bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of a novel class of early exon ALK rearrangements across two pan tumor sequencing databases.
Authors: Leong Matthew , Li Xiaomo Li , Mercer Jacob , Fields Paul A. , Abraham Eden , Nakasaki Manando , Heneidi Saleh , Gayhart Matthew , Khullar Gaurav , Vail Eric
Published: June 2023

e15105

Background: ALK-rearrangements are a powerful and highly prevalent driver in cancer biology. The advent of potent tyrosine kinase inhibitors has led to the development of numerous laboratory tests and FDA approved therapies for the detection and treatment of ALK-positive cancers. The vast majority of reported ALK rearrangements involve exon 20 with fusions involving exons 17-19 occasionally reported. While less common, ALK-rearrangements occurring earlier in the ALK gene have not been well characterized. This is largely because most clinically available biomarker testing is only optimized for detection of rearrangements involving the common regions. To this end, we aimed to investigate the prevalence of early exon ALK rearrangements (eALKr). Methods: Two pathology databases, including the Tempus deidentified database, were retrospectively queried to characterize eALKr, which were defined as any rearrangement that involved ALK exons 1-16. Demographic information and tumor type were recorded. Identification of eALKr was performed via targeted amplicon-based RNA sequencing, targeted hybrid capture DNA sequencing and whole transcriptome sequencing. Select specimens with early ALK fusions were also evaluated for ALK detection using available FDA-approved methods (immunohistochemistry [IHC] and fluorescent in-situ hybridization [FISH]). Results: Out of the specimens that underwent NGS testing in the two databases, the prevalence of eALKr was 0.05% (73/143,959) across the databases and 10.3% (73/709) for all tumors with ALK rearrangements. Across the 73 rearrangements, there were 58 unique 5’ partner genes of which 46 were only identified once. eALK rearrangements were identified in all exons from exons 1 to 16 except for exon 13, with exon 4 being the most common. Prostatic, breast and ovarian serous carcinoma were the most common tumor types (Table 1). In three cases IHC and FISH were unreliable due to the early rearrangement location (0/3 IHC positive, 1/3 FISH positive). FISH was negative for samples with rearrangements in exon 2 and 4 and showed an atypical (deletion) pattern with a rearrangement involving exon 12. Conclusions: A non-trivial number of eALKr were detected within two database using next generation sequencing (0.05% of total cancer cases; 10.3% of ALK fusion cases). These novel eALK rearrangements were predominantly in tumor types not normally associated with ALK fusions. FDA approved testing modalities are only optimized to detect common ALK rearrangements, which in part may be a reason why eALK-rearrangements are poorly characterized, with limited prevalence and treatment data available. [Table: see text]
Heterofunctional polymeric degrading chimeras (HYDRAC) for targeted protein degradation.
Authors: Wang Max Mu , Truica Mihai Ioan , Gattis Brayley , Abdulkadir Sarki , Gianneschi Nathan
Published: June 2023

e15106

Background: Here we utilize a novel proteomimetic polymer platform technology which packages peptide side chains as globular structures with benificial emergent properties for the development of compounds capable of targeted protein degradation. We refer to this class of compounds as Heterofunctional polYmeric DegRAding Chimeras (HYDRACs) and demonstrate the selective degradation of the oncoprotein Myc as a proof-of-concept. Methods: HYDRACs were generated using ring-opening metathesis polymerization (ROMP), resulting in structures highly resistant to proteolysis. Heterofunctional constructs were designed incorporating secondary sequences in addition to Myc-targeting H1. The degron sequence (RRRG) was incorporated to engage endogenous cellular machinery for targeted protein degradation. Immunofluorescence/confocal microscopy were used to assess cellular penetration; cellular viability assays coupled with use of both cellular (isogenic pairs, Myc-dependent and independent lines) and biochemical (scrambled H1 sequence, homopolymers, monomeric peptides) controls were used to assess tumor antiproliferative effects and Myc-specific responses. Myc target engagement was assessed via immunoprecipitation assays, NMR, circular dichroism, and fluorescence polarization with protein degradation monitored by Western blot. A CRISPR knockdown library screen of all known E3 ligases and mass spec was used to identify pathways responsible for HYDRAC-induced protein degradation. Results: A library of HYDRACs with narrow polydispersity and predetermined degrees of polymerization were generated. These compounds are cell-penetrating with antiproliferative effects at micromolar IC50s. HYDRAC treatment resulted in significant decreases in Myc protein levels, which was rescued by inhibition with MG132 and MLN4924. RNAseq showed selective overexpression of Myc pathway genes. Biophysical analysis showed strong HYDRAC-Myc interactions, supported by the presence of Myc protein following pull-down of biotin-terminated HYDRACs. Identification of E3 ligases implicated in the degradation pathway using a combination of a CRISPR knock down library and mass spectrometry are ongoing. Mice bearing Myc-CaP tumors showed delayed tumor growth following treatment with Myc-degrading HYDRACs. Studies on biodistribution and pharmacokinetic profile of the compound, as well as the ability to degrade other proteins of interest using combinations of degron sequences are ongoing. Conclusions: The presented work demonstrates the feasibility of HYDRACs for targeted protein degradation of Myc, providing rationale for further studies in Myc-driven tumor models. This successful demonstration of this first-in-class compound also opens the door for the development of HYDRACs targeting other proteins of interest and incorporation of different E3 ligase recruiters for tissue or disease-specific activity.
Droplet digital PCR as an alternative method for detecting MET amplification in patients with non-small cell lung cancer.
Authors: Su Jun-wei , Weng Cheng-Di , Yang Jin-Ji
Published: June 2023

