The advantage of one‐step nucleic acid amplification for the diagnosis of lymph node metastasis in colorectal cancer patients

Abstract Generally, the postoperative examination of lymph nodes (LNs) is based on a microscopic examination of one hematoxylin and eosin (HE)‐stained slide; however, an examination of only one part of the LN might lead to incorrect staging of the tumor due to tissue allocation bias. Although multilevel sectioning and the use of immunohistochemistry (IHC) have improved the detection of micrometastases in LNs, this approach is laborious, time‐consuming, and costly. A novel molecular technique for the detection of LN metastases of tumors, called one‐step nucleic acid amplification (OSNA), is a rapid and semi‐quantitative examination quantifying the number of cytokeratin 19 (CK‐19) mRNA copies derived from a tumor. OSNA is already in clinical use for the diagnosis of LN metastasis in breast cancer patients; however, the use of OSNA is under investigation with promising results for colorectal cancer (CRC). The present review assessed recent studies on OSNA vs a histopathological examination and its implications for CRC staging and treatment. A total of 16 studies of OSNA in CRC yielded by a PubMed search were reviewed. Among them, seven studies evaluating the diagnostic performance revealed that OSNA had a high specificity (96.8%), high concordance rate (96.0%), and negative predictive value (98.6%) in a pooled assessment. In addition, four studies examining the utility of OSNA in sentinel LNs (SLNs) and two studies focusing on upstaging in pathologically node‐negative CRC patients were also reviewed. Multicenter prospective studies with a large cohort of CRC patients are warranted to reveal the benefits of OSNA in the future.

on whether or not the cancer has spread to regional LNs and how many LNs are involved. Isolated tumor cells (ITCs) in LNs are defined as single cancer cells or small clusters of tumor cells measuring ≤0.2 mm and classified as N0 (i+). Micrometastases are defined as tumor clusters measuring >0.2 mm but ≤2.0 mm in their greatest dimension and classified as N1 (mic). Although the prognostic value of ITCs is unclear, a recent systematic review and meta-analysis showed micrometastasis to be a significantly poor prognostic factor. 3 Per the AJCC 8th edition, the micrometastasis may be designated as N1 (mic), but that may be better considered standard positive LNs. Because of the strong prognostic relevance of LN metastases in CRC, the presence of occult tumor cells, defined as micrometastases or ITCs within regional LNs that are not detected on a conventional histopathologic examination using hematoxylin and eosin (HE) staining has been suspected to be a marker of systemic tumor spread in these patients. 4 Therefore, the detection of occult disease may help identify patients with node-negative CRC who are at a high risk of tumor recurrence and who might benefit from adjuvant therapy.
Combining routine HE staining with tumor-specific immunohistochemistry (IHC) using step sectioning specimens or molecular detection techniques, such as reverse transcriptase polymerase chain reaction (RT-PCR), provides a higher sensitivity than routine histology alone. 5,6 However, these methods are burdensome and time-consuming, so a quick, highly sensitive, and specific diagnostic technique that enables a prompt intraoperative examination is necessary.
A new molecular technique called one-step nucleic acid amplification (OSNA), which is a rapid and semi-quantitative intraoperative procedure for quantifying the number of cytokeratin  mRNA copies in LNs, has been employed to assess the LN progression of tumors. OSNA was first reported in breast cancer patients and has been shown to be effective for detecting nodal metastases. 7 OSNA can assess the occurrence of metastasis in the whole LN regardless of tissue allocation biases and estimate the total volume of tumor cells semi-quantitatively. This is one important advantage of OSNA over a pathological assessment.
Although the validity of OSNA assay for detecting LN metastasis has also been widely reported in patients with other types of malignancies, such as CRC, lung cancer, gastric cancer, head and neck squamous cell carcinoma (HNSCC), and thyroid cancer, its clinical benefit has not been established. 8,9 In addition, several studies have recently evaluated OSNA for LN metastasis in CRC, focusing on pathological stage II disease, sentinel LN (SLN) metastasis, and lateral pelvic LN (LPLN) metastasis.
We herein review studies relevant to OSNA in CRC and discuss perspectives on the future applications of this assay.

| LITER ATURE S E ARCH
A search for medical reports relevant to OSNA in colorectal cancer published before the end of June 2020 was made through PubMed using the keywords of "OSNA" and "colorectal cancer" or "colon cancer" or "rectal cancer." Reports written in languages other than English were excluded. Review articles were also excluded. The 16 studies obtained by the PubMed search are shown in Table 1.

