Virchows Arch.2016 Oct;469(4):385-94. doi: 10.1007/s00428-016-1990-1.
Molecularly determined total tumour load in lymph nodes of stage I-II colon cancer patients correlates with high-risk factors. A multicentre prospective study.
Aldecoa I1, Atares B2, Tarragona J3, Bernet L4, Sardon JD5, Pereda T6, Villar C7, Mendez MC8, Gonzalez-Obeso E8, Elorriaga K9, Alonso GL10, Zamora J11, Planell N12, Palacios J13, Castells A14, Matias-Guiu X3, Cuatrecasas M15,16.
1Pathology Department, Centre de Diagnòstic Biomèdic (CDB), Hospital Clínic, University of Barcelona, Escala 3, Planta 5. Villarroel 170, Barcelona, 08036, Spain. 2Pathology Department, Alava University Hospital, Vitoria-Gasteiz, Spain. 3Pathology Department, Hospital Arnau de Vilanova, Lleida, Spain. 4Pathology Department, Hospital L. Alcanyis, Xativa, Spain. 5Surgery Department, Alava University Hospital, Txagorritxu, Spain. 6Pathology Department, Hospital Costa del Sol, Marbella, Spain. 7Pathology Department, Hospital Reina Sofia, Cordoba, Spain. 8Pathology Department, Hospital Severo Ochoa, Leganes, Madrid, Spain. 9Pathology Department, Hospital Onkologikoa, San Sebastian, Spain. 10Pathology Department, Hospital 12 Octubre, Madrid, Spain. 11Biostatistic Unit, Hospital Ramon y Cajal, Madrid, Spain. 12Gastroenterology Department and Bioinformatics Unit, CIBERehd, IDIBAPS, Hospital Clinic, University of Barcelona, Barcelona, Spain. 13Pathology Department, Hospital Ramon y Cajal, Madrid, Spain. 14Gastroenterology Department, Hospital Clinic, University of Barcelona, IDIBAPS, CIBERehd, Barcelona, Spain. 15Pathology Department, Centre de Diagnòstic Biomèdic (CDB), Hospital Clínic, University of Barcelona, Escala 3, Planta 5. Villarroel 170, Barcelona, 08036, Spain.16CIBERehd, and Banc de Tumors-Biobanc Clinic-IDIBAPS-XBTC, Hospital Clinic, Barcelona, Spain. email@example.com.
Stage I-II (pN0) colorectal cancer patients are surgically treated although up to 25 % will eventually die from disease recurrence. Lymph node (LN) status is an independent prognostic factor in colorectal cancer (CRC), and molecular tumour detection in LN of early-stage CRC patients is associated with an increased risk of disease recurrence and poor survival. This prospective multicentre study aimed to determine the relationship between LN molecular tumour burden and conventional high-risk factors in stage I-II colon cancer patients. A total of 1940 LN from 149 pathologically assessed pN0 colon cancer patients were analysed for the amount of tumour cytokeratin 19 (CK19) messenger RNA (mRNA) with the quantitative reverse transcription loop-mediated isothermal amplification molecular assay One-Step Nucleic Acid Amplification. Patient’s total tumour load (TTL) resulted from the sum of all CK19 mRNA tumour copies/μL of each positive LN from the colectomy specimen. A median of 15 LN were procured per case (IQR 12;20). Molecular positivity correlated with high-grade (p < 0.01), mucinous/signet ring type (p = 0.017), male gender (p = 0.02), number of collected LN (p = 0.012) and total LN weight per case (p < 0.01). The TTL was related to pT stage (p = 0.01) and tumour size (p < 0.01) in low-grade tumours. Multivariate logistic regression showed independent correlation of molecular positivity with gender, tumour grade and number of fresh LN [AUC = 0.71 (95 % CI = 0.62-0.79)]. Our results show that lymph node CK19 mRNA detection correlates with classical high-risk factors in stage I-II colon cancer patients. Total tumour load is a quantitative and objective measure that may help to better stage early colon cancer patients.
Colorectal carcinoma (CRC) is the second leading cause of neoplasia in developed countries. It represents the third carcinoma after prostate and lung in men, and after breast and lung cancer in women 1. Stage I-II CRC patients (pN0) are surgically treated, but about 10-20% stage II CRC patients recur from disease. Thus, stage II CRC patients poses a complex therapeutical challenge. Although the favorable impact of chemotherapy in stage III disease supports its use in high-risk stage II CRC cases, there is not enough evidence to support a systematic use of adjuvant chemotherapy, due to its toxicity and high costs 2.
Pathological lymph node (LN) staging is performed by microscopic LN analysis of few hemathoxilin and eosin (HE) stained 2-4 µm LN sections. This workup has not changed much since its conception 3, mainly due to the good correlation between HE results and clinical outcome 4. Nevertheless the increased diagnosis of early CRC, in part due to the implementation of CRC population-based screening programs, have demonstrated the weakness of the traditional HE LN staging to estimate prognosis and recurrence risks 2,5. The enhanced sensibility of molecular techniques, compared to the traditional HE analysis, has shown that tumor burden can be detected in LNs of 25-50% CRC patients that have been pN0 assessed with HE 6–10. These deficiencies have important consequences in stages I-II CRC (pN0) patients, treated with curative-intended surgery only 11.
Nevertheless, the prognostic value of the molecular detection of occult tumor burden in LN of stage I-II CRC (pN0) is still controversial 7,9,10, and the benefit of adjuvant chemotherapy in this patients, which harbor occult LN metastases detected by molecular methods is still unknown 2,12,13. In addition, neoplastic spread to LNs may happen in early stages of the disease 14, implying a potential negative prognosis in selected early stage tumors 6,7,9,10. Thus, patients with higher recurrence risk are both clinically and histologically difficult to identify 15, stressing an adequate pathological LN staging as a key factor for the correct therapeutic management of CRC 7,9,10.