e15107

Background: Mesenchymal-epithelial transition (MET) amplification is associated with resistance to epidermal growth factor receptor tyrosine kinase inhibitors in lung cancer patients and poor prognosis. Fluorescence in situ hybridization (FISH) is the most commonly used method for detecting MET amplification but is not possible in cases where tissue samples are unavailable. Here, we evaluated the utility of droplet digital PCR (ddPCR) in detecting MET amplification in non-small cell lung cancer (NSCLC) patients. Methods: We evaluated a c-Met: EFTUD ddPCR assay of the c-Met gene and using EFTUD as the reference gene to determine c-Met amplification in plasma cfDNA samples from 80 advanced NSCLC patients. A total of 84 plasma samples were collected for MET amplification detection using ddPCR. Paired tissue samples using FISH were to evaluate the correlation between the MET-ddPCR ratio and MET FISH. Results were correlated with clinical findings. Results: We found that ddPCR had an 88.1% concordance rate with FISH in detecting MET CNG, with an area under the curve (AUC) of 0.84. Eleven patients were positive for MET amplification by FISH prior to MET inhibitor treatment, and these patients had a partial response rate (PR) of 45.5% and a median progression-free survival (PFS) of 12.1 months. However, MET amplification identified by ddPCR failed to distinguish significant clinical outcomes. The PR rate and median PFS for ddPCR-positive vs. negative patients were 57.1% vs. 50.0% and 12.1 months vs. 2.1 months (P = 0.276). Conclusions: Our results suggest that ddPCR is a reliable alternative to detect MET amplification in plasma cfDNA samples. Met status determined in cfDNA may have potential as a predictive factor for PFS after c-Met inhibitor therapy.
Cardiovascular adverse events associated with second generation Bruton tyrosine kinase inhibitor therapy: A systematic review and meta-analysis.
Authors: Proskuriakova Ekaterina , Jasaraj Ranjit , Gaire Suman , Raut Anuradha , Reddy Vijay Ketan , Shrestha Dhan , Kovalenko Iuliia , Khosla Paramjeet Grewal
Published: June 2023

e15109

Background: Cardiovascular adverse events (CVAE) is a frequent adverse effect of first-generation bruton tyrosine kinase inhibitor (BTKi) that significantly reduce the usage of these medications. This led to the development of second-generation BTKi (acalbrutinib and zanubrutinib) that are more selective and potent with presumably better safety profile than first-generation. CVAE associated with second-generation BTKis in the clinical practice has been sporadically reported. Hence, we attempted to pool the reported CVAE in this meta-analysis. Methods: Systematic searches of PubMed, PubMed Central, Cochrane library for clinical trials, Scopus, and Embase were conducted from inception until January 14, 2023. This meta-analysis performed per PRISMA protocol and registered in PROSPERO database (CRD42023392406). All randomized clinical trials including acalabrutinib or zanabrutinib in treatment arm with standard of care as control arms were included. The literature found from search engines were imported in the covidence platform and screened independently by 2 reviewers in title/abstract and full text phase. Conflicts were resolved by a third reviewer. Included studies were thoroughly reviewed by all reviewers and data from them extracted in a Microsoft Excel. Analysis performed using RevMan 5.4 meta-analysis software. Odds ratio was used as an effect measure with 95% confidence intervals (CI) using random effect model. Results: A total of ten trials (with 3048 patients, 1710 in treatment arm) were included in this meta-analysis. Cardiovascular events leading to death were reported in 4 studies. In the second generation BTKis group cardiovascular death was 0.94% (7/744), while in the control group it was 1.7% (12/705). Using the random effect model, a pooled analysis on death due to cardiovascular events were statistically insignificant across two groups (OR 0.46, 95% CI 0.19 to 1.11; n = 2039; I2 = 57%). Similarly, pooled analysis for any bleeding/hemorrhage from eight studies (OR 2.12, 95% CI 0.91 to 4.97; n = 2611; I2 = 93%), major bleeding from seven studies (OR 1.12; 95% CI 0.66 to 1.89, n = 2658, I2 = 28%), acute coronary syndrome from four studies (OR 3.13; 95% CI 0.65 to 15.13, n = 1585, I2 = 0%) were also statistically not significant. Conclusions: Treatment with acalabrutinib and zanabrutinib did not show any significant differences in cardiovascular events between the group when compared with standard treatment as a control group, meta-analyzing the data from published trials. As there is no evidence of worse cardiovascular outcomes or superiority of the second-generation BTKis compared to standard treatments in terms of safety based on available evidence, further large-scale controlled trials are required to support that new-generation BTKis are superior in safety and better tolerated.
Examination of in vitro and in vivo antitumor activity of Menin-MLL inhibitor, D0060-319.
Authors: Xu Huan , Dai Yazhao , Li Li , Zhu Hui
Published: June 2023

