bluuebunny/crossref_metadata_2025 huggingface dataset:
Drug-free biodegradable nanoparticles alleviate myeloid cell-induced immunosuppression and inhibit metastasis.
Authors: Ma Jeffrey , Raghani Ravi , Zhang Yining , Orbach Sophia , Murthy Tushar , Podojil Joseph , Elhofy Adam , Shea Lonnie
Published: June 2022

e14555

Background: Metastasis is responsible for most cancer-related deaths and depends on the formation of a metastatic niche at distal sites. The metastatic niche consists primarily of immunosuppressive myeloid cells, such as neutrophils and monocytes. These cells are recruited to distal sites in response to cancer-induced immune dysregulation and promote the recruitment and survival of disseminated tumor cells. We have employed drug-free poly(lactide-co-glycolide) (PLGA) nanoparticles, ONP-302, to modulate myeloid cell phenotypes and trafficking to disrupt the formation of the metastatic niche. The anti-metastatic efficacy of ONP-302 nanoparticles was evaluated in a translationally relevant mouse model of orthotopic triple-negative breast cancer. Methods: ONP-302 nanoparticles were evaluated in the syngeneic 4T1 model of triple-negative breast cancer in female BALB/c or RAG1KO BALB/c mice. ONP-302 nanoparticles intravenously injected into tumor-bearing mice every 3 days starting 1 day after tumor inoculation. Lungs were extracted 14 and 21 days after inoculation for analysis by single cell RNA sequencing and quantification of metastases by luminescent imaging, respectively. In resection studies, primary tumors were resected 11 days after inoculation and tissues were extracted 42 days after resection for quantification of metastases. Results: ONP-302 nanoparticles significantly reduced primary tumor growth and completely inhibited the formation of lung metastases. ONP-302 was also effective at suppressing metastasis when applied following surgical resection. In mice which received ONP-302 after surgical resection, lung metastases were undetectable up to 42 days after resection, while 44% of control mice developed metastases. These findings suggest that ONP-302 targets both primary tumor growth and metastatic colonization. Single cell RNA sequencing was employed to analyze ONP-302 modulation of the lung metastatic niche. The lungs of ONP-302 treated mice had lower neutrophil infiltration and a higher proportion of monocytes and dendritic cells compared to control mice. Gene expression pathways associated with inflammatory interferon and tumor necrosis factor responses were upregulated in neutrophils, monocytes, and dendritic cells from ONP-302 treated mice. The upregulation of these pathways suggests that ONP-302 treatment prevents the formation of an immunosuppressive niche and promotes anti-tumor cytotoxicity. Adaptive immunity was essential for ONP-302 efficacy, as T and B cell deficient RAG1KO mice demonstrated complete loss of ONP-302 efficacy both in slowing primary tumor growth and preventing metastasis. Conclusions: Our findings demonstrate that our drug-free PLG nanoparticles, ONP-302, impede cancer metastasis as a monotherapy by inhibiting the accumulation of immunosuppressive myeloid cells.
Severe irAEs in patients with immune checkpoint inhibitor therapy: Experience in a Mexican cancer center.
Authors: Bonilla Molina Diana , Nehmad Cecilia , Tovar Cabrera Juan Manuel , Enciso López Ervin Saúl , Camarin Sanchez Efrain Isaias , Shveid Gerson Daniela , Rodriguez Benigno , Serrano Villamayor Omar , López Zepeda Lorena , Blanco Vazquez Yazmin Carolina , Camacho Limas Christian Patricio , Martínez-Herrera José Fabián , Olivares Guillermo , Gerson Cwilich Raquel
Published: June 2022

e14559

Background: A relationship has been found between a better response to treatment and the presence of irAEs. Different potential factors for developing them have been described, including the type of immunotherapy and their combination. Rare (< 1%) and potentially life-threatening irAEs have been reported, which due to severity lead to treatment discontinuation, and a prolonged effective response is seen in these patients if they survive the irAE. Methods: Observational study in patients with solid tumors who received treatment with immune checkpoint inhibitor and their characteristic, that presented irAEs grade 3 and 4 using the CTCAE version 4, from January to December 2021 in a Mexican cancer center. Results: irAEs grade 3 or 4 were observed in 7 (5.6%) of 125 patients, with an age range of 60 to 69 years, most of the patients had a diagnosis of metastatic melanoma (43%) and secondly urothelial carcinoma with 29%. The predominant treatment was pembrolizumab monotherapy (56%), with an average of 5 applications after those who presented irAEs. The rare irAEs presented were myocarditis (2.4%), myositis (1.6%), myasthenia gravis and interstitial nephritis, both with 1%. 70% of these patients were managed with three boluses of methylprednisolone and subsequently prednisone 1 mg/kg as maintenance. The median appearance of these irAEs was 72 days. Myocarditis and myositis had an earlier onset with an average of 32 days. One of the seven patients presented myasthenia gravis, myositis and myocarditis concomitantly, with complete response of the neoplasia, however, due to the prolonged use of immunosuppressants to control irAEs, the disease recurred at 7 months. Two of the three patients with myocarditis had a complete response and remain disease free. 57% survived more than 90 days, of which 75% are still alive. Conclusions: Among the 125 patients who received an immune checkpoint inhibitor in our cancer center in 2021, the most prevalent serious irAE was myocarditis, with a higher incidence than that reported by other centers, and a prolonged therapeutic response was found in the patients who presented it.
Inferior survival outcomes with concomitant immune checkpoint inhibitors and chronic opioid use.
Authors: Varghese Aaron , Gallo Steven , Argentieri Deanna , Harvey Bethany W , Catalfamo Kayla , Attwood Kristopher , Zsiros Emese
Published: June 2022

