A spectroscopic liquid biopsy for the earlier detection of multiple cancer types.

BACKGROUND: A rapid, low-cost blood test that can be applied to reliably detect multiple different cancer types would be transformational. METHODS: In this large-scale discovery study (n = 2092 patients) we applied the Dxcover® Cancer Liquid Biopsy to examine eight different cancers. The test uses Fourier transform infrared (FTIR) spectroscopy and machine-learning algorithms to detect cancer. RESULTS: Area under the receiver operating characteristic curve (ROC) values were calculated for eight cancer types versus symptomatic non-cancer controls: brain (0.90), breast (0.76), colorectal (0.91), kidney (0.91), lung (0.91), ovarian (0.86), pancreatic (0.84) and prostate (0.86). We assessed the test performance when all eight cancer types were pooled to classify 'any cancer' against non-cancer patients. The cancer versus asymptomatic non-cancer classification detected 64% of Stage I cancers when specificity was 99% (overall sensitivity 57%). When tuned for higher sensitivity, this model identified 99% of Stage I cancers (with specificity 59%). CONCLUSIONS: This spectroscopic blood test can effectively detect early-stage disease and can be fine-tuned to maximise either sensitivity or specificity depending on the requirements from different healthcare systems and cancer diagnostic pathways. This low-cost strategy could facilitate the requisite earlier diagnosis, when cancer treatment can be more effective, or less toxic. STATEMENT OF TRANSLATIONAL RELEVANCE: The earlier diagnosis of cancer is of paramount importance to improve patient survival. Current liquid biopsies are mainly focused on single tumour-derived biomarkers, which limits test sensitivity, especially for early-stage cancers that do not shed enough genetic material. This pan-omic liquid biopsy analyses the full complement of tumour and immune-derived markers present within blood derivatives and could facilitate the earlier detection of multiple cancer types. There is a low barrier to integrating this blood test into existing diagnostic pathways since the technology is rapid, simple to use, only minute sample volumes are required, and sample preparation is minimal. In addition, the spectroscopic liquid biopsy described in this study has the potential to be combined with other orthogonal tests, such as cell-free DNA, which could provide an efficient route to diagnosis. Cancer treatment can be more effective when given earlier, and this low-cost strategy has the potential to improve patient prognosis.

Quantitative assessment of glioblastoma phenotypes in vitro establishes cell migration as a robust readout of Crk and CrkL activity.

The expression levels of CT10 regulator of kinase (Crk) and Crk-like (CrkL) are elevated in many human cancers, including glioblastoma (GBM), and are believed to contribute to poor prognosis. Although Crk and CrkL have been proposed as therapeutic targets in these tumors, the lack of a reliable, quantitative assay to measure Crk and CrkL activity has hindered development of inhibitors. Here, we knocked down Crk, CrkL, or both using siRNAs in a human GBM cell line, U-118MG, to determine the respective, quantitative contributions of Crk and CrkL to cellular phenotypes. The combined use of specific and potent Crk and CrkL siRNAs induced effective knockdown of CrkII, CrkI, and CrkL. Whereas Crk knockdown did not affect cell morphology, proliferation, adhesion, or invasion, CrkL knockdown caused shrinkage of cells and inhibition of cell proliferation, adhesion, and invasion. Crk/CrkL double knockdown resulted in more pronounced morphological alterations and more robust inhibition of proliferation, adhesion, and invasion. Furthermore, Crk/CrkL double knockdown completely blocked cell migration, and this effect was rescued by transient overexpression of CrkL but not of Crk. Quantification of protein levels indicated that CrkL is expressed more abundantly than CrkII and CrkI in U-118MG cells. These results demonstrate both the predominant role of CrkL and the essential overlapping functions of Crk and CrkL in U-118MG cells. Furthermore, our study indicates that migration of U-118MG cells depends entirely on Crk and CrkL. Thus, impedance-based, real-time measurement of tumor cell migration represents a robust assay for monitoring Crk and CrkL activities.

Genomic answers for children: Dynamic analyses of >1000 pediatric rare disease genomes.

