Metabolic Lipids in Melanoma Enable Rapid Determination of Actionable BRAF-V600E Mutation with Picosecond Infrared Laser Mass Spectrometry in 10 s.

Rapid molecular profiling of biological tissues with picosecond infrared laser mass spectrometry (PIRL-MS) has enabled the detection of clinically important histologic types and molecular subtypes of human cancers in as little as 10 s of data collection and analysis time. Utilizing an engineered cell line model of actionable BRAF-V600E mutation, we observed statistically significant differences in 10 s PIRL-MS molecular profiles between BRAF-V600E and BRAF-wt cells. Multivariate statistical analyses revealed a list of mass-to-charge (m/z) values most significantly responsible for the identification of BRAF-V600E mutation status in this engineered cell line that provided a highly controlled testbed for this observation. These metabolites predicted BRAF-V600E expression in human melanoma cell lines with greater than 98% accuracy. Through chromatography and tandem mass spectrometry analysis of cell line extracts, a 30-member "metabolite array" was characterized for determination of BRAF-V600E expression levels in subcutaneous melanoma xenografts with an average sensitivity and specificity of 95.6% with 10 s PIRL-MS analysis. This proof-of-principle work warrants a future large-scale study to identify a metabolite array for 10 s determination of actionable BRAF-V600E mutation in human tissue to guide patient care.

Dual Laser and Desorption Electrospray Ionization Mass Spectrometry Imaging Using the Same Interface.

For a more comprehensive characterization of molecular heterogeneities of matter, multimodal mass spectrometry imaging must be developed to take advantage of the complementarity of information available through different ionization mechanisms. We report the design, implementation, and performance validation of a laser desorption imaging interface composed of add-on components that adapt a commercial Desorption Electrospray Ionization Mass Spectrometry (DESI-MS) imaging interface for dual imaging of Picosecond Infrared Laser Mass Spectrometry (PIRL-MS) with DESI-MS. The interface utilizes hardware elements and data analysis pipelines already established for DESI-MS imaging, and was further validated in cancer margin assessments using human medulloblastoma cancers. The PIRL-MS images were robust and reproducible across multiple experimental runs on independently prepared xenograft tumors, and could be segmented into cancer and healthy regions in concordance with pathology using a variety of supervised and unsupervised clustering methods. The spectral quality and complexity obtained with this interface were examined with infiltrating and noninfiltrating tumors, and were comparable to other mass spectrometry analysis interfaces. The average PIRL-MS spectra from spatially resolved images could be used for robust cancer m/z model building to classify medulloblastoma cancer from healthy tissue without any misclassifications, an observation that held true over close to 70 sampling data points. While the unsupervised spectral analysis methods suggested a slight suppression of signal in the phospholipid range compared to the hand-held configuration, these changes were insufficient to hamper utility in cancer margin assessment with spatially resolved data obtained with our interface. Dual PIRL-MS and DESI-MS imaging of consecutive sections, as suggested by multivariate loading plots, revealed highly complementary molecular information with m/z values identifiable with one desorption method sufficient to reveal cancer regions being absent in another, further emphasizing the need for effective hardware and software interfaces for dual mass spectrometry imaging.

Enhancing glioblastoma treatment using cisplatin-gold-nanoparticle conjugates and targeted delivery with magnetic resonance-guided focused ultrasound.

Glioblastoma (GBM) is the most common and aggressive primary brain tumor resulting in high rates of morbidity and mortality. A strategy to increase the efficacy of available drugs and enhance the delivery of chemotherapeutics through the blood brain barrier (BBB) is desperately needed. We investigated the potential of Cisplatin conjugated gold nanoparticle (GNP-UP-Cis) in combination with MR-guided Focused Ultrasound (MRgFUS) to intensify GBM treatment. Viability assays demonstrated that GNP-UP-Cis greatly inhibits the growth of GBM cells compared to free cisplatin and shows marked synergy with radiation therapy. Additionally, increased DNA damage through γH2AX phosphorylation was observed in GNP-UP-Cis treated cells, along with enhanced platinum concentrations. In vivo, GNP-UP-Cis greatly reduced the growth of GBM tumors and MRgFUS led to increased BBB permeability and GNP-drug delivery in brain tissue. Our studies suggest that GNP-Cis conjugates and MRgFUS can be used to focally enhance the delivery of targeted chemotherapeutics to brain tumors.

