostic value of micrometastatic cells detected in SLN.

The aim of the present study was to quantify the detection and false-negative rates achieved by ex vivo sentinel node mapping in CRC, and to assess the factors influencing these rates.

Section snippets

Patient selection

All patients were prospectively selected for SLNM according to a preoperative and intra-operative staging protocol, if they met the following criteria:

1.
Biopsy-confirmed colorectal cancer.
2.
No evidence of metastatic disease at preoperative staging (chest X-ray, liver ultrasound or abdominal CT scan).
3.
No advanced rectal tumor (pelvic MRI).
4.
Elective surgery.
5.
No prior colorectal resection.
6.
No (previously undetected) liver metastases found at surgery.
7.
No peritoneal carcinosis or locally advanced disease

Study population and demographics

From January 2005 to December 2007, 69 patients were enrolled for the study, including 38 males and 31 females, with a mean age of 67.4 (±12.4) years. Patients' demographic and pathological staging details are summarized in Table 1. Four patients had two colon tumors. There were three pT4 tumors with microscopic invasion of the serosal layer not seen during surgery. Neo-adjuvant radiotherapy (45 Gy) and concomitant chemotherapy were given in 3 of the 16 rectal cancer patients. No complete

Discussion

The aim of this study was to establish the sentinel node detection rate and the prognostic factors influencing the false-negative rate in colorectal cancer. Ex vivo SLN identification was done in the vast majority of patients by injecting patent blue dye into the submucosa of the tumor. The procedure was simple and inexpensive, and took only a few minutes for one of the surgeons to complete in the operating room, immediately after resecting the specimen.

The in-depth search on the few SLN

Conflict of interest

The senior author Prof. Giovanni Zaninotto certify that the authors of the submitted article “Factors affecting false-negative rates on ex vivo sentinel lymph node mapping in colorectal cancer” do not have financial or personal relationships with other people, organizations or commercial associations (e.g., consultancies, stock ownership, equity interests, patent-licensing arrangements) that might pose a conflict of interest in connection with the submitted article.

Funding source

The work was supported by a grant from the Regione Veneto, Ricerca Sanitaria Finalizzata, 2007.

Acknowledgments

In memory of Prof. Carlo Tremolada (1944–2006).

