Cells In Cerebrospinal Fluid Samples Research Articles

Combination of Cytologic Findings and Circulating Tumor DNA From Cerebrospinal Fluid Revealed SCLC Transformation in Patients With Leptomeningeal Metastases of Lung Adenocarcinoma

IntroductionTransformation to SCLC is a resistance mechanism to tyrosine kinase inhibitor in EGFR-mutated lung adenocarcinoma (LUAD). Nevertheless, the clinical and molecular features of SCLC transformation in LUAD with leptomeningeal metastases (LM) are scarce. MethodsWe retrospectively collected 237 patients with NSCLC who underwent lumbar puncture owing to suggestion of LM. All SCLC transformation in cerebrospinal fluid (CSF) was confirmed by two experienced pathologists using cytologic evaluation. CSF circulating tumor DNA (ctDNA) was tested by next-generation sequencing. ResultsTumor cells in CSF samples were found in 111 patients (111 of 237, 46.8%), and eight cases (eight of 111, 7.2%) were identified as having SCLC cells in CSF. Seven patients carried the EGFR mutation, including four patients with EGFR exon 19 deletion and three patients with EGFRexon21 L858R mutation. Another patient harbored ERBB2 insertion. Seven of these patients were resistant to targeted therapy. CSF ctDNA analysis reported that TP53 and RB1 mutations were common. The median time from the diagnosis of advanced NSCLC to SCLC transformation found in CSF was 9.7 months (95% confidence interval [CI]: 4.0–17.5 mo). The median overall survival since the initial diagnosis of metastatic NSCLC was 15.3 months (95% CI: 1.2–29.4 mo). The median overall survival after SCLC transformation detected in CSF was 5.0 months (95% CI: 4.0–5.9 mo). ConclusionsSCLC transformation may be revealed in CSF by both cytologic evaluation and ctDNA, not just in tissue that underwent rebiopsy. SCLC transformation of CSF is informative for resistance mechanism in patients with LUAD with LM on tyrosine kinase inhibitor progression, which was associated with poor survival.

Serum and Cerebrospinal Fluid Testing in Optic Neuropathy Patients with Malignant Tumors.

Purpose To evaluate the value of serum and cerebrospinal fluid (CSF) testing in optic neuropathy (ON) patients with malignant tumors. Methods Fourteen patients clinically diagnosed as ON with malignant tumors but without intracranial or orbital mass in MRI were included in this study. Detailed medical records including medical history, complete ophthalmic examination, colour fundus photography, visual field test, orbital MRI examination, serum and CSF testing data were collected and analyzed. The diagnosis of paraneoplastic optic neuropathy (PON) based on the 2004 recommended criteria of the paraneoplastic syndrome- Euronetwork consortium for paraneoplastic neurological disorders, and current adaption for neuropathies. All patients underwent serum tests for pathogens and autoantibodies including antinuclear antibodies, anticardiolipin antibodies, antineutrophil cytoplasmic antibodies, AQP4-Ab and MOG-Ab, as well as CSF tests for malignant cells under microscope. Serum paraneoplastic antibodies were detected in PON patients. Monkey cerebellar tissue-based assay was used to detect unknown serum anti-neuron antibodies in PON patients with negative paraneoplastic antibody testing results. Results Fourteen ON patients were classified as four groups based on their clinical and MRI characteristics, as well as serum and CSF testing results: [1] definite PON, 6 cases (11 eyes); [2] possible PON, 3 case (5 eyes); [3] meningeal carcinomatosis-associated optic neuropathy (MCON), 4 cases (6 eyes); [4] infiltrative optic neuropathy (ION), 2 cases (2 eyes). Malignant cells were found under microscope in CSF samples from MCON and ION patients, contrast to no malignant cells in CSF samples from PON cases. All 14 ON patients with malignant tumors showed negative results in serum tests for pathogens and autoantibodies. Serum paraneoplastic antibodies were tested in PON patients, anti- CV2, anti-Yo, and anti- amphiphysin were detected positive in 2, 1, and 1 case, respectively, in definite PON group, whereas no serum paraneoplastic antibody detected in possible PON group. Two unknown serum antineuronal antibodies (an anti- Purkinje cell antibody and an anti-granular cell antibody) were detected using monkey cerebellar tissue-based assay in 2 of 5 PON patients with negative paraneoplastic antibody test results. Conclusions Serum and CSF tests are of great importance in differentiating different subtypes of ON with malignant tumors. Current diagnosis of PON still depends on combination of clinical and MRI manifestations, as well as serum and CSF tests. Tissue-based assay may help to detect new biomarkers for ON etiology and diagnosis.