e15119

Background: Rearrangements of the mixed-lineage leukemia (MLL) gene result in the production of MLL fusion proteins, which occurs in 5-10% of acute leukemia in adults and > 70% of infant leukemia, respectively. Menin protein, encoded by the MEN1 gene, interacts with MLL fusion proteins to induce leukemia. D0060-319, a Menin-MLL inhibitor, shows a significant antitumor effect in vivo and in vitro. Methods: The fluorescence polarization (FP) binding assay to assess inhibition of D0060-319 to Menin-MLL interaction. The cross reactivity of 44 molecular targets was tested to assess their off-target binding potential. The anti-proliferative ability of D0060-319 was evaluated by CCK-8 in different cell lines. The in vivo anti-tumor effect of D0060-319 was evaluated in a human MV4-11 and MOLM-13 cell mouse xenograft tumor model. Briefly, five million MV4-11 cells or 500 thousand MOLM-13 cells were subcutaneously implanted the right flank of CB-17 SCID mice to establish cell mouse xenograft tumor model. When tumor volume reached 100-120mm3, the animals were grouped and administrated with D0060-319 at the dose of 25, 50, 75, 100 mg/kg or vehicle at twice daily for 21days. Tumor measurements and body weights were performed twice weekly to assess the antitumor efficacy and tolerability of D0060-319. Results: D0060-319 competitively binds Menin to inhibit the interaction with MLL fusion protein with IC50 of 7.46nM, has high antiproliferative activity in MV4-11 and MOLM-13 cell lines containing MLL fusion proteins with IC50 values of 4.0nM and 1.7nM, respectively. However, in Kasumi-1, K562, HL-60 and KG-1 cell lines lacking MLL rearrangement, D0060-319 antiproliferative activity with IC50 > 10 μM, indicating selectivity for leukemia cell lines containing MLL fusion proteins active. In the MV4-11 cell mouse xenograft tumor model, D0060-319 significantly inhibited tumor growth at the dose of 25mg/kg, with a tumor growth inhibition (TGI) rate of 82.97%, and other doses (50, 75 and 100mg/kg) tumor completely regressed. In the MOLM-13 cell mouse xenograft tumor model, a dose-dependent anti-tumor effect was observed after 14 days of administration, with TGIs of 81.08%, 88.47% and 90.26% at the dose of 50, 75 or 100 mg/kg, respectively. In both CDX models, no bodyweight loss was observed, suggesting that D0060-319 was well tolerated. Furthermore, off-target screening assays showed that D0060-319 did not cross-react with any of the 44 molecular targets at 10 μM, indicating that D0060-319 is highly selective, high-affinity for the MLL-binding pocket on Menin. Conclusions: In MLL-rearranged leukemia, D0060-319 showed high antitumor activity and good tolerability both in vitro and in vivo.
Phase 1, randomized, open-label, single-dose, crossover study to evaluate the bioequivalence of four formulations of oral rivoceranib tablets in healthy subjects.
Authors: Weyer Janelle , Meng Xianzhang , Finis Lynda , Strickland Bill , Park CheolHee , Jang Seong
Published: June 2023

e15122

Background: Rivoceranib is a novel oral tyrosine kinase inhibitor that potently and selectively inhibits VEGFR2. Rivoceranib is being investigated for indications targeted towards solid malignancies as either monotherapy or in combination with other anticancer therapies. Herein, we assessed the bioequivalence of a single dose of rivoceranib administered as 4 different formulations in healthy subjects. Methods: This single-center, open-label, randomized, single-dose, 4-way crossover study evaluated the bioequivalence of 4 different formulations of rivoceranib oral tablets in healthy adults. Each subject participated in 4 treatment periods, where they were randomized to 1 of 4 sequences: ABCD, BDAC, CADB, and DBAC (Formulation A = rivoceranib 250 mg tablet/clinical formulation used in the pivotal phase 3 study, Formulation B = rivoceranib 200 mg tablet/clinical formulation used in early clinical studies, Formulation C = rivoceranib 250 mg tablet/formulation to be developed for future use, Formulation D = rivoceranib 250 mg tablet/to-be-marketed formulation). Results: Of the 60 subjects enrolled, 66.7% were male, 88.3% were white, and median age was 43 years. The median plasma rivoceranib Tmax was similar following all treatments (2 hours post-dose). The 90% CIs around the geometric mean ratios (GMRs) of plasma rivoceranib AUC0-t, AUC0-inf, and Cmax for Formulation B vs. Formulation A and Formulation D vs. Formulation A were within the 80-125% reference interval, demonstrating bioequivalence between Formulation B and Formulation A as well as Formulation D and Formulation A. The 90% CIs around the GMRs of plasma rivoceranib AUC0-t, AUC0-inf, and Cmax for Formulation C vs. Formulation A were slightly outside of the 80%-125% reference interval. Conclusions: Formulations B (clinical formulation used in early clinical studies) and D (to-be-marketed formulation) were bioequivalent to Formulation A (clinical formulation used in the pivotal phase 3 study). Formulation C (formulation to be developed for future use) and Formulation A were similar, but the difference was slightly outside of the bioequivalence criteria. It remains to be evaluated whether the difference in bioavailability between Formulation C and Formulation A is clinically meaningful. Clinical trial information: NCT05287360 . [Table: see text]
Preclinical efficacy of NXP900, a YES1/SRC kinase inhibitor in esophageal squamous cancer models.
Authors: Dash Sweta , Nyswaner Katherine , Harvey Mungo J.B. , King Ben , Woods Allison , Carragher Neil O. , Unciti Asier , Poradosu Enrique , Brognard John
Published: June 2023