PURPOSE: This study aimed to provide comprehensive diagnostic and candidate analyses in a pediatric rare disease cohort through the Genomic Answers for Kids program. METHODS: Extensive analyses of 960 families with suspected genetic disorders included short-read exome sequencing and short-read genome sequencing (srGS); PacBio HiFi long-read genome sequencing (HiFi-GS); variant calling for single nucleotide variants (SNV), structural variant (SV), and repeat variants; and machine-learning variant prioritization. Structured phenotypes, prioritized variants, and pedigrees were stored in PhenoTips database, with data sharing through controlled access the database of Genotypes and Phenotypes. RESULTS: Diagnostic rates ranged from 11% in patients with prior negative genetic testing to 34.5% in naive patients. Incorporating SVs from genome sequencing added up to 13% of new diagnoses in previously unsolved cases. HiFi-GS yielded increased discovery rate with >4-fold more rare coding SVs compared with srGS. Variants and genes of unknown significance remain the most common finding (58% of nondiagnostic cases). CONCLUSION: Computational prioritization is efficient for diagnostic SNVs. Thorough identification of non-SNVs remains challenging and is partly mitigated using HiFi-GS sequencing. Importantly, community research is supported by sharing real-time data to accelerate gene validation and by providing HiFi variant (SNV/SV) resources from >1000 human alleles to facilitate implementation of new sequencing platforms for rare disease diagnoses.

Requirement for Crk and CrkL during postnatal lens development.

The Crk and CrkL adaptor proteins have SH2 and SH3 domains and play essential overlapping, as well as distinct, roles in many biological processes, ranging from cell structure and motility to proliferation. Conditional ablation of both Crk and CrkL in neuronal progenitor cells, using a Nestin-Cre transgene, resulted in severe defects in postnatal eye development, including progressive eye closure, lens rupture, and retinal malformation. These phenotypes were not observed in the presence of a single wild-type allele of either Crk or CrkL. We found that the lens in knockout mice started to rupture and disintegrate between postnatal days 7 and 12, although the structure of the retina was relatively well maintained. As the lens deteriorated further, the outer nuclear layer in the posterior of the retina enlarged and developed ruffles. Cre recombination occurred in the lens and retina of the knockout mice. Furthermore, the posterior lens capsule of the knockout mouse was thinner at postnatal days 0.5 and 3, suggesting that the defective lens capsule caused rupturing of the lens near the posterior pole. These results indicate that Crk and CrkL play essential overlapping roles in postnatal lens development.

Dok-7 regulates neuromuscular synapse formation by recruiting Crk and Crk-L.

Agrin, released by motor neurons, promotes neuromuscular synapse formation by stimulating MuSK, a receptor tyrosine kinase expressed in skeletal muscle. Phosphorylated MuSK recruits docking protein-7 (Dok-7), an adaptor protein that is expressed selectively in muscle. In the absence of Dok-7, neuromuscular synapses fail to form, and mutations that impair Dok-7 are a major cause of congenital myasthenia in humans. How Dok-7 stimulates synaptic differentiation is poorly understood. Once recruited to MuSK, Dok-7 directly stimulates MuSK kinase activity. This unusual activity of an adapter protein is mediated by the N-terminal region of Dok-7, whereas most mutations that cause congenital myasthenia truncate the C-terminal domain. Here, we demonstrate that Dok-7 also functions downstream from MuSK, and we identify the proteins that are recruited to the C-terminal domain of Dok-7. We show that Agrin stimulates phosphorylation of two tyrosine residues in the C-terminal domain of Dok-7, which leads to recruitment of two adapter proteins: Crk and Crk-L. Furthermore, we show that selective inactivation of Crk and Crk-L in skeletal muscle leads to severe defects in neuromuscular synapses in vivo, revealing a critical role for Crk and Crk-L downstream from Dok-7 in presynaptic and postsynaptic differentiation.

Fibroblast Growth Requires CT10 Regulator of Kinase (Crk) and Crk-like (CrkL).

