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Tissue fusion transcript panel gene coverage (55 genes)

Purpose:The clinical utility of plasma cell-free DNA (cfDNA) has not been assessed prospectively in patients with glioblastoma (GBM). We aimed to determine the prognostic impact of plasma cfDNA in GBM, as well as its role as a surrogate of tumor burden and substrate for next-generation sequencing (NGS).Experimental Design:We conducted a prospective cohort study of 42 patients with newly diagnosed GBM. Plasma cfDNA was quantified at baseline prior to initial tumor resection and longitudinally during chemoradiotherapy. Plasma cfDNA was assessed for its association with progression-free survival (PFS) and overall survival (OS), correlated with radiographic tumor burden, and subjected to a targeted NGS panel.Results:Prior to initial surgery, GBM patients had higher plasma cfDNA concentration than age-matched healthy controls (mean 13.4 vs. 6.7 ng/mL, P < 0.001). Plasma cfDNA concentration was correlated with radiographic tumor burden on patients' first post-radiation magnetic resonance imaging scan (ρ = 0.77, P = 0.003) and tended to rise prior to or concurrently with radiographic tumor progression. Preoperative plasma cfDNA concentration above the mean (>13.4 ng/mL) was associated with inferior PFS (median 4.9 vs. 9.5 months, P = 0.038). Detection of ≥1 somatic mutation in plasma cfDNA occurred in 55% of patients and was associated with nonstatistically significant decreases in PFS (median 6.0 vs. 8.7 months, P = 0.093) and OS (median 5.5 vs. 9.2 months, P = 0.053).Conclusions:Plasma cfDNA may be an effective prognostic tool and surrogate of tumor burden in newly diagnosed GBM. Detection of somatic alterations in plasma is feasible when samples are obtained prior to initial surgical resection.

Circulating tumor DNA next-generation sequencing gene coverage (74 genes)

Tissue next-generation sequencing gene coverage (152 genes)

Plasma cell-free DNA (cfDNA) concentration (ng/mL) is correlated with (A), total radiographic tumor burden (contrast-enhancing tumor + T2/FLAIR non-enhancing tumor) and (B), contrast-enhancing tumor burden at the first post-radiation MRI scan in patients with newly diagnosed glioblastoma

Complete list of variants detected in 20 subjects who had both tissue and plasma next generation sequencing

Frameless neuronavigation allows neurosurgeons to visualize and relate the position of surgical instruments to intracranial pathologies based on preoperative tomographic imaging. However, neuronavigation can often be inaccurate. Multiple factors have been proposed as potential causes, and new technologies are needed to overcome these challenges. To evaluate the accuracy of neuronavigation systems compared to near-infrared (NIR) fluorescence imaging using Second Window Indocyanine Green, a novel technique, and to determine factors that lead to neuronavigation errors. A retrospective analysis was conducted on 56 patients who underwent primary resections of intracranial tumors. Patients received 5 mg/kg ICG approximately 24 h preoperatively. Intraoperatively, neuronavigation was used to plan craniotomies to place the tumors in the center. After craniotomy, NIR imaging visualized tumor-specific NIR signals. The accuracy of neuronavigation and NIR fluorescence imaging for delineating the tumor boundary prior to durotomy was compared. The neuronavigation centers and NIR centers were 23.0 ± 7.7 % and 2.6 ± 1.1 % deviated from the tumor centers, respectively, relative to the craniotomy sizes. In 12 cases, significant changes were made to the planned durotomy based on NIR imaging. Patient position was a significant predictor of neuronavigation inaccuracy on both univariate and multivariate analysis, with the prone position having significantly higher inaccuracy (29.2 ± 8.1 %) compared to the supine (16.2 ± 8.1 %, p value

