bluuebunny/crossref_metadata_2025 huggingface dataset:
An electrochemical molecular assay for the detection of cancer-associated Thomsen-Friedenreich glycans.
Authors: Moraes Silva Saimon , Abeyrathne Chathurika , Li Miaosi , Cossins Luke Robert , Samudra Anushka , Greene Wren , Moulton Simon
Published: June 2023

e15046

Background: Abnormal glycosylation is a fundamental characteristic in cancer cells, where expression of glycans such as Thomsen-Friedenreich (T), Tn, and sialyl-Tn (STn) are related to particular tumour patterns. T, Tn and STn antigens are unusually short and truncated O-glycans expressed on glycoproteins synthesized within cancer cells and not normally found in healthy tissue or circulating within the blood. The association between blood circulating T-glycans and cancer progression and poor prognosis is now well-known and highlights great potential as biomarkers for cancer screening, diagnosis, and surveillance of diseases during treatment and following remission. Despite this potential, there is currently no established molecular assay for detecting or quantifying these biomarkers in blood biopsy samples. Methods: Using a test strip format reminiscent of a blood glucose test, a new electrochemical molecular assay is introduced for the detection of short O-glycans directly in unprocessed bodily fluids. This assay is based on a self-assembling sensing interface consisting of two glycoproteins, recombinant human lubricin (LUB) and peanut agglutin (PNA). In this interface, the LUB functions as both an anti-fouling layer which mitigates electrode passivation in blood and as a crucial component in the biosensing mechanism for generating readable electrical signals in the presence of target glycans. The PNA, a plant-based lectin, presents high binding specificity towards the T glycoform and functions as the sensor recognition element which is bound to the LUB through T glycans naturally present in the highly glycosylated protein backbone. In this sensing interface, electrical signals are generated by engineering a redox reporter, methylene blue into the LUB/PNA interfacial complex, where the electrical signal is altered by the capture of target glycans by PNA and its impact on the molecular dynamics of the LUB/PNA interfacial complex. Results: Using this test strip, it is possible to detect short cancer-related glycans in concentrations as low as nanomolar directly in small volumes of unprocessed whole blood. These test strips likewise show comparable results to an ELISA test developed in parallel for the detection of short O-glycans. The tests carried out with retrospective blood plasma samples from patients presenting three different tumour types, including prostate (18 patients), colorectal (15 patients), and breast cancer (6 patients), and plasma samples from healthy donors (10 different donors) showed differentiation of response signals between healthy and diseased patients. Conclusions: These findings highlight the clinical potential of this new disposable test strip for the detection of tumour associated glycans, which can be incorporated into point-of-care devices, for cancer monitoring and perhaps one day in screening and diagnosis.
Importance of clinical decision making in precision oncology: Discordance in recommendation between next generation sequencing test vendor and molecular tumour board in India.
Authors: Sirohi Bhawna , Davis Sissmol , R I Anu , Suryavanshi Moushumi , Dawood Shaheenah S. , Panda Pankaj Kumar , Nag Shona Milon , Das Arunangshu , Rohatgi Nitesh , Popat Sanjay , Shah Riyaz N.H. , Thampy Cherian , Parikh Aparna Raj , Yadav Siddhartha , Mehta Prashant , Singh Randeep , Mukherji Deborah , Shilpakar Ramila , Mullapally Sujith Kumar , Mathew Aju
Published: June 2023

e15050

Background: Molecular tumour boards (MTB) facilitate discussions among medical professionals, scientists, bioinformaticians and geneticists for clinical application of precision oncology, via interpretation of various genomic sequencing studies, most commonly Next Generation Sequencing (NGS). NGS test vendor recommendation may differ from the MTB recommendations. We aim to study the discordance rate between these recommendations. Methods: MTB discussions between April 1, 2021 and December 31, 2021, were retrospectively evaluated. MTBs which assessed NGS reports with or without significant genetic mutations but with drug recommendations were included. NGS testing was done with multiple vendors. Patients from all over India, Nepal, Bangladesh, Dubai and Abu Dhabi were discussed. We excluded MTBs which assessed NGS data without any drug recommendations. We compared therapeutic recommendations provided in the NGS report with the first-choice recommendation in the MTB discussion. Recommendation discordance was defined as disagreement between the NGS recommendation and the first MTB recommendation that either instructed its implementation either immediately or on disease progression. Results: 70 MTB data was studied. 49 MTBs assessing NGS reports of 49 unique patients (median age 54 years; range, 32-82) were included in the study. 13 patients had lung cancer, 10 colorectal, 6 breast, 7 hepatopancreatobiliary, 4 carcinoma of unknown primary and 9 others. 17 did not provide any treatment recommendations and hence were excluded. 4 were discussions based on NGS reports previously discussed in an MTB that was already included in the study, hence were excluded. Recommendation discordance rate was 49% (24 of 49). Conclusions: To our knowledge, this is the first study that assessed discordance rate in recommendations between NGS report and MTB from a low- and middle-income country setting. Accurate clinical translation of complex genomic data is at the forefront of personalized cancer care.
Generalizable FGFR prediction across tumor types using self supervised learning.
Authors: Parmar Chaitanya , Ramon Albert Juan , Stone Nicole L. , Triantos Spyros , Greshock Joel , Standish Kristopher
Published: June 2023