CT10 regulator of kinase (Crk) and Crk-like (CrkL) are the cellular counterparts of the viral oncogene v-Crk Elevated levels of Crk and CrkL have been observed in many human cancers; inhibition of Crk and CrkL expression reduced the tumor-forming potential of cancer cell lines. Despite a close relationship between the Crk family proteins and tumorigenesis, how Crk and CrkL contribute to cell growth is unclear. We ablated endogenous Crk and CrkL from cultured fibroblasts carrying floxed alleles of Crk and CrkL by transfection with synthetic Cre mRNA (synCre). Loss of Crk and CrkL induced by synCre transfection blocked cell proliferation and caused shrinkage of the cytoplasm and the nucleus, formation of adherens junctions, and reduced cell motility. Ablation of Crk or CrkL alone conferred a much more modest reduction in cell proliferation. Reintroduction of CrkI, CrkII, or CrkL individually rescued cell proliferation in the absence of the endogenous Crk and CrkL, suggesting that Crk and CrkL play overlapping functions in regulating fibroblast growth. Serum and basic FGF induced phosphorylation of Akt, MAP kinases, and S6 kinase and Fos expression in the absence of Crk and CrkL, suggesting that cells lacking Crk and CrkL are capable of initiating major signal transduction pathways in response to extracellular stimuli. Furthermore, cell cycle and cell death analyses demonstrated that fibroblasts lacking Crk and CrkL become arrested at the G1-S transition and undergo a modest apoptosis. Taken together, our results suggest that Crk and CrkL play essential overlapping roles in fibroblast growth.

Crk and CrkL are required for cell transformation by v-fos and v-ras.

Crk and CrkL are SH2- and SH3-containing cytosolic adaptor proteins that can induce anchorage-independent growth of fibroblasts. Crk and CrkL play key roles in maintaining cytoskeletal integrity, cell motility and migration. We investigated the role of these two proteins in oncogenic transformation induced by v-fos and v-ras oncogenes using cell lines and fibroblasts carrying conditional alleles of Crk or CrkL. Transformation was assessed by cell morphology, saturation density and anchorage-independent growth in soft agar. We found that cell lines expressing v-fos or v-ras in the absence of Crk or CrkL displayed no evident morphological alterations and reduced anchorage-independent growth compared to those retaining Crk and CrkL. Similarly, overexpression of v-fos in mouse embryonic fibroblasts conferred a growth advantage and induced morphological changes, both of which were abrogated in the absence of either Crk or CrkL. In contrast, Crk, but not CrkL, contributed to v-ras-induced transformation of embryonic fibroblasts. These results suggest that both Crk and CrkL are required for the acquisition of cellular transformation by v-fos, whereas Crk plays a more prominent role than CrkL in v-ras-induced transformation.

Crk1/2 and CrkL form a hetero-oligomer and functionally complement each other during podocyte morphogenesis.

Activation of the slit diaphragm protein nephrin induces actin cytoskeletal remodeling, resulting in lamellipodia formation in podocytes in vitro in a phosphatidylinositol-3 kinase-, focal adhesion kinase-, Cas-, and Crk1/2-dependent fashion. In mice, podocyte-specific deletion of Crk1/2 prevents or attenuates foot process effacement in two models of podocyte injury. This suggests that cellular mechanisms governing lamellipodial protrusion in vitro are similar to those in vivo during foot process effacement. As Crk1/2-null mice developed and aged normally, we tested whether the Crk1/2 paralog, CrkL, functionally complements Crk1/2 in a podocyte-specific context. Podocyte-specific CrkL-null mice, like podocyte-specific Crk1/2-null mice, developed and aged normally but were protected from protamine sulfate-induced foot process effacement. Simultaneous podocyte-specific deletion of Crk1/2 and CrkL resulted in albuminuria detected by 6 weeks postpartum and associated with altered podocyte process architecture. Nephrin-induced lamellipodia formation in podocytes in vitro was CrkL-dependent. CrkL formed a hetero-oligomer with Crk2 and, like Crk2, was recruited to tyrosine phosphorylated nephrin. Thus, Crk1/2 and CrkL are physically linked, functionally complement each other during podocyte foot process spreading, and together are required for developing typical foot process architecture.

Pangenome graphs improve the analysis of structural variants in rare genetic diseases.

