Delta-like Protein 3 Expression Research Articles

Methylation-driven gene DLL3 is a potential prognostic biomarker in ocular melanoma correlating with metastasis.

Ocular melanoma is an aggressive malignancy with a high rate of metastasis and poor prognosis. Increasing evidence indicated that DNA methylation plays an important role in the occurrence and development of ocular melanoma. Hence, exploring new diagnostic and prognostic biomarkers at the genetic level may be beneficial to the prognosis of patients with ocular melanoma. We collected DNA methylation and gene expression profiles of human UM (uveal melanoma) and CM (conjunctival melanoma) samples from various datasets. We conducted differential methylation and expression analyses to screen the potential biomarkers. Correlation analysis was performed to investigate the relationships between the expression level of DLL3 (delta-like protein 3) and the methylation level of its corresponding CpGs. We explored the prognostic and diagnostic value of DLL3 in UM and CM. Functional annotation and GSEA (gene set enrichment analysis) were applied to get insight into the possible biological roles of DLL3. A cohort of 60 ocular melanoma patients as well as UM and CM cell lines were used to validate our findings in bioinformatic analyses. We found that DLL3 was a methylation-driven gene correlating with UM metastasis. The CpGs of DLL3 are mainly located in the gene body and their methylation level positively correlated to DLL3 expression. Multivariate Cox regression analysis revealed that DLL3 was an independent protective factor for UM patients. High DLL3 expression significantly prolonged the overall survival and disease-free survival of UM patients. DLL3 also showed a promising power to distinguish CM from normal tissues. Functional annotation exhibited that DLL3 may suppress UM progression through modulating immune activities and down-regulating various signaling pathways. External datasets, biospecimens, and cell lines further validated the aberrant expression and prognostic role of DLL3 in ocular melanoma. Methylation-driven gene DLL3 could serve as a new potential diagnostic and prognostic biomarker in ocular melanoma. Our findings may contribute to improving the clinical outcomes of patients with UM or CM.

Characteristics of Notch signaling pathway and its correlation with immune microenvironment in SCLC

ObjectivesTo explore the characteristics of Notch pathway in small cell lung cancer (SCLC), clarify the expression of delta-like protein 3 (DLL3) in SCLC patients undergoing surgical treatment, and explore the correlation between DLL3 expression and clinicopathological features, prognosis, and immune microenvironment. MethodsA total of 134 patients who received surgical treatment and were diagnosed as SCLC by postoperative histopathology were enrolled. All exon gene sequencing was performed in tumor tissues and adjacent normal tissues of 28 patients to clarify the genomic characteristics of SCLC. The expressions of DLL3 and programmed cell death 1 ligand 1 (PDL1) were detected by immunohistochemistry (IHC) in 101 postoperative pathological specimens. ResultsNotch receptor mutations are common, but the overall mutation rate of Notch ligand family is not high and mutually exclusive. The mutation status of Notch and Notch1 gene were not related to the prognosis. Notch1 gene mutation was significantly negatively correlated with the expression of PD-L1. DLL3 was highly expressed in SCLC and had no significant correlation with prognosis. There was a positive correlation between DLL3 expression and PD-L1 expression. Patients with positive DLL3 expression and no NOTCH 1 gene mutation had higher PD-L1 expression. SCLC patients with such characteristics may be more likely to benefit from immunotherapy. ConclusionThis study preliminarily explored the genomic characteristics and immune microenvironment of surgical resection of SCLC in China, and found the correlation between Notch gene mutation, DLL3 expression and PD-L1 expression, which provided a new idea for selecting appropriate treatment strategies for SCLC in the future.

ASCL1 and DLL3 expressions and their clinicopathological implications in surgically resected pure small cell lung cancer: A study of 247 cases from the National Cancer Center of China.