e15133

Background: NXP900 (eCF506) is a novel potent and selective SRC family kinase (SFK) inhibitor, (IC50 of 0.47 nM against YES1). NXP900 locks its target into its native “closed” conformation (type 1.5 inhibitor), thereby inhibiting both kinase activity and complex formation with protein partners (Temps et al. Cancer Res. 2021, 81, 5438-5450). In contrast, multi-kinase inhibitors, including dasatinib and bosutinib, block SRC in the active “open” conformation (type 1 inhibitors) promoting the association of SFK and signaling partners via allosteric facilitation of conformational protein activation (Higuchi et al. Cell Rep 2021, 34, 108876). YES1 gene amplification is prevalent in several squamous cancers including esophageal squamous cell carcinoma (ESCC), lung SCC, head and neck SCC, and bladder cancer, indicating that YES1 would be an attractive target in these cancer types. Moreover, crosstalk between YES1 and the Hippo pathway suggests that NXP900 may have a therapeutic potential in squamous cancers with Hippo pathway alterations. Methods: Animal strain: CD1 Nude mice (Charles River). Human ESCC cancer cell lines: KYSE70, OE21, KYSE410, KYSE30 and OE19 cells (Sigma), TE5, and TE14 (Riken). Xenograft tumors were generated by subcutaneous implantation on the right lower flank of the thigh at a cell density of 2x106 to 1x107 cells/mouse at 0.1 mL Matrigel/cell dilution volume per injection. Experiment groups: Vehicle, NXP900 (40 mg/kg); treatment: QD for 28 days, oral gavage. Colony formation assay: 500 cells of 4 cell lines (KYSE70, OE21, KYSE410 and KYSE30) and 1500 cells of 3 cell lines (OE19, TE5 and TE14) were seeded in 24 well plates in triplicates; and treated with NXP900 for 14 days followed by staining with crystal violet. Results: NXP900 strongly inhibited cell proliferation of KYSE70, KYSE410, KYSE30, OE21, OE19, TE5 and TE14 in a colony formation assay. NXP900 significantly inhibited the activating phosphorylation of SRC in KYSE70 cells at 10 nM, while no effect was observed on the inhibitory tyrosine residues on YES1/FYN at 250 nM. Oral administration of NXP900 in a KYSE70 xenograft mouse model with YES1 gene amplification, resulted in an average decrease of tumor volume of 71% while in the vehicle control group tumor volume increased by 472% (p ≤ 0.001), demonstrating significant tumor regression. On Day 27, a mean increase from baseline in body weight of 2.0% and 3.5% was seen in the NXP900 and control groups respectively, indicating good tolerability. Conclusions: NXP900 is a novel potent and selective YES1/SRC kinase inhibitor with subnanomolar IC50 against YES1. NXP900 has marked antitumor efficacy in esophageal carcinoma models in vitro and in vivo providing substantial proof of concept for targeting solid tumors with YES1 and Hippo pathway alterations. IND enabling studies for NXP900 have been completed.
Effects of namodenoson on pancreatic carcinoma: Preclinical evidence.
Authors: Fishman Pnina , Bareket-Samish Avital , Itzhak Inbal
Published: June 2023

e15134

Background: Namodenoson, an A3 adenosine receptor (A3AR) agonist, is currently in a phase 3 trial for the treatment of advanced liver cancer. The mechanism of action of namodenoson involves de-regulation of the Wnt and NF-κB signaling pathways, followed by an increase in pro-apoptotic proteins and Fas-ligand, resulting in tumor growth inhibition. As the Wnt signaling pathway is also highly implicated in pancreatic carcinogenesis, we examined the anti-growth effect of namodenoson on pancreatic carcinoma cell lines and investigated the molecular mechanism involved. Methods: BxPC-3 human pancreatic carcinoma cells were cultured in the presence and absence of 0.01, 0.1, and 1 nM namodenoson. A combined treatment of namodenoson (0.1 nM) and gemcitabine (0.2 µM) was also studied. 3[H]-thymidine proliferation and MTT assays were used to monitor cell growth, and Western blot analyses were performed to identify the involved regulatory cell growth proteins. Results: Results of the 3[H]-thymidine proliferation assays demonstrated significant dose-dependent inhibition of the growth of BxPC-3 cells with namodenoson (1 nM: 67.4% ± 1.7%, p < 0.001; 0.1 nM: 53.7% ± 6.3%, p < 0.05; 0.01 nM: 27.9 % ± 2.3%, p < 0.005). The MTT results revealed that a combined treatment with namodenoson plus gemcitabine had an additive inhibitory effect (namodenoson: 48.6% ± 1.4%; gemcitabine: 44.4% ± 0.7%; namodenoson plus gemcitabine: 65.4% ± 1.4%; p < 0.001 for all). Western blot analyses showed that namodenoson treatment was associated with downregulation of the Wnt pathway regulatory proteins including p-Akt, NF- κB, GSK-3β, and β-catenin. Conclusions: Our findings showed that nanomolar concentrations of namodenoson inhibit the growth of pancreatic carcinoma via A3AR activation and de-regulation of the Wnt/β-catenin pathway, both as a monotherapy and in combination with gemcitabine. Thus, these results support a potential role for namodenoson in treating pancreatic cancer, thereby opening a novel therapeutic opportunity for this disease.
Programmed cell death ligand 1 (PD-L1) expression landscape and its relationship with tumor mutational burden (TMB) in a community-based cancer center patient population in rural central Nebraska.
Authors: Copur Mehmet Sitki , Peterson Tonya , Doornbos Kathryn , Tun Soe Min , Springer Carlene R. , Robbins Leslie , Arbogast Jessica , Muske Chandra , Buescher Lisa , Sukup Jordan , Buescher Lisa , Horn Adam , Wedel Whitney , Lintel Nicholas , Brunt Kathryn , Duckert Randall , Lavudi Swathi
Published: June 2023