e14564

Background: Immune checkpoint inhibitors (ICI) are monoclonal antibodies that promote immune recognition of tumor cells to facilitate their elimination. Opioids are a major facet of pain management in cancer patients. Since chronic opioid use results in immunosuppression, the purpose of this study was to evaluate the survival outcomes of cancer patients on concurrent ICI and opioid therapy. Methods: This single-institute retrospective study from an NCI-designated comprehensive cancer center evaluated all patients in the primary or recurrent setting who underwent ICI therapy (anti-PD-1/PD-L1/CTLA-4) from 1 January 2020 to 18 June 2021. Demographic, clinicopathologic, and co-morbidity data were obtained from electronic medical records and the New York State Cancer Registry data. The New York State Prescription Monitoring Program was used to collect data about opioid prescriptions filled by patients from 30 days prior to and 30 days after the end of ICI therapy. Patient characteristics were analyzed with the Mann-Whitney U or chi-square tests. Survival analyses were performed using the Kaplan-Meier and Cox model regression, adjusting for relevant clinical factors. Results: A total of 869 patients were evaluable based on inclusion criteria and data availability. Most patients (77.15% chronic users and 64.62% occasional users) had stage III/IV disease. The most represented services included: gynecology (11.54%), soft tissue/melanoma (14.13%), thoracic (38.75%), urology (11.9%). Comorbidities did not vary between the two groups. Pre-treatment median opioid consumption was higher in the chronic users compared to occasional users (240 oral morphine equivalents (OME) vs 120, p = 0.03). Total opioid prescriptions filled was significantly higher for chronic users compared to occasional users (4275.0 vs 520.1 OME, p < 0.001). Chronic users were more likely to be on an additional anti-neoplastic agent (64% vs 44.07% for occasional users). Occasional users had significantly longer progression free (44.7 vs 16.0 months, p < 0.001) and overall (42.7 vs 16.2 months, p < 0.001) survival. When controlling for age, stage, and concurrent therapy, chronic opioid use retained a significantly negative association with PFS and OS. Conclusions: Patients on ICI therapy in the primary and recurrent setting who chronically use opioids exhibit significantly decreased PFS and OS compared to the occasional user cohort. This study provides evidence that chronic opioid use during ICI therapy results in inferior clinical outcomes.
An open-label, multicenter, phase II study of RC48-ADC combined with radiotherapy, PD-1/PD-L1 inhibitor sequential GM-CSF and IL-2 for salvage therapy in patients with HER2-expressing advanced solid tumors(PRaG3.0).
Authors: Xu Meiling , Kong Yuehong , Xing Pengfei , Chen Rongzheng , Ma Yifu , Shan Chanchan , Zhang Liyuan
Published: June 2022

e14565

Background: The development of anticancer therapy tends to focusing on pan-cancer analysis, targeted therapy and immunotherapy with the advance of molecular and tumor microenvironment. Radiotherapy produces an immunomodulatory effect that may act synergistically with PD-1/PD-L1 inhibitor in multiple malignancies. Previous phase II trial showed HFRT/SBRT combined with PD-1 inhibitor and GM-CSF (PRaG regimen) has obtained promising results(Yuehong Kong et al. ASTRO 2021). RC48-ADC is a novel HER2-targeting antibody-drug conjugate (ADC). Preclinical studies indicated that ADC elicit immunogenic cell death and selectively radiosensitize to tumours. RC48-ADC combined with HFRT, PD-1/PD-L1 inhibitor sequential GM-CSF and IL-2 (PRaG3.0 regimen) could further improve the synergistic antitumor effects, this precise combination therapy paradigm provides an exploratory path for the treatment of pan-cancer. An exploratory phase II, open-label, multi-centre, single-arm study was conducted to evaluate the initial clinical efficacy and safety of PRaG3.0 regimen for treatment of HER2-expressing advanced solid tumors. Methods: Participants with advanced, confirmed HER2-expressing(IHC3+, 2+ or 1+) solid tumors that had progressed after the standard of care or intolerance were enrolled. In the cycle, those received RC48-ADC(2.0 mg/kg d1, every 3 weeks), then HFRT (2-3 doses of 5-8Gy) was delivered for one metastatic lesion every other day, followed by GM-CSF(200 μg d3-7), sequential IL-2(2million IU d8-12), and PD-1/PD-L1 inhibitor was dosing within one week after completion of HFRT. After RC48-ADC combined with PD-1/PD-L1 inhibitor sequential GM-CSF and IL-2 for at least 6 cycles, then maintenance with PD-1/PD-L1 inhibitor was administered until disease progression or unacceptable toxicity. The primary endpoint was objective response rate (ORR). This trial is registered with ClinicalTrials.gov, number NCT05115500. Results: With the cutoff date of 11 February 2022, a total of 8 patients were enrolled. Among 6 patients who had at least one tumor assessment, involved gastric cancer(3/6), breast cancer(1/6), cervical cancer(1/6), pancreatic cancer(1/6). The ORR was 66.7% (4/6) and disease control rate (DCR) was 83.3%(5/6). One cervical cancer and one pancreatic cancer achieved complete remission. The most common treatment-related adverse events (TRAEs) were alopecia (37.5%), fatigue (25%) and hepatic damage(12.5%). No Grade 3 and higher adverse events occurred. Conclusions: The PRaG3.0 regimen manifested manageable safety profile and encouraging efficacy. So PRaG3.0 regimen is considered as a promising salvage treatment for patients with HER2-expressing advanced solid tumors. And the conclusion should be validated in more patients subsequently. Clinical trial information: NCT05115500.
Gastrointestinal adverse effects of combined checkpoint inhibitors: A systematic review and meta-analysis.
Authors: Karna Rahul , Deliwala Smit , Ramgopal Balasubramanian , Asawa Palash , Mishra Rahul , Mohan Babu P. , Grover Dheera , Kalra Tanisha , Bhalla Jaideep , Saraswati Ushasi , Jayakrishnan Thejus , Monga Dulabh K. , Dhawan Manish
Published: June 2022

