Discordance in Tumor Mutation Burden from Blood and Tissue Affects Association with Response to Immune Checkpoint Inhibition in Real-World Settings.

BACKGROUND: Tumor mutation burden (TMB), a biomarker for immune checkpoint inhibitor (CPI) response, is reported by both blood- and tissue-based next-generation sequencing (NGS) vendors. However, the agreement between TMB from blood (bTMB) and tissue (tTMB) in real-world settings, both in absolute value and association with CPI response, is not known. MATERIALS AND METHODS: This study utilizes Sarah Cannon's precision medicine platform, Genospace, to harmonize clinico-genomic data from 17 206 patients with cancer with NGS results from September 2015 to August 2021. A subset of patients have both bTMB and tTMB results. Statistical analyses are performed in R and include (1) correlation (r) and concordance (ρ) between patient-matched bTMB-tTMB pairs, (2) distribution of total bTMB and tTMB values, and (3) association of bTMB and tTMB with time to CPI therapy failure. RESULTS: In 410 patient-matched bTMB-tTMB pairs, the median bTMB (m = 10.5 mut/Mb) was significantly higher than the median tTMB (m = 6.0 mut/Mb, P < .001) leading to conflicting "high" and "low" statuses in over one-third of cases at a threshold of 10 mut/Mb (n = 410). Significant differences were observed in the distribution of bTMB values from blood-NGS vendors, with guardant health (GH) reporting higher (m = 10.5 mut/Mb, n = 2183) than Foundation Medicine (FMI, m = 3.8 mut/Mb, n = 462, P < .001). bTMB from GH required a higher threshold (≥40 mut/Mb) than bTMB from FMI (≥12 mut/Mb) in order to be associated with CPI response. CONCLUSIONS: This study uncovers variability in bTMB reporting among commercial NGS platforms, thereby evidencing a need for assay-specific thresholds in identifying patients who may respond to CPI therapy.

Quantitative Proteomic Analysis of Small and Large Extracellular Vesicles (EVs) Reveals Enrichment of Adhesion Proteins in Small EVs.

Extracellular vesicles (EVs) are important mediators of cell-cell communication due to their cargo content of proteins, lipids, and RNAs. We previously reported that small EVs (SEVs) called exosomes promote directed and random cell motility, invasion, and serum-independent growth. In contrast, larger EVs (LEVs) were not active in those assays, but might have unique functional properties. In order to identify protein cargos that may contribute to different functions of SEVs and LEVs, we used isobaric tags for relative and absolute quantitation (iTRAQ)-liquid chromatography (LC) tandem mass spectrometry (MS) on EVs isolated from a colon cancer cell line. Bioinformatics analyses revealed that SEVs are enriched in proteins associated with cell-cell junctions, cell-matrix adhesion, exosome biogenesis machinery, and various signaling pathways. In contrast, LEVs are enriched in proteins associated with ribosome and RNA biogenesis, processing, and metabolism. Western blot analysis of EVs purified from two different cancer cell types confirmed the enrichment of cell-matrix and cell-cell adhesion proteins in SEVs. Consistent with those data, we found that cells exhibit enhanced adhesion to surfaces coated with SEVs compared to an equal protein concentration of LEVs. These data suggest that a major function of SEVs is to promote cellular adhesion.

Using Technology to Enhance Cancer Clinical Trial Participation.

The COVID-19 pandemic presented many challenges to health care systems, including oncology clinical research programs. There were substantial negative effects on oncology clinical trial screening, enrollment, and study activities that forced institutions and regulatory bodies to develop innovative solutions to maintain robust and equitable participation in these trials. Digital pathology innovations at Memorial Sloan Kettering Cancer Center have streamlined the diagnostic life cycle for patients with cancer, and the seamless integration of digital pathology services with next-generation sequencing and other molecular pathology services have accelerated the time to diagnosis and receipt of molecular results. Timely access to these results, coupled with Memorial Sloan Kettering Cancer Center's knowledge engine OncoKB, enhances patient clinical trial coordination precisely and efficiently. At the Sarah Cannon Research Institute, centralized remote clinical trial matching and screening, virtual molecular tumor boards, and centralized molecular interpretation support services have empowered clinic staff to identify more efficiently potential participants in clinical research, despite the COVID-19 pandemic. In addition, the U.S. Food and Drug Administration Oncology Center of Excellence has been involved in several efforts to address challenges for patients with cancer during the COVID-19 pandemic, including writing guidance documents and participating in efforts to modernize clinical trials. The enclosed personal experience of a patient with cancer currently participating in an oncology clinical trial emphasizes the need for continued decreasing of barriers to study participation. Clinical trial advances that were accelerated by the pandemic will ultimately help patients with cancer and the greater oncology health care community.