Rare DNA alterations that cause heritable diseases are only partially resolvable by clinical next-generation sequencing due to the difficulty of detecting structural variation (SV) in all genomic contexts. Long-read, high fidelity genome sequencing (HiFi-GS) detects SVs with increased sensitivity and enables assembling personal and graph genomes. We leverage standard reference genomes, public assemblies (n = 94) and a large collection of HiFi-GS data from a rare disease program (Genomic Answers for Kids, GA4K, n = 574 assemblies) to build a graph genome representing a unified SV callset in GA4K, identify common variation and prioritize SVs that are more likely to cause genetic disease (MAF < 0.01). Using graphs, we obtain a higher level of reproducibility than the standard reference approach. We observe over 200,000 SV alleles unique to GA4K, including nearly 1000 rare variants that impact coding sequence. With improved specificity for rare SVs, we isolate 30 candidate SVs in phenotypically prioritized genes, including known disease SVs. We isolate a novel diagnostic SV in KMT2E, demonstrating use of personal assemblies coupled with pangenome graphs for rare disease genomics. The community may interrogate our pangenome with additional assemblies to discover new SVs within the allele frequency spectrum relevant to genetic diseases.

Thyroid Hormone Suppresses Medulloblastoma Progression Through Promoting Terminal Differentiation of Tumor Cells.

Hypothyroidism is commonly detected in patients with medulloblastoma (MB). A possible link between thyroid hormone (TH) signaling and MB pathogenicity has not been reported. Here, we find that TH plays a critical role in promoting tumor cell differentiation. Reduction in TH levels frees the TH receptor, TRα1, to bind to EZH2 and repress expression of NeuroD1, a transcription factor that drives tumor cell differentiation. Increased TH reverses EZH2-mediated repression of NeuroD1 by abrogating the binding of EZH2 and TRα1, thereby stimulating tumor cell differentiation and reducing MB growth. Importantly, TH-induced differentiation of tumor cells is not restricted by the molecular subgroup of MB. These findings establish an unprecedented association between TH signaling and MB pathogenicity, providing solid evidence for TH as a promising modality for MB treatment.

Hedgehog signaling regulates the generation of ameloblast progenitors in the continuously growing mouse incisor.

In many organ systems such as the skin, gastrointestinal tract and hematopoietic system, homeostasis is dependent on the continuous generation of differentiated progeny from stem cells. The rodent incisor, unlike human teeth, grows throughout the life of the animal and provides a prime example of an organ that rapidly deteriorates if newly differentiated cells cease to form from adult stem cells. Hedgehog (Hh) signaling has been proposed to regulate self-renewal, survival, proliferation and/or differentiation of stem cells in several systems, but to date there is little evidence supporting a role for Hh signaling in adult stem cells. We used in vivo genetic lineage tracing to identify Hh-responsive stem cells in the mouse incisor and we show that sonic hedgehog (SHH), which is produced by the differentiating progeny of the stem cells, signals to several regions of the incisor. Using a hedgehog pathway inhibitor (HPI), we demonstrate that Hh signaling is not required for stem cell survival but is essential for the generation of ameloblasts, one of the major differentiated cell types in the tooth, from the stem cells. These results therefore reveal the existence of a positive-feedback loop in which differentiating progeny produce the signal that in turn allows them to be generated from stem cells.

Radial glia cells are candidate stem cells of ependymoma.

Tumors of the same histologic type often comprise clinically and molecularly distinct subgroups; however, the etiology of these subgroups is unknown. Here, we report that histologically identical, but genetically distinct, ependymomas exhibit patterns of gene expression that recapitulate those of radial glia cells in the corresponding region of the central nervous system. Cancer stem cells isolated from ependymomas displayed a radial glia phenotype and formed tumors when orthotopically transplanted in mice. These findings identify restricted populations of radial glia cells as candidate stem cells of the different subgroups of ependymoma, and they support a general hypothesis that subgroups of the same histologic tumor type are generated by different populations of progenitor cells in the tissues of origin.

3-D Transducer Mounted Shear Wave Absolute Vibro-Elastography: Proof of Concept.

Quantitative tissue stiffness characterization using ultrasound (US) has been shown to improve prostate cancer (PCa) detection in multiple studies. Shear wave absolute vibro-elastography (SWAVE) allows quantitative and volumetric assessment of tissue stiffness using external multifrequency excitation. This article presents a proof of concept of a first-of-a-kind 3-D hand-operated endorectal SWAVE system designed to be used during systematic prostate biopsy. The system is developed with a clinical US machine, requiring only an external exciter that can be mounted directly to the transducer. Subsector acquisition of radio frequency (RF) data allows imaging of shear waves with a high effective frame rate (up to 250 Hz). The system was characterized using eight different quality assurance phantoms. Due to the invasive nature of prostate imaging, at this early stage of development, validation of in vivo human tissue was instead carried out by intercostally scanning the livers of n = 7 healthy volunteers. The results are compared with 3-D magnetic resonance elastography (MRE) and an existing 3-D SWAVE system with a matrix array transducer (M-SWAVE). High correlations were found with MRE (99% in phantoms, 94% in liver data) and with M-SWAVE (99% in phantoms, 98% in liver data).