e14591

Background: Combined immune checkpoint inhibitors (ICPI) have revolutionized treatment of solid tumors. Their use can lead to treatment-related adverse events (TRAEs) resulting in hospital admissions, treatment discontinuation and increased morbidity. We aim to evaluate gastrointestinal (GI) TRAEs of different combination ICPI. Methods: A comprehensive search of multiple databases from inception to October 2021 was performed. Phase 2 and 3 trials assessing safety and efficacy of combined ICPI in solid tumors were included. Trials reporting concurrent use of chemotherapy, immunosuppressants, targeted therapy, or sequential ICPI therapy were excluded. Primary outcome was pooled luminal, hepatic and pancreatic TRAEs. A meta-analysis of proportions was done for outcomes using a random-effects model. I2 % was used to assess heterogeneity. Results: Out of 3696 citations screened, 53 trials reporting TRAEs in 6186 patients were included. Data from 41 study arms of 35 trials in nivolumab plus ipilimumab (N+I), 15 study arms of 15 trials in durvalumab plus tremelimumab (D+T), 4 study arms of 3 trials in pembrolizumab plus ipilimumab (P+I). The most common indications for combined ICPI were melanoma (26.4%) and lung cancer (24.5%). Pooled GI TRAE rates from the D+T, P+I and two different dosing combinations of N+I are shown in the table. Conclusions: Our study provides pooled data of TRAEs from different combination ICPI use in solid tumors and demonstrates high incidence of all grades and ≥ 3 GI TRAEs. Combined ICPI related TRAEs should be recognized early, as it may require treatment discontinuation. Further studies are required to assess overall impact of TRAEs on treatment course and outcomes.[Table: see text]
Effects of prophylactic high and low doses of corticosteroid on the efficacy of immune checkpoint blockade in murine hepatocellular carcinoma models.
Authors: Shen Ying-Chun , Yeh Ching-Ping , Jin Lu , Chang Nai-Wen , Hsu Chia-Lang , Ou Da-Liang , Chu Ching-Liang , Cheng Ann-Lii
Published: June 2022

e14596

Background: Immune-related adverse events (irAEs) are major hurdle to immune checkpoint blockade (ICB). Corticosteroid (CS) is effective in controlling the majority of irAEs. Although recent studies suggested CS use does not jeopardize the anti-tumor efficacy of ICB, prophylactic use of CS remains prohibited with the concerns of attenuating efficacy of ICB. This study aimed to investigate the effects of CS premedication on the efficacy of ICB in murine hepatocellular carcinoma models. Methods: Anti-mCTLA-4 (9D9C, BMS) and anti-mPD-1 antibodies (4H2, ONO) were intraperitoneally (ip) administered to tumor-bearing mice (subcutaneous Hepa 1-6 model and orthotopic BNL 1MEA.7R.1 model) with or without dexamethasone (DEXA) premedication (10 and 200 μg, equivalent to minimal anti-inflammatory dosage and pulse therapy in human, respectively). Efficacy of ICB was evaluated as tumor shrinkage. Tumor-infiltrating lymphocytes (TILs) were isolated for single cell RNA-sequencing and effector function analysis through flow cytometry. Results: In the subcutaneous model, all tumors treated with ICB alone (N=7) or ICB plus DEXA 10 μg (N=5) completely regressed, but 1 out of 7 tumors treated with ICB plus DEXA 200 μg escaped. However, the tumor growth was not significantly different between groups ( P-value >0.05, multiple Mann-Whitney test). In the orthotopic model (N=5/group), the mean (± standard error) tumor weights on day 21 after tumor implantation for isotype control, ICB, ICB plus DEXA 10 μg and ICB plus DEXA 200 μg were 2.45 (± 0.54), 0.73 (± 0.18), 0.98 (± 0.65), and 0.69 (± 0.17) grams, respectively ( P-value >0.05, comparing ICB plus DEXA 10 or 200 μg with ICB). At transcriptomic level, premedication with either dosage of DEXA significantly reduced the percentage of effector memory cells and increased the percentage of exhausted effector cells in the CD8 TIL population, which appeared to counterbalance the effects of ICB. However, at protein level, premedication of DEXA 10 or 200 μg did not reduce the interferon-γ or granzyme B production of CD8 TILs in both models (Table). Conclusions: CS premedication did not attenuate the efficacy of ICB. Our study provides the scientific basis to evaluate the potential of prophylactic CS in preventing ICB-induced irAEs in clinical studies.[Table: see text]
Analytical validation of OriMIRACLE S, a personalized and tumor-informed approach to minimal residual disease surveillance.
Authors: Li Zhendong , Zhao Weirong , Gan Yanhui , Zheng Xin , Yao Jicheng , Li Feng , Wang Weifeng
Published: June 2022