ObjectiveSmall cell lung cancer (SCLC) is one of the most aggressive malignancies characterized by neuroendocrine (NE) differentiation. The Delta‐like protein 3 (DLL3), as a direct downstream target of ASCL1, is involved in NE differentiation and carcinogenesis of SCLC. This study aims to investigate the relationship between ASCL1 and DLL3 expressions and their clinicopathological implications in SCLC.MethodsA total of 247 surgically resected pure SCLC samples with limited clinical stage and follow‐up data were retrieved in this retrospective study. ASCL1 and DLL3 protein expression was detected by immunohistochemistry staining. The correlations between ASCL1 and DLL3 expressions, as well as their clinicopathological features, were analyzed by χ2 tests. Disease‐free survival (DFS) and overall survival (OS) in SCLC patients with ASCL1/DLL3 low and high expressions were compared by the Kaplan‐Meier method and log‐rank tests.ResultsASCL1 high expression was detected in 105 (42.5%) patients. Its expression was positively correlated with the clinical stage (p = 0.02) and nerve invasion (p = 0.03). DLL3 high expression was observed in 188 (72.8%) patients and was correlated with vascular invasion (p = 0.04). ASCL1 expression was positively associated with DLL3 expression (p = 0.03). In addition, DLL3 expression has a strong correlation with the expression of thyroid transcription factor‐1 (TTF1) and conventional NE markers.ConclusionASCL1 and DLL3 were highly expressed in SCLC tumor samples, and a positive correlation between these two markers was observed. Co‐analysis of ASCL1 and DLL3 may identify a distinct SCLC subgroup benefit from targeted therapy. Therefore, ASCL1 and DLL3 could be potential biomarkers served for the selection of related patients.

Delta-Like Protein 3 Expression in Paired Chemonaive and Chemorelapsed Small Cell Lung Cancer Samples.

Rovalpituzumab tesirine (Rova-T), an antibody-drug conjugate directed against Delta-like protein 3 (DLL3), is under development for patients with small cell lung cancer (SCLC). DLL3 is expressed on the majority of SCLC samples. Because SCLC is rarely biopsied in the course of disease, data regarding DLL3 expression in relapses is not available. The aim of this study was to investigate the expression of DLL3 in chemorelapsed (but untreated with Rova-T) SCLC samples and compare the results with chemonaive counterparts. Two evaluation methods to assess DLL3 expression were explored. Additionally, we assessed if DLL3 expression of chemorelapsed and/or chemonaive samples has prognostic impact and if it correlates with other clinicopathological data. The study included 30 paired SCLC samples, which were stained with an anti DLL3 antibody. DLL3 expression was assessed using tumor proportion score (TPS) and H-score and was categorized as DLL3 low (TPS < 50%, H-score ≤ 150) and DLL3 high (TPS ≥ 50%, H-score > 150). Expression data were correlated with clinicopathological characteristics. Kaplan–Meier curves were used to illustrate overall survival (OS) depending on DLL3 expression in chemonaive and chemorelapsed samples, respectively, and depending on dynamics of expression during course of therapy. DLL3 was expressed in 86.6% chemonaive and 80% chemorelapsed SCLC samples without significant differences between the two groups. However, the extent of expression varied in a substantial proportion of pairs (36.6% with TPS, 43.3% with H-score), defined as a shift from low to high or high to low expression. TPS and H-score provided comparable results. There were no profound correlations with clinicopathological data. Survival analysis revealed a trend toward a more favorable OS in DLL low-expressing chemonaive SCLC (p = 0.57) and, in turn, in DLL3 high-expressing chemorelapsed SCLC (p = 0.42) as well as in SCLC demonstrating a shift from low to high expression (p = 0.56) without being statistically significant. This is the first study to investigate DLL3 expression in a large cohort of rare paired chemonaive-chemorelapsed SCLC specimens. Comparative analysis revealed that DLL3 expression was not stable during the course of therapy, suggesting therapy-based alterations. Unlike in chemonaive samples, a high DLL3 expression in chemorelapsed samples indicated a trend for a more favorable prognosis. Our results highlight the importance to investigate DLL3 in latest chemorelapsed SCLC tumor tissue.

P66.07 ASCL1 and DLL3 Expression and Their Clinicopathological Implications in Surgically Resected Pure Small Cell Lung Cancer