We have previously demonstrated an independent association between high levels of plasma circulating cell-free DNA (ccfDNA) concentration and poor survival outcomes in patients with newly diagnosed glioblastoma. Given that somatic mutations are rarely detected in glioblastoma patient plasma, we reasoned that DNA shed by tumor (circulating tumor DNA, ctDNA) was not likely to be a driver of prognostic increases in ccfDNA. To investigate the tissue of origin for this prognostic ccfDNA, we analyzed the plasma ccfDNA methylation signatures of 23 patients with newly diagnosed glioblastoma using the Illumina Infinium EPIC array, a genome-wide DNA methylation technique covering over 850,000 CpG sites. Deconvolution of the ccfDNA methylation signatures based on published reference methylomes revealed an increased proportion of ccfDNA derived from granulocytes in glioblastoma specimens compared to healthy controls (p < 0.0001). Further, this granulocyte proportion was increased in patients with high ccfDNA levels (above median value) compared to patients with low ccfDNA (p = 0.0001). Granulocyte proportion correlated with ccfDNA concentration (Spearman r = 0.64, p = 0.001). The top two gene sets identified by differential methylation analysis followed by gene set enrichment analysis (methylGSA) between high- and low-ccfDNA specimens were “neutrophil activation involved in immune response” (GO:0002283, p = 3.4E-23) and “neutrophil degranulation” (GO:0043312, p = 3.4E-23). Additional analysis of ccfDNA fragment size identified larger fragments ( > 700 bp) as the major source of the prognostic signal (log-rank p = 0.002 for progression-free survival) compared to traditionally sized ccfDNA fragments (50-700 bp, log-rank p = 0.12). Taken together, these data suggest that granulocytes are the primary contributing source of prognostic ccfDNA in glioblastoma. Future studies are needed to determine the mechanism through which granulocyte-derived ccfDNA is associated with clinical outcomes in glioblastoma and to explore related therapeutic opportunities.

BACKGROUND GBM genomic profiling relies on sequencing a limited tumor tissue sample, which is invasive and may underrepresent molecular heterogeneity. CSF, which can be obtained less invasively, is in intimate contact with tumor lesions and may better capture the full genomic profile of the tumor. However, datasets with contemporaneously collected CSF and tissue to support this claim have been lacking. To evaluate the performance of CSF NGS, we conducted a pilot study in patients with GBM undergoing a resection for suspected recurrence following first-line chemoradiotherapy. METHODS Paired CSF and tissue samples were sequenced using a hybrid capture-based NGS assay. Clinically meaningful variants were defined as those that are potentially targetable using off-label or clinical trial options, exhibit prognostic value, or may predict response or resistance to specific treatments. RESULTS Eighteen patients were enrolled, and 13 of 18 CSF samples (72.2%) were sequenced successfully. At least one variant was detected in all CSF samples analyzed. A median of 7 variants (range 1—67) was detected per sample across 54 genes. The median variant allele fraction was 0.6% (range 0.2—72.4%). Among 38 clinically meaningful genes, 102 variants were detected; 25 (24.5%) were detected in both tissue and CSF, while 60 (58.8%) were detectable solely in CSF. Hypermutation was detected by CSF in one patient. Of the 82 variants detected in this patient’s tumor, 15 (18.3%) were identified in both tissue and CSF, 15 (18.3%) were identified only in the tissue, and 52 (63.2%) were identified only in the CSF. CONCLUSIONS CSF NGS detects clinically meaningful variants at a substantial rate and frequently identifies mutations not detected by matched tissue NGS. These results suggest that CSF may be a suitable source material for tumor profiling, overcoming the limitations of tissue, and may also provide a more comprehensive tumor profile.

PURPOSE:Multiple studies have demonstrated the negative impact of cancer care delays during the COVID-19 pandemic, and transmission mitigation techniques are imperative for continued cancer care delivery. We aimed to gauge the effectiveness of these measures at the University of Pennsylvania.METHODS:We conducted a longitudinal study of SARS-CoV-2 antibody seropositivity and seroconversion in patients presenting to infusion centers for cancer-directed therapy between May 21, 2020, and October 8, 2020. Participants completed questionnaires and had up to five serial blood collections.RESULTS:Of 124 enrolled patients, only two (1.6%) had detectable SARS-CoV-2 antibodies on initial blood draw, and no initially seronegative patients developed newly detectable antibodies on subsequent blood draw(s), corresponding to a seroconversion rate of 0% (95% CI, 0.0 TO 4.1%) over 14.8 person-years of follow up, with a median of 13 health care visits per patient.CONCLUSION:These results suggest that patients with cancer receiving in-person care at a facility with aggressive mitigation efforts have an extremely low likelihood of COVID-19 infection.