A novel amplification-based approach to enable gene expression profiling from small clinical tumor specimens.

Glioblastoma is the most common and deadly type of brain cancer. Over the past decade, several divergent genetic pathways have been implicated in the initiation, progression and clinical outcome of this disease. As our understanding of GBM expands and identifies actionable targets specific to individual tumors, there will be a pressing need for the development of new tools that will maximize the use of limited clinical samples to enable the employment of personalized care paradigms. We used PrimePCR validated assays to generate a custom real-time PCR screening panel, containing 74 previously published mRNA targets showing gene expression changes in glioblastoma, and five house-keeping genes. A cohort of 19 frozen brain specimens were analyzed, including WHO Grade II oligodendroglioma (n = 3), WHO Grade II astrocytoma (n = 2), WHO Grade III astrocytoma (n = 1), and glioblastoma (n = 13). Four normal brain samples were also analyzed. We performed RNA extraction, followed by cDNA synthesis, multiplexed pre-amplification and SYBR-based qPCR, to generate expression profiles on all samples. We demonstrated that the workflow shows high tolerance to variation in RNA quality (RIN 8.5-4) and high sensitivity in detection. cDNA input that is equivalent to 3 ng of starting RNA was sufficient to conduct accurate semiquantitative analysis of the panel of 79 assays. Using principal component analysis, we were able to accurately separate glioblastoma from low-grade glioma. The two WHO Grade III tumors analyzed clustered with glioblastoma, but showed more similarity to Grade II gliomas. In this study, we have shown the feasibility of consolidating high-throughput data into a single functional panel capable of accurately classifying glioma specimens based solely on semiquantitative gene expression profiling.

Ultrastructural and cellular basis for the development of abnormal myocardial mechanics during the transition from hypertension to heart failure.

Although the development of abnormal myocardial mechanics represents a key step during the transition from hypertension to overt heart failure (HF), the underlying ultrastructural and cellular basis of abnormal myocardial mechanics remains unclear. We therefore investigated how changes in transverse (T)-tubule organization and the resulting altered intracellular Ca(2+) cycling in large cell populations underlie the development of abnormal myocardial mechanics in a model of chronic hypertension. Hearts from spontaneously hypertensive rats (SHRs; n = 72) were studied at different ages and stages of hypertensive heart disease and early HF and were compared with age-matched control (Wistar-Kyoto) rats (n = 34). Echocardiography, including tissue Doppler and speckle-tracking analysis, was performed just before euthanization, after which T-tubule organization and Ca(2+) transients were studied using confocal microscopy. In SHRs, abnormalities in myocardial mechanics occurred early in response to hypertension, before the development of overt systolic dysfunction and HF. Reduced longitudinal, circumferential, and radial strain as well as reduced tissue Doppler early diastolic tissue velocities occurred in concert with T-tubule disorganization and impaired Ca(2+) cycling, all of which preceded the development of cardiac fibrosis. The time to peak of intracellular Ca(2+) transients was slowed due to T-tubule disruption, providing a link between declining cell ultrastructure and abnormal myocardial mechanics. In conclusion, subclinical abnormalities in myocardial mechanics occur early in response to hypertension and coincide with the development of T-tubule disorganization and impaired intracellular Ca(2+) cycling. These changes occur before the development of significant cardiac fibrosis and precede the development of overt cardiac dysfunction and HF.

"Black Is Not Monolithic": Complexities in COVID-19 Vaccine Decision-Making.