Small cell lung cancer (SCLC) is one of the most aggressive malignancies characterized by neuroendocrine (NE) differentiation. Our previous data revealed the achaete-scute homolog 1 (ASCL1) gene was a transcription factor in NE differentiation and highly expressed in SCLC. The Delta-like protein 3 (DLL3), as a direct downstream target of ASCL1, is involved in NE differentiation and carcinogenesis of SCLC. A DLL3-targeted antibody-drug conjugate, rovalpituzumab tesirine (Rova-T) has recently been developed for treatment of SCLC. This study was to investigate the relationship between ASCL1 and DLL3 protein expression and their clinicopathological implications in surgically resected pure SCLC. 247 surgically resected pure SCLC samples with limited clinical stage and follow-up data were retrieved in this retrospective study. ASCL1 and DLL3 protein expression was detected by immunohistochemistry staining in SCLC tissue microarrays. The correlation between ASCL1 and DLL3 protein expression as well as their clinicopathological features were analyzed by chi-square test. Disease-free survival (DFS) and overall survival (OS) in SCLC patients with ASCL1/DLL3 low and high expressions were compared by Kaplan-Meier method and Log-rank test. Among the 247 surgically resected pure SCLC patients, 175 (70.9%) were male and 202 (81.8%) were less than 65 years. According to the AJCC Cancer Staging Manual (seventh edition), 78 (31.6%) were stage I, 68 (27.5%) were stage II, and 101 (40.9%) were stage III. ASCL1 expression was localized in the nucleus of SCLC tumor cells, and 105 (42.5%) patients showed high expression. ASCL1 high expression was associated with clinical stage (p=0.019) and nerve invasion (p=0.030). DLL3 expression was localized in the cytoplasm and membrane of tumor cells, and DLL3 high expression was observed in 188 (72.8%) patients. DLL3 high expression was correlated with vascular invasion (p=0.045). ASCL1 expression was positively associated with DLL3 expression (p=0.030). In addition, DLL3 expression has a strong association with expression of classic NE markers, Synapsin (Syn) and Chromogranin A (CgA). Survival analysis revealed that patients with ASCL1 high expression have a worse OS (p=0.047), whereas further multivariate cox regression analysis showed neither ASCL1 nor DLL3 expression was independent prognostic factors. ASCL1 and DLL3 were highly expressed in pure SCLC tumor cells, and their expression level was positively correlated. The patients with ASCL1 and DLL3 high expression may represent a distinct subgroup of SCLC benefit from targeted therapy. Therefore, ASCL1 and DLL3 could be potential biomarkers served for selection of related patients.

Prevalence of Delta-Like Protein 3 in a Consecutive Series of Surgically Resected Lung Neuroendocrine Neoplasms.

Delta-like protein 3 (DLL3) is a protein of the Notch pathway, and it is a potential therapeutic target for high-grade lung neuroendocrine tumors (NETs), i.e., small cell lung carcinoma (SCLC) and large cell neuroendocrine carcinoma (LCNEC). However, DLL3 prevalence in lung NETs and its association with clinicopathological characteristics and prognosis remained unclear. We analyzed the immunohistochemical expression of DLL3 and its prognostic role in a consecutive series of 155 surgically resected lung NETs, including typical carcinoid (TC), atypical carcinoid (AC), LCNEC, and SCLC patients. The DLL3 expression was categorized as high (>50% positive tumor cells) or low (<50%). In addition, tumors were categorized by H-score (i.e., percentage of positive cells by staining intensity, ≥150 vs. <150). DLL3 staining was positive in 99/155 (64%) samples, and high DLL3 expression was frequently observed in high-grade tumors. In detail, 46.9% and 75% of SCLC and 48.8% and 53.7% of LCNEC specimens showed a high DLL3 expression by using H-score and percentage of positive tumor cells, respectively. Regarding low-grade NETs, only 4.9% and 12.2% TCs and 19.5% and 24.4% ACs had high DLL3 expression considering H-score and percentage of positive tumor cells, respectively. High DLL3 expression was associated with advanced American Joint Committee on Cancer (AJCC) stage, peripheral location, and chromogranin A expression in high-grade tumors (p < 0.05). In low-grade NETs, high DLL3 expression was associated with female sex, peripheral location, a higher number of mitoses, higher Ki-67 index, presence of necrosis, and pleural infiltration (p < 0.05). No association was observed between high DLL3 expression and overall survival (OS) and disease-free survival (DFS) in high-grade NETs, whereas high DLL3 expression was associated with lower DFS in ACs (p = 0.01). In conclusion, our study demonstrated a high prevalence of DLL3 expression in high-grade lung NET patients and its association with aggressive clinicopathological features. These findings confirm that DLL3 could represent a useful biomarker for target therapy in high-grade tumors. Our results also suggest that the DLL3 expression could identify a subset of AC tumors with more aggressive behavior, thus providing the basis for new therapeutic options in this group of patients.

