Background

Lung squamous cell carcinoma (LUSC) patients suffer from less targetable onco-drivers and potentially acquire clinical benefit from immune checkpoint inhibitors (ICIs). DNA content aberrations contribute to genomic instability and are initial events of tumorigenesis. We established a non-diploidy related prognostic molecular signature (NPMS) based on differentially expressed genes (DEGs) and investigated the immune features of it.

Methods

We conducted a retrospective analysis based on data downloaded from TCGA database. By integrating CNA and SNV via ABSOLUTE algorithm, we obtained the DNA ploidy status and divided patients into near-diploid and non-diploid group. DEGs were selected and gene functional enrichments were carried out. NPMS was established by all subset multivariate Cox regression to further stratify patients and validated by integrative analysis of GSE73403, GSE41271, GSE4212. Gene sets enrichment analysis was applied to further figure out the mechanism behind NPMS.

Results

Non-diploidy coincided with higher TMB and intratumor heterogeneity. Functional enrichment indicated that DEGs mainly participated in genomic instability. NPMS containing HOXB5, TINAGL1, POLR3GL, APOB, FABP6, SCARF1 was established. Patients with low score had better OS than those with high score (HR 0.60, 95%CI 0.45-0.79, p-value=0.000321). This was validated in the GEO cohorts (HR 0.57, 95%CI 0.37-0.88, p-value=0.0121). Dysregulation of DNA repair and cell cycle checkpoints were higher enriched in high score group. The immune phenotype of high score tended to be "immunologically hot", which was characterized as high density but low activity immune cells infiltrated, accompanied by expression of higher immunosuppressive factors, such as PD-1, CTLA4, IDO1. Meanwhile, the upregulation of IFN-γ signaling pathway, lipid alternation and the high correlation between them were observed.

Conclusions

High NPMS score corresponded with an "immunologically hot" feature thereby led to shorter OS. The crosstalk of upregulated IFN-γ signaling and aberrations of tumor metabolism mignt participate. It's rational to deduced that patients with high score might be the potential ICIs benefited subpopulation.

Legal entity responsible for the study

Z. Hu.

Funding

This work was supported by Horizontal Scientific Research Project of China-Japan Friendship Hospital (Grants No. 2018-HX-26).

Disclosure

All authors have declared no conflicts of interest.

Background

Prevention and early detection of distant metastasis are critical for improving the outcome of patients with lung cancer. Previously, we showed that the presence of isolated basal cell hyperplasia (BCH) in the small bronchi distant from the tumor is associated with a high risk of distant metastasis in patients with lung squamous cell carcinoma (LUSC) and adenocarcinoma. However, mechanisms underlying this phenomenon are unknown. Here we aimed to assess the mutational landscape of metastatic LUSC associated with isolated BCH.

Methods

The study included 10 (48 to 77 years old) patients with LUSC (T_1-4N_0-2M₀) divided into three groups: 1) patients with isolated BCH and metastases; 2) patients with isolated BCH but without metastases; and 3) patients without premalignant bronchial lesions and metastases. Premalignant lesions were analyzed in hematoxylin and eosin-stained sections of formalin-fixed paraffin-embedded samples of small bronchi distant (3-5 cm) from the tumor. Tumor samples were sequenced on a NextSeq 500 (Illumina) using SureSelect XT Human All Exon v7 kit (Agilent).

Results

LUSC patients with BCH and metastases showed 1.6-times more mutations than cases without premalignant bronchial lesions and metastases. Interestingly, among patients with BCH, mutations were 1.8-times more in metastatic tumors than in non-metastatic tumors confirming that the presence of hyperplasia is not absolute for distant metastasis. Metastatic LUSC more often also harbored mutations in genes involved in cell-cell adhesion (CDH8, CDH9, ITGAL, SDK1, SDK2, et al.) and signal pathways of carcinogenesis (TP53, RB1, MMP1, MMP2, EGFR, et al.). Importantly, the most frequently mutated genes in metastatic tumors were TP53 (100% of cases) and PTPRT (75% of cases).