e15057

Background: Deep Learning models have demonstrated the ability to detect tumors, classify disease state, or infer genetic biomarkers from digital whole slide images (WSI) of Hematoxylin and Eosin (H&E)-stained tissue. Such models can be deployed to improve clinical practice or efficiently recruit for clinical trials. However, the data to train these models is often very limited and image capture protocols vary between labs. We propose to use Self Supervised Learning (SSL) on large unlabeled histopathology data sets to improve generalizability and predictive performance of deep learning models on unseen data. Methods: We pre-trained a Convolutional Neural Network (CNN) using SSL on 25,120 unlabeled, digital WSIs from various sources (multiple scanners, hospital systems, labs, diseases, tissue sites). We then fine-tuned the pre-trained CNN to infer the presence or absence of select FGFR alterations in Muscle Invasive Bladder Cancer (MIBC) from the digital WSI. We applied this model (FGFRSSL) to WSIs of biopsies from other data sources and tumor types and compared with a model (FGFRTRAD) that was trained exclusively on MIBC samples without self-supervised learning. Results: The FGFRSSL model achieved an Area Under ROC Curve (AUC = 0.80) on MIBC WSIs and maintained performance on images from an independent lab (AUC=0.82). We further show that the model generalizes to Non-Muscle Invasive Bladder Cancer (NMIBC) samples with (AUC = 0.76) and Pan-Tumor WSIs (AUC: 0.83). The FGFRTRAD model trained without SSL-based pre-training achieved an Area Under ROC Curve (AUC: 0.76) on MIBC WSIs was less generalizable, showing lower performance for independent data (AUC = 0.72), NMIBC samples (AUC = 0.72), and Pan-Tumor samples (AUC = 0.64). Conclusions: We leveraged SSL pretraining to improve the reliability and generalizability of AI-based models across multiple data sources. We also demonstrate our model’s ability to infer FGFR status across multiple solid tumor types after training on only MIBC samples, suggesting the cell morphology conferred by FGFR alteration in cancer may be shared across diverse tumor types. These models represent a means for efficiently screening patients for actionable clinical biomarkers in a robust manner to guide clinical decisions and inform drug development efforts. [Table: see text]
RNA sequencing as a confirmatory assay and its impact on patient care in multiple cancer types.
Authors: Kasi Pashtoon Murtaza , Smirnova Sofia , Nikulin Vladislav , Brown Jessica H. , Almog Nava , Ogloblina Anna , Yam Clinton , Peguero Julio Antonio , Pandya Devesh Mahesh , Kerr Daniel , Fowler Nathan
Published: June 2023

e15058

Background: Increasing evidence shows the ability of RNA sequencing (RNA-seq) to detect expression of multiple biomarkers with improved sensitivity and scalability, offering a more objective and reproducible interpretation of data compared to pathological assessment methods by immunohistochemistry (IHC). Further, ongoing research demonstrating strong correlation between RNA-seq gene expression values and IHC values suggests the utility of RNA-seq as an objective diagnostic approach. Here, we highlight 4 cases where discordance in expression levels measured by IHC and RNA-seq resulted in re-evaluation of IHC values and a subsequent change in the clinical course. Methods: All patients underwent RNA and DNA sequencing of tumor tissue as part of a standard-of-care workflow. Sequencing was performed and reported with the BostonGene Tumor Portrait test, utilizing whole-exome sequencing (WES) and transcriptome sequencing data for tumor analysis. Gene expression levels were evaluated by comparison to a reference cohort of patients with a similar diagnosis. Results: In one case, a 53-year-old female was diagnosed with metastatic cholangiocarcinoma. While IHC of her biopsy reported positive HER2 (+3) expression, RNA-seq revealed low expression. A repeated IHC assay confirmed negative HER2 expression, leading to a change in therapy options. Another 61-year-old female was diagnosed with triple negative breast cancer (TNBC) (IHC: ER 0%, PR 0%, HER2 1+). Integrated WES and RNA-seq analysis detected HER2 amplification (+8 copies), ERBB2-activating mutation, high ERBB2 (HER2) expression and a HER2-enriched PAM50 subtype. IHC was repeated, now showing positive HER2 (+3) expression. In order to target HER2, systemic treatment was changed to a combination containing trastuzumab and pertuzumab, resulting in pathological complete response. In the third case of an 85-year-old female with relapsed TNBC, IHC demonstrated 1% PDL-1 staining. In contrast, RNA-seq detected high PD-L1 expression leading the treating physician to repeat IHC. Staining showed high PDL-1 (CPS50), prompting a change in therapy to an anti-PDL-1 (pembrolizumab) therapy. Lastly, while IHC of a 21-year-old female diagnosed with diffuse large B-cell lymphoma (DLBCL) showed negative CD30, subsequent RNA-seq detected relatively high CD30 expression that prompted a second IHC evaluation confirming positive CD30 expression. Eventually, the diagnosis was changed to primary mediastinal B-cell lymphoma and DA-EPOCH was selected as the therapy. Conclusions: RNA-seq is emerging as an objective tool to evaluate key diagnostic and targetable events in multiple cancer types. The use of RNA-seq can also add value as an objective measurement to confirm critical IHC findings, potentially changing clinical care.
The role of MIBG scan in the diagnosis of pheochromocytoma.
Authors: Ghomari Cherifa Fazila , Medjahedi Abdelkader
Published: June 2023

