Alexander Baraniskin

Identification of microRNAs in the cerebrospinal fluid as marker for primary diffuse large B-cell lymphoma of the central nervous system

The diagnosis of primary central nervous system lymphoma (PCNSL) depends on histopathology of brain biopsies, because disease markers in the cerebrospinal fluid (CSF) with sufficient diagnostic accuracy are not available yet. MicroRNAs (miRNAs) are regulatory RNA molecules that are deregulated in many disease types, including cancer. Recently, miRNAs have shown promise as markers for cancer diagnosis. In this study, we demonstrate that miRNAs are present in the CSF of patients with PCNSL. With a candidate approach and miRNA quantification by reverse transcription polymerase chain reaction, miRNAs with significant levels in the CSF of patients with PCNSL were identified. MiR-21, miR-19, and miR-92a levels in CSF collected from patients with PCNSL and from controls with inflammatory CNS disorders and other neurologic disorders indicated a significant diagnostic value of this method. Receiver-operating characteristic analyses showed area under the curves of 0.94, 0.98, and 0.97, respectively, for miR-21, miR-19, and miR-92a CSF levels in discriminating PCNSL from controls. More importantly, combined miRNA analyses resulted in an increased diagnostic accuracy with 95.7% sensitivity and 96.7% specificity. We also demonstrated a remarkable stability of miRNAs in the CSF. In conclusion, CSF miRNAs are potentially useful tools as novel noninvasive biomarker for the diagnosis of PCNSL.

Identification of microRNAs in the cerebrospinal fluid as biomarker for the diagnosis of glioma

Malignant gliomas are the most common and lethal primary intracranial tumors. To date, no reliable biomarkers for the detection and risk stratification of gliomas have been identified. Recently, we demonstrated significant levels of microRNAs (miRNAs) to be present in cerebrospinal fluid (CSF) samples from patients with primary CNS lymphoma. Because of the involvement of miRNA in carcinogenesis, miRNAs in CSF may serve as unique biomarkers for minimally invasive diagnosis of glioma. The objective of this pilot study was to identify differentially expressed microRNAs in CSF samples from patients with glioma as potential novel glioma biomarkers. With use of a candidate approach of miRNA quantification by reverse-transcriptase polymerase chain reaction (qRT-PCR), miRNAs with significant levels in CSF samples from patients with gliomas were identified. MiR-15b and miR-21 were differentially expressed in CSF samples from patients with gliomas, compared to control subjects with various neurologic disorders, including patients with primary CNS lymphoma and carcinomatous brain metastases. Receiver-operating characteristic analysis of miR-15b level revealed an area under the curve of 0.96 in discriminating patients with glioma from patients without glioma. Moreover, inclusion of miR-15b and miR-21 in combined expression analyses resulted in an increased diagnostic accuracy with 90% sensitivity and 100% specificity to distinguish patients with glioma from control subjects and patients with primary CNS lymphoma. In conclusion, the results of this pilot study demonstrate that miR-15b and miR-21 are markers for gliomas, which can be assessed in the CSF by means of qRT-PCR. Accordingly, miRNAs in the CSF have the potential to serve as novel biomarkers for the detection of gliomas.

Circulating U2 small nuclear RNA fragments as a novel diagnostic biomarker for pancreatic and colorectal adenocarcinoma