Conclusions

Taken together, our findings indicate that metastatic LUSC associated with isolated BCH shows a highly mutable phenotype.

Legal entity responsible for the study

Cancer Research Institute, Tomsk National Research Medical Center.

Funding

Russian Science Foundation, #20-75-10060.

Disclosure

All authors have declared no conflicts of interest.

Background

MiRNAs have been thoroughly studied for their vast roles in oncology, however, recent immune-related miRNAs have been recognized as prospective modulators in the tumor immune surveillance process. The high immunogenicity of TNBC along with its stubbornness towards effective targeted therapy shifted therapeutic interests towards immunotherapy. Accordingly, a promising approach would be targeting the immune suppressive tumor microenvironment (TME). Such an approach could be manifested in a dual manner through blocking prominent checkpoint proteins and regulating Natural Killer (NK) cells and thus modifying the TME. MiR-939-5p is a scarcely explored miRNA especially in TNBC. Therefore, our aim is to identify the expression profile of miR-939-5p in BC tissues and the role it plays in tumor suppression as well as the immunomodulatory role on checkpoint proteins and NKG2D ligands in TNBC.

Methods

Breast biopsies were collected from 40 BC patients. MDA-MB-231 cells were cultured and transfected with different oligonucleotides. Total RNA was extracted and quantified by qRT-PCR. Cellular viability, colony forming ability and migration were measured using MTT, colony forming and scratch assays, respectively.

Results

MiR-939-5p was down-regulated in TNBC tissues compared to normal tissues and other BC subtypes. Functionally, forced expression of miR-939-5p ensued a decline in cellular viability, colony forming ability and migration capacity of MDA-MB-231 cells thus highlighting miR-939-5p as a tumor suppressive miRNA in TNBC. Immunomodulation wise, miR-939-5p overexpression resulted in down-regulation of the checkpoint protein PD-L1 as well as upregulation of the shedded NKG2D ligands MICA/B in TNBC cells. Lastly, drastic repression of the immune inhibitory cytokines, TNF-α and IL-10 was detected upon miR-939-5p upregulation.

Conclusions

The present study categorized miR-939-5p as a tumor suppressing miRNA under expressed in TNBC patients and cell lines. Furthermore, it institutes the first major stride in unraveling the immunomodulatory role of miR-939-5p in advancing NK cells cytotoxicity and trimming TME in favor of TNBC eradication, thus proposing miR-939-5p as a novel therapeutic module in TNBC.

Legal entity responsible for the study

German University in Cairo.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

Background

Recently, hydrogen sulphide (H₂S) has been acknowledged as a pivotal gasotransmitter altering several oncogenic signaling pathways in breast cancer (BC). Our research group has recently identified H₂S and its synthesizing enzyme cystathionine-γ-lyase (CSE) as promising targets for BC treatment especially that their expression pattern was closely associated with aggressive tumor phenotypes. However, the motor engines responsible for such high expression pattern of CSE/H₂S in BC patients are yet to be explored. Transcriptomic analysis for CSE promoter region has revealed STAT-3 as a direct regulator for CSE. Long non-coding RNAs (lncRNAs) have recently been identified as vital post-transcriptional regulators. Yet, lncRNAs regulating H₂S production is still a virgin field. MALAT-1 is an oncogenic lncRNA tuning self-renewal capacity of BC cells. However, the role of MALAT-1 in modulating STAT-3-regulated CSE has never been probed in BC. Therefore, our aim is to investigate the role of MALAT-1 in regulating STAT-3/CSE/H₂S axis.

Methods

BC biopsies and their normal counterparts were collected from 20 BC patients. MDA-MB-231 cells were cultured and transiently transfected using MALAT-1 and CSE siRNAs by lipofection method. Total RNA was extracted using Biozol reagent, reverse transcribed and quantified using qRT-PCR. Western blot analysis was performed.