e15064

Background: Pheochromocytoma is an uncommon adrenal medulla tumor. Metaiodobenzylguanidine (MIBG) scintigraphy is the current gold standard functional imaging method for evaluating pheochromocytoma, due to its excellent 100% specificity, 80% sensitivity, and general availability. In addition to establishing an extension evaluation and monitoring medication efficacy, MIBG scintigraphy is essential to confirm the diagnosis of suspected pheochromocytoma, especially when there is a tumor-like adrenal mass whose etiology is to be determined and biopsy is contraindicated given the risk of catecholamine discharge with hypertensive flare and the risk of bleeding. Our aims are to determine the efficacy of scintigraphy in the diagnosis of pheochromocytoma by examining the epidemiological characteristic and uptake patterns of the disease. Methods: In a retrospective analysis from January 2010 to December 2020, we examined pheochromocytoma patients medical records who had been referred for MIBG scintigraphy to the regional western Algerian Nuclear Medicine department at the University Hospital Center of Tlemcen. Prior to a progressive intravenous injection of 40–80 MBq of l-MIBG, patients underwent preparation (discontinuation of interfering drugs with I-MIBG especially antihypertensive treatment, thyroid blocking, and hydration). Imaging acquisition was carried out 48 and 72 hours later. Results: Fifty patients with probable pheochromocytoma (sex ratio = 0,78; mean age = 43 [17-74] years) completed I-MIBG scintigraphy prior to adrenalectomy. The MIBG scan has a high specificity and positive predictive value (100%). It confirms the presence of pheochromocytoma in 21 cases (42%), even if the metanephrine is normal in a patient with a non-secreting pheochromocytoma. In 18 cases, the main pattern is a single, homogenous, and intense intra-abdominal uptake. For the three remaining patients, a voluminous and heterogeneous uptake with a photopenic center that was associated to a necrotic adrenal mass larger than 10 cm in size, seen on abdominal Computerized Tomography (CT). In this retrospective analysis, no cases of metastatic or multiple diseases were documented. The sensitivity and negative predictive value are 84% and 86%, respectively. In 29 patients (58%), the scan reveals no abnormalities in uptake. This is attributed, in part, to 30% false positive biology (sampling conditions of urinary and/or plasmatic metanephrines and/or normetanephrines), 20% false positive CT imaging (differential diagnosis of adrenal mass: benign or malignant adrenal nodule), and 8% false negative MIBG scintigraphy (subcentimetric, cystic and necrotic lesion). Conclusions: Due to its exceptional specificity, robust sensitivity, and whole-body staging, MIBG scintigraphy—along with biology and morphology imaging—is a crucial noninvasive diagnostic method for the management of pheochromocytoma prior to adrenalectomy.
Interim analysis examining the feasibility of incorporating laser microdissection enrichment of tumor specimens and reverse phase protein array analysis with next generation sequencing into a molecular tumor board.
Authors: Cannon Timothy Lewis , Randall Jamie , Hunt Allison L. , Nutcharoen Aratara , Deeken John F. , Bateman Nicholas W. , Corgiat Brian , Davis Justin B , Petricoin Emanuel , Conrads Thomas P.
Published: June 2023

e15068

Background: Since next generation sequencing (NGS) based profiling does not provide measures of protein abundance or activation state, clinical integration of reverse phase protein array (RPPA) with NGS could be useful for improving selection of targeted cancer therapy. We incorporated biopsy testing and proteogenomic analyses in an institutional Molecular Tumor Board (MTB) for cancer patients using CLIA-certified commercially available multi-omic platforms, including a commercially available CLIA RPPA functional protein drug target activation mapping platform. We present an interim analysis of our experience integrating functional phosphoprotein/protein based drug target activity data with NGS from 117 patients with metastatic solid tumors. Methods: Formalin-fixed, paraffin-embedded (FFPE) tumor biopsy specimens (n = 166) were obtained prospectively from patients as part of an IRB approved MTB. A representative H&E-stained tissue section for each specimen was reviewed by a board-certified surgical pathologist. Specimens with sufficient total tumor cellularity to achieve protein lysate input requirements for RPPA were thin sectioned onto laser microdissection (LMD) membrane slides for selective harvest of tumor epithelium by LMD and RPPA analysis. In parallel, specimens from each case were also submitted for NGS analysis. The multi-omic data for each patient was reviewed by a panel of clinicians and scientists as part of the Inova Institutional MTB for clinical decision-making. Results: Patient tissue specimens (median = 1 per patient; range 1-5) were reviewed to assess feasibility of enriching tumor areas via LMD prior to RPPA analysis. Specimens from 64/117 patients were used for LMD, RPPA, and NGS. 48/117 patients were dropped due to inadequate tumor cellularity, patient death, or referral cases with inadequate specimen. At this time, 7/117 patients remained in the consenting-LMD-RPPA workflow. The median time from patient consent to RPPA analysis completion was 47.4 days. During this period, specimens were outside of the standard hospital-NGS workflow (ie, utilized for sectioning and review for LMD-RPPA) for ~10 days (median). Integrated review of the RPPA and NGS data by the MTB supported a clinical recommendation change for 34/64 patients (53%) overall, with some dependency by primary disease site. Clinical trial information: U20-11-4308.Conclusions: Incorporation of RPPA analysis of LMD enriched tumor samples with NGS was feasible in this pan-cancer cohort, with the mean time to results being less than 7 weeks. RPPA data provided additional treatment considerations for 59% of patients, the outcomes for whom continue to be monitored. [Table: see text]
MOSAIC: Multi-Omic Spatial Atlas in Cancer, effect on precision oncology.
Authors: Lehar Joseph , Madissoon Elo , Chevallier Jerome , Schiratti Jean Baptiste , Kamburov Atanas , Barnes Rodrigo , Haignere Carla , Joy Anna , Dodacki Agnès , Hoffmann Caroline , Lopez Lastra Silvia , Espin Perez Almudena , Bayard Quentin , von Loga Katharina , Chaperon Hubert , Vert Jean-Philippe , Durand Eric , Soumelis Vassili , Wainrib Gilles , Clozel Thomas ,   MOSAIC Consortium
Published: June 2023