Improved non‐invasive strategies for early cancer detection are urgently needed to reduce morbidity and mortality. Non‐coding RNAs, such as microRNAs and small nucleolar RNAs, have been proposed as biomarkers for non‐invasive cancer diagnosis. Analyzing serum derived from nude mice implanted with primary human pancreatic ductal adenocarcinoma (PDAC), we identified 15 diagnostic microRNA candidates. Of those miR‐1246 was selected based on its high abundance in serum of tumor carrying mice. Subsequently, we noted a cross reactivity of the established miR‐1246 assays with RNA fragments derived from U2 small nuclear RNA (RNU2‐1). Importantly, we found that the assay signal discriminating tumor from controls was derived from U2 small nuclear RNA (snRNA) fragments (RNU2‐1f) and not from miR‐1246. In addition, we observed a remarkable stability of RNU2‐1f in serum and provide experimental evidence that hsa‐miR‐1246 is likely a pseudo microRNA. In a next step, RNU2‐1f was measured by qRT‐PCR and normalized to cel‐54 in 191 serum/plasma samples from PDAC and colorectal carcinoma (CRC) patients. In comparison to 129 controls, we were able to classify samples as cancerous with a sensitivity and specificity of 97.7% [95% CI = (87.7, 99.9)] and 90.6% [95% CI = (80.7, 96.5)], respectively [area under the ROC curve 0.972]. Of note, patients with CRC were detected with our assay as early as UICC Stage II with a sensitivity of 81%. In conclusion, this is the first report showing that fragments of U2 snRNA are highly stable in serum and plasma and may serve as novel diagnostic biomarker for PDAC and CRC for future prospective screening studies.

Current strategies in the diagnosis of diffuse large B‐cell lymphoma of the central nervous system

Lymphomas can arise within the central nervous system (CNS) as primary CNS lymphoma (PCNSL) typically involving the brain and less often the leptomeninges, eyes, and spinal cord. In contrast to PCNSL, secondary CNS lymphoma (SCNSL) is considered to originate as quasi metastasis from systemic lymphoma spreading to the CNS. Both types of CNS lymphomas are predominantly tumours of the diffuse large B‐cell type and represent aggressive diseases necessitating a rapid diagnosis. Following neuroimaging based on magnetic resonance imaging, stereotaxy and histopathological diagnosis of CNS lymphoma currently remain obligatory to plan treatment. However, progress in cytopathological, immunophenotypic, and molecular genetic analyses of the cerebrospinal fluid (CSF) has been achieved recently and potentially will facilitate lymphoma diagnosis in the future. This review describes the diagnostic procedures in patients with suspected CNS lymphomas, primarily PCNSL. In addition to a summary of the standard diagnostic work‐up, an overview and discussion of current data on different techniques for evaluation of the CSF in CNS lymphoma are given.

Diagnosis of leptomeningeal disease in diffuse large B-cell lymphomas of the central nervous system by flow cytometry and cytopathology.

Reliable detection of leptomeningeal disease has the potential of facilitating the diagnosis of central nervous system (CNS) lymphoma and is important for therapeutic considerations. Currently, the standard diagnostic procedure for the detection of lymphoma in the cerebrospinal fluid is cytopathology. To improve the limited specificity and sensitivity of cytopathology, flow cytometry has been suggested as an alternative. Here, we evaluated multi‐parameter flow cytometry in combination with conventional cytopathology in cerebrospinal fluid (CSF) samples from 30 patients with primary CNS lymphoma and seven patients with secondary CNS lymphoma. Overall, in 11 of 37 (29.7%) patients with CNS lymphoma, lymphoma cells were detected in CSF by flow cytometry, while cytopathology was less sensitive displaying unequivocally malignant CSF cells in only seven of all 37 (18.9%) patients. Six (16.2%) patients showed cytopathological results suspicious of lymphoma; however, in only one of these patients, the diagnosis of CSF lymphoma cells could be confirmed by flow cytometry. In primary CNS lymphomas (PCNSL), seven of 30 (23.3%) patients were positive for CSF lymphoma cells in flow cytometry, in contrast to four (13.3%) patients with PCNSL with definitely positive cytopathology. In summary, our results suggest that multi‐parameter flow cytometry increases the sensitivity and specificity of leptomeningeal disease detection in CNS lymphomas. Both methods should be applied concurrently for complementary diagnostic assessment in patients with CNS lymphoma.