BACKGROUND: Longstanding inequities in the USA have resulted in the disproportionate impact of COVID-19 on Black Americans. Coupled with medical mistrust, COVID-19 vaccine uptake is lower in Black populations. METHODS: We sought to understand the perspectives of Black parents on the COVID-19 pandemic, COVID-19 vaccination for themselves and their children, and trust with the medical community. Using qualitative methodology, we conducted in-depth semi-structured in-person interviews of Black parents of children admitted to the inpatient pediatric units in our tertiary academic medical center in Connecticut from July to November 2021. We used the grounded theory approach, and the constant comparative method until saturation was reached. RESULTS: We interviewed 20 parents who identified as Black; 50% were vaccinated against COVID-19. The following 5 themes and sub-themes emerged: (1) mixed feelings influenced COVID-19 vaccine decision-making ranging from much needed relief and feelings of uncertainty, distrust, and fear; (2) COVID-19 vaccine uptake was influenced by individual and family's health concerns and job or school mandates; (3) deferring the COVID-19 vaccine was influenced by the perception of risk and concerns about vaccine integrity; (4) institutional mistrust within the Black community bred by systemic racism influenced vaccine decision-making; and (5) conflicted feelings about the COVID-19 vaccine for their child. CONCLUSION: Our findings reiterate the complexities around vaccine decision-making and underscore the importance of recognizing the pervasive influence of institutional mistrust when counseling Black families about the COVID-19 vaccine.

Highly proliferative sellar chordoma with unusually rapid recurrence.

Chordomas are tumors of notochordal differentiation of low to intermediate grade malignancy. These tumors are typically slow growing, with an indolent but progressive clinical course. We present a case of a highly proliferative chordoma arising in a 73-year-old woman with unusually rapid clinical growth and aggressive histologic and immunohistochemical features. This patient had an unusually brief preclinical course and within 1 month of developing headaches presented to medical attention with diplopia. The resected chordoma showed uncommonly elevated mitotic activity, without the histologic hallmarks of de-differentiation. This proliferative activity correlated with elevated Ki67 staining (60%), B-cell leukemia/lymphoma1 (BCL1) expression (100%), and topoisomerase IIα staining (>95%). E-cadherin expression was also lost throughout the majority of the tumor. Other markers of epithelial mesenchymal transition (EMT) including vimentin, N-cadherin, Slug and Twist, were also strongly expressed in this aggressive tumor. The sellar component of the tumor recurred within a 2-month interval. The evaluation of the additional biomarkers, including makers of EMT studied in this, case may allow for identification of aggressive chordomas in which the tempo of disease is significantly more rapid than in typical cases of chordoma.

Notch promotes dynamin-dependent endocytosis of nephrin.

Notch signaling in podocytes causes proteinuria and glomerulosclerosis in humans and rodents, but the underlying mechanism remains unknown. Here, we analyzed morphologic, molecular, and cellular events before the onset of proteinuria in newborn transgenic mice that express activated Notch in podocytes. Immunohistochemistry revealed a loss of the slit diaphragm protein nephrin exclusively in podocytes expressing activated Notch. Podocyte-specific deletion of Rbpj, which is essential for canonical Notch signaling, prevented this loss of nephrin. Overexpression of activated Notch decreased cell surface nephrin and increased cytoplasmic nephrin in transfected HEK293T cells; pharmacologic inhibition of dynamin, but not depletion of cholesterol, blocked these effects on nephrin, suggesting that Notch promotes dynamin-dependent, raft-independent endocytosis of nephrin. Supporting an association between Notch signaling and nephrin trafficking, electron microscopy revealed shortened podocyte foot processes and fewer slit diaphragms among the transgenic mice compared with controls. These data suggest that Notch signaling induces endocytosis of nephrin, thereby triggering the onset of proteinuria.

Circulating tumor DNA reveals mechanisms of lorlatinib resistance in patients with relapsed/refractory ALK-driven neuroblastoma.

