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. 2019 May 8;24(11):e1172–e1179. doi: 10.1634/theoncologist.2018-0676

Analysis of DLL3 and ASCL1 in Surgically Resected Small Cell Lung Cancer (HOT1702)

Megumi Furuta a, Jun Sakakibara‐Konishi a,*, Hajime Kikuchi c, Hiroshi Yokouchi d, Hiroshi Nishihara e, Hiroyuki Minemura f, Masao Harada d, Shigeo Yamazaki h, Kenji Akie i, Yuka Fujita j, Kei Takamura c, Tetsuya Kojima k, Toshiyuki Harada l, Yoshinori Minami m, Naomi Watanabe n, Satoshi Oizumi d, Hiroyuki Suzuki g, Masaharu Nishimura a, Hirotoshi Dosaka‐Akita b, Hiroshi Isobe k; on behalf of Hokkaido Lung Cancer Clinical Study Group
PMCID: PMC6853124  PMID: 31068386

Growing evidence suggests that DLL3 has a role in the tumorigenesis of small‐cell lung cancer. This article reports on the relationship between DLL3 and ASCL1 expression in resected small‐cell lung cancer samples using immunohistochemical analysis.

Keywords: Small cell lung cancer, Delta‐like protein 3, Achaete‐scute homolog‐1, Immunohistochemistry, Surgery

Abstract

Background.

Delta‐like protein 3 (DLL3) is a Notch ligand that has an important role in the tumorigenesis of small cell lung cancer (SCLC). Recently, rovalpituzumab tesirine (Rova‐T), a DLL3‐targeted antibody‐drug conjugate, has been developed for treating SCLC. DLL3 is a transcriptional target of the achaete‐scute homolog‐1 (ASCL1) transcription factor, which is involved in pulmonary neuroendocrine cell development. However, the relationship between DLL3 and/or ASCL1 expression and the clinical features of SCLC remains unknown, especially for early‐stage resected SCLC. This study aimed to investigate the expression of DLL3 and ASCL1 in resected SCLC samples using immunohistochemical analysis.

Materials and Methods.

We collected 95 surgically resected SCLC samples, which were formalin fixed and paraffin embedded. Immunohistochemistry staining was performed to investigate the correlation between the expression of either DLL3 or ASCL1 and clinicopathological features of study patients.

Results.

Seventy‐seven (83%) of 93 immunohistochemically evaluable samples were positive for DLL3 (expression in ≥1% of tumor cells), and DLL3‐high expression (≥75%) was observed in 44 samples (47%). Sixty‐one (64%) of 95 samples were positive for ASCL1 (expression in ≥5% of tumor cells). A positive correlation was observed between DLL3 and ASCL1 expression. DLL3 and ASCL1 expression were not associated with survival in SCLC patients. DLL3 was more prevalent in patients with advanced clinical disease.

Conclusion.

DLL3 and ASCL1 were highly expressed in patients with surgically resected SCLC. DLL3 and ASCL1 may be targets for the treatment of SCLC.

Implications for Practice.

This article examines the relationship between delta‐like protein 3 (DLL3) and achaete‐scute homolog‐1 (ASCL1) protein expression with the clinical features of 95 surgically resected small cell lung cancer (SCLC). DLL3 is attracting attention because rovalpituzumab tesirine (Rova‐T), a DLL3‐targeted antibody‐drug conjugate, was developed recently. DLL3 and ASCL1 were highly expressed in patients with surgically resected SCLC. DLL3 and ASCL1 may be targets for the treatment of early‐stage SCLC, including with Rova‐T.

Introduction

Lung cancer is the leading cause of cancer‐related death worldwide, with small cell lung cancer (SCLC) accounting for approximately 15% of lung cancer cases [1], [2]. Most patients with SCLC initially respond to chemotherapy and radiotherapy but usually relapse and acquire resistant disease. The prognosis of patients with SCLC remains poor, and they frequently require multiple treatments [3].