Global serum profiling: an opportunity for earlier cancer detection.

The advances in cancer research achieved in the last 50 years have been remarkable and have provided a deeper knowledge of this disease in many of its conceptual and biochemical aspects. From viewing a tumor as a 'simple' aggregate of mutant cells and focusing on detecting key cell changes leading to the tumorigenesis, the understanding of cancer has broadened to consider it as a complex organ interacting with its close and far surroundings through tumor and non-tumor cells, metabolic mechanisms, and immune processes. Metabolism and the immune system have been linked to tumorigenesis and malignancy progression along with cancer-specific genetic mutations. However, most technologies developed to overcome the barriers to earlier detection are focused solely on genetic information. The concept of cancer as a complex organ has led to research on other analytical techniques, with the quest of finding a more sensitive and cost-effective comprehensive approach. Furthermore, artificial intelligence has gained broader consensus in the oncology community as a powerful tool with the potential to revolutionize cancer diagnosis for physicians. We herein explore the relevance of the concept of cancer as a complex organ interacting with the bodily surroundings, and focus on promising emerging technologies seeking to diagnose cancer earlier, such as liquid biopsies. We highlight the importance of a comprehensive approach to encompass all the tumor and non-tumor derived information salient to earlier cancer detection.

Direct haplotype-resolved 5-base HiFi sequencing for genome-wide profiling of hypermethylation outliers in a rare disease cohort.

Long-read HiFi genome sequencing allows for accurate detection and direct phasing of single nucleotide variants, indels, and structural variants. Recent algorithmic development enables simultaneous detection of CpG methylation for analysis of regulatory element activity directly in HiFi reads. We present a comprehensive haplotype resolved 5-base HiFi genome sequencing dataset from a rare disease cohort of 276 samples in 152 families to identify rare (~0.5%) hypermethylation events. We find that 80% of these events are allele-specific and predicted to cause loss of regulatory element activity. We demonstrate heritability of extreme hypermethylation including rare cis variants associated with short (~200 bp) and large hypermethylation events (>1 kb), respectively. We identify repeat expansions in proximal promoters predicting allelic gene silencing via hypermethylation and demonstrate allelic transcriptional events downstream. On average 30-40 rare hypermethylation tiles overlap rare disease genes per patient, providing indications for variation prioritization including a previously undiagnosed pathogenic allele in DIP2B causing global developmental delay. We propose that use of HiFi genome sequencing in unsolved rare disease cases will allow detection of unconventional diseases alleles due to loss of regulatory element activity.

Suppression of the Shh pathway using a small molecule inhibitor eliminates medulloblastoma in Ptc1(+/-)p53(-/-) mice.

Medulloblastoma is the most common malignant pediatric brain tumor. Current treatment is associated with major long-term side effects; therefore, new nontoxic therapies, targeting specific molecular defects in this cancer, need to be developed. We use a mouse model of medulloblastoma to show that inhibition of the Sonic Hedgehog (Shh) pathway provides a novel therapy for medulloblastoma. A small molecule inhibitor of the Shh pathway, HhAntag, blocked the function of Smoothened in mice with medulloblastoma. This resulted in suppression of several genes highly expressed in medulloblastoma, inhibition of cell proliferation, increase in cell death and, at the highest dose, complete eradication of tumors. Long-term treatment with HhAntag prolonged medulloblastoma-free survival. These findings support the development of Shh antagonists for the treatment of medulloblastoma.

Clinical validation of a spectroscopic liquid biopsy for earlier detection of brain cancer.