Quantification of circulating endothelial progenitor cells in human peripheral blood: Establishing a reliable flow cytometry protocol

OBJECTIVES Accurate enumeration of circulating endothelial progenitor cells (CEP) is essential for their potential application as biomarkers of angiogenesis. In this study different stem cell markers (CD34, CD133) and endothelial cell antigens (KDR/VEGFR-2, CD31) in different flow cytometric protocols were assessed for the purpose of CEP quantification. METHODS Blood samples from 19 healthy volunteers and 16 patients with different cancer types were analyzed by means of flow cytometry. Mononuclear cell gating was compared to additional gating on CD45-negative cells. CD34+/KDR+ and CD31+/CD133+ cells were analyzed in a direct immunolabeling approach. CEP were measured at different time points in individual patients and after storage of blood samples for 24h and 48h, respectively. RESULTS In contrast to previous studies, measurement of CD34+/KDR+ cells was an unreliable method for CEP enumeration, regardless of the applied gating strategy. However, detection of CD31+/CD133+ cells in a combined mononuclear cell and CD45-negative gating approach provided a reproducible method for CEP quantification. In individual blood donors, the CEP numbers were stable over time. For the first time, it was demonstrated that CEP are unstable in extracorporeal blood samples. CONCLUSION In this study, a reproducible protocol for CEP quantification was established. This protocol should facilitate future studies with the goal to further define the role of CEP as angiogenic biomarkers.

Circulating U2 Small Nuclear RNA Fragments as a Novel Diagnostic Tool for Patients with Epithelial Ovarian Cancer

BACKGROUND Ovarian cancer is the leading cause of death among malignancies in women. Despite advances in treatment, >50% of patients relapse. For disease monitoring, the identification of a blood-based biomarker would be of prime interest. In this regard, noncoding RNAs, such as microRNA (miRNA) or small nuclear RNA (snRNA), have been suggested as biomarkers for noninvasive cancer diagnosis. In the present study, we sought to identify differentially expressed miRNA/snRNA in sera of ovarian cancer patients and investigate their potential to aid in therapy monitoring. METHODS miRNA/snRNA abundance was investigated in serum (n = 10) by microarray analysis and validated in an extended serum set (n = 119) by reverse-transcription quantitative PCR. RESULTS Abundance of U2-1 snRNA fragment (RNU2-1f) was significantly increased in sera of ovarian cancer patients (P < 0.0001) and paralleled International Federation of Gynecology and Obstetrics stage as well as residual tumor burden after surgery (P < 0.0001 and P = 0.011, respectively). Moreover, for patients with suboptimal debulking, preoperative RNU2-1f concentration was associated with radiographic response after chemotherapy and with platinum resistance (P = 0.0088 and P = 0.0015, respectively). Interestingly, according to the RNU2-1f abundance dynamics, persistent RNU2-1f positivity before surgery and after chemotherapy identified a subgroup of patients with high risk of recurrence and poor prognosis. CONCLUSIONS This is the first report to suggest that a circulating snRNA can serve as an auxiliary diagnostic tool for monitoring tumor dynamics in ovarian cancer. Our results provide a rationale to further investigate whether this high-risk patient group may benefit from additional therapies that are directly applied after chemotherapy.

Detection of free immunoglobulin light chains in cerebrospinal fluids of patients with central nervous system lymphomas

