Detection of lymph node metastasis in lung cancer patients using a one-step nucleic acid amplification assay: a single-centre prospective study

Lung cancer is one of the most frequent human cancers and the leading cause of cancer deaths in the world. In the clinical management of the disease, identifying the histological classification by stages is essential for an accurate therapeutic approach for the patients [1, 2]. Diagnostic methods, such as endobronchial (EBUS) and oesophageal ultrasounds (EUS), fine-needle aspirations, or mediastinoscopies, are carried out to take samples of mediastinal lymph nodes (LNs) to perform the pre-surgical staging of the patient [3]. In patients with an early pathological stage, once the main tumour has been resected, the regional LNs are studied for the pathological staging that will define the treatment [2]. Traditionally, this step has been performed through microscopic examination of histological sections, the sensitivity of which can be increased by immunohistochemistry (IHC). Currently, the molecular detection of keratin 19 (KRT19) is a routine method in the clinical practice of breast cancer [4]. KRT19 is a type I keratin with a low molecular weight. It is part of the cytoskeleton of numerous epithelial cells of different organs, but is not detected in any of the cellular components of the LNs. Furthermore, it is highly expressed in epithelial tumours, so it is an appropriate biomarker for detecting micro- and macrometastasis. The one-step nucleic acid amplification (OSNA) assay is an automated molecular diagnostic assay that analyses the entire LN tissue. The reaction is based on rapid nucleic acid amplification technology, a loop-mediated isothermal amplification (RT-LAMP), to quantify KRT19 mRNA expression [5, 6]. The expression rate of KRT19 mRNA correlates with the size of the metastatic foci. In the last few years, the OSNA assay has been developed as an alternative intraoperative method for detecting of tumour metastasis in breast, gastric, and colorectal cancers [4, 7, 8]. Additionally, the feasibility of OSNA for other malignancies like thyroid, endometrial, head and neck and bronchopulmonary carcinomas is being investigated [9‐12]. Only a few reports have been published about the potential of the OSNA assay in non-small cell lung cancer (NSCLC) patients [10, 13‐15]. In this study we examined 705 LNs of 160 patients with lung cancer by OSNA assay. It represents the biggest cohort of patients and single measured LNs and one of only two reports outside of Japan. Furthermore, this is a single-centre study, with the subsequent advantage of a strictly standardised protocols and methodology. All LNs were divided to be evaluated by routine histological diagnosis or OSNA assay and the results were compared. We also evaluated the KRT19 expression rate in different types of primary thoracic tumours by IHC and OSNA. The purpose of the project was to test whether the OSNA assay is an accurate, feasible and adequate method for analysis of LN metastasis in lung cancer patients both in intraoperative and definitive pathological diagnoses.

Lung cancer is a disease with a high mortality. Some of the complexity associated with the pathology is due to its heterogeneous nature. Large-scale sequencing studies have revealed the complex genomic landscape of NSCLC and strong differences between lung adenocarcinomas and lung squamous-cell carcinomas [20]. However, both tumour types have an over 96% expression of KRT19, a commonly used marker for epithelial tumours [20]. In most pleural and lung tumours, positive rates for expression of KRT19 range from 70 to 100%. In small-cell carcinomas the expression rate is almost 60% [21‐23]. But, the criteria for immunohistochemical scores are not protocolled. Literature shows different evaluation ranges, and the fact is that the positive threshold is not yet properly established for KRT19 in lung cancer. Nevertheless, this biomarker is widely expressed in bronchopulmonary malignancies. Moreover, all LN metastasis from KRT19-positive primary lung tumours express the protein by IHC. Surprisingly, deposits from KRT19 negative primary neoplasms can be positive for KRT19 in LNs too [20]. This fact is commonly observed in breast cancer, where 67% of KRT19 negative primary tumours show KRT19 positivity in LNs metastasis [24]. Our results in primary lung tumours showed high KRT19 expression by OSNA and IHC, not only in number of positive cells, but also in staining intensity. Only neuroendocrine carcinomas presented lower rates. These data suggest that KRT19 is an accurate biomarker for detecting metastasis in lung malignancies and that, unlike in breast cancer, performing IHC for KRT19 from the primary tumour site prior to OSNA analysis might not be necessary. In contrast, our samples of different granulomatous diseases were negative for KRT19 both by OSNA and pathological examination.

In this context, our present findings in 160 patients demonstrated that the OSNA assay successfully predicts LN metastasis in lung cancer, showing high specificity, concordant rates and NPV by comparison with the gold standard technique. These results are in accordance with the few previous studies in NSCLC that are similar [10, 13‐15]. Furthermore, our research includes a larger number of patients and LNs analysed individually in a single-centre study. This fact implies a strictly standardised and normalised protocol for the performance of the whole process with an experienced surgical and diagnostic team in these procedures. However, this premise may have its weaknesses and limitations, so for the next step for validation of the technique in lung cancer, it would be necessary to conduct a multi-centre study in which not all variables and characteristics are controlled, in order to established our results.