e15076

Background: Precision oncology aims to first break diagnoses into biologically distinct subtypes, then pursue personalized therapies for each group of patients ( Garraway et al, J Clin Onc, 2013). This strategy is enabled by recent advances in technologies for medical imaging, molecular profiling, and artificial intelligence. Spatially resolved molecular profiling (“spatial omics”) is an emerging technology that harnesses all three of these advances ( Navarro et al, Science, 2016; Rao et al, Nature, 2021). Cancer is driven by localized interactions between tumor cells, immune and non-immune stromal components. Methods: Spatial omics studies have revealed specific cell types and pathways that control those tumor-microenvironment crosstalks ( Hunter et al, Nat Comm, 2021; Alon et al, Science, 2021), involving both innate and adaptive immunity ( Binnewies et al, Nat Med, 2018). Identifying new cancer subtypes based on the spatially localized tumor-host interactions across many patients have the potential to revolutionize immuno-oncology. The advances in utilizing AI-based methods have so far been unattainable due to a small number of patients in spatial omics studies. Results: Here we present the MOSAIC project - a collaborative initiative across industry and top oncology hospitals to build the largest collection of spatial omics data in cancer. We combine comprehensive clinical annotation with new deep profiling methods to both discover cancer subtypes and identify drug targets and biomarkers within them. MOSAIC aims to generate multimodal data for a total of 7,000 patients in seven cancer indications. Data modalities will include spatial and single cell transcriptomics and proteomics, bulk molecular profiling, pathology images, and curated clinical information. This collaboration harnesses the strengths of academia and industry to provide patient samples, generate high-quality data, develop AI-based analytical tools, and compile a resource that will eventually be made widely available for medical research. Conclusions: Here we present the workflow of the MOSAIC study, demonstrate the data processing pipelines and show spatial distributions of transcripts within the tumor microenvironment. Building upon these initial datasets, we discuss how the MOSAIC parties will collaborate to build an unprecedented database for research and discovery of new immuno-oncology therapeutic approaches.
Association of encapsulation of CDK12 inhibitor with anti-tumour activity in colon cancer cell lines.
Authors: Pérez-Segura Pedro , Diaz-Tejeiro Cristina , Ocana Alberto , Sanvicente Adrián , Arenas-Moreira María , Moreno Carlos Alonso , Nieto Jiménez Cristina
Published: June 2023

e15077

Background: Colorectal cancer (CRC) is the third most common cancer worldwide. Unfortunately, despite the incorporation of new therapeutic options, the metastatic setting is still an incurable disease. Cyclin-dependent kinases (CDKs) are a large family of proteins involved in cell cycle and transcriptional regulation, and are highly expressed in colon cancer, such as CDK12. Here, we explore the antitumor activity of a novel CDK12 inhibitor (CDK12-IN-E9) in preclinical models of colon cancer, and its liposomal formulation (LP-CDK12-IN-E9). Methods: Expression of CDK12 at a genomic level was assessed using web tools such as the Colonomics database. CDK12-IN-E9 was encapsulated in a liposomal formulation to evaluate differences between the free and the liposome form. The effect of compounds on cell viability was assessed with MTT assays. Matrigel assays were performed to study invasiveness and wound assays for migration. In addition, cell cycle and apoptosis studies were carried out by flow cytometry with 7-AAD, annexin V staining, and western-blot for specific protein expression. DNA damage and caspase degradation assays were performed by immunofluorescence. Results: Genomic mapping of CDK12 showed a higher expression in CRC than in normal tissue ( p−value = 1.424e−43). The CDK12 inhibitor was formulated in liposomes LP-CDK12-IN-E9 with the following characteristics: a polydispersity index of 0.11 ±0.02, a hydrodynamic radius of 62.98 ±10.29 nm and a zeta potential of -27.12±1.65 mV. CDK12-IN-E9 induced an antiproliferative effect in SW620, SW480 and HCT116 colon cancer cell lines. LP-CDK12-IN-E9 displayed an EC50 two times higher compared to CDK12-IN-E9 and a higher reduction of invasiveness between free inhibitor and its liposomal formulation mainly in HCT116 ( p-value < 0.001). Cell cycle studies showed no significant differences between CDK12-IN-E9 and LP-CDK12-IN-E9, but there was a profound induction of cell death at the highest doses through apoptosis mediated largely by caspases. In parallel to the effect on cell death, an increase in histone H2AX phosphorylation and an increase in cleaved caspase-3 was observed, which also implies a large increase in DNA damage in tumor cells. In vivo studies are ongoing. Conclusions: In summary, LP- CDK12-IN-E9 showed more antitumoral activity than its free formulation in CRC cell lines mainly through a caspase-dependent induction of apoptosis. This data suggests that liposomal formulation of novel chemical entities can augment its antitumoral properties.
Transformation of tissue-resident, very small embryonic-like stem cells (VSELs) into cancer stem cells (CSCs) and its role in cancer initiation via epigenetic changes.
Authors: Bhartiya Deepa , Singh Pushpa , Kaushik Ankita
Published: June 2023

