Abstract INTRODUCTION Patients with LMD have poor prognosis and limited treatment options. Oncogene amplification of primary, metastatic, and CNS metastatic tumors can be heterogeneous. Therefore, patients with LMD may benefit from assessment of clinically relevant biomarkers in CSF, which may guide the choice of a targeted therapy specifically for the LMD tumor. CNSide is a CLIA-validated laboratory-developed test ordered commercially at the discretion of physicians for CSF-TC enumeration, immunocytochemical (ICC) and fluorescence in situ hybridization (FISH) analysis of oncogene amplification. We longitudinally analyze oncogenes in CSF-TCs in patients with LMD of various primary cancers. METHODS CSF was collected from patients with suspected or confirmed LMD; 613 tests were ordered on 218 individual patients with breast (N=105 patients), lung (N=65), gastrointestinal (N=10), and other cancers. Using CNSide, CSF-TCs were isolated and tested via ICC (ER, PD-L1, and PR), and FISH (ALK, cMET, cMyc, EGFR, FGFR1, HER2, NTRK1, NTRK3, PTEN, RET, and ROS1). RESULTS In patients with lung cancer, ALK was detected in 14% (17/118) of samples, CMET in 61% (78/128), HER2 in 73% (16/22), and RET in 4% (4/90). In patients with breast cancer, HER2 was detected in 39% (65/168) of samples, FGFR1 in 32% (19/60), ER in 26% (44/168) and PR in 4% (5/120). 66 patients underwent 2+ CSF draws; 24 underwent 5+. Among these, there were 13 ICC flips (7 acquired mutations) and 58 FISH probe detection flips (26 acquired mutations). 20/66 patients (30.3%) had at least one flip in their ordered biomarkers. CONCLUSION CNSide can be used to detect oncogene amplification on CSF-TCs of patients with LMD, and mutational status of the LMD tumor may differ from the original tumor biopsy. CSF-TC analysis may provide therapeutic insights that vary from the original tumor and could open the door to additional treatment targets for patients struck with LMD.

Babesia microti is an Apicomplexan parasite that infects erythrocytes and causes the tick-transmitted infection, babesiosis. B. microti can cause a wide variety of clinical manifestations ranging from asymptomatic to severe infection and death. Some risk factors for severe disease are well-defined, an immune compromised state, age greater than 50, and asplenia. However, increasing cases of severe disease and hospitalization in otherwise healthy individuals suggests that there are unknown risk factors. The immunopathology of babesiosis is poorly described. CD4+ T cells and the spleen both play a critical role in parasite clearance, but few other factors have been found that significantly impact the course of disease. Here, we evaluated the role of several immune mediators in B. microti infection. Mice lacking TNF receptors 1 and 2, the receptors for TNFα and LTα, had a higher peak parasitemia, reduced parasite killing in infected red blood cells (iRBCs), and delayed parasite clearance compared to control mice. Mice lacking CCR2, a chemokine receptor involved in the recruitment of inflammatory monocytes, and mice lacking NADPH oxidase, which generates superoxide radicals, demonstrated reduced parasite killing but had little effect on the course of parasitemia. These results suggest that TNFR-mediated responses play an important role in limiting parasite growth, the death of parasites in iRBCs, and the clearance of iRBCs, and that the parasite killing in iRBCs is being primarily mediated by ROS and inflammatory monocytes/macrophages. By identifying factors involved in parasite killing and clearance, we can begin to identify additional risk factors for severe infection and newer therapeutic interventions.