DLL3 (delta-like protein 3) expression correlates with stromal desmoplasia and lymph node metastases in medullary thyroid carcinomas.

Medullary thyroid carcinomas (MTC) are rare and aggressive neuroendocrine tumors of the thyroid. About 70% of MTC are sporadic; approximately 50% of those harbor somatic RET mutation. DLL3 is widely expressed in many neuroendocrine tumors and has been evaluated as a potential therapeutic target. Since stromal desmoplasia in sporadic MTC has been identified as a reliable predictor of aggressive behavior and development of lymph node metastases, a possible correlation of DLL3 expression with the presence of stromal desmoplasia was of particular interest. 59 paraffin-embedded samples of sporadic MTC with (44 cases) and without (15 cases) stromal desmoplasia and known lymph node status were included. DLL3 expression was determined by immunohistochemistry; no expression (0%), low expression (1–49%) and high expression (≥50%) were correlated with clinicopathological data. The proportion of DLL3 positivity was significantly correlated with both stromal desmoplasia (P < 0.0001) and lymph node metastases (P < 0.0001). MTC without stromal desmoplasia consistently lack DLL3 expression. This is the first study to focus on MTC regarding DLL3 expression and the relationship to various factors. Our results demonstrate that expression of DLL3 in MTC represents a reliable surrogate marker for stromal desmoplasia and lymph node metastases and might be an indicator for aggressive clinical behavior. DLL3 expression in ≥50% of tumor cells virtually excludes MTC without stromal desmoplasia. DLL3 was discussed as a potential therapeutic target in malignant tumors of other locations with positive immunohistochemical reaction and might therefore be a new therapeutic option in MTC, as well.

Potential prognostic value of delta-like protein 3 in small cell lung cancer: a meta-analysis

BackgroundCurrent researches have revealed that delta-like protein 3 (DLL3) may be related with prognosis in patients with small cell lung cancer (SCLC). However, this finding remains controversial in small cell lung cancer. This meta-analysis was systematically performed to evaluate the prognostic value of DLL3 in SCLC.MethodsThe PubMed, EMBASE and Web of Science databases were retrieved to collect the eligible references. Through Stata 15.0 software, we pooled hazard ratios (HR) with 95% confidence intervals (CI) by using random or fixed-effects models to evaluate the association between DLL3 and SCLC survival results.ResultsA total of 6 inter-related studies including 645 patients were qualified. After we removed 1 study, the remaining 5 studies including 601 patients were pooled to testify that high expression of DLL3 was an inferior prognostic for patients with SCLC in Asian populations (HR = 1.37, 95% CI = 1.05, 1.69; I2 = 0.0%, p = 0.000). The pooled results showed that DLL3 might be higher expression in advanced metastasis SCLC in Asian populations (RR = 0.84, 95% CI = 0.71, 0.99; I2 = 44.7%, p = 0.039). But the expression of DLL3 was not correlated with sex (RR = 1.33, 95% CI = 0.98, 1.80; I2 = 0.0%, p = 0.064), smoking history (RR = 1.01, 95% CI = 0.58, 1.75; I2 = 72.1%, p = 0.967) and tumour stage (RR = 0.68, 95% CI = 0.44, 1.05; I2 = 66.6%, p = 0.081).ConclusionsOur meta-analysis confirms that in Asian populations, high expression of DLL3 was a potential poor prognostic biomarker for SCLC and DLL3 highly expressed in advanced stage SCLC in Asian populations.

International real-world study of DLL3 expression in patients with small cell lung cancer