e15084

Background: CSCs are postulated to initiate cancer but still no consensus on their surface markers and origin. Pluripotent VSELs exist in adult tissues, express receptors for steroid and gonadotropin hormones and are possible ‘embryonic remnants’ that could initiate cancer. Methods: Neonatal exposure to diethylstilbestrol (DES) or estradiol (E2) resulted in tumor-like changes in uterus and testes of 100 days old mice. Various studies included histological changes, VSELs enumeration by flow cytometry, and a carefully selected panel of transcripts by qRT-PCR to delineate various cellular events. Mice neonatally exposed to endocrine disruption were administered XAR (a nano-formulation of resveratrol) or transplanted niche-providing mesenchymal stromal cells on D60 and studied later on D100 for reversal/prevention of cancer phenotype. Results: Both E2 and DES resulted in increased numbers of VSELs in testis (Ctrl:0.2%; E2:0.4%; DES:1.3%) and uterus (Ctrl:18.52%; E2:35.42%; DES:40.57%). An up-regulation of pluripotent transcripts ( Oct-4, Sox-2, Nanog) was detected. Oct4A (specific marker for pluripotent VSELs) was upregulated after both DES (testis > 8 folds; uterus > 15 folds) and E2 (testis > 3 folds and uterus > 10 folds) treatment. Excessive self-renewal of VSELs was associated with dysregulated expression of DNA methyltransferases (Dnmts). Dnmt1 was downregulated while Dnmt3a, Dnmt3b, and Dnmt3L were upregulated in the testis. In the uterus, Dnmts showed increased expression after both E2 and DES treatment. Marked reduction of 5mC suggested global hypomethylation and increased Igf2 and Dlk-1 suggested affected/loss of imprinting at IGF2/H19 and DLK-1/MEG3 loci. VSELs acquired CSCs phenotype with increased expression of CD166 and ALDH1. Genomic instability in CSCs was suggested by dysregulated mismatch repair axis (DNMT-1/NP95/PCNA) and repair enzymes ( Brca-1, Rad-51, Mlh-1). Reduced expression of Pten and p53 was associated with blocked differentiation. Both reversal strategies resulted in normalization of stem cells compartment, improved differentiation (spermatogenesis/adenogenesis), reduced CSC markers, and increased p53, Pten, Np95, Sirt-1 expression. Interestingly, XAR treatment resulted in downregulation of Igf2 and Dlk1. Stem cells niche was also improved as evident by increased expression of SOX9 in testis and CD90, VIMENTIN in endometrium. Conclusions: Only immortal VSELs can transmit developmental insults to adult cells. Altered epigenetic state of VSELs resulted in cancer initiation and it was possible to prevent/reverse cancer by normalizing epigenetic state of VSELs and their niche. Targeting CSCs and pushing them back into quiescent VSELs and facilitating their normal differentiation offers novel method to win the war against cancer.
Safety and feasibility of the addition of a radiosensitizing methionine-restricted diet to radiation therapy.
Authors: Mattes Malcolm David , Leung Calvin N. , Koturbash Igor , Wen Sijin , Jacobson Geraldine M.
Published: June 2023

e15087

Background: Methionine is an amino acid necessary for cell growth and survival. Normal cells can tolerate methionine deficiency, but most cancer cells are methionine auxotrophs, requiring dietary intake since they cannot synthesize it. In vitro, methionine deficiency causes cancer cells to undergo cell cycle arrest and cell death, and in vivo a methionine restricted diet (MRD) enhances radiosensitization without significant adverse effects. Combining a MRD and radiation therapy (RT) for human malignancies has never been evaluated. The hypothesis of this Phase I study was that a MRD would be safe, and feasible to administer concurrently with curative-intent RT. Methods: Eligible patients included adults with any non-skin cancer malignancy undergoing standard RT without concurrent cytotoxic chemotherapy. The MRD consisted of low-protein cereals, grains, and breads; fruits; vegetables; margarines and oils; and simple carbohydrates. A clinical dietician developed a personalized meal plan with each subject to reduce methionine consumption to 5-10 mg/kg body weight/day, while maintaining adequate protein and caloric intake. An unlimited supply of a commercially available methionine-free protein supplement was provided to minimize hunger and weight loss. The MRD extended from 2 weeks before initiation of RT, through 2 weeks beyond completion of RT. The primary endpoint for safety was the rate of grade 3 or higher acute and late toxicities per CTCAE, over 12 months follow-up. Feasibility was assessed with a biweekly quantitative plasma amino acid panel during the MRD. The target accrual was 15 subjects. Results: Over two years, 53 patients were offered enrollment, 9 subjects enrolled, 5 completed the MRD and RT, and 4 withdrew during the MRD. The table summarizes subjects’ characteristics and outcomes. There was no grade 3 or higher adverse events attributable to the MRD. Methionine plasma levels varied over the course of treatment, and while no subject achieved the target of 13 μM, two nadired at 14 μM. The trial was closed early due to slow accrual and subjects’ difficulty maintaining the diet. Clinical trial information: NCT03574194 .Conclusions: This study suggests a MRD is safe with thoracic or abdominopelvic RT, with toxicities comparable to those expected with RT alone. However, the diet was challenging, and unacceptable to most patients with cancer. [Table: see text]
A phase 2 basket trial of an ERK1/2 inhibitor (LY3214996) in combination with abemaciclib for patients whose tumors harbor pathogenic alterations in BRAF, RAF1, MAP2K1/2 ERK1/2, and NF1.
Authors: Taza Fadi , Durm Greg Andrew , Opyrchal Mateusz , Jalal Shadia Ibrahim , Radovich Milan , Schneider Bryan P. , Turk Anita Ahmed
Published: June 2023