The Notch signaling pathway regulates tumorigenesis and can be either oncogenic or tumor‐suppressive, depending on the cellular context [4], [5]. Regarding SCLC, overexpression of Notch1 induces G1 cell cycle arrest [6], and the Notch target gene HES1 inhibits the neuroendocrine transcription factor achaete‐scute homolog‐1 (ASCL1) [7], [8], which suggests that Notch can act as a tumor suppressor in SCLC.

In mammals, there are four Notch receptors (Notch1 to Notch4) and two families of ligands, jagged (JAG1 and JAG2) and delta‐like ligands (DLL1, DLL3, and DLL4) [9]. Unlike the other activating DLL ligands, DLL3 does not bind or activate Notch receptors when presented in trans, but instead inhibits Notch signaling in cis [10]. Moreover, DLL3 is regulated directly by ASCL1 [11], [12], which is essential for development in several types of neuroendocrine cells [13], [14], [15] and correlates to tumorigenicity in SCLC [16], [17]. Altogether, ASCL1‐induced DLL3 expression might be associated with neurogenesis and SCLC carcinogenesis through modulating the Notch pathway.

Rovalpituzumab tesirine (Rova‐T; AbbVie Inc., North Chicago, IL), a recently developed DLL3‐targeted antibody‐drug conjugate, comprises a humanized anti‐DLL3 monoclonal antibody conjugated to a DNA‐damaging pyrrolobenzodiazepine dimer toxin. It has antitumor efficacy in vivo [18], and a recent phase I study found that patients with high DLL3 expression had better objective responses than those with low expression, suggesting that DLL3 expression is a potential predictive biomarker for SCLC outcomes [19].

Although growing evidence suggests that DLL3 has a pivotal role in SCLC, little is known about the prognostic influence of DLL3 and the association between protein expression of DLL3 and ASCL1, especially in early‐stage SCLC, because of the difficulty in obtaining surgical samples. Furthermore, obtaining tumor specimens is important as they can provide precise histologic information, such as pure SCLC or combined SCLC.

Therefore, the aim of this study was to investigate the expression of DLL3 and ASCL1 in resected SCLC samples using immunohistochemical analysis.

Materials and Methods

Patient Data

We included patients with primary SCLC who had undergone complete surgical resection of a primary lung tumor between January 2003 and January 2013 at institutions participating in either the Fukushima Investigative Group for Healing Thoracic Malignancy (FIGHT) or the Hokkaido Lung Cancer Clinical Study Group Trial (HOT) [20]. Patients were centrally re‐reviewed for a confirmed pathological diagnosis of pure SCLC or combined SCLC, according to the 2004 World Health Organization classification [21]. Written informed consent was obtained from patients who were still alive at the time of data accrual (from February 2013 through January 2014). The study was registered with the University Hospital Medical Information Network Clinical Trials Registry (identification number, UMIN000010117) and was approved by the institutional review board of each participating institution. All individual data were obtained from medical records and deidentified. An unidentifiable code number was assigned to each tissue sample. Stages were determined or reclassified according to the 7th edition of the TNM staging system [22].

Tissue Samples

Between January 2003 and January 2013, 156 patients were enrolled from 17 institutions. Ninety‐five of 156 samples from 11 institutions had sufficient material to assess protein expression of DLL3 and ASCL1 by immunohistochemistry (IHC), along with patient metadata and pathology reports.

IHC

DLL3 expression was assessed in 5‐μm sections cut from formalin‐fixed and paraffin‐embedded SCLC tissue blocks using anti‐DLL3 antibody (SP347; Spring Bioscience, Pleasanton, CA). Briefly, SP347 was used at 0.24 μg/mL, followed by an OptiView DAB IHC kit (Ventana, Tucson, AZ) to visualize DLL3 expression. ASCL1 expression was detected by the Vectastain ABC HRP kit (Vector Laboratories, Burlingame, CA) with a mouse monoclonal antibody (SC72.201; AbbVie Stemcentrx, South San Francisco, CA). Stained slides were then observed under a light microscope to assess positivity. DLL3 positivity was scored at 4× magnification for any cytoplasmic or membranous staining at any intensity in total tumor cells, as previously described [19]. DLL3‐high was defined as greater than or equal to 75% stained tumor cells, as used in the ongoing Rova‐T clinical trial, and DLL3‐low was defined as less than 75% positivity. DLL3‐positive was defined as greater than or equal to 1% stained tumor cells. ASCL1 positivity was defined as nuclear staining in ≥5% of all tumor cells [23], [24]. DLL3 and ASCL1 scoring was only assessed in histologically SCLC cells, not in cancer cells histologically characterized as squamous cell carcinoma, adenocarcinoma, or large cell carcinoma components. Notch1 protein expression from previously reported data was used for analysis [25].