e15141

Background: PD-L1 and TMB are the most widely used immunotherapy biomarkers to identify populations who would benefit from immunotherapy, with the higher values predicting better therapeutic efficacy. We sought to explore the occurrence and correlation of these two biomarkers in a community based cancer center patient population in rural central Nebraska. Methods: One hundred sixty consecutive cancer patients(pts) diagnosed and treated between 2018-2022 at Morrison Cancer Center were evaluated. Malignant tumor tissue specimens were analyzed for PD-L1expression and TMB (Foundation test). PD-L1 expression was classified as low ( < 1%), intermediate (1% ≥ PD-L1 < 50%), high (≥ 50%), and TMB classified as low [1–5 mutations/Megabase (muts/Mb)], intermediate (6–19 muts/Mb), high (≥ 20 muts/Mb)]. PD-L1 and TMB values were treated as continuous variables to calculate a Pearson correlation coefficient and p value. Results: Mean age at diagnosis was 64 years (range 33-89). Malignant histology diagnoses included 55 pts with lung, 18 colorectal, 12 breast, 15 unknown primary, 15 pancreas, 10 renal, 3 melanoma, 1 thyroid, 6 ovary, 5 uterus, 5 head and neck, 5 prostate, 5 biliary, and 1 vulvar. Fifty four percent (86/160) of pts had low, 33% (53/160) had intermediate, and 13% (21/160) had high PD-L1 expression score with a mean PD-L1 value of 13.8% (range 0-100). Sixty six percent (106/160) of pts had low, 30% (48/160) had intermediate, and 4% had high TMB with a mean TMB value of 5.6 muts/Mb (range 0-66). A marginal linear correlation between PD-L1 expression and TMB was observed (Pearson Correlation Coefficient = 0.172, P = 0.0270). Conclusions: PD-L1 and TMB are both important biomarkers for immunotherapy. Increasing availability and use of both PD-L1 and TMB testing in the community oncology practice beseeches the question of frequency of these two biomarkers in various cancers in the community oncology patient population as well as any association between them. The results of our study gives a glimpse of the distribution landscape of these two biomarkers for community oncology patients and suggest that PD-L1 and TMB may be independent markers with a weak association. PD-L1 and TMB may each inform use of immunotherapy by different mechanisms. [Table: see text]
Prognostic relevance of 3q amplification (AMP) in a racially diverse patient population with advanced squamous cell carcinoma of the lung.
Authors: Abu Rous Fawzi , Li Pin , Carskadon Shannon , Gutta Radhika , Potugari Bindu Rani , Gadgeel Shirish M. , Palanisamy Nallasivam
Published: June 2023

e15142

Background: Amplification (AMP) of 3q is the most common genetic alteration in squamous cell carcinoma of the lung (LUSC) with the most frequently amplified region being 3q26-3q28. It represents one of the most striking molecular differences between LUSC and adenocarcinoma. We identified a minimal common region (MCR) of AMP constituted by 25 genes within a large amplicon on chromosome 3q by analyzing data of 476 patients with LUSC from The Cancer Genome Atlas (TCGA) and extracting copy number alteration data from cBioPortal. Full 25-gene MCR AMP was detected in 46.4% of the cases and was associated with better disease-specific survival (NR vs 9.25 years, 95% CI [5.24-NR]; p = 0.011) and progression-free interval (8 vs 4.9 years, 95% [3.5-NR]; p = 0.020). The TCGA cohort was mainly comprised of Caucasians (69.7%) and patients with early-stage disease (98.7%). We conducted a retrospective pilot analysis to validate the prognostic value of MCR AMP using a racially diverse cohort with advanced LUSC. Methods: We retrieved tissue samples for 33 patients with stage III or stage IV LUSC and collected retrospective data from Electronic Medical Records. The pathologist reviewed the slides to ensure the adequacy of tumor tissue. MCR AMP was evaluated via laboratory- developed FISH probe spanning MCR region. FISH test that showed a cluster of signals with more than 4 copies was regarded as positive; we used a control probe for the 3p arm which is typically not amplified in LUSC. Results: The mean age of the cohort was 69.1 years, 57.6% were males, 39.4% were African Americans (AA) and 54.5% were Caucasians, 66.7% had stage 3, and 33.3% had stage 4. Only 6.1% were never smokers and 84.8% were former smokers. MCR AMP was detected in 54.5% (n = 18) of the cases. PD-L1 status was available for 21 (63%) cases, 6 cases (3 had MCR AMP) were PD-L1 negative (TPS < 1%), and 15 cases (9 had MCR AMP) were PD-L1 positive (TPS > 1%). Consistent with the results observed in the TCGA cohort, MCR amplified cases had a trend towards better overall survival (OS) compared to non-amplified cases (1.74 vs 0.91 years; HR = 0.57, 95% CI [0.26-1.24], p = 0.15) as well as a trend towards better progression-free survival (PFS) (0.90 vs 0.74 years; HR = 0.88, 95% CI [0.43-1.81], p = 0.73). Conclusions: This pilot analysis shows that MCR AMP carries a prognostic value in a racially diverse patient population with advanced LUSC, similar to our observations in the TCGA cohort. Further validation in a large cohort is in progress.
Copy number variation (CNV) calling of MET, MYC, and ERBB2 using PiVAT bioinformatic platform.
Authors: Yu Yao , Zou ShiPing , Wang Jing , Wang Bo , Sun Jing , Zhao Dan , Shi Xinyi
Published: June 2023