We aimed to determine the relationship between the presence of molecularly detected tumor burden in LNs from stages I-II colon cancer patients (pN0) and conventional high-risk factors.
Studies on CRC molecular staging are promising but based on complex and resource-consuming techniques 6,16–19. In our study, we have used the One Step Nucleic Acid Amplification technique (OSNA; Sysmex Corporation, Kobe, Japan), a quantitative, fast, standardized and automated approach that performs four determinations in 45 minutes. The technique has been validated for breast and colon cancer LN analysis. It is routinely used in pathology departments for breast cancer sentinel LN analysis 20–22. Its results are equivalent to extensive histological and immunohistochemical work up 8,20,21,23–27. It uses the Reverse Transcription Loop-mediated Isothermal Amplification method (RT-LAMP), which amplifies CK19 mRNA from tissue lysates 20, with 94.9-95.2% sensibility and 97.7-97.9% specificity 20,28. The OSNA assay enables a complete analysis of the LN. The results, expressed as the number of CK19 mRNA copies//μL, correlate with the size of the metastatic foci. Values of <250 copies/μL correspond to isolated tumor cells; between 250-5,000 copies/μL to micrometastases, and >5,000 copies/μL to macrometastases 20,21,23,24.
We analyzed by OSNA 2,483 LN from 149 cases, previously staged as pN0 with HE. (Fig. 1). At least 12 LNs from each patient were analyzed by OSNA. We used the total tumor load (TTL), defined as the sum of CK19 mRNA copies/μL from each positive LN of a given case 25,21. Our results showed OSNA positivity in 51% cases, with few positive LN per case, and median TTLs of 2,015 CK19 mRNA copies/μL in positive cases. OSNA molecular positivity was related to the amount of LN analysed, male genre and histological grade. The multivariant analysis showed that these factors were independent predictors of the OSNA results in stage I-II colon carcinomas 29. The unvariant analysis showed that molecular positivity also correlated to other CRC classical high-risk factors, such as mucinous or signet ring cell histology, the presence of foci of high histological grade, tumor size and male genre. Although not significant, we observed a tendency between TTL and pT stage, with 1,280 copies/µL in pT1 patients, raising to 1,790 copies/µL in pT2 cases, and to 3.080 copies/µL in pT3 stage patients 29. We also observed different TTL distribution among low and high-grade tumors. In low-grade tumors, the TTL increased with pT stage (p=0.01) and was related to tumor size (p<0.01; rho=0.27) 29. This phenomena may be explained by the fact that low-grade tumors may follow an orderly pattern of mutation and progression during its growth and disemination, while high-grade tumors may held distinct molecular and spread patterns 30. These data suggests that the TTL may imply a different clinical impact depending on other pathologic factors, which may modulate the predictive values of the OSNA results 22.
The importance of this study lies on the demonstration that the presence of occult tumor burden in HE-assessed LNs as pN0, is associated to other recognized high-risk factors in CRC, such as genre, high grade, presence of lymphatic invasion, number of LNs analysed, pT and pN stages 8,25. We have used the OSNA technique, which provides a quantitative value of the amount of tumor burden present in each LN. It is also important to stress that the presence of small amounts of tumor burden in LNs may not be clinically meaningful or imply a negative prognosis 6,21. In our colon cancer studies, the median TTL value in preinvasive lesions and stage I carcinomas was 1,275 copies/µL 31. Although we have scant follow-up data, one of the pN0 patients that recurred had a TTL of 47.760 copies/µL, and two patients with histologically confirmed LN metastases had 560,000 and 41,160 copies/µL, respectively 31. Additionally, after 2-year follow up, four OSNA positive cases that had disease recurrence harbored a median TTL of 4,375 copies/µL 29.
The prognostic relevance of the molecular detection of LN metastases has been already underlined in CRC and breast cancer 6,7,9,21,22. Yet, some authors have demonstrated the correlation between the amount of metastatic burden and prognosis, stressing the need to quantify the TTL to elucidate the prognostic significance of the molecular results. A recent study has demonstrated that OSNA assessed TTL significantly increases with histological pN stage in CRC: with TTL values of 1.550 CK19 mRNA copies/μL in pN0, 24.050 copies/μL in pN1 and 90,600 copies/μL in pN2 patients 25. Other studies have shown similar results using different molecular targets. One of them was based on the quantification by RT-PCR of Guanylyl cyclase C, an epithelial intestinal receptor which is overexpressed in CRC cells. They showed that as well as its positivity, the quantification of the metastatic volume may be a relevant predictive factor 6. Yet, another study based on the usefulness of the quantification of by RT-PCR of CEA mRNA in LNs of stage II colon carcinomas, found that greater micrometastasis volume correlated both worse 5-year disease free and overall survival 32.
The actual challenge of OSNA LN staging in CRC is to obtain clinically significant and robust data concerning its ability to predict disease recurrence. In order to do so, OSNA results must be integrated not only with pathological factors such as tumor size, budding and stage, but also with molecular factors. These data will lead to the development of working nomograms and algorithms, which will give the importance to each of the relevant factors, in order to estimate disease recurrence and the need of adjuvant therapy.
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Fig. 1: a) Fresh lymph node dissection from mesocolon fat; white arrow shows a lymph node; b) RD-100i platform for OSNA analysis. It uses the Reverse Transcription Loop-mediated Isothermal Amplification (RT-LAMP) method, for amplification of CK19 mRNA from LN tissue lysates.