Background: Diagnostic delays impact the quality of life and survival of patients with brain tumors. Earlier and expeditious diagnoses in these patients are crucial to reduce the morbidities and mortalities associated with brain tumors. A simple, rapid blood test that can be administered easily in a primary care setting to efficiently identify symptomatic patients who are most likely to have a brain tumor would enable quicker referral to brain imaging for those who need it most. Methods: Blood serum samples from 603 patients were prospectively collected and analyzed. Patients either had non-specific symptoms that could be indicative of a brain tumor on presentation to the Emergency Department, or a new brain tumor diagnosis and referral to the neurosurgical unit, NHS Lothian, Scotland. Patient blood serum samples were analyzed using the Dxcover® Brain Cancer liquid biopsy. This technology utilizes infrared spectroscopy combined with a diagnostic algorithm to predict the presence of intracranial disease. Results: Our liquid biopsy approach reported an area under the receiver operating characteristic curve of 0.8. The sensitivity-tuned model achieves a 96% sensitivity with 45% specificity (NPV 99.3%) and identified 100% of glioblastoma multiforme patients. When tuned for a higher specificity, the model yields a sensitivity of 47% with 90% specificity (PPV 28.4%). Conclusions: This simple, non-invasive blood test facilitates the triage and radiographic diagnosis of brain tumor patients while providing reassurance to healthy patients. Minimizing time to diagnosis would facilitate the identification of brain tumor patients at an earlier stage, enabling more effective, less morbid surgical and adjuvant care.

PTHrP treatment fails to rescue bone defects caused by Hedgehog pathway inhibition in young mice.

The advent of molecular targeted therapies offers the hope of therapeutic advance in the fight against cancer. However, this hope is tempered by recent findings that certain targeted therapies may have unique side effects. The Hedgehog (HH) pathway is a potential target for treatment of several cancers, including basal cell carcinoma and a subset of medulloblastoma. Recent clinical trials in adults have shown responses to HH pathway inhibition in both basal cell carcinoma and medulloblastoma. However, concerns have been raised about the use of HH pathway inhibitors in children because of the role the HH pathway plays in development. Indeed, young mice treated with the HH pathway inhibitor HhAntag developed severe bone defects, including premature differentiation of chondrocytes, thinning of cortical bone, and fusion of the growth plate. In an effort to lessen the severity of bone defects caused by HhAntag, we treated young mice simultaneously with HhAntag and parathyroid hormone-related protein (PTHrP), which functions downstream of Indian Hedgehog to maintain chondrocytes in a proliferative state. The results show that whereas treatment with PTHrP causes a significant increase in trabecular bone, it does not prevent fusion of the growth plate induced by HhAntag.

A call to action: Issuing a diversity and inclusion challenge to research organizations.

Individuals who identify as Black, indigenous, and people of color face well-documented health disparities. A root cause is the lack of empiric evidence for or against the use of various treatments in their medical management. This communication proposes a new benchmarking strategy for evaluating racial and ethnic representation in clinical research that can be compared across institutions with the intent of increasing accountability for diversity and inclusion among organizations that conduct clinical research.

Statins repress hedgehog signaling in medulloblastoma with no bone toxicities.

The Hedgehog (Hh) pathway plays an indispensable role in bone development and genetic activation of the pathway results in medulloblastoma (MB), the most common malignant brain tumor in children. Inhibitors of Hh pathway (such as vismodegib and sonedigib), which are used to treat MB, cause irreversible defects in bone growth in young children. Cholesterol is required for the activation of the Hh pathway, and statins, inhibitors of cholesterol biosynthesis, suppress MB growth by repressing Hh signaling in tumor cells. Here, we investigate the role of cholesterol biosynthesis in the proliferation and Hh signaling in chondrocytes, and examine the bone development in mice after statin treatment. Statins significantly inhibited MB growth in young mice, but caused no defects in bone development. Conditional deletion of NADP steroid dehydrogenase-like (NSDHL), an enzyme necessary for cholesterol biosynthesis, suppressed cholesterol synthesis in chondrocytes, and disrupted the growth plate in mouse femur and tibia, indicating the important function of intracellular cholesterol in bone development. Hh pathway activation and the proliferation of chondrocytes were inhibited by statin treatment in vitro; however, statins did not impair bone growth in vivo due to insufficient penetration into the bone. Our studies reveal a critical role of cholesterol in bone development, and support the utilization of statins for treatment of MB as well as other Hh pathway-associated malignancies.