e15088

Background: LY3214996 (L) is a potent inhibitor of ERK1/2 kinases that can potentially obviate resistance mechanisms in cancers that harbor BRAF, RAS, NF1, MAP2K1/2, and other MAPK alterations. Abemaciclib (A) is a CDK 4/6 inhibitor. Studies have shown that concomitant inhibition of the MAPK pathway exemplified by ERK inhibitors may augment the antitumor activity of CDK4/6 inhibitors. In this biomarker-informed phase 2 study, we assess the clinical efficacy of L in combination with A. Methods: The study enrolled patients with advanced unresectable or metastatic cancer with no other standard of care options available. The malignancy must harbor pathogenic alterations in BRAF, RAF1, MAP2K1/2, ERK1/2, or NF1, in whom standard treatments failed to provide clinical benefit. L was administered at a dose of 200 mg orally QD in combination with A at 150 mg BID hours on Days 1 through 28 of a 28-day cycle until progression, toxicity, or withdrawal. The primary endpoint is the objective response rate. Secondary outcomes include evaluation of safety and tolerability, progression-free survival (PFS), and duration of the overall response. Additionally, tumor samples were collected for organoid development. Results: Between September 1, 2020, and February 1, 2023, 12 patients were enrolled. Primary malignancies included colorectal (6), lung (1), breast (2), pancreas (1), uterus (1), and thyroid (1). The pathogenic alterations included NF1 (4), BRAF (6), and MAP2K1/2 (2). There were no CR or PR observed. Stable disease was achieved in 4 patients (33%) with a median disease control rate of 6.5 months. Pathogenic alterations in patients with stable disease included BRAF, MAP2K1/2, and NF1. Most common treatment-related adverse events of any grade included fatigue (41%), rash (41%), nausea (33%), vomiting (33%), anemia (25%), anorexia (25%), and increased creatinine (25%). There were no grade 4 or 5 adverse events. Conclusions: The combination of L+A is safe and promising in patients whose tumors harbor pathogenic alterations in BRAF, MAP2K1/2, and NF1. Enrollment is ongoing, and efficacy and toxicity outcomes continue to be assessed. (NCT04534283). Clinical trial information: NCT04534283 .
Capturing age-related changes in the tumor microenvironment of luminal breast cancer using the multiplex immunohistochemistry technique, MILAN.
Authors: Lambrechts Yentl , Antoranz Martinez Asier , De Schepper Maxim , Geukens Tatjana , Vos Hanne , Berben Lieze , Messiaen Julie , Marcelis Lukas , Van Herck Yannick , Neven Patrick , Smeets Ann , Punie Kevin , Desmedt Christine , Bosisio Francesca Maria , De Smet Frederik , Hatse Sigrid , Wildiers Hans , Floris Giuseppe
Published: June 2023

e15089

Background: The ‘immune landscape’ of luminal breast tumors is poorly described to date. We applied the ‘multiple iterative labeling by antibody ‘neodeposition’ (MILAN) technique for in-depth characterization of the tumor microenvironment of luminal-like breast cancer (BC) in relation to age. Methods: Newly diagnosed patients with early luminal BC (grade 2/3, ER+, HER2-, tumor size ≥1,5 cm) were prospectively included in UH Leuven between March 2014 and November 2015 and divided into 3 distinct age categories (young: 35-45 yrs.; middle: 55-65 yrs.; old: ≥70 yrs.). 55 patients (young: n = 12; middle: n = 16; old: n = 27) were included in the cohort. From each patient up to 2 cores from the tumor center (TC) and 2 cores from the invasive front (IF) were sampled from formalin-fixed and paraffin-embedded tumor resection specimens and embedded in tissue microarrays. The MILAN workflow included 20 rounds of three-color indirect immunofluorescence (FITC, TRITC, and Cy5 channel combined with 58 antibodies), image acquisition, and removal/stripping of the antibodies before any other stain. The MILAN data analysis pipeline was applied to study the expression of phenotypic and functional proteins at single-cell level including spatial resolution. These were compared between the different age groups using Wilcoxon's test (FDR correction). Results: We identified 1,5 million cells, of which 59% were epithelial cells (cancer and normal), 29% were immune cells, and the remaining 12% were endothelial or stromal cells. Within the immune cell fraction, T-helper (Th) cells, B-cells, T-cytotoxic (Tcy) cells, and NK-cells significantly decreased with aging while monocytes significantly increased in TC and IF (Table 1). The evaluation of the functional markers in the inflammatory cells revealed a decreased expression with aging of PD-L1 in Tcy cells (p < 0.001 for TC and IF), Th cells (p = 0.003 for IF), T-regulatory (Treg) cells (p = 0.038 for TC), monocytes (p < 0.001 for TC and IF), and B-cells (p = 0.023 for TC). Perforin expression significantly decreased with aging in Th cells (p < 0.05 for TC and IF) and monocytes (p < 0.001 for TC and IF). OX-40 expression significantly decreased with aging in Th cells (p = 0.001 for TC), Treg cells (p = 0.002 for TC), and B-cells (p = 0.012 for TC). Conclusions: Our results suggest important compositional and functional changes in the tumor inflammatory microenvironment during aging in patients with luminal BC. Further analysis on other cell types is warranted. [Table: see text]
Genomics-based screening of novel therapeutic targets and biomarkers for lung cancer.
Authors: Daigo Yataro , Takano Atsushi , Nakamura Yusuke
Published: June 2023

e15093

Background: Since the number of lung cancer patients who show objective response to standard treatments is still limited, development of new anti-cancer agents with minimum risk of side effects and specific cancer biomarkers is urgently required. Methods: We have been screening new therapeutic targets and their companion biomarkers for lung cancer as follows; i) To identify overexpressed genes in lung cancers by the gene expression profile analysis, ii) To verify the candidate genes for their scarce expression in normal tissues, iii) To validate the clinicopathological importance of their protein expression by tissue microarray covering hundreds of lung cancers, and iv) To characterize their function for the growth and/or survival of lung cancer cells by siRNAs. Results: We identified dozens of candidate oncoproteins and selected a lung cancer cell division-associated protein 1 (LCDAP1). Immunohistochemical analysis showed that LCDAP1 was expressed in 171 of 267 (64%) non-small cell lung cancer (NSCLC) tissues but not in normal lung epithelia. LCDAP1 expression was significantly associated with poor prognosis for NSCLC patients. To investigate its function, we screened LCDAP1-binding protein in lung cancer cells and found an interaction of LCDAP1 with a member of 5'-to-3' DNA helicase that was also up-regulated in lung cancer cells. Knockdown of either of LCDAP1 or its binding partner expression with siRNAs significantly inhibited the growth of cancer cells. Conclusions: Our genomics-based screening could be useful to develop diagnostic and prognostic biomarkers as well as therapeutic targets for small molecules, monoclonal antibodies, and nucleic acid drugs against lung cancer.
A first-in-human, open-label, phase I trial of daily oral PCLX-001, an NMT inhibitor, in patients with relapsed/refractory B-cell lymphomas and advanced solid tumors.
Authors: Sangha Randeep S. , Jamal Rahima , Spratlin Jennifer L. , Kuruvilla John , Sehn Laurie Helen , Weickert Michael , Berthiaume Luc G. , Mackey John Robert
Published: June 2023