Diagnosis of central nervous system (CNS) lymphoma depends on histopathology of brain biopsies, because no reliable disease marker in the cerebrospinal fluid (CSF) has been identified yet. B‐cell lymphomas such as CNS lymphomas are clonally restricted and express either kappa or lambda immunoglobulin light chains. The aim of this study was to find out a potential diagnostic value of free immunoglobulin light chains released into the CSF of CNS lymphoma patients. Kappa (κ) and lambda (λ) free immunoglobulin light chains (FLC) were measured in CSF and serum samples collected from 21 patients with primary and secondary CNS lymphomas and 14 control patients with different neurologic disorders. FLC concentrations and ratios were compared between patient groups and were further analyzed in correlation with clinical, cytopathological, and radiological findings. FLC concentrations for all patients were lower in CSF when compared to serum. In patients with CNS lymphoma, the FLC ratios in CSF were higher (range 392–0.3) compared to control patients (range 3.0–0.3). Irrespective of cytopathological proven lymphomatous meningitis, in 11/21 lymphoma CSF samples the FLC ratios were markedly above 3.0 indicating a clonally restricted B‐cell population. Increased FLC ratios in CSF were found in those patients showing subependymal lymphoma contact as detected in magnetic resonance imaging. In summary, this is the first report demonstrating that a significant proportion of patients with CNS lymphomas display a markedly increased FLC ratio in the CSF.

Blood‐based detection of RAS mutations to guide anti‐EGFR therapy in colorectal cancer patients: concordance of results from circulating tumor DNA and tissue‐based RAS testing

An accurate blood‐based RAS mutation assay to determine eligibility of metastatic colorectal cancer (mCRC) patients for anti‐EGFR therapy would benefit clinical practice by better informing decisions to administer treatment independent of tissue availability. The objective of this study was to determine the level of concordance between plasma and tissue RAS mutation status in patients with mCRC to gauge whether blood‐based RAS mutation testing is a viable alternative to standard‐of‐care RAS tumor testing. RAS testing was performed on plasma samples from newly diagnosed metastatic patients, or from recurrent mCRC patients using the highly sensitive digital PCR technology, BEAMing (beads, emulsions, amplification, and magnetics), and compared with DNA sequencing data of respective FFPE (formalin‐fixed paraffin‐embedded) tumor samples. Discordant tissue RAS results were re‐examined by BEAMing, if possible. The prevalence of RAS mutations detected in plasma (51%) vs. tumor (53%) was similar, in accord with the known prevalence of RAS mutations observed in mCRC patient populations. The positive agreement between plasma and tumor RAS results was 90.4% (47/52), the negative agreement was 93.5% (43/46), and the overall agreement (concordance) was 91.8% (90/98). The high concordance of plasma and tissue results demonstrates that blood‐based RAS mutation testing is a viable alternative to tissue‐based RAS testing.

Circulating U2 small nuclear RNA fragments as a novel diagnostic biomarker for primary central nervous system lymphoma

BACKGROUND Primary central nervous system lymphomas (PCNSLs) are highly aggressive tumors. Chemotherapy has improved prognosis significantly; however, early diagnosis is crucial for effective treatment. Presently, the diagnosis of PCNSL depends on histopathology of tumor biopsies. We have previously demonstrated differential expression of microRNAs in cerebrospinal fluid (CSF) samples from patients with PCNSL. Based on promising findings about circulating U2 small nuclear RNA fragments (RNU2-1f) as novel blood-based biomarkers for pancreatic, colorectal, and lung cancer, we investigated RNU2-1f in the CSF of PCNSL patients. METHODS CSF was collected from patients with PCNSL (n = 72) and control patients with various neurologic disorders (n = 47). Sequential CSF samples were collected from 9 PCNSL patients. RNU2-1f levels were measured by real-time polymerase chain reaction. RESULTS Measurement of RNU2-1f levels in CSF enabled the differentiation of patients with PCNSL from controls with an area under the curve (AUC) of 0.909 with a sensitivity of 68.1% and a specificity of 91.4%. The diagnostic accuracy was further improved by combined determination of RNU2-1f and miR-21, resulting in AUC of 0.987 with a sensitivity of 91.7% and a specificity of 95.7%. In consecutive measurements of RNU2-1f, which were performed in 9 patients at different stages of the disease course, RNU2-1f CSF levels paralleled the course of the disease. CONCLUSIONS Our data suggest that the measurement of RNU2-1f detected in CSF can be used as a diagnostic marker and also as a possible marker for treatment monitoring. These promising results need to be evaluated within a larger patient cohort.