Tumor-Specific and Tumor-Agnostic Molecular Signatures Associated With Response to Immune Checkpoint Inhibitors.

PURPOSE: Next-generation sequencing (NGS) testing is being incorporated into routine standard of care for patients with cancer. Immune checkpoint inhibitors (CPIs) are approved for use in both tumor-specific and tumor-agnostic indications. We sought to determine tumor type-specific or tumor-agnostic correlations between mutations detected by NGS and response to CPIs. MATERIALS AND METHODS: A retrospective analysis of 26,004 patient records with NGS data available was conducted. Time to treatment failure and overall survival analyses were performed. Hazard ratios and associated statistics were computed in the R programming language. The study was considered exempt from internal review board review and data were considered nonhuman subjects. RESULTS: Response to CPIs varied between tumor types with melanoma and lung cancer performing relatively better on CPIs than other tumor types. Within tumor types, response to CPIs was stratified by mutations in specific genes. Tumor-agnostic markers including high tumor mutation burden and microsatellite instability-high were also associated with longer time to treatment failure on CPIs. Importantly, within the high tumor mutation burden and microsatellite instability-high groups, mutations in individual genes correlate with response to CPIs. CONCLUSION: The results from commercial NGS panels may be used to stratify patients for response to CPIs. In tumors where CPIs show relatively low efficacy, there may be distinct patient populations-based on gene mutation status-that are predicted to have better response to CPIs. Likewise, there may be distinct patient populations who do relatively worse on CPIs within tumor types known to respond well to CPIs.

Next-Generation Sequencing of Patients With Breast Cancer in Community Oncology Clinics.

PURPOSE: Molecular biomarkers informing disease diagnosis, prognosis, and treatment decisions in patients with breast cancer are being uncovered by next-generation sequencing (NGS) technologies. In this study, we survey how NGS is used for patients with breast cancer in real-world settings with a focus on physician behaviors and sequencing results. METHODS: We conducted a retrospective analysis of patients with breast cancer who received NGS testing from commercial vendors as part of standard of care from 2014 to 2019. A total of 2,635 NGS reports from 2,316 unique breast cancer patients were assessed. Hormone receptor and human epidermal growth factor receptor 2 statuses were abstracted from patient medical records. Comparative gene amplification and mutation frequencies were analyzed using Pearson's correlation and Lin's concordance statistics. RESULTS: The number of physicians ordering NGS tests for patients with breast cancer increased more than six-fold from 2014 to 2019. Tissue- and plasma-based tests were ordered roughly equally by 2019, with plasma-based testing ordered most frequently in hormone receptor-positive subtypes. Patients with triple-negative breast cancer were most likely to receive NGS testing. Gene amplifications including ERBB2 were detected less frequently in our real-world data set as compared to previous genomic landscape studies, whereas the opposite was true for gene mutations including ESR1. Pathogenic mutations in the PI3K pathway (38.6%) and DNA damage repair pathway (11.0%) were frequently reported. Alterations were also reported across other cellular pathways. CONCLUSION: Overall, we found that an increasing number of physicians in community settings are adopting NGS in the care of patients with breast cancer. Discrepancies between our real-world NGS data and previous genomic landscape studies are likely owed to the prevalence of plasma-based testing in community oncology clinics, as the reference data were from tissue-based NGS alone.

Molecular Profiling in Drug Development: Paving a Way Forward.