CNSide is a platform that detects and characterizes tumor cells in the cerebrospinal fluid (CSF) of patients with leptomeningeal disease (LMD). The platform was validated per College of American Pathologists (CAP) and Clinical Laboratories Improvement Amendment (CLIA) guidelines and run as a commercial Laboratory Developed Test (LDT) at Biocept in San Diego, CA. The platform allows CSF tumor cell (CSF-TC) enumeration and biomarker characterization by fluorescent in situ hybridization (FISH). We performed a multicenter retrospective chart review of HER2 FISH CNSide test results that were commercially ordered on 26 patients by physicians for LMD breast cancer patients between April 2020 and October 2022. We show that HER2 is amplified on CSF tumor cells in 62% (16/26) of LMD breast cancer patients. 10/26 (38%) patients had discordant HER2-positivity between the primary tumor tissue and CSF-TC; of these, 35% (9/26) of the patients displayed HER2 amplification on the CSF-TCs, however were categorized as HER2 negative on the primary tumor. Of the 27% (7/26) patients with a HER2 positive primary tumor, one patient showed a HER2 negative LMD tumor. Two patients, 8% (2/26) had a HER2 equivocal primary tumor; of these, one demonstrated a HER2 negative, and one a HER2 positive LMD tumor. Serial analysis (at least 4 longitudinal tests) of HER2 status of the CSF-TC throughout therapy was available for 14 patients and demonstrated that HER2 status of the LMD changed in 29% (4/14) during their treatment course and impacted care decisions. Our data suggests that CSF-TC HER2 FISH analysis in LMD breast cancer patients may be discordant to the primary tumor sample and the discovery of HER2 positivity in the CSF may open doors to anti-HER2 targeted therapy options for LMD patients.

Leptomeningeal disease (LMD) is a devastating complication for patients with advanced cancer. Diagnosis and monitoring the response to therapy remains challenging due to limited sensitivity and specificity of standard-of-care (SOC) diagnostic modalities, including cerebrospinal fluid (CSF) cytology, MRI, and clinical evaluation. These hindrances contribute to the poor survival of LMD patients. CNSide is a CLIA-validated test that detects and characterizes CSF-derived tumor cells and cell-free (cf) DNA. We performed a retrospective analysis on the utility of CNSide to analyze CSF obtained from advanced non-small cell lung cancer (aNSCLC) patients with suspected LMD treated at the Huntsman Cancer Institute in Salt Lake City, UT. CNSide was used to evaluate CSF from 15 patients with aNSCLC. CSF tumor cell quantification was performed throughout treatment for 5 patients. CSF tumor cells and cfDNA were characterized for actionable mutations. In LMD-positive patients, CNSide detected CSF tumor cells in 88% (22/25) samples versus 40% (10/25) for cytology (matched samples). CSF tumor cell numbers tracked response to therapy in 5 patients where CNSide was used to quantify tumor cells throughout treatment. In 75% (9/12) of the patients, genetic alterations were detected in CSF, with the majority representing gene mutations and amplifications with therapeutic potential. The median survival for LMD patients was 16.1 m (5.2-NR). We show that CNSide can supplement the management of LMD in conjunction with SOC methods for the diagnosis, monitoring response to therapy, and identifying actionable mutations unique to the CSF in patients with LMD.

Abstract LM is diagnosed via clinical evaluation, neuroaxis neuroimaging (MRI brain and spine), and CSF cytology. These methods have limited sensitivity and specificity, and lack the ability to quantitatively measure the LM’s response to treatment. These hindrances create challenges in LM diagnosis and management for physicians. CNSide is a commercially available Laboratory Developed Test (LDT) that is analytically validated for the detection and characterization of tumor cells in the CSF, and run commercially in Biocept’s CLIA-certified, CAP-accredited laboratory. Physicians order CNSide testing at their discretion for patients that have a suspicion of LM. To evaluate the performance of CNSide for LM disease management in a controlled setting, Biocept is conducting a prospective, multi-center clinical study (FORESEE, NCT05414123) involving patients previously diagnosed with NSCLC or Breast Cancer and a suspicion or diagnosis of LM per standard of care (SOC) management, including clinical assessment, radiographic evaluation, and CSF cytology. At baseline and at designated time points throughout treatment, matched CSF samples will be analyzed in parallel with standard of care CSF cytology and CNSide at Biocept along with time matched neuroimaging and clinical evaluation. The primary end point of the trial is the proportion of decision points during LM treatment in which physicians indicated that CNSide aided in their decision making. Physicians will also indicate which component of the test aided in management, modeling other similar studies that measure the effect of serum biomarker results on clinical management of cancer. Secondary end points include diagnostic comparison to clinical evaluation, cytology, and radiographic results, and determining the sensitivity, specificity, PPV and NPV of CNSide in the detection of CSF tumor cells (cytology being the reference standard).