Results

Screening showed an upregulation of MALAT-1, STAT-3 and CSE in aggressive BC tissues. Upon knocking down of MALAT-1, a marked repression of STAT-3 and CSE was observed nominating MALAT-1 as a novel upstream lncRNA regulating STAT-3/CSE axis. On the other hand, CSE siRNAs resulted in the induction of MALAT-1 expression level, STAT-3 transcript and phosphorylated forms in attempt to restore H₂S levels in BC cells.

Conclusions

MALAT-1 was identified as the first lncRNA regulating STAT-3/CSE/ H₂S in BC. Furthermore, this study underscores the significance of dual targeting of MALAT-1 and CSE for efficient repression of H₂S levels in BC cells by-passing the compensatory mechanism exercised by CSE.

Legal entity responsible for the study

German University in Cairo.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

Background

Patient-reported adverse events may be a useful adjunct for assessing a drug’s tolerability in DPOT. A review of DPOT registered on clinicaltrials.gov showed increasing use of PROs over time, but overall use remained limited. Little is known about the reasons for limited PRO use. We conducted a global online survey of key stakeholders to understand attitudes towards PROs in DPOT.

Methods

A 35-question survey of clinicians, trial managers, statisticians, funders and regulators involved in DPOT in hospitals, academia or industry distributed via professional bodies. Questions focussed on prior experience of designing, conducting and reporting DPOT with PROs, attitudes towards benefits and barriers to PRO use and their potential role in defining tolerable doses. Adaptive questioning was used. No identifiable data was collected.

Results

112 responses from 15 September to 30 November 2020; 103 trialists [48 clinicians (42.9%), 38 statisticians (34.0%), 17 trial managers (15.2%)], 7 regulators (6.3%) and 2 funders (1.8%). Most had worked in DPOT for 6-10 years (35, 31.3%). Most worked in the United Kingdom (65, 58.0%) or United States (22, 19.6%). Most trialists had no prior experience designing (73, 70.9%), conducting (52, 50.5%) or reporting (88, 85.4%) PROs in DPOT. Respondents strongly agreed/ agreed that PROs could identify new toxicities (75, 67.0%), provide data regarding frequency (86, 76.8%) and duration (81, 72.3%) of toxicities, especially in agents with moderate, chronic or delayed toxicities (77, 68.8%). Respondents agreed that PROs should be reviewed when making dose-escalation decisions (61, 54.5%), when determining the maximum tolerated dose (63, 56.3%) and recommended phase II dose (76, 67.9%). Top 5 concerns were: lack of guidance regarding PRO selection (73/103, 70.9%), missing PRO data (79/112, 70.5%), overburdening staff (68/103, 66.0%) or patients (57/103, 55.3%), and analysis and publication (58/112, 51.8%).

Conclusions

Key stakeholders reported minimal experience collecting and analysing PROs in DPOT. However, there was broad support for using PROs to inform selection of tolerable doses. Guidelines are needed to standardise selection, analysis and reporting, and increase efficiency of PRO collection in DPOT.

Legal entity responsible for the study

Christina Yap.

Funding

Has not received any funding.

Disclosure

A. Minchom: Honoraria (institution), Advisory/Consultancy: Merck; Honoraria (institution), Advisory/Consultancy: Faron; Honoraria (institution), Advisory/Consultancy: Novartis; Honoraria (institution), Advisory/Consultancy: Bayer; Honoraria (institution), Advisory/Consultancy: Janssen. All other authors have declared no conflicts of interest.

Background

Immune check-point inhibitors (ICPi) are routinely used in cancer treatment in the United Kingdom and are associated with immune-related adverse events (irAE). Most patients with autoimmune disease (AID) are often excluded from clinical trials investigating ICPi, due to hypothetical increased risks of AID flare or severe irAE, but these risks have never been conclusively verified. We present experience using ICPi at a UK tertiary cancer centre, comparing the irAE profile and efficacy in patients with pre-existing AID to those without.