ObjectivesExpression of the Notch-family ligand delta-like protein 3 (DLL3), a potential therapeutic target in small cell lung cancer (SCLC), has not been assessed in the real-world setting. To identify the real-world utility of DLL3 as an SCLC therapeutic target, we performed the largest retrospective international noninterventional study to date to evaluate DLL3 prevalence in SCLC patients. Materials and MethodsDLL3 expression was assessed using immunohistochemistry in archived histological and cytological specimens (independent and paired) and correlated to patient demographics, clinical disease characteristics, and survival. The primary endpoint was the proportion of patients with DLL3 expression in ≥25 % of tumor cells. DLL3 expression concordance was assessed in paired specimens. ResultsIndependent tumor specimens were collected from 1073 patients. The mean age at biopsy was 66 years (SD, 10); 682 (64 %) patients were male. Paired specimens were collected from 36 patients. The mean age at biopsy was 62 years (SD, 11); 16 (44 %) patients were male. Most patients had ECOG performance status of 0–1, were smokers/ex-smokers, and received ≥1 prior therapy. Positive DLL3 expression (defined as ≥25 % of tumor cells) was identified in 895/1050 (85 %) patients with 1 specimen and evaluable DLL3 expression; 719/1050 (68 %) patients had high DLL3 expression (defined as ≥75 % of tumor cells). DLL3 expression concordance was 88 % between paired specimens (n = 17; Cohen’s kappa P value, .9412). There was no significant difference in median overall survival from SCLC diagnosis for evaluable patients with nonmissing data based on DLL3 expression (negative DLL3 expression [n = 139], 9.5 months; positive DLL3 expression [n = 747], 9.5 months; all evaluable patients [n = 893, 9.5 months). ConclusionThese real-world epidemiologic findings indicate that DLL3 is robustly expressed across SCLC disease stages and remains stable despite treatment, consistent with available clinical trial data. There was no prognostic role for DLL3 observed in this study for overall survival.

Prevalence of DLL3, CTLA-4 and MSTN Expression in Patients with Small Cell Lung Cancer.

IntroductionImmune-based and antibody-drug conjugate therapies have shown promise in the treatment of patients with small cell lung cancer (SCLC). However, better predictive biomarkers are needed for selection of the appropriate SCLC patients for these advanced therapies and also for evaluation of the efficacy of these treatments.ObjectiveThe aim of this study was to examine the expression of delta-like protein 3 (DLL3), cytotoxic T lymphocyte-associated protein 4 (CTLA-4), and mesothelin (MSTN) in patients with SCLC and compare them with those patients’ clinical characteristics.MethodsImmunohistochemical analyses of DLL3, CTLA-4 and MSTN expression were performed in 38 samples from patients with SCLC.ResultsWe found that positive expression in patients of the biomarkers was as follows: for DLL3, 100% (38/38), for CTLA-4, 89.5% (36/38) and for MSTN 81.5% (31/38). The median survival time was 17.9 months in the DLL3 high expression group and 23 months in the DLL3 low expression group. Patients with a high expression of DLL3 showed a poorer prognosis than those with a low expression of DLL3 (HR=3.4; 95% CI, 1.34–8.6; p=0.01).ConclusionThe expression of DLL3, CTLA-4 and MSTN was not correlated with patients’ age, sex, smoking status, stage, and tumor metastasis. The fact that there was a higher expression of DLL3, CTLA-4, and MSTN in SCLC suggested that these molecules could be used as predictive biomarkers for SCLC.

P2.12-18 Prevalence of DLL3 Expression and Its Prognostic Role in Extensive Stage Small Cell Lung Cancer

Extensive stage small cell lung cancer (ES-SCLC) is a highly aggressive disease with a very poor prognosis, and there is a highly unmet need to develop new treatment strategy for them. Rovalpituzumab tesirine (Rova-T), an antibody drug conjugate directed against Delta-like protein 3 (DLL-3) has shown good efficacy in phase I and II clinical trials for the patients with ES-SCLC. We herein examined the prevalence of DLL3 protein expression and its prognostic role in ES-SCLC patients. Tumor samples of ES-SCLS were subjected to immune-histochemical staining for DLL3 (AbbVie Stemcentrx). Expression in at least 50% of cancer cells were defined as DLL3-high. The association of patients’ characteristics including gender, metastatic sites, and smoking history with DLL3 expression was analyzed by medical chart review. The correlation of DLL3 protein expression status with chemotherapy effect (PFS, OS and response rate) was analyzed. All the statistical analyses were performed by SPSS ver. 18.0 program. A total of 56 patients' sample were included in our analysis. The median age was 54 with a range of 44 to 86, and the majority of patients (N=49, 89.1%) are male. Above 90 percent of patients (N=50, 90.9%) received etoposide and cisplatin as chemotherapeutic regimen, and among the 47 patients evaluable for the response, the objective response rate (ORR) was 61.7% (1 complete response and 28 partial response). The median progression free survival (PFS) and overall survival (OS) was 175 days (95% confidence interval (CI), 130.4∼220.0) and 250 days (95% CI, 184.0∼316.0), respectively. DLL-3 high expression was observed in 91% of patients (N=50). It is not correlated with any of the patients’ characteristics and response to chemotherapy. In addition, there was no difference in PFS (242.0 vs. 165.0 days, p=0.900) or OS (160.0 vs. 250.0 days, p=0.975) according to DLL-3 expression. Our study showed that DLL3 high expression was found in almost all tumor samples in ES-SCLC. Also, its expression was not correlated with the patients’ characteristics and prognosis of them. Further studies are warranted.