e15013

Background: The presence of minimal residual disease (MRD) after curative-intent therapies in solid tumors is recognized as a risk factor for relapse. Glycoprotein-based tumor markers lack sensitivity and specificity for clinical application. Circulating tumor DNA (ctDNA) based MRD detection which can identify disease relapse ahead of radiological imaging has shown promising performance. The objective of this study was to develop and validate OriMIRACLE S (Minimal Residual Circulating Nucleic Acid Longitudinal Detection in Solid Tumor), a highly sensitive and specific tumor-informed assay for MRD detection via plasma cell-free DNA. Methods: Tumor-specific somatic single nucleotide variants (SNVs) were identified by whole exome sequencing of tumor tissue and matched germline DNA. Clonal SNVs were selected by OriSelector algorithm for patient-specific multiplex PCR-based NGS assay for MRD tracking. The limit of detection (LOD), specificity, sensitivity, repeatability, and reproducibility of this assay were determined experimentally with a series of standard references and in silico. Standard references were developed as following for assessing assay performance. The somatic variant references were two commercialized standards each containing five positive sites at variant allele frequency [VAF] of 0.1% and 1%, and a wild type sample. The single nucleotide polymorphism (SNP) references were made by blending three gDNA samples (160 sites) with gDNA samples without these SNPs to VAFs of 0%, 0.005%, 0.01%, 0.02%, 0.05% and 0.1%. And cfDNA references were made by titrating the clinical cfDNA mixture from five patients containing 31 positive sites with mononucleosomal HEK293 DNA to VAFs of 0.01-0.3% and 0.1-3%. Results: Site level sensitivity of ctDNA samples were 68.8% to 100% with 10 to 50ng input ctDNA (3,000 to 15,000 haploid genome equivalents). Site level reproducibility and repeatability were 98.0% and 97.0% using somatic variant references, respectively. The detection of three positive sites was sufficient to achieve a nearly 100% overall sample level sensitivity and specificity (6,000 haploid genome equivalents of gDNA samples) determined either by calculating a binomial probability or in silico with customized panels have 21 to 30 variants. The LOD of this assay was as low as 0.01% with the algorithm of OriCleverClover which could filter process and sequencing-related artifacts. Conclusions: OriMIRACLE S has been analytically validated as a highly sensitive and specific method to detect low quantities of ctDNA in plasma. The low limit of detection at a VAF of only 0.01% with low DNA input. OriMIRACLE S has wide-ranging potential applications in the clinical setting and drug development in a real-world scenario.
Large-scale whole-genome sequencing identifies genetic alterations in circulating hepatocellular carcinoma cells.
Authors: Zhou Zhihang , Xu Tao , Zou Heng , Yang Zihan , Yang Mengsu
Published: June 2022

e15019

Background:Hepatocellular carcinoma (HCC) is one of the most lethal malignancies. Most of the patients have large tumor burden, vascular invasion, or metastasis at initial diagnosis. Liver biopsy is usually inaccessible in HCC patients due to the high risk of hemorrhage and tumor dissemination. Circulating tumor cells (CTCs) are malignant cells shed from the tumor tissue into the blood or lymphatic vessel. The fact that CTCs are live tumor cells makes them distinct from any of the existing cancer biomarkers. The biophysical property-based enrichment methods make downstream process easier as the isolated CTCs are not tagged with antibodies and the cell viability can be largely remained. Our objective is to isolate CTCs using a novel inertial focusing-base microfluidic device (CTC-100) and determine both their clinical value and the genomic features. Methods: We enriched and isolated CTCs in 155 HCC patients, 51 cirrhosis patients and 10 healthy donors. These patients were enrolled from Sep, 2020 to Feb, 2022 in the Second Affiliated Hospital of Chongqing Medical University (Chongqing, China). This method enriches CTCs according to the cell size and is label-free. The enriched CTCs are then validated via immunostaining. The CD45-EpCAM+DAPI+ cells with size larger than 15μm are considered as epithelial CTCs, and CD45-EpCAM-DAPI+ cells are considered mesenchymal. The CTCs were then micromanipulated from the slide and the pooled CTCs from each patient were then subjected to MALBAC whole genome amplification and whole genome sequencing, which has been finished in CTCs from 12 patients receiving surgery and 4 without tumor biopsy. The other samples are under processing. Results: Our data revealed that the number of total CTCs or mesenchymal CTCs were significantly higher in HCC patients than that in cirrhosis or healthy donors. The detection rate of CTC in HCC patients was 100%. The EpCAMpositive CTCs accounted only for 16.6% (298/1798) of the total CTCs. Moreover, the number of CTCs was positively associated with macrovessel invasion and tumor size (P < 0.01). Most of the mutated genes in CTC samples were accordant with corresponding cancer tissues. We have detected 24 CTC-specific mutated genes, including FBXL22, USP3, MIR1827, S100A1. We then figure out the frequently mutated genes in CTC samples. Finally, we cluster the SNVs in CTCs according to clinicopathological parameters including macrovessel invasion, distant metastasis, tumor size. The results showed that part of the mutated genes might be associated with tumor invasion or metastasis. Conclusions: The novel microfluidic platform can efficiently detect CTCs in HCC patients. CTC number is associated with macrovessel invasion and tumor size. Finally, the SNV but not CNV in CTC samples is tightly consistent with that in tumor tissue. We also find out some CTC-specific mutated genes that might be involved in HCC metastasis. Clinical trial information: ChiCTR2100051584.
Genomic profile by liquid biopsy of Peruvian patients with lung cancer.
Authors: Galvez-Nino Marco , Roque Perez Katia , Castro Melanie Wendy , Ruiz Rossana , Valdivieso Natalia , Coanqui Ofelia , Olivera Hurtado de Mendoza Mivael , Amorin Edgar , Mas Luis
Published: June 2022