Follow - up prognostic study on two imported patients with human African trypanosomiasis

To investigate the prognosis of two rare imported patients with human African trypanosomias (HAT) after treatment in a follow-up study, and to evaluate the therapeutic efficacy, so as to provide insights into the treatment of imported HAT patients. The white blood cells in cerebrospinal fluid samples and the trypomastigotes in cerebrospinal fluid and blood samples were monitored in an imported case with Trypanosoma brucei rhodesiense infection 1, 3, 11 and 25 months post-treatment and in an imported case with T. brucei gambiense infection 1, 3, 8 and 12 months post-treatment to evaluate the therapeutic efficacy and prognosis. There were 1, 1, 4 and 2 white blood cells in per μL of cerebrospinal fluid in the case with T. brucei rhodesiense infection 1, 3, 11 and 25 months post-treatment, and there were 3, 6, 4 and 3 white blood cells in per μL of cerebrospinal fluid in the case with T. brucei gambiense infection 1, 3, 8 and 12 months post-treatment. In addition, no trypomastigotes were identified in the cerebrospinal fluid or blood samples of either case with T. brucei rhodesiense or T. brucei gambiense infection. Following standardized treatment, two imported cases with human African trypanosomiasis cases recover satisfactorily, without any signs of relapse.

Macrophages in Giemsa-stained cerebrospinal fluid specimens predict carcinomatous meningitis.

Carcinomatous meningitis is a condition in which tumor cells spread to the subarachnoid space. Leukocyte counting and typing of cerebrospinal fluid (CSF) cell components are performed manually or using flow cytometry. However, a detailed analysis of these variables using cytological specimens has not yet been reported. The present study analyzed cytological specimens using Giemsa staining and whole slide imaging with computer-assisted image analysis (CAIA) to clarify the characteristics of the leukocyte population in CSF, especially in carcinomatous meningitis. Manual evaluation was performed using 280 Giemsa-stained cytological CSF specimens. For 49 samples, CAIA was used for the whole area of Papanicolaou (Pap) staining, and Giemsa-stained specimens of the same samples were imaged using a virtual slide scanner. The nuclear morphology of the leukocytes was assessed, and the total leukocyte and leukocyte subset (lymphocytes, neutrophils and macrophages) counts were evaluated. Then, the number and percentage of each leukocyte subset population were evaluated. The total leukocyte count was significantly higher in Giemsa-stained specimens compared with in Pap-stained specimens. The percentage of macrophages was significantly higher in samples from patients with non-hematological tumors compared with in samples from patients without tumors, which was confirmed by manual evaluation of the specimens. In addition, the cut-off value of the percentage of macrophages that could discriminate between the tumor history negative cases and cytologically tumor positive cases was determined, revealing that a higher proportion of macrophages reflected the existence of atypical/malignant epithelial tumor cells in CSF samples. Thus, atypical cell screening and analysis of the background characteristics of the leukocyte population should be the focus of cytological specimen screening, especially not to miss carcinomatous meningitis.