Activating point mutations in Anaplastic Lymphoma Kinase (ALK) have positioned ALK as the only mutated oncogene tractable for targeted therapy in neuroblastoma. Cells with these mutations respond to lorlatinib in pre-clinical studies, providing the rationale for a first-in-child Phase 1 trial (NCT03107988) in patients with ALK-driven neuroblastoma. To track evolutionary dynamics and heterogeneity of tumors, and to detect early emergence of lorlatinib resistance, we collected serial circulating tumor DNA samples from patients enrolled on this trial. Here we report the discovery of off-target resistance mutations in 11 patients (27%), predominantly in the RAS-MAPK pathway. We also identify newly acquired secondary compound ALK mutations in 6 (15%) patients, all acquired at disease progression. Functional cellular and biochemical assays and computational studies elucidate lorlatinib resistance mechanisms. Our results establish the clinical utility of serial circulating tumor DNA sampling to track response and progression and to discover acquired resistance mechanisms that can be leveraged to develop therapeutic strategies to overcome lorlatinib resistance.

Variability in timing of spontaneous calcium release in the intact rat heart is determined by the time course of sarcoplasmic reticulum calcium load.

BACKGROUND: Abnormalities in intracellular calcium (Ca) cycling during Ca overload can cause triggered activity because spontaneous calcium release (SCR) activates sufficient Ca-sensitive inward currents to induce delayed afterdepolarizations (DADs). However, little is known about the mechanisms relating SCR and triggered activity on the tissue scale. METHODS AND RESULTS: Laser scanning confocal microscopy was used to measure the spatiotemporal properties of SCR within large myocyte populations in intact rat heart. Computer simulations were used to predict how these properties of SCR determine DAD magnitude. We measured the average and standard deviation of the latency distribution of SCR within a large population of myocytes in intact tissue. We found that as external [Ca] is increased, and with faster pacing rates, the average and SD of the latency distribution decreases substantially. This result demonstrates that the timing of SCR occurs with less variability as the sarcoplasmic reticulum (SR) Ca load is increased, causing more sites to release Ca within each cell. We then applied a mathematical model of subcellular Ca cycling to show that a decrease in SCR variability leads to a higher DAD amplitude and is dictated by the rate of SR Ca refilling following an action potential. CONCLUSIONS: Our results demonstrate that the variability of the timing of SCR in a population of cells in tissue decreases with SR load and is dictated by the time course of the SR Ca content.

Cognitive Behavioral Therapy (CBT) Telehealth Augmented With a CBT Smartphone Application to Address Type 2 Diabetes Self-Management: A Randomized Pilot Trial.

PURPOSE: The purpose of the study was to investigate the feasibility and acceptability of phone-delivered cognitive behavioral therapy (CBT) combined with an adjunctive smartphone application CBT MobileWork-DM© to improve self-management of type 2 diabetes mellitus (T2DM). METHODS: Participants were 12 patients with T2DM on antihyperglycemic medication and had an A1C level of 8 or greater. A randomized controlled pilot study assessed treatment as usual (TAU) T2DM care versus a phone-delivered CBT (6, 8, or 12 weekly sessions) augmented with a CBT skills practice smartphone application. The CBT telehealth intervention addressed T2DM self-management and diabetes distress. Electronic and self-report medication taking, diabetes-related distress, and A1C were assessed at baseline and post-intervention. RESULTS: After 16 weeks, a decrease in A1C and distress levels was observed in all 3 CBT phone groups and TAU group. The group with the most improvement was the 12-week CBT group, which had the greatest mean decrease in A1C (-2.33) and diabetes distress (-31.67). The TAU group exhibited a mean decrease of -2.15 and -21 for A1C and diabetes distress, respectively. The overall rate of completion for phone CBT sessions across the 3 CBT groups was 83%. CONCLUSION: This study demonstrates that telehealth CBT augmented with a smartphone application is feasible and acceptable. Patients demonstrated improvements in both T2DM management and distress.

Health-Related Quality of Life and Toxicity After Definitive High-Dose-Rate Brachytherapy Among Veterans With Prostate Cancer.