e15027

Background: The optimal treatment of advanced NSCLC patients is currently led by genomic profiling with the goal of detecting actionable mutations with an approved targeted therapy. However, tissue DNA samples are not always available to perform this assays. In this context, liquid biopsy has emerged as a valuable tool for providing the best treatment option. Methods: Retrospective study held at Instituto Nacional de Enfermedades Neoplasicas (INEN), Lima - Peru, between January 2019 and September 2021 to assess the genomic profile of 190 newly diagnosed advanced NSCLC patients through liquid biopsy. Blood samples were profiled with the FoundationOne Liquid Biopsy assay. We describe frequency of pathogenic and targetable genetic alterations, as well as the survival outcomes. Genomic and clinical data were recorded from clinical files. Results: The median age was 63 years (26-90), 66.3% were women, 15.3% smokers, 26.3% had exposure to biomass, 22.6% had a first-degree family history of cancer. The most frequent histological type was adenocarcinoma (86.8%). Regarding genomic profile, 183 patients (96.3%) had at least one pathogenic genetic alteration, the most frequent altered genes were TP53 (54.7%), EGFR (50.5%), DNMT3A (17.9%), KRAS (9.5%) and NF1 (6.8%). 116 patients (61%) had a targetable mutation: EGFR Ex19del/ins (27.4%), EGFR L858R/L861Q (17.9%), ALK rearrangements (5.8%), BRAF V600E (3.2%), RET fusions (1.6%), METex14 skipping (1.6%), KRAS G12C (1.6%), ROS 1 (1.1%), and NTRK (0.5%). Of these, 71 patients (65.5%) were able to access target therapy. For the latter, estimated median PFS and OS were 14.0 months (9.3 – 18.6) and 17.8 months (8.2 – 27.4), respectively. Conclusions: Peruvian NSCLC patients represent a population with a high frequency of pathogenic and targetable mutations, being EGFR mutations and ALK rearrangements the most prevalent ones. Despite limited access, a great proportion of patients received target therapy, achieving remarkable survival.
Assessment of tumour infiltrating subpopulations and pathological complete response using multiplex immunohistochemistry and digital pathology in early HER2+ breast cancer.
Authors: Hurley Camille , Lacroix Laetitia , Sheehan Katherine , Lucas Mairi , Buckley Rachel , Blümel Anna , Toomey Sinead , Hennessy Bryan T. , Crown John , Sautes-Fridman Catherine , O'Connor Darran
Published: June 2022

e15041

Background: The treatment of HER2+ breast cancer (BC) has improved substantially with the use of targeted therapies, and patients achieving pathological complete response (pCR) following neoadjuvant therapy have significantly improved disease free survival. However, there is currently no way to determine which patients are most likely to respond to neoadjuvant HER2-targeting treatment. Growing evidence indicates tumour infiltrating lymphocytes influence HER2+ BC outcome. We hypothesize that a deeper investigation of the immune landscape of HER2+ patient tumours may improve patient stratification and identify an immune profile associated with pCR. Methods: 3plex immunohistochemistry panels identifying immune cell subpopulations and tertiary lymphoid structures were applied to HER2+ BC tumors (pre-treatment N=19, post-treatment N=9) of patients of the neoadjuvant TCHL clinical trial (NCT01485926), who received HER2-targeting therapy. Quantitative analysis of CD3+, CD20+, DC-Lamp+, CD4+, and CD8+ immune cells on multi-labelled whole-slide images of TCHL tumor sections was performed using artificial intelligence image analysis models. Patient-specific densities of each cell type in tumor regions were determined to facilitate assessment of pre-treatment differences in target immune subpopulations in complete, partial, and non-responders. Results: Statistical analysis demonstrated a trend for higher densities of each immune subpopulation in pre-treatment biopsies of pCR vs. non-pCR patients. Patient categorisation based on median threshold demonstrated patients achieving pCR are more frequently high for each immune cell type assessed, while non-pCR patients are more frequently in the low category. Investigation of association of pre-treatment immune infiltrate with clinicopathological features demonstrated trends for higher levels of T-cell infiltration in later stage and hormone receptor negative patients. Conclusions: Multiplex IHC is an optimal technique for the concurrent assessment of multiple target markers in patient tissue, which can be quantitatively analyzed in a high throughput manner using AI models. The findings of this preliminary study support recent evidence regarding the role of tumor infiltrating immune cells in HER2+ BC outcome and warrant further investigation in a larger cohort.
Comparison of 3 PCR-based assays for microsatellite instability detection in formalin-fixed paraffin-embedded tissues of patients with colorectal cancer.
Authors: Yang Jun , Zhang Biao , Guan Wenyan , Pu Xiaohong , Miao Shuying , Fan Zhiwen , He Lu
Published: June 2022

e15042

Background: Routine assessment of microsatellite instability (MSI) status in colorectal cancer (CRC) patients can help with initial screening for Lynch syndrome, prediction of tumor prognosis and selection of patients for immunotherapy. Immunohistochemistry (IHC) and PCR-based approaches represent the current clinical laboratory standard for the evaluation of MSI status. Both Bethesda and Promega marker panels have been used widely in the world and the Bethesda panel and the six mononucleotides panel (NR21/BAT25/NR24/NR27/MONO27/BAT26) have been approved by the National Medical Products Administration (NMPA) in China. The Idylla MSI Test, a fully automated system based on real-time PCR, which incorporates 7 novel MSI loci (ACVR2A/BTBD7/DIDO1/MRE11/RYR3/SEC31A/SULF2) can perform MSI analysis within 150min using 1-3 tissue slides. The aim of this study was to evaluate the performance of the three 3 PCR-based assays for MSI detection including Idylla MSI Test, Bethesda and that six mononucleotides panel. Methods: One hundred and fifty-three formalin-fixed paraffin-embedded (FFPE) tumor tissues from patients with CRC were collected and their MSI status were analyzed using 3 PCR-based assays, respectively, i.e. the Idylla MSI, “2B3D” NCI panel and the six mononucleotides panel. All samples were previously evaluated by IHC method. Results: In the all 153 patients, IHC identified 27 cases to be MMR-deficient (dMMR), and 126 to be MMR-proficient (pMMR). Using IHC as a reference, the Idylla MSI had a higher consistency that showed overall agreement, sensitivity and specificity as 93.46%, 92.89% and 93.65%, respectively, while the corresponding values of the six mononucleotides panel were 84.31%, 92.59% and 82.54% respectively, and the “2B3D” NCI panel showed a sensitivity of 92.16%, a specificity of 88.89% and an overall concordance of 92.86%. The PCR-based approaches identified 11 more MSI-H cases than IHC while 22 dMMR cases identified by IHC were detected by all the 3 assays, 2 dMMR by 2 assays, 1 dMMR by one assay only and 1 dMMR by neither assays above. Moreover, the Idylla MSI showed advantages in ease of use, turnaround time (TAT) and small dose of tissue input. Conclusions: Our findings support that the Idylla MSI assay provides a rapid, reliable, and ease-to-use solution to MSI detection in Chinese CRC patients with high sensitivity and specificity. The inconsistent cases between IHC and PCR-based approaches need a further analysis.
Discovery of potent, orally active KIF18A inhibitors targeting CIN-high cancer cells.
Authors: Zhang Rumin , Cogan Derek , Eng Christina , Phillips Aaron , Carty Marysol Chu , Feinberg Tamar , Verma Akanksha , Tourdot Richard , Swiston Albert , Bettigole Sarah , Drutman Scott , Su Michael
Published: June 2022