Abstract A52: Systemic anti-PD-1 immunotherapy results in PD-1 blockade on T cells in the cerebrospinal fluid

Abstract Immune checkpoint inhibitors have shown promising clinical efficacy in many types of cancers, including some with metastasis into the central neural system (CNS). However, little is known about the CNS penetration of immune checkpoint inhibitors and their bioactivity on the locoregional T cells. Here, we analyzed the CNS and systemic concentration of pembrolizumab (Pembro) in high-grade glioma patients, who were concurrently treated with intravenously administered Pembro and locoregionally delivered chimeric antigen receptor (CAR) T cells. A total of 99 paired cerebrospinal fluid (CSF) and serum samples were collected from 10 patients during multicycle treatment of CAR T cell and Pembro. We discovered that after the first Pembro infusion, CSF antibody concentrations were increased slower than serum concentrations. However, CSF antibody concentrations reached steady-state 24 hours after Pembro infusion and were maintained stable over the 21-day infusion cycles. Although average CSF Pembro concentrations (3.2nM) were only 1% of serum levels (329nM), we observed effective PD-1 blockade on the T cells in CSF samples of all analyzed patients. The blocking effect was found on both endogenous T cells and locoregionally administered CAR T cells. Further, PD-1 on CD3/CD28 beads-activated T cells was completely blocked after incubation with cell-depleted CSF samples, suggesting the bioactivity of Pembro that penetrated into the CSF. Using a dose-titration assay, we also confirmed that CSF Pembro concentrations were sufficient to ameliorate the cytotoxic activity of CAR T cells against repetitive tumor challenges. Together, for the first time, we showed the dynamic Pembro levels in the CSF throughout multiple treatment cycles and confirmed that systemically administered immune checkpoint inhibitors can penetrate into the CNS and elicit blocking effect on T cells. Our findings add strength to the rationale for combining immune checkpoint inhibitors with locoregionally delivered CAR T cells and other types of immunotherapy for the treatment of brain tumors. Citation Format: Dongrui Wang, Jana Portnow, Vivi Tran, Vivian Chiu, Alfonso Brito, Darya Alizadeh, Renate Starr, Julie Kilpatrick, Paige McNamara, Stephen J. Forman, Behnam Badie, Timothy W. Synold, Christine E. Brown. Systemic anti-PD-1 immunotherapy results in PD-1 blockade on T cells in the cerebrospinal fluid [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A52.

Cellular origin and diagnostic significance of high‐fluorescent cells in cerebrospinal fluid detected by the XE‐5000 hematology analyzer

The Sysmex XE-5000 is a blood and body fluid analyzer able to differentiate cells into polymorphonuclear, mononuclear, and high-fluorescent cells (HFC). The identity of HFC in cerebrospinal fluid (CSF) has been uncertain; however, compatible with their high nucleic acid content, HFC could represent intrathecal tumor cells. Here, we studied the cellular origin and the diagnostic significance of HFC in CSF. Results of CSF examinations with the XE-5000 were analyzed in 65 CSF samples with and 126 CSF samples without tumor cells, as defined by manual microscopy of CSF cytospin preparations. The XE-5000 detected HFC in 51 of 65 tumor cell-positive and in 33 of 126 tumor cell-negative CSF samples (sensitivity: 78.5%, specificity: 73.8%, positive likelihood ratio: 3.0, negative likelihood ratio: 0.29). The percentages of HFC and tumor cells in CSF samples correlated (r²=0.41, P<0.0001). Tumor cells escaped detection by the XE-5000 especially in CSF samples with a low percentage of tumor cells. While this study identifies tumor cells as the predominant correlate of HFC in CSF, it suggests that measuring HFC is not an appropriate diagnostic test for intrathecal tumor cells. However, if HFC are incidentally detected in CSF, further evaluation by CSF microscopy seems mandatory.