Background We aimed to determine whether plasma cell-free DNA (cfDNA) concentration is associated with survival in patients with isocitrate dehydrogenase (IDH) wild-type glioblastoma (GBM). Methods Pre-operative and post-chemoradiotherapy blood samples were prospectively collected from patients with newly diagnosed IDH wild-type GBM. Patients underwent surgical resection or biopsy and received adjuvant radiotherapy with concomitant temozolomide. Cell-free DNA (cfDNA) was isolated from plasma and quantified using SYBR Green-based q polymerase chain reaction (qPCR). Results Sixty-two patients were enrolled and categorized into high vs. low cfDNA groups relative to the pre-operative median value (25.2 ng/mL, range 5.7–153.0 ng/mL). High pre-operative cfDNA concentration was associated with inferior PFS (median progression-free survival (PFS), 3.4 vs. 7.7 months; log-rank P = .004; hazard ratio [HR], 2.19; 95% CI, 1.26–3.81) and overall survival (OS) (median OS, 8.0 vs. 13.9 months; log-rank P = .01; HR, 2.43; 95% CI, 1.19–4.95). After adjusting for risk factors, including O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation status, pre-operative cfDNA remained independently associated with PFS (HR, 2.70; 95% CI, 1.50–4.83; P = .001) and OS (HR, 2.65; 95% CI, 1.25–5.59; P = .01). Post-hoc analysis of change in cfDNA post-chemoradiotherapy compared to pre-surgery (n = 24) showed increasing cfDNA concentration was associated with worse PFS (median, 2.7 vs. 6.0 months; log-rank P = .003; HR, 4.92; 95% CI, 1.53–15.84) and OS (median, 3.9 vs. 19.4 months; log-rank P < .001; HR, 7.77; 95% CI, 2.17–27.76). Conclusions cfDNA concentration is a promising prognostic biomarker for patients with IDH wild-type GBM. Plasma cfDNA can be obtained noninvasively and may enable more accurate estimates of survival and effective clinical trial stratification.

Frameless neuronavigation allows neurosurgeons to visualize and relate the position of surgical instruments to intracranial pathologies based on preoperative tomographic imaging. However, neuronavigation can often be inaccurate. Multiple factors have been proposed as potential causes, and new technologies are needed to overcome these challenges.To evaluate the accuracy of neuronavigation systems compared to near-infrared (NIR) fluorescence imaging using Second Window Indocyanine Green, a novel technique, and to determine factors that lead to neuronavigation errors.A retrospective analysis was conducted on 56 patients who underwent primary resections of intracranial tumors. Patients received 5 mg/kg ICG approximately 24 h preoperatively. Intraoperatively, neuronavigation was used to plan craniotomies to place the tumors in the center. After craniotomy, NIR imaging visualized tumor-specific NIR signals. The accuracy of neuronavigation and NIR fluorescence imaging for delineating the tumor boundary prior to durotomy was compared.The neuronavigation centers and NIR centers were 23.0 ± 7.7 % and 2.6 ± 1.1 % deviated from the tumor centers, respectively, relative to the craniotomy sizes. In 12 cases, significant changes were made to the planned durotomy based on NIR imaging. Patient position was a significant predictor of neuronavigation inaccuracy on both univariate and multivariate analysis, with the prone position having significantly higher inaccuracy (29.2 ± 8.1 %) compared to the supine (16.2 ± 8.1 %, p value 0.001) or the lateral (17.9 ± 5.1 %, p value = 0.003) positions.Patient position significantly affects neuronavigation accuracy. Intraoperative NIR fluorescence imaging before durotomy offers an opportunity to readjust the neuronavigation image space to better align with the patient space.