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There is considerable evidence that colon cancer patients with micrometastases have a prognosis comparable to that of patients with macrometastases. However, the prognostic value of isolated tumor cells (ITCs) is less clear, with conflicting results in the literature, and is still debated. Moreover, there is a subgroup of patients who undergo curative resections without evidence of nodal disease, who unfortunately succumb to relapse. There is growing evidence that sentinel lymph-node mapping with ultra-staging can lead to up-staging of a significant number of patients and that these patients show significantly lower disease-free survival and overall survival compared to true node-negative patients. However, there is still no consensus on its usefulness in daily clinical practice, as the results of studies are difficult to compare due to the enormous clinical diversity observed in studies, involving patient selection and pathological analysis, among others. This chapter takes a detailed look at the definition of micrometastases and ITCs, and clarifies some controversies. It also reviews the findings in the literature of studies on the impact of micrometastasis and ITCs in colorectal cancer resection specimens. In addition, it summarizes the results of sentinel lymph-node mapping and ultra-staging studies in colon cancer for lymph node-negative colon cancer patients. Finally, it contains recommendations for standardized histopathological analysis of lymph nodes in colorectal cancer resections and a proposal for standardized analysis of sentinel lymph nodes.
Impact of lymph node ratio on survival of colorectal cancer patients
2013, International Journal of Surgery
Citation Excerpt :
Hence, an ideal staging criterion for N status should not be affected by the extent of lymphadenectomy. Node ratio has been proposed as a solution to this problem. 6–17 Firstly, our study confirmed that nodal involvement is among the strongest prognostic factors for colorectal cancer patients: pN and NR significantly stratified patient survival (Fig. 1) and multivariate analysis showed that these indicators were both independent prognostic factors.
This study analyzed the prognostic significance of the staging system based on the metastatic Node Ratio (NR) compared with the TNM staging system in patients with colorectal cancer.
We reviewed the data of 444 patients who underwent colorectal resection for cancer between January 2005 and December 2011. NR categories NR0 (0%), NR1 (1–19%) and NR2 (≥20%) were determined by the best cut-off approach at log-rank test. To compare the prognostic power of the NR versus pN, we plotted these different factors against the mortality estimates. Additionally, we evaluated the relationship between these variables and the extent of lymphadenectomy.
Both the NR and the pN classification significantly stratified patient outcomes (p < 0.0001), but the NR system seems to better discriminate prognostic subgroups than the pN. Furthermore, NR is less dependent on the extent of lymphadenectomy than pN.
NR is a simple and reliable tool to stratify survival of colorectal cancer patients and it seems to have a higher prognostic power than the current pN system, because it is less dependent on the extent of lymphadenectomy.
Ex vivo sentinel lymph node investigation in colorectal cancer
2013, Journal of Coloproctology
In Brazil, about 26,000 cases of colorectal cancer are diagnosed per year. Pa- tients considered at the early stage of disease (without lymph node) evolve with tumor relapse or recurrence in up to a quarter of cases, probably due to understaging.
Research on ex vivo sentinel lymph node in patients with colorectal adenocarcinoma.
We studied 37 patients who underwent curative surgical resection. The marker used to identify lymph nodes was patent blue dye injected into the peritu- moral submucosa of the open surgical specimen immediately after its removal from the abdominal cavity.
Ex vivo identification of sentinel lymph node with marker occurred in 13 (35.1%) patients. The sensitivity was 40% and 60% false negative. The detailed histological examina- tion of sentinel lymph nodes with multilevel section and immunohistochemistry showed metastasis in one (4.3%) individual, considered ultra-staging.
The ex vivo identification of sentinel lymph node had questionable benefits, and worse results when include patients with rectal cancer. Restaging of one patient was possible after multilevel section and immunohistochemistry of the sentinel lymph node, but more research is needed to evaluate the role of micrometastases in patients with colorectal cancer.
No Brasil, a cada ano são diagnosticados cerca de 26.000 casos de câncer colorre- tal. Pacientes com estadiamento considerado inicial, sem linfonodo metastático, evoluem com recorrência ou recidiva do tumor em até um quarto dos casos, por provável subesta- diamento. Objetivo: pesquisar sobre linfonodo-sentinela ex vivo em pacientes com adeno- carcinoma colorretal.
Foram estudados 37 pacientes, submetidos à cirurgia oncológica com ressecção caráter curativo. O marcador de linfonodos utilizado foi o corante azul patente, injetado na submucosa peritumoral da peça cirúrgica aberta imediatamente depois de sua retirada da cavidade abdominal.
A identificação ex vivo de linfonodo-sentinela com o marcador ocorreu em 13 (35,1%) pacientes. A sensibilidade do método foi de 40% e o falso negativo de 60%. O exame histológico pormenorizado dos linfonodos-sentinela com multissecção e imu- noistoquímica diagnosticou metástase em um (4,3%) indivíduo, sendo considerado ultra--estadiamento.
A identificação de linfonodo-sentinela ex vivo apresenta benefícios questioná- veis, e piores resultados quando são incluídos pacientes com câncer de reto. Foi possível reestadiamento de um paciente depois da realização de multissecção e imunoistoquímica de linfonodos-sentinela, mas mais trabalhos são necessários para estabelecer a importân- cia das micrometástases em pacientes com câncer colorretal.
Lymph node staging in colorectal cancer: Revisiting the benchmark of at least 12 lymph nodes in R0 resection
2012, Journal of the American College of Surgeons
Sentinel-lymph-node procedure in colon and rectal cancer: A systematic review and meta-analysis
2011, The Lancet Oncology
Citation Excerpt :
In 26 studies,14–19,28,29,34–37,41,43,45,48,51,53–56,60,61,62,64,65 the four quadrant method was used; 20 studies20–23,25–27,30–32,38,39,44,46,47,49,50,57,58,63 did not specify the number of peritumoral injections. In most studies14–17,19–24,27,29–34,37,39,41,43–49,51–54,56–61,63–65 immunohistochemistry staining was done on the sentinel lymph node when the haematoxylin and eosin staining result was negative. In two studies only,40,43 RT-PCR was used after a negative haematoxylin and eosin staining result.
No consensus exists on the validity of the sentinel-lymph-node procedure for assessment of nodal status in patients with colorectal cancer. We aimed to assess the diagnostic performance of this procedure.
We searched Embase and PubMed databases for studies published before March 20, 2010. Eligible studies had a prospective design, a sample size of at least 20 patients, and reported the rate of sentinel-lymph-node positivity. Individual patient data were requested for localisation and T-stage stratification. A subset of reports with high methodological quality was selected and analysed.
We identified 52 eligible studies, which included 3767 sentinel-lymph-node procedures (2961 [78·6%] colon and 806 [21·4%] rectal carcinomas). Most tumours 2339 (62·1%) were stage T3 or T4. 1887 (50·1%) of patients were male, 1880 (49·9%) female. Mean overall weighted-detection rate was 0·94 (95% CI 0·92–0·95), at a pooled sensitivity of 0·76 (0·72–0·80) with limited heterogeneity (χ²=286·08, degrees of freedom=51; p=0·003). A mean weighted upstaging of 0·15 (95% CI 0·12–0·19) was noted. Individual patient data were available from 19 studies that included 1168 patients. Analysis of these data showed no significant difference in sensitivity between colon (0·86 [95% CI 0·83–0·90]) and rectal cancer (0·82 [0·77–0·88]; p=0·23). Also, there was no dependency of sensitivity on T stage for both colon (pT1: 0·79 [95% CI 0·73–0·84], pT2: 0·76 [0·62–0·90], pT3: 0·73 [0·59–0·87], pT4: 0·73 [0·53–0·93]) and rectal cancer (T1 or T2: 0·81 [0·52–0·94] vs T3 or T4: 0·80 [0·51–0·93]). The subgroup of eight studies with high methodological quality showed a mean detection rate of 0·96 (95% CI 0·90–0·99) for colonic tumours and 0·95 (0·75–0·99) for rectal tumours, and a mean sensitivity of 0·90 (95% CI 0·86–0·93) for colonic tumours and 0·82 (0·60–0·93) for rectal tumours.
The sentinel-lymph-node procedure shows a low sensitivity, regardless of T stage, localisation, or pathological technique. For every patient diagnosed with colon or rectal cancer without clinical evidence of lymph-node involvement or metastatic disease, this procedure in addition to conventional resection should be considered, since the prognostic information provided by this technique could be clinically significant.
Cancer Center Amsterdam Foundation, Amsterdam, Netherlands.
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^☆: Oral presentation at the 13th Congress of the European Society of Surgical Oncology, Venice, Italy, 29 November–2 December 2006.