e15145

Background: CNV in tumor-associated genes such as MET, MYC and ERBB2 amplification can lead to cancer progression and poor survival in multiple cancers, and is associated with treatment regimen decision. Whole genome sequencing (WGS) and comprehensive genomic profiling (CGP) panels covering hundreds of genes are currently used to detect CNV in hospitals and CLIA-accredited laboratories. However, cost and complex workflow of WGS/CGP restrict their application in clinical molecular diagnostics. CGP panel covering dozens of genes based on targeted amplicon sequencing (TAS) method is preferred, but it is not ideal to detect CNV accurately due to unique methodology. Here we established a CNV calling toolkit in PiVAT bioinformatic platform to solve CNV calling problems in small-size TAS panel. Methods: 46 CNV positive (CNV+) and 14 CNV negative (CNV-) samples were sequenced using MGISEQ-2000 with a paired end, 2x100 read length protocol and adapter sequences were trimmed from 3’ ends of each read. PiVAT’s CNV calling method is based on double coverage normalization, including one per-sample normalization and one per-amplicon normalization. CNV- samples were used as normalization references. MET, MYC and ERBB2 copy number was investigated to determine sensitivity, specificity, precision, and accuracy of the overall test. Copy number deletion is defined as ratio < 0.8 and amplification is defined as ratio > 1.2. PiVAT CNV output calls were compared against excepted copy number to calculate and plot the concordance. CNVkit and Control-FREEC, 2 other CNV calling tools, were chosen as comparators to compare ERBB2, MET and MYC copy number levels with PiVAT’s CNV caller. Results: Sequencing results from all 46 CNV+ samples showed high coverage uniformity, with > 90% of sites having base coverage depth > 20% of mean coverage for all samples. Average mapping rate and on-target rate of sequencing run, analyzed by PiVAT, were 98.98±0.41% and 98.39±0.45%. Results from PiVAT showed a sensitivity of 97.83% and a specificity of 100% for MET, MYC, ERBB2 amplifications. PiVAT estimated copy number more accurately compared with CNVkit and Control-FREEC. PiVAT’s R2, measuring degree of difference from expected copy number, was greater than CNVkit and Control-FREEC for MET, MYC and ERBB2, with values at 0.78, 0.69 and 0.96, respectively. In contrast, CNVkit and Control-FREEC were on average 77.38% and 164.04% worse. For CNVkit and Control-FREEC, many of MET, MYC and ERBB2 CNV calls were seen at a much lower magnitude than expected. Conclusions: Our studies have shown that PiVAT is a reliable tool for calling CNVs. PiVAT’s sensitivity and accuracy were calculated to be > 97%. And PiVAT outperformed other CNV calling tools, CNVkit and Control-FREEC. This demonstrates that PiVAT’s CNV caller can be used to provide CNV results from TAS panel, and guide relevant cancer diagnosis and clinical decision more accurately.
The landscape of ATM alteration in Chinese solid tumor patients.
Authors: Li Yan , Li Qingcong , Zhang Yaxuan , Lai Yuqing , Yang Zhouchao , Li Yueqin , Liao Yuehan , Yin Lu , Wu Huiting , Zhang Zhengqiong , Hu Libo , Li Ling
Published: June 2023

e15147

Background: The Ataxia-telangiectasia mutated (ATM) gene is an oncosuppressor, which plays an important role in the repair of DNA double-strand breaks (DSBs). The mutation profiles of ATM gene in Chinese solid tumor patients can help understand pathogenesis, prognosis, and identify targets for therapy. Methods: Using commercial NGS assays, we retrospectively analyzed genomic DNA alterations in normal-paired samples from 1351 patients with malignancies from January 2021 to October 2022, and alterations including single base substitution, short and long insertion/deletions, copy number variations, gene fusions and rearrangements. Results: ATM gene was altered in 6.8% (92 of 1351) of all solid tumors. The frequency of ATM variations was assessed in multiple cancer types, including non-small cell lung cancer 22% (35/159), endometrial carcinoma 15% (2/13), biliary tract tumor 12.1% (7/58), gastric cancer 10.7% (3/28), prostatic cancer 8.3% (1/12), breast cancer 6.7% (2/30), colorectal cancer 6.5% (11/168), thyroid cancer 6.2% (5/80), cervical carcinoma 5.7% (2/35), prostatic cancer 3.9% (2/51), liver cancer 3% (2/66). A total 102 genetic alterations were identified in 92 patients, including 70 missense mutations (69%), 14 nonsense (14%), 9 splicing (9%), 3 frameshift (3%), 2 deletion (2%), 2 copy number variation (2%). Among these patients, 2 had pathogenic germline ATM heterozygous mutations, including one lung adenocarcinoma and one pancreatic cancer. Furthermore, in our cohort, ATM mutation carriers also owned other actionable mutation, the most frequent one was TP53 (41%), followed by APC (29%), KRAS (23%), MLL3 (22%) and BRCA2 (20%). Conclusions: Our findings indicate that ATM alterations are widely mutated in Chinese multiple cancer types, with specific mutations differing largely among different tumor types. ATM alterations warrant further investigation as predictive biomarkers of response to PARP inhibitors or platinum-based therapy.
Frequency of somatic mutations in solid tumor malignancies from inner city minorities: A single center study.
Authors: Cuartas Mesa Maria Cristina , Bemis Thomas Stiles , Tamkus Deimante , Gupta Shweta
Published: June 2023

e15149

Background: The treatment landscape for solid tumor malignancies continues to change with identification of predictive biomarker targets by next generation sequencing (NGS). However, data from individuals from racial and ethnic minorities are often underrepresented. To bridge this gap, we examined the frequency of actionable biomarker findings with a treatment target outlined in NCCN guidelines in our inner city minority population at John H. Stroger, Jr. Hospital of Cook County. Methods: We examined patients who had FoundationOne CDx or FoundationOne Liquid CDx reports for our adult patients with a solid tumor malignancy between July 2021 through December 2022. We categorized patients as having an actionable biomarker finding if there was a finding reported with a treatment target with indication in a patient’s tumor type as outlined in NCCN guidelines. Statistics were done using t-test, z-test, and chi square tests. Results: A total of 313 patients had a FoundationOne CDx or FoundationOne Liquid CDx report for a solid tumor malignancy. Median age for the population was 64.6 years (range 20-89 years). The majority of patients were African American (50%), followed by Hispanic (31%), Caucasian (13%) and Asian (6%). There were 149 patients (47.6%) that had an actionable biomarker target in their tumor type. Among the 50 total lung adenocarcinoma patients, 20 (40%) had a driver mutation, with 12 (60%) being African American. In 43 colorectal tumors were tested, 16 (37.2%) were KRAS WT, with 6 being African American, 6 Hispanic, and 4 white. Among 27 prostate cancer samples tested, 7 (25.9%) harbored a homologous recombination repair (HRR) gene mutation. Of the patients without an actionable biomarker in their tumor type, 47 patients (15%) had an actionable biomarker finding approved in another tumor type. Of the patients with biomarker targets in another tumor type, mutations in PIK3CA and HRR genes were among the most frequent findings. There were no statistically significant differences in frequency of actionable biomarkers in patient tumor type between gender (P = 0.31) or race (P = 0.63). Conclusions: Our findings showed that nearly half of our patients had a targetable somatic mutation in their tumor type. Even in patients without an actionable biomarker in their specific cancer, those patients may harbor a biomarker finding with an approved target in another cancer. While sample sizes were small to investigate mutational profiles for specific tumor types, future analysis with more samples will allow for better characterization. These findings support increasing NGS testing to better characterize the tumor mutational profiles for our racially underrepresented population, as well as for identifying potential biomarkers for future studies in other tumor types.
PD-L1 expression in patients with multiple tumors and their correlation with genomic mutations.
Authors: Lin Guomin , Yuan Shaohua , Pang Fei
Published: June 2023