Intraoperative distinction of lesional tissue versus normal brain parenchyma can be difficult in neurosurgical oncology procedures. We report the successful, real-time visualization of central nervous system (CNS) lymphoma using the 'Second Window Indocyanine Green' (SWIG) method for two patients who underwent craniotomy for pathology that was determined to be large B cell lymphoma. Indocyanine green (ICG), when administered intravenously the day prior to cranial surgery, is a re-purposed fluorophore that may afford safe, immediate visual confirmation of on-target tissue resection, thereby providing a valuable adjunct to intraoperative navigation and decreasing reliance on frozen pathology analysis. These first reported cases of SWIG for lymphoma in the CNS indicate that further study of fluorophores to improve biopsy targeting and yield is warranted.

Background Plasma cell-free DNA (cfDNA) concentration is lower in glioblastoma (GBM) compared to other solid tumors, which can lead to low circulating tumor DNA (ctDNA) detection. In this study, we investigated the relationship between multimodality magnetic resonance imaging (MRI) and histopathologic features with plasma cfDNA concentration and ctDNA detection in patients with treatment-naive GBM. Methods We analyzed plasma cfDNA concentration, MRI scans, and tumor histopathology from 42 adult patients with newly diagnosed GBM. Linear regression analysis was used to examine the relationship of plasma cfDNA concentration before surgery to imaging and histopathologic characteristics. In a subset of patients, imaging and histopathologic metrics were also compared between patients with and without a detected tumor somatic mutation. Results Tumor volume with elevated (>1.5 times contralateral white matter) rate transfer constant (Kep, a surrogate of blood–brain barrier [BBB] permeability) was independently associated with plasma cfDNA concentration (P = .001). Histopathologic characteristics independently associated with plasma cfDNA concentration included CD68+ macrophage density (P = .01) and size of tumor vessels (P = .01). Patients with higher (grade ≥3) perivascular CD68+ macrophage density had lower volume transfer constant (Ktrans, P = .01) compared to those with lower perivascular CD68+ macrophage density. Detection of at least 1 somatic mutation in plasma cfDNA was associated with significantly lower perivascular CD68+ macrophages (P = .01). Conclusions Metrics of BBB disruption and quantity and distribution of tumor-associated macrophages are associated with plasma cfDNA concentration and ctDNA detection in GBM patients. These findings represent an important step in understanding the factors that determine plasma cfDNA concentration and ctDNA detection.

PURPOSE: Intraoperative molecular imaging with tumor-targeting fluorophores offers real-time detection of neoplastic tissue. The Second-Window-Indocyanine-Green (SWIG) technique relies on passive accumulation of indocyanine-green (ICG), a near-infrared fluorophore, in neoplastic tissues.In this study, we explore the ability of SWIG to detect neoplastic tissue and to predict postoperative Magnetic Resonance Imaging (MRI) findings intraoperatively. PROCEDURES: Retrospective data were collected from 36 patients with primary high-grade gliomas (HGG) enrolled as part of a larger trial between October 2014 and October 2018. Patients received systemic ICG infusions at 2.5–5mg/kg 24 h preoperatively. Near-infrared fluorescence was recorded throughout the case and from biopsy specimens. The presence/location of residual SWIG signal after resection was compared to the presence/location of residual gadolinium enhancement on postoperative MRI. The extent of resection was not changed based on near-infrared imaging. RESULTS: All 36 lesions demonstrated strong near-infrared fluorescence (signal-to-background = 6.8 ± 2.2) and 100% of tumors reaching the cortex were visualized before durotomy. In 78 biopsy specimens, near-infrared imaging demonstrated higher sensitivity and accuracy than white-light for diagnosing neoplastic tissue intraoperatively. Furthermore, near-infrared imaging predicted gadolinium enhancement on postoperative MRI with 91% accuracy, with visualization of residual enhancement as small as 0.3cm(3). Patients with no residual near-infrared signal after resection were significantly more likely to have complete resection on postoperative MRI (p-value < 0.0001). CONCLUSIONS: Intraoperative imaging with SWIG demonstrates highly sensitive detection of HGG tissue in real-time. Furthermore, post-resection near-infrared imaging correlates with postoperative MRI. Overall, our findings suggest that SWIG can provide surgeons with MRI-like results in real-time, potentially increasing resection rates.