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Factors affecting false-negative rates on ex vivo sentinel lymph node mapping in colorectal cancer☆

Abstract

Purpose

Methods

Results

Conclusions

Introduction

Section snippets

Patient selection

Study population and demographics

Discussion

Conflict of interest

Funding source

Acknowledgments

Lancet

Am J Surg

Levamisole and fluorouracil for adjuvant therapy of resected colon carcinoma

N Engl J Med

Efficacy of adjuvant fluorouracil and folinic acid in B2 colon cancer. International Multicentre Pooled Analysis of B2 Colon Cancer Trials (IMPACT B2) Investigators

J Clin Oncol

Colon cancer survival is associated with increasing number of lymph nodes analyzed: a secondary survey of intergroup trial INT-0089

J Clin Oncol

Clinical significance of colorectal cancer: metastases in lymph nodes < 5 mm in size

Ann Surg Oncol

Clearance technique for the detection of lymph nodes in colorectal cancer

Br J Surg

Lymphatic staging in colorectal cancer: pathologic, molecular, and sentinel node techniques

Dis Colon Rectum

Intraoperative lymphatic mapping and the sentinel node concept in colorectal carcinoma

Br J Surg

Technical details of sentinel lymph node mapping in colorectal cancer and its impact on staging

Ann Surg Oncol

Validation of lymphatic mapping in colorectal cancer: in vivo, ex vivo, and laparoscopic techniques

Ann Surg Oncol