1748P - Phase I open-label study evaluating the safety, pharmacokinetics, and preliminary efficacy of ABBV-181 and rovalpituzumab tesirine (ROVA-T) in patients with small cell lung cancer

1748P - Phase I open-label study evaluating the safety, pharmacokinetics, and preliminary efficacy of ABBV-181 and rovalpituzumab tesirine (ROVA-T) in patients with small cell lung cancer

Abstract 3171: Delta-like protein 3 expression in Merkel cell carcinoma

Abstract Purpose: Delta-like protein 3 (DLL3) is being developed as a predictive biomarker to identify small cell lung cancer (SCLC) patients for treatment with the DLL3-targeting antibody-drug conjugate rovalpituzumab tesirine (Rova-T). Merkel cell carcinoma (MCC) is an aggressive neuroendocrine carcinoma of the skin, associated with Merkel cell polyomavirus. Given the neuroendocrine features of SCLC and MCC, we sought to evaluate DLL3 expression, its association with common clinicopathological factors, and its prognostic role in MCC. Methods: A total of 65 formalin-fixed and paraffin-embedded MCC cases were consecutively identified from an institutional biospecimen repository and were cut at 5 µm. Each case was stained for DLL3 protein with SC16.65 mouse monoclonal antibody at 3 µg/mL with the FLEX+ system (DAKO). Slides were then scanned at 20X with AT2 (Leica/Aperio) and read out for percentage of positive tumor cells with 1+, 2+, and 3+ intensity side-by-side with isotype control (IgG2a). Any subcellular staining pattern (membranous, cytoplasmic or punctate) was counted as overall positive. Polyomavirus status was determined by immunohistochemistry (IHC) using an antibody that binds to MCPyV large T-antigen (sc-136172, Santa Cruz Biotechnology, Inc.). A generalized additive model was used to visualize the trend between common clinical factors and overall survival (OS). A multivariable Cox proportional hazards model was applied to assess the association between DLL3 expression and OS. The association between DLL3 expression and other clinicopathological factors was evaluated with median and t tests. Results: A total of 67 patients were included with a median age of 74 years. Thirty-six (55%) had stage I/II disease; 29 (45%) had stage III/IV disease. Thirty-five (52%) patients died from MCC. Among the 65 cases with successful DLL3 stain, the median H-score of DLL3 expression was 60 (interquartile range: 30-100). Fifty-eight cases (89%) had ≥1% tumor cells positive for DLL3 expression with any intensity, of which the median DLL3 expression was 50% (interquartile range: 25-70%). Thirty-four cases (52%) had ≥50% tumor cells positive for DLL3 expression with any intensity. Older age significantly predicted shorter overall survival (p = 0.03). However, higher AJCC stage did not predict shorter OS (p = 0.3). H-score of DLL3 expression was not associated with AJCC stages (p = 0.4). However, higher H-score of DLL3 expression was associated with higher polyomavirus nuclear expression (p = 0.02) when it was dichotomized to 0/1+ and 2+/3+. In a multivariable Cox regression analysis, H-score of DLL3 expression did not predict OS of patients with MCC (p = 0.8). Conclusions: DLL3 overexpression is very common in MCC by IHC. DLL3 expression was significantly associated with Merkel cell polyomavirus expression but was not prognostic for OS in this cohort. The high levels of DLL3 expression in a subset of MCC may potentially be used to select patients to receive Rova-T. Citation Format: Hao Xie, Kumiko Isse, Yan Sun, Johanna Ramoth, Dorothy M. French, Laura R. Saunders, Aaron S. Mansfield. Delta-like protein 3 expression in Merkel cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3171.