Purpose: The clinical utility of plasma cell-free DNA (cfDNA) has not been assessed prospectively in patients with glioblastoma (GBM). We aimed to determine the prognostic impact of plasma cfDNA in GBM, as well as its role as a surrogate of tumor burden and substrate for next-generation sequencing (NGS). Experimental Design: We conducted a prospective cohort study of 42 patients with newly diagnosed GBM. Plasma cfDNA was quantified at baseline prior to initial tumor resection and longitudinally during chemoradiotherapy. Plasma cfDNA was assessed for its association with progression-free survival (PFS) and overall survival (OS), correlated with radiographic tumor burden, and subjected to a targeted NGS panel. Results: Prior to initial surgery, GBM patients had higher plasma cfDNA concentration than age-matched healthy controls (mean 13.4 vs. 6.7 ng/mL, P < 0.001). Plasma cfDNA concentration was correlated with radiographic tumor burden on patients' first post-radiation magnetic resonance imaging scan (ρ = 0.77, P = 0.003) and tended to rise prior to or concurrently with radiographic tumor progression. Preoperative plasma cfDNA concentration above the mean (>13.4 ng/mL) was associated with inferior PFS (median 4.9 vs. 9.5 months, P = 0.038). Detection of ≥1 somatic mutation in plasma cfDNA occurred in 55% of patients and was associated with nonstatistically significant decreases in PFS (median 6.0 vs. 8.7 months, P = 0.093) and OS (median 5.5 vs. 9.2 months, P = 0.053). Conclusions: Plasma cfDNA may be an effective prognostic tool and surrogate of tumor burden in newly diagnosed GBM. Detection of somatic alterations in plasma is feasible when samples are obtained prior to initial surgical resection.

BACKGROUND Liquid biopsy has been not been widely utilized in patients with glioblastoma (GBM) compared to other solid tumors. However, the clinical utility of plasma cell-free DNA (cfDNA) in GBM has not been assessed prospectively or at the time of initial diagnosis. METHODS We conducted a prospective cohort study of patients with newly diagnosed GBM. Whole blood was collected in Streck® tubes at baseline prior to initial surgical resection and longitudinally during the course of adjuvant chemoradiotherapy. Plasma cfDNA concentration (ng/mL) was quantified by qPCR for a 115 bp amplicon of the human ALU repeat element, correlated with radiographic tumor burden by volumetry at multiple time points using Spearman rank correlation, and assessed for its impact on progression-free (PFS) and overall survival (OS) by Cox regression. RESULTS Prior to initial resection, GBM patients (N=42) had higher plasma cfDNA concentration compared to age-matched healthy controls (N=42) (mean 13.43 vs. 6.70 ng/mL, p< 0.001). Plasma cfDNA concentration was correlated with radiographic tumor burden on subjects’ first post-radiation MRI scan (r=0.77, p=0.003) and tended to rise prior to or concurrently with radiographic tumor progression. Pre-operative plasma cfDNA concentration above the mean (>13.4 ng/mL) was associated with inferior PFS (median 4.9 vs. 9.5 months, p=0.038) and OS (median 8.9 vs. 14.8 months, p=0.078). The impact on PFS persisted after adjusting for age, extent of resection, performance status, MGMT promoter methylation, and IDH1/2 mutational status (HR 2.48, 95% CI 1.1–6.1, p=0.046). CONCLUSIONS Plasma cfDNA may be an effective prognostic tool and noninvasive surrogate of tumor burden in newly diagnosed GBM. Tumor tissue samples from our cohort have been subjected to targeted next generation sequencing (NGS), and baseline plasma samples have been sent to Guardant Health for NGS. Plasma NGS results and concordance with matched tissue NGS will be included at time of presentation.