As researchers learn more about tumor biology and the molecular mechanisms involved in tumorigenesis, metastasis, and tumor evolution, clinical trials are growing more complex and patient selection for clinical trials is becoming more specific. Rather than exploit certain phenotypic characteristics of tumor cells (e.g., rapid cell division and uncontrolled cell growth), pharmaceuticals targeting the genotypic causes of tumorigenesis are emerging. The sequencing of the human genome, advances in chemical techniques, and increased efficiency in drug target identification have changed the way drugs are developed. Now, more precise drugs targeting specific mutations within individual genes are being used to treat narrow patient populations harboring these specific driver mutations, often with greater efficacy and lower toxicity than traditional chemotherapeutic agents. This precision in drug development relies not only on the ability to design exquisitely specific pharmaceuticals but also to identify (with the same level of precision) the patients who are most likely to respond to those therapies. Robust screening techniques and adequate molecular oncology education are required to match the appropriate patient to precision therapies, and these same screening techniques provide the data necessary to advance to the next generation of drug development.

Protein kinase A activity is regulated by actomyosin contractility during cell migration and is required for durotaxis.

Dynamic subcellular regulation of protein kinase A (PKA) activity is important for the motile behavior of many cell types, yet the mechanisms governing PKA activity during cell migration remain largely unknown. The motility of SKOV-3 epithelial ovarian cancer (EOC) cells has been shown to be dependent both on localized PKA activity and, more recently, on mechanical reciprocity between cellular tension and extracellular matrix rigidity. Here, we investigated the possibility that PKA is regulated by mechanical signaling during migration. We find that localized PKA activity in migrating cells rapidly decreases upon inhibition of actomyosin contractility (specifically, of myosin ATPase, Rho kinase, or myosin light-chain kinase activity). Moreover, PKA activity is spatially and temporally correlated with cellular traction forces in migrating cells. Additionally, PKA is rapidly and locally activated by mechanical stretch in an actomyosin contractility-dependent manner. Finally, inhibition of PKA activity inhibits mechanically guided migration, also known as durotaxis. These observations establish PKA as a locally regulated effector of cellular mechanotransduction and as a regulator of mechanically guided cell migration.

Cyclopamine inhibition of the sonic hedgehog pathway in the stomach requires concomitant acid inhibition.

The Shh pathway has been implicated in gastric carcinogenesis, and inhibition of this pathway has been shown to inhibit tumour growth in gastric cell lines. Assessing the in vivo efficacy of Shh pathway antagonists in blocking Shh signaling in the stomach is important for clinical trial design, but has not been previously investigated. We investigated the in vivo efficacy of a Shh antagonist, cyclopamine, in correlation to the secondary effects induced by this treatment on gastrin levels and acid secretion. Gastrin has been shown to induce Shh production, processing and activity, which is believed to be mediated by acid secretion. We tested this hypothesis and showed that hypergastrinaemia induces Shh production in vivo, and confirmed that this effect on Shh is mediated by acid secretion. We showed that cyclopamine treatment induces both hypergastrinaemia and Shh, and does not inhibit Gli-1. Inhibition of the effect of hypergastrinaemia on the Shh pathway, in cyclopamine-treated mice, was demonstrated by use of lansoprazole which concomitantly inhibited Gli-1, and did not increase Shh production. Therefore, this evidence suggests that hypergastrinaemia, via increased acid secretion, may increase expression of Shh and that Shh antagonists may require concomitant acid inhibition to successfully inhibit a pathway known to be up-regulated in gastric carcinogenesis.

The mechanical microenvironment regulates ovarian cancer cell morphology, migration, and spheroid disaggregation.

There is growing appreciation of the importance of the mechanical properties of the tumor microenvironment on disease progression. However, the role of extracellular matrix (ECM) stiffness and cellular mechanotransduction in epithelial ovarian cancer (EOC) is largely unknown. Here, we investigated the effect of substrate rigidity on various aspects of SKOV3 human EOC cell morphology and migration. Young's modulus values of normal mouse peritoneum, a principal target tissue for EOC metastasis, were determined by atomic force microscopy (AFM) and hydrogels were fabricated to mimic these values. We find that cell spreading, focal adhesion formation, myosin light chain phosphorylation, and cellular traction forces all increase on stiffer matrices. Substrate rigidity also positively regulates random cell migration and, importantly, directional increases in matrix tension promote SKOV3 cell durotaxis. Matrix rigidity also promotes nuclear translocation of YAP1, an oncogenic transcription factor associated with aggressive metastatic EOC. Furthermore, disaggregation of multicellular EOC spheroids, a behavior associated with dissemination and metastasis, is enhanced by matrix stiffness through a mechanotransduction pathway involving ROCK, actomyosin contractility, and FAK. Finally, this pattern of mechanosensitivity is maintained in highly metastatic SKOV3ip.1 cells. These results establish that the mechanical properties of the tumor microenvironment may play a role in EOC metastasis.