Detection of occult cerebrospinal fluid involvement during maintenance therapy identifies a group of children with acute lymphoblastic leukemia at high risk for relapse

We aimed at assessing the clinical significance of the levels of acute lymphoblastic leukemia (ALL) cells in samples of cerebrospinal fluid (CSF) during therapy. We studied 990 CSF samples from 108 patients, at the time of diagnosis (108) and at each time of intrathecal therapy (882). The proportions of leukemic cells in CSF samples were assessed by flow cytometry (FCM). Patients with central nervous system (CNS) involvement at diagnosis (FCM+) showed predominantly a T-ALL, and higher percentages of known negative prognostic factors: high risk group, higher white blood cell counts, normal karyotype, and the BCR-ABL fusion gene. No differences in relapse free survival (RFS) and overall survival (OS) were observed between FCM+ versus FCM- at diagnosis. Patients with CNS involvement during therapy showed significantly older age, and higher frequencies of T-cell leukemia. We found a significantly higher RFS in patients with FCM+ during therapy. The detection of subclinical CNS disease by FCM during maintenance was associated with significantly lower 3-years RFS and 3-years OS. A sensitive methodology like FCM can be applied for a close follow-up of the levels of ALL in CFS samples, and may identify a group of patients at high risk for relapse.

Detection of donor-derived CMV-specific T cells in cerebrospinal fluid in a case of CMV meningoencephalitis after cord blood stem cell transplantation

Cytomegalovirus (CMV) meningoencephalitis is a rather rare complication after allogeneic stem cell transplantation. We describe here the case of a 59-year-old man with acute myeloid leukemia who developed CMV meningoencephalitis after cord blood transplantation. The patient presented with a sudden onset of neurological symptoms, such as convulsion, on day 37. The analysis of cerebrospinal fluid (CSF) sample revealed an increase in the number of cells, which were of donor (cord blood) origin, consisting mainly of T cells. No bacteria were detected in the CSF sample. Real-time PCR analysis revealed that the CSF sample was positive for CMV, but was negative for HHV-6, adenovirus, or BK virus. The patient was diagnosed with CMV meningoencephalitis and received cidofovir. His neurological symptoms were gradually improved and completely disappeared by day 60. CMV-specific dextramer-positive CD8(+) T cells were detected in the peripheral blood and CSF samples, with the frequency being much higher in the CSF. To our knowledge, this is the first report on the appearance of CMV-specific T cells in CSF samples from a patient with CMV meningoencephalitis. Cord blood-derived CMV-specific T cells may develop early after transplantation, enter the intrathecal compartment, and likely contribute to the regulation of CMV-meningoencephalitis.

Neutrophils in cerebrospinal fluid without pleocytosis

Neutrophils in cerebrospinal fluid (CSF) samples are commonly considered a pathological feature; however, there is little information on the frequency and significance of these cells in CSF samples without pleocytosis. Therefore, the frequency and possible clinical significance of neutrophils in CSF was investigated. In a retrospective study comprising 1556 consecutive CSF samples, cytologies and patient records were reviewed. Five hundred thirty-eight CSF samples without pleocytosis were identified. Neutrophils were detected in 35.5% of these samples. The presence of neutrophils was associated with sepsis (P < 0.01), recent epileptic seizure (P < 0.0001), and blood contamination (P < 0.01). Amongst patients without CSF pleocytosis, CNS infections were not more frequent if neutrophils were present. Neutrophils are frequently observed in CSF with normal leukocyte counts. As sepsis but not CNS infection occurred more frequently in these patients, we conclude that in the absence of CSF pleocytosis, neutrophils are not indicative of CNS infections.

Immunocytochemical localization of carcinoembryonic antigen in cerebrospinal fluid with metastatic carcinoma of the stomach: Report of four cases

A study was undertaken to evaluate the cellular findings of gastric carcinoma in cerebrospinal fluid (CSF). Immunocytochemical localization of carcinoembryonic antigen (CEA) was performed on four cases of metastatic gastric carcinoma cells in CSF samples. A positive peroxidase-antiperoxidase (PAP) reaction was obtained in all cases, with intense staining for CEA in the CSF samples as well as in the paraffin-embedded tissue sections from the primary gastric tumors. Cellular morphology and the results of immunoperoxidase staining can be studied simultaneously. We believe that the PAP method for CEA increases diagnostic accuracy of cytology in the CSF.