Methods

This was a single centre retrospective analysis of electronic records of solid tumour patients who received treatment with an ICPi from January 2017 to December 2020. From a total of 562 patients, 15 had pre-existing AID. These were compared to those without AID in a 1:3 matched cohort for tumour-type and treatment.

Results

The AID cohort had inflammatory bowel disease (N=5), psoriasis (N=3), rheumatoid arthritis (N=2), polymyalgia rheumatica (N=2), autoimmune endocrinopathies (N=4), lupus (N=1) and sarcoidosis (N=1). 3 patients had 2 co-existing AID. All 15 patients had well controlled AID. 9 were on hormone replacement, immunosuppressive (prednisolone <10mg, n=2, mesalazine, n=1), or topical agents. 6 were not on AID treatment. The table below lists patient demographics, irAE and outcomes. 3 out of 15 AID patients had irAE possibly linked to their underlying AID (1 psoriasis, 1 RA, 1 colitis; no G3 or higher). No significant differences were found in the number of G3 or higher irAE, treatment discontinuation due to irAE, response to treatment, or survival between cohorts. Table: 31P

Patient demographics, irAE and outcomes

Conclusions

Exacerbation of pre-existing AID was infrequent and did not result in severe toxicity. Patients with well-controlled AID could be considered for entry in clinical trials involving ICPi.

Legal entity responsible for the study

The authors.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

Background

The hormonal therapy is among the most effective treatment of the hormone-dependent breast cancer however its efficiency is limited by the acquired resistance to the hormonal drugs. Previously we have shown the exosomes involvement in the transferring of the hormonal resistance from the resistant to the sensitive cells, and here the study of the features of the exosomes of the resistant cells was performed.

Methods

Experiments were performed on the MCF-7 breast cancer cells, MCF-7/T resistant subline developed under long-term tamoxifen treatment, and MCF-7/exoT resistant subline developed under resistant exosomes treatment. Exosomes were prepared from the conditioned medium by the differential ultracentrifugation, and exosome imaging was carried out by transmission electron microscope. The analysis of exosomal microRNAs was performed by HiSeq2500 and at least 5 million reads per samples were obtained. Library preparation was carried out with NEBNext® Small RNA Library Prep Set for Illumina® (E7330S). More than 2500 miRNAs were identified in the exosomal samples. DNA methylation was evaluated by the RRBS (Reduced Representation Bisulfite Sequencing) method.

Results

The analysis of the exosomal microRNAs revealed 471 microRNAs over-expressed in the exosomes of the resistant cells. Among them, three DNMT1/3-targeting microRNAs - miR-148b-3р, miR-193a and miR-383, were identified. The subsequent analysis of the cellular proteins revealed the decreased expression of DNMT1/ DNMT3 both in the primary-resistant MCF-7/T cells and in the exosome-treated MCF-7/exoT cells. The suppression of DNMT1/3 correlated with the hypomethylation of particular CpG islands in DNA of the resistant cells.

Conclusions

Taken together, the results obtained demonstrate the important role of the DNA (de)methylation in the exosome-mediated transferring and maintaining of the hormone resistance in the breast cancer cells.

Legal entity responsible for the study

The authors.

Funding

The Russian Science Foundation, project 19-15-00245.

Disclosure

All authors have declared no conflicts of interest.

Background

Atezolizumab plus nanoparticle albumin-bound (nab)-paclitaxel prolonged progression-free survival (PFS) and overall survival (OS) among patients with unresectable locally advanced TNBC but its use is hampered by the lack of reliable predictors of tumor response. The study evaluates whether PD-L1 mRNA copies per ml in plasma-derived exosomes may predict response to anti-PD-L1 antibodies early in the course of therapy.

Methods

Seventy-seven consecutive patients with unresectable locally advanced TNBC treated with atezolizumab plus nab-paclitaxel were studied by blood draws at baseline, 28 days and 56 days after initiation of treatment. Exosomal PD-L1 mRNA in plasma was determined using Bio-Rad QX100 digital droplet PCR system and exoRNeasy kit. Objective responses were defined following the RECIST criteria v.1.1.