Abstract INTRODUCTION Patients diagnosed with Leptomeningeal Metastasis (LM) have limited treatment options. However, they may benefit from targetable therapy. LM breast cancer patients with HER2 positive primary tumors treated with IT Trastuzumab experienced clinical benefit (Malani, 2020), and improved craniospinal PFS (Figura, 2019). HER2 amplification of the primary and metastatic tumors can be heterogeneous. Biocept’s CNSideTM is a CLIA validated Laboratory Developed Test and is used commercially at the discretion of Physicians to detect CSF tumor cells (CSF-TCs). We analyzed HER2 amplification on CSF-TCs in LM patients with breast-, upper GI cancer as well as Non-Small Cell Lung Cancer (NSCLC). METHODS CSF was collected from patients with suspected or confirmed LM having Breast Cancer (N=334 patients, N=181 evaluable), NSCLC (N=28 patients) or upper GI cancer (N=4 patients, N=2 evaluable). CSF TCs were tested for HER2 amplification by FISH using CNSide. RESULTS For the breast cancer patients, 38% (68/181) had HER2 FISH detected on CSF TCs, 60% (109/181) had a HER2 negative primary tumor of which 35% (38/109) had HER2 positive CSF-TCs. Furthermore, 8% (14/181) had an equivocal HER2 primary tumor, 21% (3/14) of which had HER2 positive CSF-TCs. For the NSCLC patients, 50% (14/28) showed HER2 amplification on the CSF-TCs. For the upper GI cancer patients, one patient had an equivocal HER2 primary tumor, and HER2 positive CSF-TCs. CONCLUSION HER2 amplification is detected on CSF-TCs of patients with LM having breast cancer, upper GI cancer or NSCLC cancer. For NSCLC patients, HER2 positivity in the LM can be associated with drug resistance. Patients who have a HER2 negative primary tumor can develop HER2 positive LM, allowing for an anti-HER2 targetable treatment. Additional studies are needed to establish the clinical utility of CNSide in the detection of HER2 on the CSF-TCs to direct treatment of anti-HER2 therapy in patients with LM.

Point-of-care (POC) serological testing provides actionable information for several difficult to diagnose illnesses, empowering distributed health systems. Accessible and adaptable diagnostic platforms that can assay the repertoire of antibodies formed against pathogens are essential to drive early detection and improve patient outcomes. Here, we report a POC serologic test for Lyme disease (LD), leveraging synthetic peptides tuned to be highly specific to the LD antibody repertoire across patients and compatible with a paper-based platform for rapid, reliable, and cost-effective diagnosis. A subset of antigenic epitopes conserved across Borrelia burgdorferi genospecies and targeted by IgG and IgM antibodies, were selected based on their seroreactivity to develop a multiplexed panel for a single-step measurement of combined IgM and IgG antibodies from LD patient sera. Multiple peptide epitopes, when combined synergistically using a machine learning-based diagnostic model, yielded a high sensitivity without any loss in specificity. We blindly tested the platform with samples from the U.S. Centers for Disease Control & Prevention (CDC) LD repository and achieved a sensitivity and specificity matching the lab-based two-tier results with a single POC test, correctly discriminating cross-reactive look-alike diseases. This computational LD diagnostic test can potentially replace the cumbersome two-tier testing paradigm, improving diagnosis and enabling earlier effective treatment of LD patients while also facilitating immune monitoring and surveillance of the disease in the community.