Selection of radiolabeled gastrin analogs for peptide receptor-targeted radionuclide therapy.

UNLABELLED: The gastrin/cholecystokinin-2 (CCK-2) receptor has been identified as a possible target for peptide receptor radionuclide imaging and therapy. Several radiolabeled peptides binding to this receptor have been explored in animal models and clinical trials but either low tumor uptake or high renal retention has been found. The aim of this study was to identify a peptide with improved tumor-to-kidney pharmacodynamics when compared with current candidates. METHODS: A small peptide-chelator library of 34 compounds based on the C-terminal sequences of CCK-8 or minigastrin was constructed. The peptides were radiolabeled with (111)In with high labeling efficiency (>90%), as determined by high-performance liquid chromatographic analysis. The labeled peptides were screened by assessing tumor and kidney uptake in pancreatic xenograft nude mouse models, including AR42J. An extensive biodistribution analysis was performed on the lead candidate from the library. RESULTS: Minigastrin analogs containing a pentaglutamate sequence showed the highest tumor uptake but very high renal retention. CCK analogs showed the lowest tumor and renal uptake. Deletion of the pentaglutamate sequence in the gastrin analogs lowered the tumor uptake by a factor of 3 but decreased the kidney uptake by a factor of 20. Insertion of histidine residues in the sequence reduced kidney uptake by a further factor of almost 2-fold. In AR42J tumor-bearing mice, the peptide with the sequence DOTA-HHEAYGWMDF-NH(2) (DOTA is tetraazacyclododecane tetraacetic acid) showed the highest tumor-to-kidney ratio of all peptides studied, with saturable uptake in target organs and low uptake by nontarget tissues other than the kidney. CONCLUSION: This peptide is a worthwhile candidate for clinical studies to determine whether it is suitable for use in peptide receptor-targeted radionuclide therapy.

Formulation development and manufacturing of a gastrin/CCK-2 receptor targeting peptide as an intermediate drug product for a clinical imaging study.

A DOTA-gastrin analogue (APH070) which, when labelled with (111)In, has high affinity for the gastrin/CCK-2 receptor (3nM) and low tumour to kidney ratio in animal models, has been formulated and manufactured for a clinical study. Oxidation of the peptide methionine residue greatly reduces receptor affinity, therefore development work focused on producing a stable intermediate drug product (iDP) whilst ensuring that the formulation, container, closure and manufacturing process did not inhibit the extremely sensitive radiolabelling reaction (itself a source of oxidation). Stress testing revealed that APH070 was stable at 2-8 degrees C at pH 6-9. Addition of an antioxidant (monothioglycerol) to the peptide formulation reduced stability when compared to buffer alone. Use of FluroTec (4023/50) stoppers (rather than FluroTec Plus (4110/40)) increased both the stability and radiolabelling efficiency of APH070. Long term stability (6 months) of the final formulation (1mg/ml APH070 in 0.01 M pH 7.2 phosphate buffer) stored at 5 degrees C in type I glass vials with FluroTec (4023/50) stoppers was 98.6+/-0.2% and 98.4+/-0.1% for upright and inverted samples, respectively. Clinical scale radiolabelling of the final formulation routinely achieves the specification of >85% (111)In-APH070 (unoxidised) stable for up to 2h after dilution with 0.9% w/v saline solution. Specific uptake of the radiopharmaceutical in CCK-2R-expressing AR42J tumours in nude mice has been demonstrated.

The biological and therapeutic importance of gastrin gene expression in pancreatic adenocarcinomas.