Statistical Analysis

The correlation between DLL3 or ASCL1 expression and categorical variables was analyzed using the chi‐square test or Fisher's exact test, as appropriate. Survival curves were estimated using the Kaplan‐Meier method, and differences in survival distributions were evaluated using the log‐rank test. The interrelationship between DLL3 and ASCL1 or Notch1 was analyzed using Spearman's rank analysis. The level of significance was set at p < .05. Statistical analyses were conducted with JMP software (JMP Pro version 11.0.0; SAS Institute, Cary, NC).

Results

DLL3 and ASCL1 Expression in SCLC

Patient characteristics are shown in Table 1. The median patient age was 70 years, 74 patients (77.9%) were male, and 81 patients (85.2%) were current or former smokers. The numbers of patients with pure SCLC and combined SCLC were 66 (69.5%) and 29 (30.5%), respectively. Seventy‐one, 13, and 11 patients had clinical disease stage (cStage) I, II, and III (TNM, 7th edition), respectively.

Table 1. Clinical characteristics of patients included in this study.

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Abbreviations: cStage, clinical disease stage; ECOG PS, Eastern Cooperative Oncology Group performance status; SCLC, small cell lung cancer.

Ninety‐three and 95 samples were evaluable for DLL3 and ASCL1 expressions by IHC staining, respectively. Representative images of DLL3 and ASCL1 staining and histograms detailing the distribution of expression are shown in Figure 1. DLL3 was localized in the cytoplasm and membrane of tumor cells, whereas ASCL1 was localized in the nucleus (Fig. 1C). Seventy‐seven out of 93 patients (83%) with any stage and 66 of 82 patients (80%) with cStage I or II were positive for DLL3 staining in at least 1% of tumor cells. Forty‐four patients (47%) with any stage and 35 patients (43%) with cStage I or II showed DLL3‐high expression (expression ≥75% of tumor cells; Table 2). Sixty‐one of 95 patients (64%) with any stage and 52 of 84 patients (62%) with cStage I or II showed positive expression for ASCL1 in at least 5% of tumor cells (Table 2).

Figure 1.

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Delta‐like protein 3 (DLL3) and achaete‐scute homolog‐1 (ASCL1) expression in small cell lung cancer (SCLC). (A): DLL3 protein expression levels, as determined by immunohistochemistry, in representative samples of positive (98%) and negative (0%) tumor specimens. (B): ASCL1‐positive (95%) and ‐negative (0%) tumor specimens. SCLC specimens were stained with an anti‐DLL3 or anti‐ASCL1 antibody (scale bars, 100 μm, right corner inset, lower magnification images). (C): Subcellular location of DLL3 and ASCL1 expression (red arrow, positive cell; yellow arrow, negative cell). (D): Histogram illustrating the distribution of DLL3 expression of any intensity in the 93 SCLC tumor specimens (arrow, proposed cutoff value for high DLL3 expression). (E): Histogram illustrating the distribution of ASCL1 expression of any intensity in the 95 SCLC tumor specimens (arrow, proposed cutoff value for positive expression of ASCL1).

Table 2. Patients according to rate of DLL3 or ASCL1 expression.

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Abbreviations: ASCL1, achaete‐scute homolog‐1; cStage, clinical disease stage; DLL3, delta‐like protein 3.