Despite our high concordance rate, 27 LNs showed discordant results in 21 patients. Regarding −macrometastasis, 4 LNs were OSNA positive and pathological examination was negative, with two of them belonging to the same patient who underwent an N2 stage with other affected LNs. This case could be due to a sampling bias, as it was the only OSNA negative discordance with a positive histological diagnostic. LN metastasis are usually diagnosed with a postoperative pathological examination of permanent sections. However, with this method, small areas of metastasis between slices may go undetected, as may micro-micrometastasis or ITCs. Single tumour cells or small aggregates of tumour cells can be overlooked among the lymphocytes and sinusoidal histiocytes that could lead to a false-negative assessment [25]. Moreover, only a small part of the node is analysed each time. The great advantage of the OSNA assay is its ability to examine the whole LN as compared with the pathological examination, which scans only several sections. Furthermore, histological examination procedures for LNs are not standardised, and the inter-observer reproducibility of measuring metastatic tumour volume can occur [26]. This could lead to an under-stage pathology and the subsequent inappropriate treatment. Although KRT19 is present in healthy epithelial cells [10], the protocol for LN resection and processing in the laboratory prior to OSNA analysis avoids any potential false positives due to contamination. Likewise, the presence of benign epithelial inclusions is exceptional in mediastinal LNs, as well as its likelihood to cause a false-positive result. The differences and similarities between histopathological procedures and OSNA assay is shown in comparison Table 6.

The high sensitivity of the OSNA assay also enables detecting of occult −micrometastasis missed by pathological examinations. We detected 22 −micrometastasis with a negative KRT19-IHC. The significance of these findings is not clear, nor are the implications in treatment decisions. Patients with pathological pN0 or pN1 diseases have heterogeneous outcomes [27‐29]. Maybe these occult −micrometastasis are the key to explaining why some of these cases are classified as pN0 or pN1 after surgery, by means of conventional methods, progressing with worse clinical course. In breast cancer there are discrepancies about the presence of these microdeposits and whether they have do or do not have an impact on disease-free survival (DFS) and overall survival (OS) [30‐32]. The same conflicting results are shown for lung cancer. Rena et al. [33] showed that LN ITCs and micrometastasis in pathological stage I NSCLC do not affect long term DFS. In contrast, other groups concluded that patients with ITCs or micrometastasis in LNs had higher recurrence rates and worse survival rates in early-stage NSCLC [34, 35]. Therefore, the cut-off point of the KRT19 copy number which is pathologically significant is critical. In this work we used the established parameters for breast cancer [4]. Do we need validation studies for the KRT19 mRNA copy numbers set up as cut-off for negative micro- and −macrometastasis in lung cancer? Or are breast cancer approximations accurate enough values applied to mediastinal LNs? The cut-off level of the OSNA assay used in previous studies in gastric, colorectal and lung cancer was also established at 250 copies/μl, considering that the KRT19 mRNA expression level is similar among the various types of malignancies. However, in head and neck cancer, new cut-off levels of 131 and 300 copies/μl were proposed by different studies [9, 36]. Head and neck tumours are squamous cell carcinomas and expression of KRT19 is a poor prognostic marker [37]. Most breast, gastric, colorectal, and between the 40 and 60% of lung malignancies, are adenocarcinomas. However, 20–30% are epidermoids and, thus, the cut-off point established for lung cancer could depend on the histological type of the tumour. For all these reasons, the limits of detection of the assay in bronchopulmonary malignancies must be established, monitoring the progression of patients with conflicting results, micrometastasis deposits and presence of ITCs. Furthermore, in lung carcinomas with low or no expression of KRT19, such as small cell carcinomas, the inclusion of a second or third marker could minimise false negatives and improve the specificity of the test.

On the other hand, in colorectal cancer, the amount of tumour burdens or total tumour loads detected by OSNA were showed to be an accurate LN pathological staging with potential prognostic implications [38]. Moreover, studies show that OSNA positivity in stage II colon cancer patients who are pN0 by H&E is associated with classical high-risk factors and has a prognostic value, suggesting that OSNA could be detecting patients as under-staged by histological techniques [39]. The total tumour burden pooled by RT-LAMP has not yet been analysed in lung cancer, but nevertheless could be a promising approach for future studies.

Our findings show that the OSNA assay is a fast, feasible and accurate technique. It is a methodology that improves cost-effectiveness, accuracy and management of patients with an adequate clinical practice in pre-staging and pathological diagnosis. Furthermore, it is able to detect macrometastatic foci and small metastatic lesions and ITCs that are not identified by conventional techniques. The clinical and prognostic significance of such findings would also need to be evaluated with prospective studies, including a sufficient number of patients and an adequate follow-up period. The analysis of the whole LN by the OSNA assay could overcome the limitations of sample bias and the histological examination. In addition, the remaining LN lysate has proved to be adequate for subsequent molecular analysis [40]. In conclusion, this work addresses the validity of the procedure as an adequate method for diagnosis of LN metastasis in lung cancer.