e15094

Background: Myristoylation, the N-terminal modification of proteins with the fatty acid myristate, regulates multiple membrane-bound signal transduction pathways driving cancer cell biology. This modification is catalyzed by two N-myristoyltransferases (NMT), NMT1 and NMT2. Aberrant NMT expression has been identified in cancer cells and inhibition of myristoylation represents a novel anticancer treatment strategy. PCLX-001 is an oral, highly bioavailable, small molecule NMT inhibitor with strong affinity for both NMT1 and NMT2 proteins (IC50 of 5nM and 8nM, respectively). In vitro, hematologic cancer cell lines were exquisitely sensitive to PCLX-001. PCLX-001 regressed subcutaneous tumors in xenograft models derived from lymphoma cell lines, as well as in a refractory DLBCL patient derived xenograft model. PCLX-001 also demonstrated anticancer activity in multiple solid tumor xenograft models. Based on pre-clinical data, we hypothesized that PCLX-001 would be safe, tolerable, and active as a novel oral cancer therapeutic. Methods: Patients with relapsed/refractory B-cell lymphomas and advanced solid tumors are being enrolled in a multicenter, open label, standard phase I dose escalation design to determine feasibility, maximum tolerated dose (MTD), and pharmacokinetics of PCLX-001 monotherapy. Seven dose levels are being studied: 20 mg, 40 mg, 70 mg, 100 mg, 140 mg, 200 mg, and 280 mg. Dose limiting toxicity (DLT) is defined as: grade (Gr) 4 platelets, Gr 3 platelets with bleeding, Gr 4 ANC ≥ 7 days, febrile neutropenia, or clinically significant > Gr 3 non-hematologic toxicity. Results: Eighteen evaluable patients (3 relapsed B-cell lymphoma; 15 advanced solid tumor) have been accrued through dose level six (200 mg cohort) without DLT. No attributable Gr 3 or 4 toxicities have been identified. Noncompartmental pharmacokinetic (PK) analyses demonstrate rapid oral absorption, a terminal half-life of approximately 10 hours, rapid achievement of steady state, and no induction of metabolism. PCLX-001 trough plasma concentrations at higher dose cohorts exceed the EC90 required to inhibit cultured cancer cells. Conclusions: PCLX-001 is safe and well tolerated up to the 200 mg daily dose. Our results support the ongoing development of PCLX-001 as an oral daily therapy for the treatment of patients with cancer. Updated study results will be presented, summarizing the safety, MTD, and PK outcomes in PCLX-001 treated patients. Preliminary clinical activity will be determined in 20 patient dose expansion cohorts of relapsed/refractory B-cell lymphomas, and selected advanced solid tumors following the identification of PCLX-001 MTD. Clinical trial information: NCT04836195 .
PUF: A potential repressor of oncogenes.
Authors: Aslam Hafiz , Muzaffar Mahvish , Lee Myon Hee
Published: June 2023

e15095

Background: RNA-binding proteins (RBPs) play a crucial role in orchestrating the function of RNAs. The regulation of RBPs is critical in various biological processes, including tumorigenesis. The PUF (Pumilio/FBF) RBPs are of particular interest due to their function in stem cells and tumor-initiating cells. Its has been shown that PUF proteins inhibit the translation of target genes by binding to a regulatory element (PBE) in the 3’ untranslated regions (3’UTRs) of their target mRNA. PUF proteins are required to maintain normal stem cells and potentially inhibit the formation of tumor-initiating cells by repressing the expression of oncogenes. Bioinformatics analysis has identified many oncogenes as potential targets of PUF, and pilot studies have confirmed that ERK, p38, JAK2, RUNX2, and RET mRNAs specifically interact with PUF protein. The objective of this study is to investigate the function of human PUF proteins in the initiation and progression of cancer cells. Methods: In worms (C. elegans), 100 % PUF mutants form tumors. Bioinformatics were performed to identify PUF target genes, it specifically binds UGUAnAUA sequences on target mRNA 3'UTRs. Yeast three-hybrid assay were performed to test the physical interaction between PUF protein and its target mRNA. The novel function of human PUF proteins in the initiation and progression of cancer cells was investigated using CRISPR/Cas9 knockout cancer cell lines and western Blot/IHC was done to determine their expression. Results: The results of the study showed that PUF proteins play a crucial role in inhibiting the formation of tumor-initiating cells. Loss of PUF function and activation of ERK signaling were found to promote the formation of these cells. Bioinformatics analysis revealed that many oncogenes are potential targets for PUF repression. The study also demonstrated that PUF represses the expression of ERK and p38 mRNAs via their 3’UTRs. These findings suggest that PUF may have therapeutic potential as a target for cancer treatment. Conclusions: In worms and human cells, normal PUF proteins promote the cell proliferation of stem cells, however, stem cells begin to differentiate, PUF proteins are required to complete differentiation. Therefore, in the differentiating cells, PUF mutation cannot complete differentiation, instead, return to undifferentiating cells, resulting in tumors. Bioinformatics and biochemical analyses revealed that many PUF repressing targets (~ > 30%) are oncogene and some labs including us found that PUF knockdown enhanced oncogene expression. PUF can be a potential biomarker, as low PUF expression may initiate tumors. Therapeutically, conditional PUF expression in tumor-initiating cells may suppress tumor progression by suppressing oncogene.
Association of cachexia with loss of function mutations in STK11/LKB1 for NSCLC.
Authors: Infante Rodney E. , Ahn Chul , Minna John D. , Iyengar Puneeth
Published: June 2023