e15152

Background: Immune checkpoint inhibitors (ICIs) is effective in treating tumors expressing PD-L1, however, the reported response rate was low. Inter-tumoral heterogeneity might be one of the reasons for the insensitive of ICIs. In patients with multiple tumors (defined as co-occurrence of more than one malignant lesion in a single or multiple sites), the situation is more complicated. Methods: A total of 67 patients who provided tumor tissues from at least two anatomical sites for assessment of PD-L1 expression by immunohistochemistry (22C3 and/or 28-8 antibodies) was enrolled in this study. Cells with ≥1% staining was defined as a positive result. The sequencing data of 61 patients was also retrospectively reviewed. Targeted sequencing was performed with panels of up to 450 cancer-related genes (at least 47 genes in common). Matched peripheral blood samples were also tested as control to exclude germline mutations. Results: At sample level, the PD-L1 positivity by cancer type was as follow: cervical cancer/melanoma/adrenal gland carcinoma (100% [1/1]), thymic cancer (100% [5/5]), colorectal cancer (80% [8/10]), esophageal squamous cell carcinoma (62% [8/13]), ovarian cancer (57% [4/7]), biliary tract cancer (50% [3/6]), lymph node metastasis (50% [1/2]), pancreatic cancer (50% [2/4]), lung squamous cell carcinoma (43% [3/7]), endometrial cancer (40% [4/10]), breast cancer (29% [2/7]), gastric cancer (25% [2/8]), lung adenocarcinoma (15% [4/26]) and liver cancer (14% [1/7]). All the provided tissues were positive or negative for PD-L1 in 42 patients and were partly positive and partly negative in 25 patients. At patient level, differentially mutated genes between the two above-mentioned groups of patients included CCND1 (10.5% vs 39.1%, P = 0.0116), FGF19 (7.9% vs 30.4%, P = 0.0324), FGF3/4 (7.9% vs 34.8%, P = 0.0143) and TERT (21.1% vs 0%, P = 0.0199). Tumor mutation burden and microsatellite instability status were comparable between the two groups. Consistent with previously reported (not specifically recruiting patients with multiple tumors), EGFR exon 19 deletion was preferentially mutated in PD-L1 negative tumors (16.7% [4/24] vs 0% [0/7], P = 0.5497) in non-small-cell lung cancer, and PD-L1 expression was correlated with EGFR wild-type (wild-type: 75% [3/4] vs mutated: 45% [9/20], P = 0.5901) and ALK mutation (mutated: 25% [1/4] vs wild-type: 5% [1/20], P = 0.3116) in lung adenocarcinoma, although statistical significance was not reached. Conclusions: For patients with multiple tumors, inter-tumoral heterogeneity of PD-L1 expression is common. Several actionable gene mutations were found to be more commonly occurred in PD-L1-heterogenous tumors. Physicians should carefully consider PD-L1 and genomic assessments by case in order to select appropriate patients for ICI treatments.
Identifying anatomopathological factors associated with a Oncotype DX 21-gene recurrence score higher than 25 in menopaused women.
Authors: Guerif Clara , Jamelot Mathieu , Kebir Fatima , Zilberman Sonia , Seknazi Lauren , Barthere Xavier , De Forceville Louise , Theodore Claire , Touboul Cyril , Buob David , Gligorov Joseph , Benderra Marc Antoine
Published: June 2023

e15156

Background: Oncotype DX recurrence score is a predictive assay clinically used to estimate the risk of distant recurrence for patients with hormone receptor positive (HR+) early breast cancer (EBC) and to predict the benefit of adjuvant chemotherapy, based on molecular factors of the tumor itself. This retrospective study examines the factors that are linked to a Recurrent score (RS) higher than 25, indicating adjuvant chemotherapy for menopaused women with hormone receptor positive early breast cancer. Methods: 157 patients diagnosed with HR+, HER 2 negative early breast cancer who underwent Oncotype DX testing during their treatment between November 2018 and December 2022 were included in the study. Of these 157 patients, 114 were older than 50 years old and with complete data. Univariate and multivariate logistic regression analysis were used to select pathological factors associated with a RS higher than 25. The receiver operating characteristic (ROC) curve was used to assess the optimal threshold of Estrogen receptor (ER), Progesterone receptor (RP) and Ki67. Results: Median age was 65 years (IQR: 59-71). 57 patients (47.5%) had a T1 tumor and 48% of patients had negative lymph nodes. Most of the patients had a Scarff-Bloom-Richardson (SBR) grade 1 (12%) or grade 2 tumor (70%), and 18% of the patients had grade 3 breast cancer. The cell proliferation marker Ki67 was higher than 30 % for 28 patients (23%). The RS was higher than 25 for 17 patients out of 120 patients. Multivariate logistic regression analysis showed that Ki67 higher than 30% was significant associated with RS > 25 (p = 0.01). To predict a RS > 25, the optimal threshold were respectively 80% for the PR, 90% for the ER and 30% for the Ki67. The area under the ROC curve (AUC) were respectively 0.70, 0.60 and 0.88. With the threshold of 30% for Ki67, the sensitivity was 0.76 and specificity 0.89. Conclusions: The use of the Oncotype DX test is validated and useful to better decide the absence of chemotherapy indication in adjuvant situation in some patients with HR+ cancers. However, access to it remains difficult and expensive in some countries with limited resources. Ki67 and SBR grade could be used as an indication for adjuvant chemotherapy but cannot replace Oncotype DX.
Comprehensive genomic profiling and therapeutic implications for patients with advanced cancers: The experience of an academic hospital.
Authors: Teuwen Laure-Anne Marie Nicole , Roets Evelyne , D'Hoore Pieter , Pauwels Patrick , Prenen Hans
Published: June 2023