The gastrin gene is expressed widely in pancreatic adenocarcinomas and the study aimed to assess its role in both the resistance of cancer cells to apoptosis and the sensitivity of cells to chemotherapeutic agents. Two human pancreatic cell lines, PAN1 and BXPC3, expressed gastrin at both the RNA and protein levels and are shown to be representative of human pancreatic adenocarcinomas in terms of gastrin expression. Inhibition of endogenous gastrin production by tumor cells was achieved with neutralizing gastrin antiserum and transfection with a gastrin antisense plasmid. Gastrin antiserum synergized with both taxotere and gemcitabine in inhibiting the in vitro growth of the PAN1 cell line with the inhibitory effect of the antiserum increasing from 12.7% to 70.2% with taxotere (P < 0.05) and 28.6% with gemcitabine (P < 0.01) after controlling for the effects of the cytotoxics. Synergy was only achieved with taxotere in BXPC3 cells with the inhibitory effect of gastrin antiserum increasing from 22.9% to 50.0% (P < 0.005). Cells transfected with gastrin antisense had reduced in vitro growth in low serum conditions and were poorly tumorigenic in nude mice at an orthotopic site. Gastrin antisense-transfected PAN1 cells had increased sensitivity to the antiproliferative effects of both gemcitabine (IC50 of > 100 microg/ml reduced to 0.1 microg/ml) and taxotere (IC50 of 20 microg/ml reduced to < 0.01 microg/ml) when compared with vector controls. The increased sensitivity of PAN1 antisense coincided with increased caspase-3 activity and reduced protein kinase B/Akt phosphorylation in response to both gemcitabine and taxotere. Gastrin gene circumvention may be an optimal adjunct to chemotherapeutic agents, such as taxotere and gemcitabine, in pancreatic adenocarcinoma.

AMPK activity regulates trafficking of mitochondria to the leading edge during cell migration and matrix invasion.

Cell migration is a complex behavior involving many energy-expensive biochemical events that iteratively alter cell shape and location. Mitochondria, the principal producers of cellular ATP, are dynamic organelles that fuse, divide, and relocate to respond to cellular metabolic demands. Using ovarian cancer cells as a model, we show that mitochondria actively infiltrate leading edge lamellipodia, thereby increasing local mitochondrial mass and relative ATP concentration and supporting a localized reversal of the Warburg shift toward aerobic glycolysis. This correlates with increased pseudopodial activity of the AMP-activated protein kinase (AMPK), a critically important cellular energy sensor and metabolic regulator. Furthermore, localized pharmacological activation of AMPK increases leading edge mitochondrial flux, ATP content, and cytoskeletal dynamics, whereas optogenetic inhibition of AMPK halts mitochondrial trafficking during both migration and the invasion of three-dimensional extracellular matrix. These observations indicate that AMPK couples local energy demands to subcellular targeting of mitochondria during cell migration and invasion.

KRAS-MEK Signaling Controls Ago2 Sorting into Exosomes.

Secretion of RNAs in extracellular vesicles is a newly recognized form of intercellular communication. A potential regulatory protein for microRNA (miRNA) secretion is the critical RNA-induced silencing complex (RISC) component Argonaute 2 (Ago2). Here, we use isogenic colon cancer cell lines to show that overactivity of KRAS due to mutation inhibits localization of Ago2 to multivesicular endosomes (MVEs) and decreases Ago2 secretion in exosomes. Mechanistically, inhibition of mitogen-activated protein kinase kinases (MEKs) I and II, but not Akt, reverses the effect of the activating KRAS mutation and leads to increased Ago2-MVE association and increased exosomal secretion of Ago2. Analysis of cells expressing mutant Ago2 constructs revealed that phosphorylation of Ago2 on serine 387 prevents Ago2-MVE interactions and reduces Ago2 secretion into exosomes. Furthermore, regulation of Ago2 exosomal sorting controls the levels of three candidate miRNAs in exosomes. These data identify a key regulatory signaling event that controls Ago2 secretion in exosomes.

3-Dimensional Patient-Derived Lung Cancer Assays Reveal Resistance to Standards-of-Care Promoted by Stromal Cells but Sensitivity to Histone Deacetylase Inhibitors.