BACKGROUND Oligodendroglioma is a rare primary brain tumor characterized by mutation in the IDH1/IDH2 genes and codeletion of chromosomes 1p and 19q. Although median overall survival extends beyond 10 years for patients with oligodendroglioma who receive adjuvant radiation and alkylating chemotherapy, the disease uniformly relapses and has no other effective treatments. The CIC gene, which encodes a high-mobility group box transcriptional repressor, is mutated in about 70% of oligodendrogliomas and is associated with worse prognosis and overexpression of cyclin D1 (CCND1). Cyclin D1 assembles with the cyclin-dependent kinases CDK4 and CDK6 to cause phosphorylation of Rb protein and subsequent cell cycle progression from the G1 to S phase. Therapeutic inhibition of CDK4/6 activity, which leads to Rb dephosphorylation and cell cycle arrest, has revolutionized the care of breast cancer. The current phase 2 study will investigate the efficacy and safety of abemaciclib, a selective and potent small molecule inhibitor of CDK4 and CDK6 with significant brain-brain barrier penetration, in patients with 1p/19q codeleted oligodendroglioma that has recurred after standard radiation and alkylating chemotherapy. METHODS This protocol (NCT03969706) has a single-arm, open-label, single-stage phase 2 design. Subjects will be treated with abemaciclib 200mg tablet twice daily on 28-day cycles until disease progression or unacceptable toxicity. Tumor response will be assessed according to modified Response Assessment in Neuro-Oncology (mRANO) criteria. The primary objective of the study is to determine the efficacy of abemaciclib for recurrent oligodendroglioma. The primary endpoint is the progression-free survival status of the subject at 6 months after enrollment (PFS-6). The alternative hypothesis is that the proportion of subjects without an event at 6 months will be ³85%. We will test this hypothesis against a null hypothesis of PFS-6 = 50%. With enrollment of 10 planned subjects, the power to detect this difference is 82% (one-sided alpha = 0.05).

INTRODUCTION: To improve surgical resection of brain tumors, our lab has pioneered a novel fluorescent dye technique, Second-Window Indocyanine-Green (SWIG), that relies on passive delivery and accumulation of indocyanine-green (ICG) in neoplastic tissue via the enhanced permeability and retention effect. We hypothesize that SWIG can provide early localization of brain metastasis prior to dural opening and can improve identification of surgical margins. METHODS: Subjects were prospectively enrolled in clinical trial after informed consent. Approximately 24 hours prior surgery, subjects were infused intravenously with 2.5mg/kg or 5mg/kg of ICG. Intraoperatively, a dedicated near-infrared (NIR) camera was used to detect ICG signal. After bone flap removal, the NIR imaging system was positioned above the presumed location of tumor. Additional NIR images were obtained after dural opening, corticectomy, and after conventional white-light surgical resection. RESULTS: We enrolled 50 patients with 51 total intraparenchymal brain metastases (23 lung, 7 breast, 8 GU/GI, 4 melanoma, and 7 others). Prior to dural opening, NIR signal was identified in 35 patients at an average depth of 4.3mm with SBR = 5.3 + 3.7. In the seven patients where NIR signal could not be identified prior to dural opening, tumor depth was an average of 8.4mm from cortical surface. Upon dural opening and tumor identification, all 51 tumors demonstrated strong NIR signal with SBR = 6.2 + 2.8. With white light alone, sensitivity/specificity/PPV/NPV for tumor detection was 83%, 94%, 98%, 57%. With NIR, sensitivity/specificity/PPV/NPV for tumor detection was 100%, 29%, 85%, 100%. DISCUSSION: NIR fluorophores are superior to visible light fluorophores in their depth of penetration. All contrast-enhancing brain metastasis accumulate ICG using our SWIG technique, and NIR fluorescence could be used to localize brain metastasis prior to dural opening. NIR fluorophores are likely to represent the next phase in tumor visualization given the rapid growth of fluorophores targeted to systemic cancers.