e15099

Background: Cancer cachexia (CC), a wasting syndrome of muscle and adipose tissue resulting in weight loss, occurs in 50% of non-small cell lung cancer (NSCLC) patients. Management of CC is limited by the absence of biomarkers and knowledge of molecules that drive its phenotype. The evolving landscape of increased genomic testing of human tumors and derived cell lines has further given us an opportunity to leverage these findings to identify a genetic biomarker that regulates CC development through a novel in vivo human NSCLC cachexia screen. Methods: To identify such molecules, we injected 54 human non-small cell lung cancer (NSCLC) lines into immunodeficient mice, 17 of which produced an unambiguous phenotype of cachexia (n = 10) or non-cachexia (n = 7). Whole exome sequencing of tumors was conducted to identify candidate cachexia-associated genes and gene variant analysis was conducted to identify gene variants that associated with NSCLC-associated CC. CRISPR/Cas9 targeted gene silencing in human NSCLC and mouse cancer lines verified if target genes conferred the cachexia phenotype. Circulating tumor DNA analysis of a 246 NSCLC patient cohort was used to confirm candidate gene variants association with cancer-related weight loss. Results: Whole exome sequencing revealed that 8 of 10 cachexia lines, but none of the non-cachexia lines, possessed mutations in STK11/LKB1 (p = 2x10-12), a regulator of nutrient sensor AMP kinase. Silencing of STK11/LKB1 in human NSCLC and murine colorectal carcinoma lines conferred a cachexia phenotype after cell transplantation into immunodeficient and immunocompetent models, respectively. This host wasting was associated with an alteration in the immune cell repertoire of the tumor microenvironments that led to increases in local mRNA expression and serum levels of CC-associated cytokines resulting in host adipose and muscle tissue wasting. Mutational analysis of circulating tumor DNA from 246 NSCLC patients identified 89% concordance between STK11/LKB1 mutations and weight loss at cancer diagnosis (OR = 17 ± 1, p = 0.004). Conclusions: The current data provides evidence that tumor STK11/LKB1 loss of function is a driver of CC in NSCLC, simultaneously serving as the first genetic biomarker for this wasting syndrome.
Base-edited live microbiome therapeutics with target-aid -live biotherapeutic products (LBPS) with synergistic effects of anti-PD-1 antibodies in a syngeneic model of mice.
Authors: Okumura Ryo , Mitsunobu Hitoshi , Ishii Chiaki , Miyazaki Shoko , Tamiya Taiga , Bernier Francois
Published: June 2023

e15104

Background: Base editing without DNA cleaving is a technique that introduces point mutations using enzymes that induce base conversions. Generally, only the point mutation occurs, achieving a high level of precision. Base editing can easily be applied to bacteria, for which DNA cleaving is often lethal. With Bio Palette's core technology of base editing, Target-AID, we can quickly achieve the required therapeutic effects by making pinpoint modifications to bacteria. Immune checkpoint inhibitors (ICIs) targeting CTLA-4, PD-1, or PD-L1 have shown outstanding therapeutic outcomes in various cancers. However, the therapeutic efficacy of ICIs-based immunotherapy has been limited. Recent studies have revealed that the human gut microbiome controls the efficacy of ICIs. Bacterial flagellin derived from Enterococcus spp. has been suggested to enhance immune responses to cancer through activation of TLR5 signaling (1). As a regulatory mechanism of flagellin secretion in Enterococcus spp, FliD, FlgK, FlgN, or FlgM are known as negative regulators. Thus, if the flagellar expression can be enhanced by making the negative regulator functionally deficient, it can be expected to increase the efficacy and response rate of ICIs when combination with a base-edited strain. Methods: As human TLR5 luciferase reporter assay, HEK293T cells were transfected with human TLR5-expression vector. BALB/cAnN mice (6–8 weeks’ old) were injected subcutaneously with 3 x 106 cells for murine colon cancer cell line CT-26. Six days post inoculation, the mice were randomized based on tumor volume into treatment and vehicle control groups (n = 10 per each group). Anti-mouse PD-1 antibody was dosed 5 mg/kg, q3d, iv, for up to 3 weeks either alone or in combination with each of various strains (1 x 109 CFU/mouse, po). Body weight and tumor size were routinely measured until endpoint. Results: To achieve the increased extracellular secretion of Flagellin, we created fliD dysfunctional strain because deletion of the flagellar cap component FliD was expected to promote extracellular secretion of Flagellin monomers. Comparing the wild-type strains we used, E. casseliflavus activated TLR5 compared to E. gallinarum. E. casseliflavus with a stop codon introduced into the fliD gene, BP5040 strain, was found to increase TLR5 activation by approximately 4.9-fold when compared to wild type strain. In vivo experiments indicated that antitumor effect was observed with BP5040 alone. Strikingly, a remarkable antitumor effect was also demonstrated when BP5040 was combined with an anti-PD-1 antibody. Conclusions: To achieve the desired therapeutic effect, we performed base editing without inserting foreign genes. As a result, BP5040 strain is found to be a potential candidate for the treatment of immuno-oncology, which was created using our LBP platform technology.