A positive correlation was observed between DLL3 and ASCL1 expression (R = 0.723, p < .0001; Figure 2). As shown in Figure 1A–C, DLL3 and ASCL1 were expressed in similar regions of the tumor specimen and coexpressed in the majority of tumor cells. Although DLL3 is a Notch ligand and ASCL1 is suppressed by Notch target genes [7], [8], there were no correlations between DLL3 or ASCL1 expression with Notch1 expression using the tumor specimens from our previous study (supplemental online Fig. 1) [25]. Moreover, we obtained data regarding the expression of neuroendocrine (NE)‐specific proteins, including chromogranin A, synaptophysin, and CD56, based on pathological reports of each surgical specimen when possible. There was significant correlation between the expression of ASCL1 and synaptophysin (p = .001), but there was no correlation between the expression of DLL3 and NE‐specific proteins (supplemental online Table 1).

Figure 2.

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Relationship between the expression of DLL3 and ASCL1. DLL3 and ASCL1 expression levels in small cell lung cancer tumor specimens showed a significant correlation (R = 0.723, p < .001).

Abbreviations: ASCL1, achaete‐scute homolog‐1; DLL3, delta‐like protein 3.

Correlation Between DLL3 or ASCL1 Expression and Clinicopathological Features

Next, we evaluated the association of clinicopathological features with high expression of DLL3 and expression of ASCL1. DLL3‐high expression was significantly more prevalent in patients with clinical lymph node status (cN) 2–3 than in those with cN 0–1 (p = .006; Fisher's exact test) and in patients with cStage III or IV than in those with cStage I or II (p = .022; Fisher's exact test; Table 3). No other clinicopathological features were correlated with DLL3 expression. ASCL1 expression was correlated with pure SCLC histology (p = .003; Table 3).

Table 3. Relationship between expression of DLL3 or ASCL1 and clinical and clinicopathological characteristics.

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Abbreviations: ASCL1, achaete‐scute homolog‐1; cN, clinical lymph node status; cStage, clinical disease stage; cT, clinical tumor classification; DLL3, delta‐like protein 3; ECOG PS, Eastern Cooperative Oncology Group performance status; SCLC, small cell lung cancer.

Prognostic Value of DLL3 and ASCL1 Expression

In the analysis of all study patients, the overall survival was 24.4 months in the DLL3‐high group and 33.3 months in the DLL3‐low group, with no significant difference existing between these two groups in terms of survival (p = .160; Fig. 3A). To exclude any bias of cStage in the DLL3 expression analysis, we analyzed patients with cStage I and II or cStage III and IV separately. Similar to the analysis of all patients, there was no correlation between DLL3 positivity and overall survival in patients with cStage I and II (p = .182) or with cStage III and IV (p = .641; Fig. 3C and supplemental online Fig. 2A). ASCL1 expression was not associated with survival in the entire study population (p = .096), in patients with pure SCLC (p = .111), and in patients with cStage I and II (p = .139; Fig. 3B and D and supplemental online Fig. S2B).

Figure 3.

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Kaplan‐Meier survival curves of all study patients. Survival curves for all patients with small cell lung cancer (SCLC) who underwent surgical resection, stratified by the expression of DLL3 (A) and ASCL1 (B) (n = 93 and n = 95, respectively). (C): Survival curves for patients with clinical stage III or IV SCLC who underwent surgical resection, stratified by the expression of DLL3 (n = 11). (D): Survival curves for patients with pure SCLC who underwent surgical resection, stratified by the expression of ASCL1 (n = 41).

Abbreviations: ASCL1, achaete‐scute homolog‐1; CI, confidence interval; DLL3, delta‐like protein 3; NA, not applicable; OS, overall survival.

Discussion

In this study, we demonstrated that both DLL3 and ASCL1 were highly expressed in patients with SCLC, with approximately half of these patients having DLL3‐high expression levels. Moreover, there was positive correlation between the expression of DLL3 and ASCL1. This is the first study to analyze the DLL3 and ASCL1 expression levels in surgically resected, early‐stage SCLC tumor specimens.