PURPOSE: High-dose-rate (HDR) brachytherapy (BT) is a well-tolerated and effective treatment for prostate cancer. There is limited research, however, investigating toxicity outcomes with HDRBT treatment among veterans. The objective of this study is to assess the impact on health-related quality of life (hrQOL) and physician-graded toxicities associated with HDRBT as monotherapy among veterans treated at Edward Hines, Jr. Veterans Affairs Hospital in Hines, Illinois. METHODS: Between 2016 and 2019, 74 veterans with low- or intermediate-risk prostate cancer were treated with HDRBT as monotherapy with 27 Gy in 2 fractions, delivered over 2 implants. Veteran-reported hrQOL in the genitourinary (GU), gastrointestinal (GI), and sexual domains was assessed using the International Prostate Symptoms Score (IPSS) and Expanded Prostate Cancer Index Composite (EPIC-26) questionnaire. Mixed linear effect models were used to assess differences in the hrQOL scores at follow-up compared with baseline scores. Statistically significant differences in hrQOL scores from baseline were further assessed for clinical significance, using minimal clinically important difference (MCID) evaluations. RESULTS: Median follow-up was 18 months. Veterans reported declines in GU, GI, and sexual hrQOL scores immediately after treatment, with the IPSS and EPIC-26 hrQOL scores all displaying significant decrease from baseline over time. The majority of the declines in hrQOL scores met criteria for MCID. These hrQOL scores trended toward a return to baseline, with the EPIC-26 urinary obstruction score returning to baseline at the 18-month follow-up assessment and the EPIC-26 bowel score returning to baseline at the 12-month follow-up. The IPSS, urinary incontinence, and sexual scores did not return to baseline at 18 months. The grade 2 maximum physician-graded GU, GI, and sexual toxicity rates were 65%, 5%, and 53%, respectively. There was 1 incidence of grade 3 GU toxicity but no grade 3 GI or sexual toxicity. CONCLUSIONS: HDRBT as monotherapy is a well-tolerated treatment option for veterans with low- or intermediate-risk prostate cancer, with favorable veteran-reported and physician-graded toxicities. Veterans should be educated about HDRBT as an option when counseled regarding treatment for localized prostate cancer.

Computed tomography versus magnetic resonance imaging in high-dose-rate prostate brachytherapy planning: The impact on patient-reported health-related quality of life.

PURPOSE: High-dose-rate (HDR) prostate brachytherapy uses volumetric imaging for treatment planning. Our institution transitioned from computed tomography (CT)-based planning to MRI-based planning with the hypothesis that improved visualization could reduce treatment-related toxicity. This study aimed to compare the patient-reported health-related quality of life (hrQOL) and physician-graded toxicity outcomes of CT-based and MRI-based HDR prostate brachytherapy. METHODS: From 2016 to 2019, 122 patients with low- or intermediate-risk prostate cancer were treated with HDR brachytherapy as monotherapy. Patients underwent CT only or CT and MRI imaging for treatment planning and were grouped per treatment planning imaging modality. Patient-reported hrQOL in the genitourinary (GU), gastrointestinal (GI), and sexual domains was assessed using International Prostate Symptom Score and Expanded Prostate Cancer Index Composite Short Form-26 questionnaires. Baseline characteristics, changes in hrQOL scores, and physician-graded toxicities were compared between groups. RESULTS: The median follow-up was 18 months. Patient-reported GU, GI, and sexual scores worsened after treatment but returned toward baseline over time. The CT cohort had a lower baseline mean International Prostate Symptom Score (5.8 vs. 7.8, p = 0.03). The other patient-reported GU and GI scores did not differ between groups. Overall, sexual scores were similar between the CT and MRI cohorts (p = 0.08) but favored the MRI cohort at later follow-up with a smaller decrease in Expanded Prostate Cancer Index Composite Short Form-26 sexual score from baseline at 18 months (4.9 vs. 19.8, p = 0.05). Maximum physician-graded GU, GI, and sexual toxicity rates of grade ≥2 were 68%, 3%, and 53%, respectively, with no difference between the cohorts (p = 0.31). CONCLUSION: Our study shows that CT- and MRI-based HDR brachytherapy results in similar rates of GU and GI toxicity. MRI-based planning may result in improved erectile function recovery compared with CT-based planning.

Lunatic Fringe-mediated Notch signaling is required for lung alveogenesis.