e15046

Background: KIF18A is a kinesin assisting chromosome congression and alignment during mitosis. It is nonessential in normal, euploid cell division but essential for a subset of aneuploid cancer cells with high chromosomal instability (CIN-high). KIF18A knockdown in these cells leads to mitotic arrest, cytostasis and cell death. As a result, KIF18A is an attractive anticancer target. Methods: Volastra drug discovery efforts identified and validated KIF18A as a promising target using genetic knockdown and pharmacological inhibition with a tool compound. High-throughput screening identified an ATP-noncompetitive hit that was optimized for potency, selectivity, and pharmacokinetic properties. Breadth of efficacy studies against a panel of cancer cell lines were used to formulate responder hypotheses and aid in patient selection. Mechanistic profiling and cell fate determination upon KIF18A inhibition was used to identify target engagement and clinical response biomarkers. Results: Our lead candidates bind to KIF18A with sub-nM potency and exhibit long (̃2 days) drug-target residence time. They are ATP and microtubule uncompetitive. They are highly selective for KIF18A and have no known off-target effects on related kinesins and in an in vitro safety screen. They are orally bioavailable in preclinical species, well tolerated and demonstrate tumor regression in mouse xenograft models. In contrast to many agents that disrupt the mitotic machinery, KIF18A inhibition does not impact growth of proliferating normal cells. Breadth of efficacy studies performed on a large panel of human cancer cell lines reveal sensitivity to KIF18A inhibitors across multiple tumor types including high-grade serous ovarian, as well as subtypes of breast and lung cancer. Conclusions: Volastra’s research efforts have culminated in the discovery of orally active, highly potent, selective, and efficacious KIF18A inhibitors as development candidates.
Development of heterobifunctional proteomimetic polymers for delivery of MYC inhibitory peptides and targeted MYC degradation.
Authors: Truica Mihai Ioan , Wang Max Mu , Gattis Brayley , Abdulkadir Sarki , Gianneschi Nathan
Published: June 2022

e15049

Background: The c-Myc(Myc) oncogene plays critical roles in tumorigenesis and therapeutic resistance, being dysregulated in up to 70% of all human cancers. Heterodimerization with the closely related bHLH-LZ protein Max is required for oncogenic transformation, yet the development of small molecule inhibitors has been challenging since disordered Myc and Max monomers lack suitable binding pockets, while the Myc-Max interface is flat and extensive. Myc inhibitory peptides derived from the first helix (H1) of the bHLH-LZ region have overcome the limited binding affinity of small molecules. However, poor cellular penetrance and extremely limited bioavailability have precluded clinical translation of peptide-based approaches. Aim: To address and improve the limitations and low potential for clinical translation of Myc inhibitory peptides via a versatile peptide delivery platform. This novel platform technology is referred to as the Protein-Like Polymer (PLP) and it is based on packaging peptides together as high-density brush polymers. Methods: We generated H1-PLPs using ring-opening metathesis polymerization and characterized them using HPLC, LC-MS, NMR, and GPC. We also explored the potential of PLPs to be designed as heterobifunctional compounds by copolymerizing with secondary side chain peptides (nuclear localization sequences and degron sequence). For biological studies, immunofluorescence and 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-based selectivity of response. Myc target engagement was assessed via immunoprecipitation assays and dual-reporter luciferase-based assays. Results: Our studies show that heterobifunctional PLPs employing H1 peptide and a degron sequence are cell-penetrating and co-localize with Myc in the nucleus. They display antiproliferative effects with submicromolar IC50 values in a Myc-dependent manner. The PLPs lead to Myc/Max complex disruption, promote significant levels of Myc degradation and prevent binding of Myc/Max heterodimers to the Myc/Max consensus canonical DNA binding site. Selectivity studies reveal that antiproliferative effects are dependent on intact H1 sequence. Animal studies using transplanted Myc-CaP model, which mimics the clinical course of human castration resistant prostate cancer are ongoing. Conclusions: We present a novel peptide delivery platform that addresses the challenges inherent to peptide delivery approaches. The presented work demonstrates the feasibility of this platform in delivering Myc inhibitory peptides and provides rationale for further in vivo studies in Myc-dependent tumor models.
Systems biology basis of permanent tumor regression with normal tissue protection: Experimentally validated signaling pathway framework.
Authors: Kumari Bindu , Sakode Chandrasekhar , Raghavendran L. , Roy Prasun Kumar
Published: June 2022