Abstract BACKGROUND Metastatic involvement of the CSF by non-CNS neoplasms surpasses that of primary brain tumors, although conventional glioblastoma (GBM) can occasionally be identified in the CSF. Here, we apply Biocept’s CNSide test to examine a patient CSF sample with GBM, verified with an antibody against mutant p53. The p53 pathway is deregulated in 84% of glioblastoma patients and point mutations lead to expression of mutant p53 protein. METHODS A CSF sample from 65-year-old male patient, with GBM diagnosed by MRI & CT, and prior identified p53 mutation on R273C by NGS was collected into Biocept’s CSF collection tubes at Providence Saint John’s Health Center. CSF-cells were incubated, 2 days post collection, with a proprietary antibody cocktail, including anti-CD9, followed with a biotinylated secondary, which enables enrichment of capture antibody labeled cells in a streptavidin coated microfluidic device. Cells were fixed and permeabilized immunofluorescence was performed against mutant p53, CD45, fluorescently labeled streptavidin, for capture antibody detection, and DAPI. Microfluidic chips were scanned and analyzed on a Bioview system. The specificity of the mutant p53 antibody was verified using the same test on tumor cells with either wildtype p53 vs various p53 mutations. RESULTS CSF analysis by the CNSide platform detected 3 tumor cells / mL. Immunofluorescence confirmed strong expression of mutant p53 protein on about 1/3 of cells identified as tumor cells. Besides the patient R273C mutation, we demonstrated detection of P223L/V274F, E285K and Q331R mutations on tumor cell lines. DISCUSSION Our work suggests that a microfluidic based GBM capture test paired with appropriate biomarkers can be used as sensitive means to detect GBM cells in CSF and may be useful for diagnostics and treatment monitoring. Further, we demonstrated the utility of a mutant p53 antibody to identify tumor cells. Technical and clinical studies are needed to substantiate this hypothesis.

Abstract INTRODUCTION Patients with leptomeningeal metastasis (LM) have limited treatment options and a poor outcome. However, they may benefit from targeted therapy. LM patients with HER2-positive primary breast cancer treated with IT trastuzumab demonstrated clinical benefit (Malani, 2020), and improved PFS (Figura, 2019). HER2 amplification in the primary and metastatic tumors can be divergent. Biocept’s CNSideTM is a CLIA validated test that can detect CSF tumor cells (CSF-TCs) and interrogate those cells with FISH and NGS. We analyzed HER2 amplification on CSF-TCs in LM patients with breast cancer, non-small cell lung cancer (NSCLC) and upper GI cancer. METHODS CSF was collected from patients with suspected or confirmed LM with breast cancer (N =134 patients), NSCLC (Nf28 patients) or upper GI cancer (Nf2 patients). CSF TCs were tested for HER2 amplification by FISH using CNSide. RESULTS HER2 amplification in CSF-TCs was detected in 46% (76/164) of all patients. Of the breast cancer patients, 37% (49/134) flipped HER2 status in the LM tumor, 38% (41/108) switched from a HER2 negative or equivocal primary tumor to HER2 positive CSF-TCs, and 27% (7/26) from a HER2 positive primary tumor to HER2 negative CSF-TCs. For the NSCLC patients, 50% (14/28) showed HER2 amplification on the CSF-TCs. For the upper GI cancer patients, HER2 amplification in CSF-TCs was detected in both patients, one patient had an equivocal HER2 primary tumor and HER2 positive CSF-TCs. CONCLUSION HER2 amplification was detected in a substantial fraction of CSF-TCs from patients with LM from breast, upper GI, and NSCL cancers. This finding may have important therapeutic (justifying the use of intra-CSF trastuzumab), prognostic (HER2 positivity in NSCLC is associated with poorer prognosis and a higher frequency of CNS metastases) and pathophysiologic implications (a potential role of HER2 amplification in the genesis of CNS metastases). Additional investigations are underway.