In our study, DLL3‐high expression levels were observed in 44 (47%) of the surgically resected specimens, which was similar to the expression rates of 67%, which was determined in a phase I trial of Rova‐T [19], and 32%, which was shown in a DLL3 IHC study from Japan [26]. In these studies, an anti‐DLL3 mouse monoclonal antibody was used and the cutoff rate was 50%, whereas in our study an anti‐DLL3 rabbit monoclonal antibody was used and the cutoff rate was 75%, which was in line with the protocol being used in several ongoing Rova‐T clinical trials. One study [18] evaluated DLL3 expression of normal lung samples, lung squamous cell carcinoma, lung adenocarcinoma, large cell neuroendocrine cell carcinoma (LCNEC), and SCLC. No normal lung specimen or lung squamous cell carcinoma tumor cells stained positively, and 3 of 82 (3.7%) lung adenocarcinoma had DLL3 expression, whereas 37 of 57 (65%) LCNEC, 120 of 167 (72%) treatment‐naïve SCLC, and 17 of 20 (85%) recurrent and treatment‐refractory SCLC had DLL3 expression, respectively. Furthermore, aside from SCLC, DLL3 protein expression was also strongly correlated with ASCL1 protein expression in small cell bladder cancer, and DLL3 was positive (expression in ≥1% of tumor cells) in 68% of patients, with 58% having expression in >10% of tumor cells [27]. Collectively, these findings indicate that DLL3 is highly expressed in small cell cancers, whereas its expression is low in normal lung or non‐small cell lung cancer. Because DLL3‐high expression was correlated to the response to Rova‐T treatment in a phase I trial [19], the results presented in several studies might be helpful for the development of DLL3‐targeted therapy. However, a phase III trial assessing the efficacy of Rova‐T versus topotecan as a second‐line treatment in patients with advanced SCLC and high levels of DLL3 resulted in stopping enrollment because of the shorter survival in the Rova‐T arm [28]. Other clinical studies for Rova‐T as first‐line maintenance therapy (NCT03033511) or combination therapy with immunotherapy (NCT03026166) are also ongoing. We need further data from ongoing studies to discuss the efficacy of Rova‐T in SCLC.

We showed that the expression of DLL3 was not related to the survival of patients with SCLC in our study population, which is consistent with another report from Japan that evaluated DLL3 expression in patients with SCLC and limited or extensive disease [26]. DLL3 itself may have little impact on the survival of patients with SCLC based on those data; however, patients with DLL3‐high or ASCL1‐positive expression tended to have worse survival. Therefore, adjuvant therapy with Rova‐T might have potential for patients with DLL3‐high or ASCL1‐positive expression. Our study showed that DLL3‐high expression was more prevalent in patients with lymph node metastasis and advanced cStage. However, the numbers of patients with advanced cStage was small; therefore, more investigation into this topic is required.

ASCL1 has been shown to promote tumor growth and is a therapeutic target for lung cancer with NE features [11], [12]. Moreover, DLL3 promotes the growth of murine lung cancer cells [29]; however, there are few analyses regarding the role of DLL3 in the tumorigenesis of human lung cancer. It has already been reported that DLL3 is a downstream target of ASCL1 [11], [12] and that the mRNA expression of both genes is highly correlated [18]. We also found that there was a positive correlation between DLL3 and ASCL1 protein expression, which supports the above previous results.

SCLC can be pathologically divided into pure SCLC and combined SCLC [21]. In this study, ASCL1 was expressed more in pure SCLC samples than in combined SCLC samples. To the best of our knowledge, there are no reports analyzing differences in ASCL1 expression between pure SCLC and combined SCLC samples, partly because of the difficulty in obtaining large tissue samples from patients, because surgery is rarely performed in SCLC cases. The genetic or molecular difference between pure SCLC and combined SCLC remains unknown. Moreover, the cell origin of combined SCLC remains unclear. SCLC components and non‐SCLC components in combined SCLC have been reported to share almost 75% common mutations and showed similar genetic background, suggesting that the SCLC components and non‐SCLC components are derived from common precursors [30]. Moreover, this also implies that one component of combined SCLC arises from other components with subsequent acquisition of oncogenic change and microenvironment in combined SCLC [30]. In our study, ASCL1 expression was lower in combined SCLC, and we hypothesize that ASCL1 expression may be lost during oncogenic change and through different microenvironments in combined SCLC. Recent molecular studies on SCLC cell lines suggest that SCLC could comprise two distinct subgroups with different expression patterns of ASCL1 and NEUROD1, which are associated with classic and variant SCLC, respectively, and which may distinguish SCLC heterogeneity using different genetic programs [31], [32], [33], [34]. These differences may influence therapeutic outcomes. For example, there have been reports that ASCL1‐enriched tumors were more sensitive to Bcl2 inhibition, whereas MYC‐driven variant tumors were more sensitive to an Aurora kinase inhibitor [32], [34], [35]. Pathological classification has no impact on the current treatments for SCLC, the relationship between pathological classification and subtypes in human SCLC cell lines remains unknown. However, the correlation between pure SCLC pathology and ASCL1 in this study indicates that pure SCLC possesses features of classic SCLC and may allow for multiple therapeutic options based on the expression levels of ASCL1.