e15066

Background: Clinicians generally deal with malignant tumors in progression phase only, but there is a reverse complimentary process, namely permanent spontaneous regression of tumors, a well-known phenomenon occurring across human populations at 22%-46% rate (Scandinavian and Wisconsin Screening Registries: 0.33 million and 2.95 million populace respectively; Lancet Oncol.12: 1118-24, 2011; J.Nat.Cancer.Inst.36: 37-47, 2006). Probing such a phenomenon can illuminate how one can induce a permanent regression process on human tumors therapeutically. Here we develop a systems biology approach for this process, that we validate by experimental findings. Methods: Mathematical System Biology Formulation: For permanent tumor regression, we develop a coupled differential equations-based cell kinetic analysis, keeping normal tissue protected. Thereby, we mathematically model the temporal variation of the level of three essential tumor-lysis components: DNA replication-blockade, Antitumor Lymphocyte, Cytokine interleukin-2. Experimental Investigation: We investigate the temporal process of spontaneous tumor regression of melanoma (in Liebechov pigs) and analyze the microarray signal using IPA platform Gene expression signature: Using IPA analysis, we find the signaling pathways associated with the permanent tumor regression. Results: Mathematical System Biology Formulation: We find the temporal behavior of tumor-lysis components are: (1) DNA replication-blockade activation: Unimodal inverted U function (2) Antitumor Lymphocyte activation: Bimodal M function; (3) Interleukin-2 activation as Stationary step function. Experimental findings: Our microarray analysis gives temporal pattern of gene-expression levels corresponding to above three components: G2/M DNA damage checkpoint regulation (genes CCNB1, CDC2), Chemokine signaling activation - Interleukin 2 ( GRB2, PIK3CG), T Lymphocyte Receptor Signaling (CCR5, PRF1). These three temporal components respectively show Unimodal, Bimodal, and Stationary activation. Validation: We show that the three mathematically-derived temporal activation functions are well corroborated by corresponding experimental temporal pathway activation data (Smirnov-Kolmogorov statistical test satisfied; α = 5%). Conclusions: A novel tri-phasic activation of three antitumor signaling pathways (DNA blockade, Interleukin 2, Cytotoxic T cell) can permanently regress melanoma tumor in the experimental setting if the pathways are activated at different time points and not simultaneously; These temporally orchestrated patterns are much different from conventional chemotherapy/ immunotherapy protocols where the temporal activations are simultaneous and empirically chosen. Optimal temporal profiling of administration of antitumor agents is thus crucial from a therapeutic standpoint.
A pancancer analysis of CDK12 alterations in Chinese population.
Authors: Lou Luyan , Zhang Yanrui , Liu Huanhuan , Liang Xiayuan , Lou Feng , Cao Shanbo , Wang Huina
Published: June 2022

e15068

Background: Cyclin-dependent kinases ( CDKs) are key regulators of both cell cycle progression and transcription. Analysis of mutation detection of CDK12 in pan-cancer could contribute to understand the complexity and diversity of genetic alterations present in CDK12 mutant cancers. Herein, we provide a pan-cancer analysis of CDK12 alterations in Chinese population. Methods: Using gene-panel target-capture next generation sequencing, we analyzed 17509 tumor tissue or plasma ctDNA samples from different patients. Testing included analysis of single nucleotide variants, insertions/deletions, fusions and amplifications. CDK12 variants of uncertain significance and synonymous variants excluded. Results: In total, 0.95% (167/17509) of pan-cancer patients harbored CDK12 mutation, and 32 patients carried at least two CDK12 mutations. The most common genetic alteration type was truncating mutations (128, 64%), followed by amplications (53, 26%), missense mutations (9, 5%) and splicing mutations (9, 5%). In our cohort, the highest CDK12 mutation frequency tumor type was prostate cancer(11.30%), followed by breast cancer (3.96%), bladder cacner (3.16%), stomach cancer (1.84%) and colorectal cacner (1.13%). The TCGA pan-cancer cohort confirmed that the CDK12-altered cancer was associated with shorter overall survival (81.11 vs 133.00 mos, p = 5.30×10−18) in prostate cancer. Tumor mutation burdens(TMB) were calculated for 137 samples of all the 167 samples. The median TMB was 7.66 mut/Mb with 38%≥10 mut/Mb and 17%≥20 mut/Mb. including a patient with hypermutated in colorectal cancer(TMB: 88.24 Mut/Mb), harboring three CDK12 mutations. Conclusions: Genomic alterations in CDK12 are widespread among Chinese patients, with alteration rates and alteration types varying by cancer type. We identified prostate cancer as the cancer type with the highest prevalence of CDK12 oncogenic alterations. Additional analyses are ongoing.
Predictive factors for successful growth of patient derived xenografts (PDX).
Authors: Hernandez-Guerrero Tatiana , Doger Bernard , Garcia-Foncillas Jesus , Wick Michael Jude , Moreno Victor
Published: June 2022

e15069

Background: PDXs have become a core component of translational cancer research. Developing these models can become challenging since little is known about which factors influence engraftment rates. We sought to determine which clinical, pathological, or molecular factors may predict better engraftment rates in PDXs. Methods: Between March 2017 and January 2021, biopsies obtained from patients with primary or metastatic cancer were implanted into athymic nude mice. Statistical analyses were performed to identify factors that could correlate with final engraftment defined as achievement of at least three passes and sampling of PDXs tumors. We focused on clinical (patient factors) pathological (patients’ tumor samples) and molecular characteristics (patients’ tumor samples) analyzed either by immunohistochemistry (IHC) or next generation sequencing (NGS). Results: 585 tumor samples were collected and implanted. 21 failed to engraft due lack of malignant cells. Of 564 tumor-positive samples, 187 (33.2%) PDXs achieved successful growth at time of analysis (Feb, 21). The following clinical characteristics were correlated with engraftment: systemic antibiotics within 2 weeks of sampling: (38.1% (72/117) antibiotics- group vs 30.7% (115/260) no-antibiotics) (p = 0.048); systemic steroids within 2 weeks (41.5% (34/48) the steroids-receiving group vs 31.7% (153/329) no-steroids) (p: 0.05). For women, menopausal status was predictive: 34.9% (95/177) in postmenopausal achieved growth, Vs 20,4% (10/39) for premenopausal (p = 0.031). Baseline LDH levels: 74.9% (140/187) LDH levels above the upper limit of normality (ULN) against 25.1% (47/187) with normal LDH (p = 0.034). Tumor grade: Grade 1: 25.4% (47/187); grade 2: 34.8% (65/187) and grade 3: 40.1% (75/187) tumors achieved successful growth (p = 0.043). Similarly, higher ki67 levels were also correlated with better engraftment rates: (low (Ki67 < 15%): 8.9% (9/45) achieved growth, Vs high (Ki67 > 15%): 31% (35/113) (p:0.002). Presence of lymphovascular invasion in tumor sample was also predictive: 42.2% (97/230) with lymphovascular Vs 26.9% (90/334) of samples with no invasion (p = 0.0001). Likewise, 41.8% (59/141) of neural invasion-positive samples achieved growth against 30.3% (128/428) (p = 0.008). Mismatch repair deficient tumors showed better engraftment rates: 62.1% (18/29) achieved growth vs 40.8% (75/184) of proficient tumors (p = 0.026). 84 PDX were breast models, among which 57.9% (11/19) ER negative models grew, Vs 15.4% (10/65) of ER positive models (p = 0.0001). Conclusions: tumors with higher grade and Ki67, lymphovascular and/or perineural invasion, with dMMR and ER expression negative have higher chance of PDX development. Some clinical characteristics can also interfere with PDXs development such as use of steroids or antibiotics prior sampling.
Paeoniflorin, ASK1-TF, and SOCS3 and association with radiation enteritis.
Authors: Yang Mi , Sheng Lei , Fei Wenjing , Hu Liang , Liu Wen-Tao , Ma Jianxin
Published: June 2022