Abstract 4029: Analysis of DLL3 and ASCL1 in surgically resected small cell lung cancer (HOT1702)

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 and has shown improved clinical response in patients with high DLL3 expression. 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 formalin-fixed and paraffin-embedded SCLC samples, which were surgically resected at 11 institutions participating in either the Hokkaido Lung Cancer Clinical Study Group Trial (HOT) or the Fukushima Investigative Group for Healing Thoracic Malignancy (FIGHT) between 2003 and 2013. Immunohistochemistrystaining 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 and ASCL1 were more prevalent in patients with advanced clinical disease and pure SCLC, respectively. Conclusion:DLL3 and ASCL1 were highly expressed in surgically resected SCLC patients. Citation Format: 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 Minami, Naomi Watanabe, Satoshi Oizumi, Hiroyuki Suzuki, Masaharu Nishimura, Hirotoshi Dosaka-Akita, Hiroshi Isobe. Analysis of DLL3 and ASCL1 in surgically resected small cell lung cancer (HOT1702) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4029.

Prognostic value of delta-like protein 3 combined with thyroid transcription factor-1 in small-cell lung cancer.

Rovalpituzumab tesirine is a promising delta-like protein 3 (DLL3)-targeted antibody-drug conjugate for the treatment of small-cell lung cancer (SCLC). Thyroid transcription factor-1 (TTF-1) and DLL3 protein are associated with SCLC, and may be used to identify patients, who respond to the DLL3-targeted therapy. However, little is known about the expression pattern of the DLL3 protein, and the prognostic value of DLL3 and TTF-1 for SCLC. A total of 335 patients with SCLC were identified, including 11 patients with paired biopsy of primary site and lobectomy specimens, and 37 patients with paired specimens of primary and metastatic site. The DLL3 expression levels of individuals were evaluated using the anti-DLL3 antibody. No differences in DLL3 expression levels were observed in paired biopsy and lobectomy specimens (P=0.774), and paired primary and metastatic sites (P=0.472). SCLC cases with high DLL3 expression levels were more frequent in male patients (P=0.041), smokers (P=0.023) and patients with positive TTF-1 expression (P=0.006) compared with DLL3-low SCLC. DLL3-high SCLC exhibited worse overall survival compared with DLL3-low SCLC (log-rank test, P=0.007). Patients with TTF-1+ SCLC experienced a significantly worse overall survival compared with patients with TTF-1- SCLC (P<0.001). DLL3-low/TTF-1- was defined as a distinct molecular subgroup of SCLC with optimal prognosis (P<0.001). DLL3-low/TTF-1- was an independent prognostic marker for SCLC (P=0.001). In conclusion, the present study, to the best of our knowledge, provided novel evidence for SCLC intratumoral and intertumoral homogeneity with the identification of DLL3 protein levels. Therefore, it is reliable to use biopsy specimens to evaluate DLL3 expression levels for identification of patients who may benefit from DLL3-targeted therapy. In addition, DLL3 and TTF-1 are two protein markers with potential clinical value in risk stratification for patients with SCLC.

DLL3 is regulated by LIN28B and miR-518d-5p and regulates cell proliferation, migration and chemotherapy response in advanced small cell lung cancer

Delta-like protein 3 (DLL3) has been reported as a biomarker in various human tumors. However, the biological function and mechanism of it in advanced small cell lung cancer (SCLC) is rarely reported. This study was devised innovatively to explore the role of DLL3 in the progression of SCLC. Immunohistochemistry (IHC) analysis was used to examine DLL3 expression in paraffin-embedded SCLC tumor samples. Upregulation of DLL3 reduced chemotherapy sensitivity. Kaplan-Meier analysis was used to analyze the progression-free survival or overall survival of SCLC patients with high or low level of DLL3. The negative association between DLL3 expression and the PFS or OS rate of SCLC patients was identified. Relative high level of DLL3 was determined in SCLC cell lines by using qRT-PCR analysis. Loss-of function assays were performed to detect the biological functions of the silencing of DLL3 in SCLC. As a result, silencing of DLL3 led to the proliferative and migratory inhibition of SCLC cells and reversed EMT process. Mechanistically, DLL3 mRNA was stabilized by the RNA-binding protein lin-28 homolog B (LIN28B). Further mechanism investigation revealed that LIN28B and DLL3 are two downstream targets of miR-518d-5p. Finally, rescue assays demonstrated that LIN28B and miR-518d-5p could regulate DLL3-mediated cell proliferation and migration. Collectively, our present study revealed a novel molecular pathway in SCLC, which providing a new insight in exploring the therapeutic strategy for SCLC.

