Comparison of four DLL3 antibodies performance in high grade neuroendocrine lung tumor samples and cell cultures

Small cell lung carcinoma (SCLC) and large cell neuroendocrine carcinoma (LCNEC) are high-grade pulmonary neuroendocrine tumors, with a poor prognosis. Advances in therapy have not been significant in the last three decades [1]. Small cell lung carcinoma represents 13–15% of the primary lung malignancies [2] and occurs in the elderly population, almost exclusively in smokers, with a 5-year survival rate of 7% [3]. At the time of diagnosis, patients usually present with distant metastases; they receive the platinum-etoposide combination chemotherapy as the standard treatment regimen [4, 5]. Response to the therapy is very high, but so is the recurrence. The only approved second-line treatment is topotecan, which has a low response rate (5–24%), with an overall survival < 7 months [6]. The progress in therapy for adenocarcinoma and squamous cell carcinoma of the lung have been achieved by identifying the targetable molecular changes in the former, and by applying immune checkpoint inhibitors in both. The effectiveness of immune checkpoint inhibitors is less in SCLC, unlike non-small cell lung carcinomas (NSCLC). The programmed death-ligand 1 (PD-L1) is expressed only in a small percentage of SCLC. On the other hand, the most common genetic changes in SCLC, TP53 and RB1 mutations and amplifications of SOX2 and SRSF1 are not targetable as these are tumor suppressor gene mutations [7]. The two main therapeutic approaches used for more than three decades for LCNEC are the SCLC-like treatment and the non-neuroendocrine carcinomas-like treatment that is based on resection and/or platinum-based protocols. Recent reports have demonstrated the possibility of a gene expression-based therapy for LCNEC [8, 9]. LCNEC with RB1 mutations, as in SCLC, might best respond to an SCLC-like chemotherapy, whereas LCNEC tumors with PTEN loss and/or PI3KCA activating mutations might be optimally treated with platinum-based therapy [8, 9].

Recently, Saunders et al. were able to identify the overexpression of delta-like ligand 3 (DLL3) using tumor-initiating cells obtained from SCLC and LCNEC patient-derived xenografts [10]. The DLL3, normally expressed in fetal brain, plays a physiological role in development [11‐13]. It is the only member of the Notch receptor ligand family that inhibits the Notch receptor pathway [10]. Notch inactivation is found in most of the SCLC cells, and correlates to the gene expression of the neuroendocrine markers [14]. Furthermore, the expression of DLL3 is closely related to neuroendocrine differentiation, and the expression of the transcription factor achaete-scute homolog 1 (ASCL1), which is an important oncogenic driver for SCLC [15], regulates growth and survival of the SCLC cells and is found in 75% of SCLC cells [16]. Some SCLC cells, which express POU class 2 homeobox 3 (POU2F3), do not present neuroendocrine features [17]. Saunders et al., demonstrated DLL3 protein expression by immunohistochemistry in 65% of LCNEC cells and 72% of treatment-naïve SCLC cells, whereas the normal lung parenchyma was completely negative [10]. Thus, DLL3 protein has emerged as very promising drug target.

A recent first-in-human, first-in-class, open-label phase 1 study using rovalpituzumab tesirine, an antibody-drug conjugate directed against DLL3, demonstrated significant antitumor activity in patients with SCLC [18], especially when they have high expression of DLL3. It has already been shown that the overall expression of DLL3 in SCLC is high [10, 19]. However, several DLL3 antibodies are available, but correlation studies are yet to be conducted. The antibody used in clinical trials is bound to a specific platform, which is not available in all pathology laboratories, so a search for the adequate substitution is necessary. Unlike the release of new drugs in the USA, which is usually accompanied by a companion test, the European Medicines Agency (EMA) allows use of any validated test. In other words, companies or pathology laboratories can develop their own test, which than has to be validated.