e15076

Background: Radiation enteritis is one of the main adverse effects of radiotherapy, presenting with a poorly understood etiology and limited options for therapy. Intestinal inflammation and ischemia are the core mechanisms of radiation enteritis. Suppressor of cytokine signaling 3 (SOCS3) is an endogenous “inflammation brake”. We hypothesized that paeoniflorin, a pinane monoterpene bitter glycoside, could increase SOCS3 expression to reduce inflammation and ischemia and improve enteritis in mice. Methods: Laser doppler flowmetry was used to detect changes in intestinal blood flow. RAW264.7 and human umbilical vein endothelial cells were used to investigate the mechanism of action of paeoniflorin. Results: It was observed that radiation caused high mortality, intestinal inflammatory responses, and low blood flow in mice. Paeoniflorin effectively alleviated intestinal atrophy, prevented thrombosis, improved radiation enteritis, and reduced mortality in mice undergoing radiotherapy. In addition, paeoniflorin increased the release of growth arrest-specific gene 6 (Gas 6) and phosphorylation of the Axl receptor, subsequently inducing the expression of SOCS3 and inhibiting the expression of p-apoptosis signal-regulating kinase 1 and tissue factor in vivo and vitro. Conclusions: we suggest that paeoniflorin is potentially effective in alleviating radiation enteritis via the activation of the Gas6/Axl/SOCS3 axis and subsequent reduction in intestinal inflammation and ischemia.
The combination of the IAP antagonist, tolinapant and hypomethylating agents (HMA) is highly synergistic in in vitro models of T-cell lymphoma (TCL).
Authors: Manavalan John , Pal Ipsita , Pursley Aidan , Ward George , Smyth Tomoko , Sims Martin , Feith David , Loughran Thomas , O’Connor Owen , Marchi Enrica
Published: June 2022

e15088

Background: Peripheral T-cell lymphoma (PTCL) are a highly aggressive and heterogeneous group of non-Hodgkin lymphomas that often carry a poor prognosis with standard chemotherapy. Tolinapant is a potent antagonist of inhibitors of apoptosis proteins (cIAP1/2 and XIAP). In a phase 2 trial (NCT02503423), tolinapant showed activity against heavily pre-treated patients with TCL. We explored the sensitivity of a range of TCL lines to tolinapant, established the synergy coefficient and interrogated the mechanism of synergy between tolinapant and drugs active against PTCL including romidepsin, pralatrexate, and the HMAs; azacytidine and decitabine. Methods: A panel of 10 human TCL lines were tested in proliferation assays (CellTiterGlo) for sensitivity to tolinapant in the presence or absence of 10 ng/ml of TNFα. For combination studies, each drug was tested, in the presence or absence of TNFα. Synergy coefficient was calculated using Excess over Bliss. Additionally, the on-target effects of the drugs were measured by analyzing levels of the IAPs, acetylated histones, DNMTs and key apoptosis and necroptosis markers by Western blotting. Results: TCL Lines demonstrated varying sensitivities to tolinapant with the most sensitive cell line, ALK+ ALCL SUP-M2, having an IC50 as low as 20 nM ± 1 nM while a resistant CTCL cell line HH had an IC50 of over 20 µM. In combination experiments using both cell lines, tolinapant plus azacytidine or decitabine displayed the highest degree of synergism, compared to romidepsin or pralatrexate. Of note, a high degree of synergism was also seen with concentrations 10 folds lower of azacytidine or 100 folds lower of decitabine, added daily (Table). The combination of tolinapant and the HMAs led to a decrease in the levels of cIAP1 and DNMT3a in TCL lines, demonstrating on-target activity of tolinapant and the HMAs, respectively. In addition, both azacytidine and decitabine increased the levels of RIPK3 and MLKL by western blot analysis, indicating activation of the necroptosis pathway. Conclusions: Tolinapant has demonstrated varying cytotoxic effects against a range of TCL lines as a monotherapy and displays a very high degree of synergism in combination with the HMAs, azacytidine and decitabine, also at low concentration, in vitro. The HMAs were also able to activate the necroptosis pathway, providing a possible mechanism for the high degree of synergism displayed when combined with tolinapant in the TCL lines in vitro. These data provide the rationale to initiate phase I trials with the combination of tolinapant and HMAs. Average synergy scores at 48 hrs for drugs used in combination with tolinapant in a CTCL line HH and ALK+ ALCL line SUP-M2. Above 10 indicates synergy.[Table: see text]