DLL3 protein expression was significantly higher in Myc wild‐type genetically engineered mouse models than that in a Myc overexpressing model. TTF‐1 is also highly expressed in DLL3‐positive human SCLC samples [35]. ASCL1 expression is higher in Myc wild‐type models and in human SCLC samples with MYC‐low and TTF‐1 high expression [34], [35]. Moreover, inactivating Notch mutations were detected in human SCLC samples and induced ASCL1 activation [36]. These studies show that molecules such as MYC, TTF‐1, and Notch modulate DLL3 or ASCL1 and might contribute to treatment response or survival in SCLC.

This study has some limitations, including the small sample size (n = 95) and its retrospective, nonglobal design. Moreover, there was a limited number of deaths (n = 57, 60%). Various treatment regimens were used in a heterogeneous patient population, and it could introduce another bias.

Conclusion

DLL3 and ASCL1 were highly expressed in SCLC tumors, and DLL3 was related to lymph node metastasis and advanced cStage. A statistically significant correlation between DLL3 and ASCL1 expression was observed in our IHC analysis. Our data suggest that DLL3 and ASCL1 may be mainstream targets in the treatment of SCLC.

See http://www.TheOncologist.com for supplemental material available online.

Acknowledgments

This work was done in collaboration with Dr. Harumi Mukai (AbbVie GK, Tokyo, Japan) and Dr. Kumiko Isse (AbbVie Stemcentrx, South San Francisco, CA). We thank AbbVie Stemcentrx for performing immunohistochemistry assays.

Author Contributions

Conception/design: Megumi Furuta, Jun Sakakibara‐Konishi

Provision of study material or patients: Megumi Furuta, Hiroyuki Minemura, Masao Harada, Shigeo Yamazaki, Kenji Akie, Yuka Fujita, Kei Takamura, Tetsuya Kojima, Toshiyuki Harada, Yoshinori Minima, Naomi Watanabe, Hiroyuki Suzuki

Collection and/or assembly of data: Megumi Furuta, Hajime Kikuchi, Hiroshi Yokouchi, Hiroshi Nishihara, Hiroyuki Minemura, Masao Harada, Shigeo Yamazaki, Kenji Akie, Yuka Fujita, Kei Takamura, Tetsuya Kojima, Toshiyuki Harada, Yoshinori Minima, Naomi Watanabe

Data analysis and interpretation: Megumi Furuta, Jun Sakakibara‐Konishi, Satoshi Oizumi, Masaharu Nishimura, Hirotoshi Dosaka‐Akita, Hiroshi Isobe

Manuscript writing: Megumi Furuta, Jun Sakakibara‐Konishi

Final approval of manuscript: Megumi Furuta, Jun Sakakibara‐Konishi, Hajime Kikuchi, Hiroshi Yokouchi, Hiroshi Nishihara, Hiroyuki Minemura, Masao Harada, Shigeo Yamazaki, Kenji Akie, Yuka Fujita, Kei Takamura, Tetsuya Kojima, Toshiyuki Harada, Yoshinori Minima, Naomi Watanabe, Satoshi Oizumi, Hiroyuki Suzuki, Masaharu Nishimura, Hirotoshi Dosaka‐Akita, Hiroshi Isobe

Disclosures

The authors indicated no financial relationships.

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