Distal lung development occurs through coordinated induction of myofibroblasts, epithelial cells, and capillaries. Lunatic Fringe (Lfng) is a beta(1-3) N-acetylglucosamine transferase that modifies Notch receptors to facilitate their activation by Delta-like (Dll1/4) ligands. Lfng is expressed in the distal lung during saccular development, and deletion of this gene impairs myofibroblast differentiation and alveogenesis in this context. A similar defect was observed in Notch2(beta-geo/+)Notch3(beta-geo/beta-geo) compound mutant mice but not in Notch2(beta-geo/+) or Notch3(beta-geo/beta-geo) single mutants. Finally, to directly test for the role of Notch signaling in myofibroblast differentiation in vivo, we used ROSA26-rtTA(/+);tetO-CRE(/+);RBPJkappa(flox/flox) inducible mutant mice to show that disruption of canonical Notch signaling during late embryonic development prevents induction of smooth muscle actin in mesenchymal cells of the distal lung. In sum, these results demonstrate that Lfng functions to enhance Notch signaling in myofibroblast precursor cells and thereby to coordinate differentiation and mobilization of myofibroblasts required for alveolar septation.

In situ tissue pathology from spatially encoded mass spectrometry classifiers visualized in real time through augmented reality.

Integration between a hand-held mass spectrometry desorption probe based on picosecond infrared laser technology (PIRL-MS) and an optical surgical tracking system demonstrates in situ tissue pathology from point-sampled mass spectrometry data. Spatially encoded pathology classifications are displayed at the site of laser sampling as color-coded pixels in an augmented reality video feed of the surgical field of view. This is enabled by two-way communication between surgical navigation and mass spectrometry data analysis platforms through a custom-built interface. Performance of the system was evaluated using murine models of human cancers sampled in situ in the presence of body fluids with a technical pixel error of 1.0 ± 0.2 mm, suggesting a 84% or 92% (excluding one outlier) cancer type classification rate across different molecular models that distinguish cell-lines of each class of breast, brain, head and neck murine models. Further, through end-point immunohistochemical staining for DNA damage, cell death and neuronal viability, spatially encoded PIRL-MS sampling is shown to produce classifiable mass spectral data from living murine brain tissue, with levels of neuronal damage that are comparable to those induced by a surgical scalpel. This highlights the potential of spatially encoded PIRL-MS analysis for in vivo use during neurosurgical applications of cancer type determination or point-sampling in vivo tissue during tumor bed examination to assess cancer removal. The interface developed herein for the analysis and the display of spatially encoded PIRL-MS data can be adapted to other hand-held mass spectrometry analysis probes currently available.

Ectopic notch activation in developing podocytes causes glomerulosclerosis.

Genetic evidence supports an early role for Notch signaling in the fate of podocytes during glomerular development. Decreased expression of Notch transcriptional targets in developing podocytes after the determination of cell fate suggests that constitutive Notch signaling may oppose podocyte differentiation. This study determined the effects of constitutive Notch signaling on podocyte differentiation by ectopically expressing Notch's intracellular domain (NOTCH-IC), the biologically active, intracellular product of proteolytic cleavage of the Notch receptor, in developing podocytes of transgenic mice. Histologic and molecular analyses revealed normal glomerular morphology and expression of podocyte markers in newborn NOTCH-IC-expressing mice; however, mice developed severe proteinuria and showed evidence of progressive glomerulosclerosis at 2 wk after birth. Features of mature podocytes were lost: Foot processes were effaced; expression of Wt1, Nphs1, and Nphs2 was downregulated; cell-cycle re-entry was induced; and the expression of Pax2 was increased. In contrast, mice with podocyte-specific inactivation of Rbpsuh, which encodes a protein essential for canonical Notch signaling, seemed normal. In addition, the damaging effects of NOTCH-IC expression were prevented in transgenic mice after simultaneous conditional inactivation of Rbpsuh in murine podocytes. These results suggest that Notch signaling is dispensable during terminal differentiation of podocytes but that constitutive (or inappropriate) Notch signaling is deleterious, leading to glomerulosclerosis.

BMP signaling mediates glioma stem cell quiescence and confers treatment resistance in glioblastoma.