e15162

Background: Next generation sequencing (NGS) can be used to detect tumor-specific genomic alterations, with the aim to identify actionable genomic alteration and provide additional treatment options for patients with advanced cancer. While smaller sequencing panels are being applied broadly, controversy exists around the routine implementation of comprehensive genomic profiling with large multigene NGS panels. Indeed, there is an ongoing discussion about the indications as well as timing of comprehensive genomic profiling in patients with advanced solid tumors. Therefore, there is a need for more data to increase understanding of the potential indications and added clinical benefit of routine implementation of broad comprehensive genomic profiling. However, available studies have often been reporting on a small patient population, which complicates the interpretation and translation to the clinical setting. This study aims to describe the feasibility of performing broad NGS panels in patients with advanced cancer in an academic hospital setting, as well as the targetable alterations detected. Methods: In this retrospective study, we analysed genomic alterations in solid tumor biopsies of 464 patients with locally advanced or metastatic cancer. Next generation sequencing was performed using the Foundation Medicine assay (FoundationOneCDx). The therapeutic implications of the genomic alterations were determined using the OncoKB classification. Results: The FoundationOne CDx was successfully applied in 464/521 patients (89%) with a range of cancer types. The most common altered genes were TP53 (61%), followed by KRAS (20%), CDKN2A (20%), TERT (16%), APC (16%), PTEN (14%), PIK3CA (14%) and CDKN2B (14%). Among 419 patients with successfully analyzed tumor mutational burden (TMB), the median TMB was 2.52 (range 0 – 67) mutations/Mb (mut/Mb). Forty-three patients presented with a high-TMB ( > = 10 mut/Mb) of which eight had microsatellite instability. Out of 126 patients with an actionable target (e.g. ERBB2, KRAS, PIK3CA, FGFR2), 40 patients received matched treatment (8.6%), of which 17 were included in clinical trials. Conclusions: This single-center retrospective study shows that broad application of NGS is feasible in an academic center, increases the detection of actionable genomic alterations, and opens opportunities to provide treatment with immune checkpoint inhibitors in case of high tumor mutational burden and microsatellite instability status regardless of tumor histology. The study brings to attention that strategies such as inclusion in clinical (basket) trials and development of new targeted drugs are necessary to improve matched treatment rates. Further research is needed to assess the possible clinical benefit of broad application of NGS and genotype matched treatment.
m6A mediated upregulation of lncRNA CASC9 in head and neck squamous cell carcinoma.
Authors: Karuvantevida Noushad , Arumugam Paramasivam
Published: June 2023

e15165

Background: Long noncoding RNAs (lncRNAs) are dysregulated in many cancers including head and neck squamous cell carcinoma (HNSCC). Many recent studies have shown that abnormal expression of lncRNA CASC9 (CASC9) is involved in the pathophysiological processes of cancers. N6-methyladenosine (m6A) represents the most common methylation modification of lncRNAs and has been shown to play important roles in the development of different cancer types. However, the potential role of m6A mediated upregulation of CASC9 in HNSCC and its molecular mechanisms remain largely unknown. In the present study, we identified the molecular mechanisms of CASC9 in promoting the malignancy of HNSCC. Methods: We used real-time polymerase chain reaction (qPCR) to identify the level of CASC9 expression in HNSCC clinical samples and cell lines. The oncogenic role of CASC9 in HNSCC was analyzed using online tool UALCAN. RNA immunoprecipitation sequencing (MeRIP-seq) was performed to determine the m6A modification of CASC9. The relationship between CASC9 expression and patients prognosis was analysed using Kaplan-Meier plotter. Loss of function studies used to evaluate cell proliferation, viability, and migration of HNSCC cell lines. Results: Results revealed that m6A mediated CASC9 expression was significantly upregulated in HNSCC tissues than those in the paired adjacent non-tumor tissues. Similarly, m6A mediated CASC9 was significantly overexpressed in the HNSCC cell lines compared with normal cells. The upregulated CASC9 expression was significantly associated with advanced stages and closely correlated to the unfavorable prognosis. m6A was highly enriched within CASC9 and enhanced its RNA stability. Knockdown of CASC9 remarkably inhibited the proliferation, migration and promoted the apoptosis of HNSCC cells. Mechanistically, we found that IGF2BP2 (insulin-like growth factor 2 mRNA binding protein 2), m6A reader could recognize the m6A site in CASC9 and enhance its stability. Conclusions: Overall, this work highlights for the first time that m6A modification enhance CASC9 stability may serve as a potential prognostic biomarker and therapeutic target in HNSCC.