There is a growing recognition that current preclinical models do not reflect the tumor microenvironment in cellular, biological, and biophysical content and this may have a profound effect on drug efficacy testing, especially in the era of molecular-targeted agents. Here, we describe a method to directly embed low-passage patient tumor-derived tissue into basement membrane extract, ensuring a low proportion of cell death to anoikis and growth complementation by coculture with patient-derived cancer-associated fibroblasts (CAF). A range of solid tumors proved amenable to growth and pharmacologic testing in this 3D assay. A study of 30 early-stage non-small cell lung cancer (NSCLC) specimens revealed high levels of de novo resistance to a large range of standard-of-care agents, while histone deacetylase (HDAC) inhibitors and their combination with antineoplastic drugs displayed high levels of efficacy. Increased resistance was seen in the presence of patient-derived CAFs for many agents, highlighting the utility of the assay for tumor microenvironment-educated drug testing. Standard-of-care agents showed similar responses in the 3D ex vivo and patient-matched in vivo models validating the 3D-Tumor Growth Assay (3D-TGA) as a high-throughput screen for close-to-patient tumors using significantly reduced animal numbers. Mol Cancer Ther; 15(4); 753-63. ©2016 AACR.

Protein kinase A activity and anchoring are required for ovarian cancer cell migration and invasion.

Epithelial ovarian cancer (EOC) is the deadliest of the gynecological malignancies, due in part to its clinically occult metastasis. Therefore, understanding the mechanisms governing EOC dissemination and invasion may provide new targets for antimetastatic therapies or new methods for detection of metastatic disease. The cAMP-dependent protein kinase (PKA) is often dysregulated in EOC. Furthermore, PKA activity and subcellular localization by A-kinase anchoring proteins (AKAPs) are important regulators of cytoskeletal dynamics and cell migration. Thus, we sought to study the role of PKA and AKAP function in both EOC cell migration and invasion. Using the plasma membrane-directed PKA biosensor, pmAKAR3, and an improved migration/invasion assay, we show that PKA is activated at the leading edge of migrating SKOV-3 EOC cells, and that inhibition of PKA activity blocks SKOV-3 cell migration. Furthermore, we show that while the PKA activity within the leading edge of these cells is mediated by anchoring of type-II regulatory PKA subunits (RII), inhibition of anchoring of either RI or RII PKA subunits blocks cell migration. Importantly, we also show--for the first time--that PKA activity is up-regulated at the leading edge of SKOV-3 cells during invasion of a three-dimensional extracellular matrix and, as seen for migration, inhibition of either PKA activity or AKAP-mediated PKA anchoring blocks matrix invasion. These data are the first to demonstrate that the invasion of extracellular matrix by cancer cells elicits activation of PKA within the invasive leading edge and that both PKA activity and anchoring are required for matrix invasion. These observations suggest a role for PKA and AKAP activity in EOC metastasis.

Bioluminescence imaging of human embryonic stem cells transplanted in vivo in murine and chick models.

Research into the behavior, efficacy, and biosafety of stem cells with a view to clinical transplantation requires the development of noninvasive methods for in vivo imaging of cells transplanted into animal models. This is particularly relevant for human embryonic stem cells (hESCs), because transplantation of undifferentiated hESCs leads to tumor formation. The present study aimed to monitor hESCs in real time when injected in vivo. hESCs were stably transfected to express luciferase, and luciferase expression was clearly detected in the undifferentiated and differentiated state. When transfected hESCs were injected into chick embryos, bioluminescence could be detected both ex and in ovo. In the SCID mouse model, undifferentiated hESCs were detectable after injection either into the muscle layer of the peritoneum or the kidney capsule. Tumors became detectable between days 10-30, with approximately a 3 log increase in the luminescence signal by day 75. The growth phase occurred earlier in the kidney capsule and then reached a plateau, whilst tumors in the peritoneal wall grew steadily throughout the period analysed. These results show the widespread utility of bioluminescent for in vivo imaging of hESCs in a variety of model systems for preclinical research into regenerative medicine and cancer biology.