Despite advances in therapy, glioblastoma remains an incurable disease with a dismal prognosis. Recent studies have implicated cancer stem cells within glioblastoma (glioma stem cells, GSCs) as mediators of therapeutic resistance and tumor progression. In this study, we investigated the role of the transforming growth factor-ß (TGF-ß) superfamily, which has been found to play an integral role in the maintenance of stem cell homeostasis within multiple stem cell systems, as a mediator of stem-like cells in glioblastoma. We find that BMP and TGF-ß signaling define divergent molecular and functional identities in glioblastoma, and mark relatively quiescent and proliferative GSCs, respectively. Treatment of GSCs with BMP inhibits cell proliferation, but does not abrogate their stem-ness, as measured by self-renewal and tumorigencity. Further, BMP pathway activation confers relative resistance to radiation and temozolomide chemotherapy. Our findings define a quiescent cancer stem cell population in glioblastoma that may be a cellular reservoir for tumor recurrence following cytotoxic therapy.

Picosecond Infrared Laser Desorption Mass Spectrometry Identifies Medulloblastoma Subgroups on Intrasurgical Timescales.

Medulloblastoma (MB) is a pediatric malignant brain tumor composed of four different subgroups (WNT, SHH, Group 3, Group 4), each of which are a unique biological entity with distinct clinico-pathological, molecular, and prognostic characteristics. Although risk stratification of patients with MB based on molecular features may offer personalized therapies, conventional subgroup identification methods take too long and are unable to deliver subgroup information intraoperatively. This limitation prevents subgroup-specific adjustment of the extent or the aggressiveness of the tumor resection by the neurosurgeon. In this study, we investigated the potential of rapid tumor characterization with Picosecond infrared laser desorption mass spectrometry (PIRL-MS) for MB subgroup classification based on small molecule signatures. One hundred and thirteen ex vivo MB tumors from a local tissue bank were subjected to 10- to 15-second PIRL-MS data collection and principal component analysis with linear discriminant analysis (PCA-LDA). The MB subgroup model was established from 72 independent tumors; the remaining 41 de-identified unknown tumors were subjected to multiple, 10-second PIRL-MS samplings and real-time PCA-LDA analysis using the above model. The resultant 124 PIRL-MS spectra from each sampling event, after the application of a 95% PCA-LDA prediction probability threshold, yielded a 98.9% correct classification rate. Post-ablation histopathologic analysis suggested that intratumoral heterogeneity or sample damage prior to PIRL-MS sampling at the site of laser ablation was able to explain failed classifications. Therefore, upon translation, 10-seconds of PIRL-MS sampling is sufficient to allow personalized, subgroup-specific treatment of MB during surgery. SIGNIFICANCE: This study demonstrates that laser-extracted lipids allow immediate grading of medulloblastoma tumors into prognostically important subgroups in 10 seconds, providing medulloblastoma pathology in an actionable manner during surgery.

ID1 Is Critical for Tumorigenesis and Regulates Chemoresistance in Glioblastoma.

Glioblastoma is the most common primary brain tumor in adults. While the introduction of temozolomide chemotherapy has increased long-term survivorship, treatment failure and rapid tumor recurrence remains universal. The transcriptional regulatory protein, inhibitor of DNA-binding-1 (ID1), is a key regulator of cell phenotype in cancer. We show that CRISPR-mediated knockout of ID1 in glioblastoma cells, breast adenocarcinoma cells, and melanoma cells dramatically reduced tumor progression in all three cancer systems through transcriptional downregulation of EGF, which resulted in decreased EGFR phosphorylation. Moreover, ID1-positive cells were enriched by chemotherapy and drove tumor recurrence in glioblastoma. Addition of the neuroleptic drug pimozide to inhibit ID1 expression enhanced the cytotoxic effects of temozolomide therapy on glioma cells and significantly prolonged time to tumor recurrence. Conclusively, these data suggest ID1 could be a promising therapeutic target in patients with glioblastoma. SIGNIFICANCE: These findings show that the transcriptional regulator ID1 is critical for glioblastoma initiation and chemoresistance and that inhibition of ID1 enhances the effect of temozolomide, delays tumor recurrence, and prolongs survival.