| THE IDENTIFI C ATI ON OF AN OP TIMAL mRNA MARK ER FOR THE OS NA A SSAY IN CRC
At present, the validity of the OSNA assay targeting CK19 mRNA for detecting LN metastasis has been widely reported in patients with various types of tumors, including CRC. Yamamoto et al reported the background for the identification of CK19 mRNA as an optimal marker for the OSNA assay in CRC. 13 Ninety-eight candidate mRNAs selected from the genome-wide expressed sequence tag database were evaluated by quantitative RT-PCR using a mixture of metastasis-positive LNs and another mixture of metastasis-negative LNs in CRC patients. Thereafter, the three identified candidates (CK19, CEA, and CK20) were examined by an OSNA assay, and CK19 mRNA was found to have the best diagnostic performance. The cut-off value for discriminating positive from negative LNs was set at 75-500 copies/µL, with 96.4% sensitivity and 100% specificity.

| D IAG NOS TI C PERFORMAN CE OF THE OS NA A SSAY IN CRC
Among the 16 studies listed in Table 1, the seven comparing the diagnostic performance between the OSNA and a pathological examination for the detection of LN metastasis in CRC are shown in Table 2. The calculated values of sensitivity, specificity, concordance, positive predictive value (PPV), and negative predictive value (NPV) in a pooled analysis were 90.4%, 96.8%, 96.0%, 79.8%, and 98.6%, respectively. These values are similar to those of breast cancer previously reviewed by Tamaki et al. 6 It should be noted that the methodology of the histopathological evaluation for detection of the LN metastasis differed among studies. For example, a standard pathological examination using HE staining with a single 4-µm-thick tissue section was used in some studies, 15,19,21 while a more precise pathological evaluation for the detection of LN metastasis, such as macrometastasis, micrometastasis, and isolated tumor cells (ITCs), was attempted, and the diagnostic accuracy was compared between an intensive pathological evaluation and the OSNA assay in others. [10][11][12]23 12 The fact that the pooled PPV of OSNA (79.8%) was lower than other values as shown in Table 2, shows that the false-positive rate of OSNA is high. This may be because the OSNA assay can detect some LN metastases of CRC that cannot be detected by histopathological examinations due to tissue allocation biases or insufficient performance for the detection of micrometastasis.
The studies listed in Table 2

| THE OS NA A SSAY FOR THE DE TEC TI ON OF CRC ME TA S TA S IS IN S LN S
In CRC, the SLN concept was introduced in 1999 by Joosten et al 27 in order to reduce the false negative result rates and to understand the importance of the LN involvement for further therapy and the survival rate of these patients. SLNs are considered to be the LNs located closest to the tumor in the lymphatic drainage pathways, bearing the highest risk of tumor involvement. 27 Using lymphatic mapping, two to four such LNs may be identified.

| THE OS NA FOR THE PRED IC TI ON OF LPLN ME TA S TA S IS IN REC TAL C AN CER
In Western countries, preoperative chemoradiotherapy (CRT) with total mesorectal excision (TME) is the standard therapy for rectal cancer. 36 However, in Japan, TME plus lateral pelvic LN dissection (LLND) is performed for advanced low rectal cancer. 37

| FUTURE PER S PEC TIVE S
The LN status remains the most significant prognostic factor and an shown to be a significant prognostic factor. 45,46 An accurate diagnostic tool, such as the OSNA, seems effective for determining the treatment strategy after the evaluation of extended LN metastasis, as an accurate diagnosis can be achieved rapidly (intraoperatively if necessary), regardless of the preoperative treatment.
The main advantage of an OSNA assay over a pathological examination is that it provides objective and semi-quantitative data on the tumor volume in the whole LN rapidly with little to no effort by pathologists and with low inter-observer variability. As described above, an OSNA assay can help improve the treatment outcome of CRC patients in several respects, such as in the detection of highrisk stage II disease, the SLN analysis and the prediction of LPLN metastasis. Multicenter prospective studies with a large cohort of CRC patients are warranted to confirm such advantages of an OSNA assay, and technical refinements of the OSNA system are essential before it can be applied as a new standard.

D I SCLOS U R E S
Conflicts of interest: Authors declare no conflicts of interest or financial ties to disclosure.