Our aim was to investigate four different antibodies for their reliability to detect DLL3 expression in high-grade neuroendocrine tumors of the lung and well characterized cell cultures. Furthermore, we aimed to analyze if there is a correlation between DLL3 expression with the TP53 mutation or Rb1 loss, which might aid in directing cases of LCNEC for a possible DLL3 staining and eventual treatment.

Recently, first-in-human, first-in-class, open-label phase 1 study of a DLL3-targeted antibody-drug conjugate was published [18], paving the way for the treatment of patients with SCLC, expressing DLL3 on the tumor cells. Since there has not been any significant improvement in therapy for this group of patients for many decades, and they are usually diagnosed at a later stage, this represents a tremendous step forward. Keeping this in mind, and the fact that therapy decision will be based on immunohistochemical DLL3 expression, we tested four different antibodies, VenA, NovA, TherA, and AbcA to evaluate their adequacy for this evaluation.

Using VenA (antibody used in the clinical trial) as a reference antibody, we have shown that none of the other three antibodies can reliably be used for the DLL3 test. Differences in expression with respect to VenA were observed, regardless of the cut-off values (≥75%, or ≥ 50%, or ≥ 25%), that were applied. This was true for samples from patients with SCLC as well as with LCNEC.

In our study, different results in DLL3 expression in SCLC, applying 50% as a cut-off, were obtained in study cohort and validation cohort (45.8% vs 78%, respectively). These might be best explained as the matter of sampling issue: in the study cohort, the samples from the resection material were analyzed, while in the validation cohort small biopsy samples dominated.

To evaluate the specificity of all these antibodies, we used SCLC cell cultures with known expression of neuroendocrine markers, DLL3 and Notch1/3. Testing the antibodies on these cell cultures, using smears and cytoblocks, we showed that VenA is the only antibody that reliably stains tumor cells known to be DLL3 positive, and is also negative in cells that do not express DLL3. Thus, cell lines NCI-H69a and NCI-H69s can serve as a control for other antibodies. It is known that batches of antibodies can vary quite substantially in specificity; cell blocks from these cell cultures provide reference standards. These are superior to in-house produced tissue references, as the amount of tumor tissue in SCLC biopsies is very limited.

Although LCNEC was excluded in the clinical trial due to a small sample size, we have tested its DLL3 expression in this study using a larger sample size. Approximately 50% of the tumors were positive, and the expression values (scores) were higher in several cases compared to SCLC. Therefore, DLL3-based toxin-coupled antibody treatment might be an option for these patients, if they express DLL3. Interestingly, new treatment options have been proposed for LCNEC, such as chemotherapy based on an SCLC regimen for cases with RB1 loss and cisplatin-based chemotherapy for cases with loss of PTEN or activating mutation of PI3KCA [8, 9]. As shown here, there is no correlation between RB1 loss and DLL3 expression in LCNEC. Therefore, anti-DLL3 therapy is another new option for these patients. This investigation fails to answer whether the positive statistical correlation between RB1 loss and DLL3 expression is a real association or only because most SCLC cases show this loss. However, as RB1-loss is typically found in neuroendocrine marker expressing SCLC this might point to the associated DLL3-expression in this set of carcinomas.

Finally, our study has several limitations. We have used two cohorts, with different sample types (resections vs small biopsies). Both cohorts were rather small. We have used archived tissue, which is not optimal for protein expression analysis, but is a real-life situation.

DLL3 immunohistochemical evaluation will be mandatory for the decision about an application of the DLL3-targeted antibody-drug conjugate in patients with SCLC. The EMA, upon release of the drug, usually requires the use of a validated test. Therefore, several antibodies, if successfully validated, might be used. In this study, we have shown the superiority of the antibody used in the clinical study in detecting DLL3 expression. Furthermore, we have demonstrated that, like for other antibodies, positive and negative probes are mandatory. We have established cell cultures as test samples which can be used for further DLL3 antibodies to be tested. We have also provided evidence, that patients suffering from LCNEC express DLL3 in high proportions, which might provide basis for studies of new therapy options in this group of patients.