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

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

DLL3 regulates the migration and invasion of small cell lung cancer by modulating Snail.

Delta‐like protein 3 (DLL3) is a ligand of Notch signaling, which mediates cell‐fate decisions and is tumor‐suppressive or oncogenic depending on the cellular context. Previous studies show that DLL3 is highly expressed in small cell lung cancer (SCLC) but not in normal lung tissue, suggesting that DLL3 might be associated with neuroendocrine tumorigenesis. However, its role in SCLC remains unclear. To investigate the role of DLL3 in tumorigenesis in SCLC, we performed loss‐of‐function and gain‐of‐function assays using SCLC cell lines. In vitro analysis of cell migration and invasion by transwell assay showed that DLL3 knockdown reduced migration and invasion of SCLC cells, whereas DLL3 overexpression increased these activities. In addition, DLL3 positively regulated SNAI1 expression and knockdown of SNAI1 attenuated the migration and invasion ability of SCLC cells. Moreover, upregulated DLL3 expression induced subcutaneous tumor growth in mouse models. These results indicate that DLL3 promoted tumor growth, migration and invasion in an SCLC model by modulating SNAI1/Snail.

Delta-like Protein 3 Prevalence in Small Cell Lung Cancer and DLL3 (SP347) Assay Characteristics.

Delta-like protein 3 (DLL3) is a protein that is implicated in the Notch pathway. To present data on DLL3 prevalence in small cell lung cancer and staining characteristics of the VENTANA DLL3 (SP347) Assay. In addition, the assay's immunoreactivity with other neoplastic and nonneoplastic tissues is outlined. Individual formalin-fixed, paraffin-embedded specimens of small cell lung cancer and tissue microarrays comprising neoplastic and nonneoplastic tissues were procured. Sections were cut and stained with DLL3 (SP347) assay. The slides were examined to determine prevalence, staining characteristics, and immunoreactivity. Cytoplasmic and/or membranous staining was observed in 1040 of 1362 specimens of small cell lung cancer (76.4%). Homogenous and/or heterogeneous and partial and/or circumferential granular staining with varied intensities was noted. Immunoreactivity was also observed in other neoplastic and nonneoplastic tissues. Our study findings provided the profile of DLL3 staining characteristics that can be used for determining the level of DLL3 expression in small cell lung cancer.

Upregulated delta-like protein 3 expression is a diagnostic and prognostic marker in endometrial cancer: A retrospective study.

Upregulated delta-like protein 3 (DLL3) functions as a Notch ligand and has been a target for cancer therapy. The present study assessed DLL3 expression as a tumor marker for endometrial cancer.RNA-Seq expression data and clinicopathologic records from 545 patients with endometrial cancer were downloaded from The Cancer Genome Atlas database. Mann–Whitney U and logistic regression tests were applied to associate the level of DLL3 expression with clinical variables from the patients. Kaplan–Meier curves and log-rank tests were performed to compare overall survival of patients stratified by different levels of DLL3 expression. Multivariate Cox regression tests were used to analyze independent predictors for endometrial cancer. DLL3 expression was upregulated in endometrial cancer tissues compared to para-carcinoma tissues (P = .0003). High DLL3 expression was associated with the age of patients (odds ratio [OR] = 1.74), advanced stages of the International Federation of Gynecology and Obstetrics system (OR = 2.9), grade III/IV (OR = 5.1), myometrial invasion (OR = 2.2), pelvic involvement (OR = 12.9), and para-aortic lymph node metastasis (OR = 9.9) (all P ≤ .001). Furthermore, upregulated DLL3 expression was also associated with a median overall survival of 112 months (HR = 1.85, confidence internal 1.202–2.846, P = .005). The multivariate analysis showed that DLL3 overexpression and advanced tumor stages, grades, and lymph node metastases were all independent prognostic predictors for endometrial cancer.The DLL3 expression could be a potential and novel tumor marker for early diagnosis and an independent predictor of poor survival for patients with endometrial cancer.