Results

At baseline, patients with complete and partial response (CR+PR, n=28) displayed a significantly higher number of PD-L1 mRNA copies per ml compared to ones with stable or progressive disease (SD+PD, n=49) (mean value: 785.6±121.1 vs 114.7±31.4, p<0.001). In patients with CR and PR mean PD-L1 copies/ml were 747.6±121.1 and 125.4 at baseline vs 2 months, respectively (p=0.001). In patients with stable disease the mean ± s.e.m. values were 270±71.1 vs 217.5±17.3 copies per ml (p=0.614) while in progressive disease PD-L1 mRNA levels were 122.1±31.2 vs 494.3±46.2 copies per ml at baseline vs 2 months, respectively (p<0.001). Patients with an increase of PD-L1 mRNA copies per ml were also characterized by significantly shorter PFS (p=0.007) and shorter OS (p=0.001) (OS: 5 months (range 2-7 months, 95%CI 1.1-6.1) vs more than double (range 8-15 months).

Conclusions

Despite the study’s limitations, our data suggest exosomal PD-L1 is significantly associated with outcome and response to atezolizumab plus nab-paclitaxel.

Legal entity responsible for the study

The authors.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

Background

Ewing’s sarcoma (ES) is an aggressive and rare malignancy that primarily afflicts children and young adults. Patients with metastases have the worst prognosis. We have previously demonstrated that locally delivered mesenchymal stromal/stem cells (MSCs) can control ES growth releasing tumor necrosis factor-related apoptosis inducing ligand (TRAIL). However, considering the nature of the disease, new MSC strategies for metastatic ES are needed, accounting that ES can express high levels of the disialoganglioside GD2. To optimize the MSC affinity for tumors, we recently developed a bi-functional (BF) strategy where MSCs expressing TRAIL were further modified by a truncated anti-GD2 chimeric antigen receptor (GD2 tCAR). Here, anti-GD2 BF MSCs delivering a soluble variant of TRAIL (sTRAIL) were challenged in several in vitro and in vivo models, including a metastatic ES xenotransplant.

Methods

Co-expression in MSCs of GD2 tCAR together with sTRAIL was obtained by lentiviral vectors. The BF MSC binding to GD2-positive ES cells was verified in a cell-to-cell interaction assay. Cytotoxicity by BF MSCs was assessed by 2D and 3D cocultures. Tumor targeting and killing by BF MSCs was then investigated in a metastatic ES xenotransplant by in vivo imaging and ddPCR.

Results

In vitro data demonstrated both tumor affinity and killing of BF MSCs. The in vivo model closely mimicking the disseminated ES, with lung and liver as the main metastatic sites, demonstrated that MSCs were able to counteract ES growth in the lung with a significant reduction in tumor signal. As for the liver, a slight though not significant antitumor effect was observed. Evidence on engraftment of BF MSCs into ES metastatic sites was also provided indicating that GD2 tCAR ameliorated the tumor targeting of BF MSCs.

Conclusions

Our work represents the first attempt to target metastatic ES by MSCs delivering an anticancer molecule. With the limitation of a monotherapy approach, BF MSCs promise to pave the way for an improved therapeutic delivery of TRAIL to treat metastatic ES and other deadly GD2-positive malignancies.

Legal entity responsible for the study

University of Modena and Reggio Emilia.

Funding

This work was supported in part by grants from the Association ASEOP and from MIUR “Dipartimenti Eccellenti” 2017.

Disclosure

G. Golinelli: supported by an AIRC fellowship for Italy Grant. G. Grisendi, C. Spano, G. Casari: Honoraria (self): Rigenerand Srl. M. Dominici: Honoraria (self), Advisory/Consultancy, Leadership role: Rigenerand Srl. All other authors have declared no conflicts of interest.