Cerebrospinal Fluid Monitoring in Childhood Leukemia: Laboratory Medicine’s Central Role in an Ongoing Oncology Success Story

May 9, 2022, 10:17 AM by Alexandra E. Kovach

Cure of childhood leukemia is arguably one of the greatest medical achievements of the last century.1-3 B-lymphoblastic leukemia (B-ALL), the most common cancer of childhood, was previously a uniformly fatal disease. Famously, pathologist Sidney Farber’s4-6 and others’7 introduction of folate antagonists produced cures and led to the recognition of the central nervous system (CNS), specifically the cerebrospinal fluid (CSF), as a sanctuary site for disease.8,9 Prophylactic intrathecal (IT) chemotherapy remains standard of care today, having largely replaced cranial irradiation.10-13 Indeed, the largest incremental improvement in leukemia outcomes over the last century, even compared to improvements in supportive therapy, is attributable to CNS therapy.8,14 Moreover, staging of CSF at initial leukemia is a significant independent prognostic factor15 that requires accurate manual CSF total nucleated cell (TNC) differential counts and blast identification.16 

Skilled clinical laboratory professionals are therefore crucial to accurate CSF staging and monitoring, the development of standardized cytocentrifugation having been fundamental to this practice.17-21 In the U.S., modern Children’s Oncology Group (COG) clinical trials as well as institutional standard-of-care (SOC) protocols for ALL (B-cell and T-cell) utilize CSF status categories based on the absence or presence of blasts on manual review of cytomorphology on concentrated CSF cytospin slides stained with Wright Giemsa and, when blasts are present, the TNC based on unconcentrated manual count by hematocytometer chamber, which has its roots in older protocols.22-24 Current categories predictive of clinical behavior are: CNS1 = no blasts present, CNS2 = blasts present in a sample with a TNC <5 cells/mL, and CNS3 = blasts present in a sample with a TNC ³5 cells/mL.25 CSF TNC and cytomorphologic evaluation is repeated at each lumbar puncture (LP) prior to intrathecal chemotherapy administration to ensure CSF clearance or maintained absence of blasts.26,27 

Lymphoblasts, while often stereotypical and reproducibly identifiable, can show significant morphologic variation, particularly in body fluid preparations such as CSF. Typical lymphoblasts (Figure 1A) are slightly larger than small mature lymphocytes with an increased nuclear:cytoplasmic ratio, smooth to notched nuclear contours, finely dispersed chromatin, and inconspicuous to absent small nucleoli.  The scant cytoplasm may show occasional small pale vacuoles and/or fine granules. However, sample centrifugation, age, temperature and staining as well as interval intrathecal therapy and leukemia genetics may affect cellular morphology of lymphoblasts (Figure 1B, 1C) as well as that of normal CSF cells, which include small mature lymphocytes (Figure 1D) and monocytes. Inherent challenges with CSF blast morphology have led many pathology groups to pair cytospin evaluation with CSF flow cytometry for immunophenotyping and therefore definite cell identification (Figure 1E-H, corresponding right panels). Some groups are utilizing flow only in morphologically challenging cases, while others are running flow by routine.28,29 Flow may also provide a prognostic staging advantage over conventional cytology.30,31 

Figure 1. Spectrum of lymphoblast morphology in cerebrospinal fluid cytomorphology preparations.  

Left column: Photomicrographs. A. B lymphoblast at diagnosis with typical morphology, B. T lymphoblast at diagnosis resembling mature lymphocyte, C. B lymphoblasts at relapse resembling abnormal immature monocytes, possible due to centrifugation and/or prior therapy, D. Normal mature lymphocyte (all images: 1,000x magnification).  
Right column: Corresponding flow cytometry data confirming the lineage of each cell population. E. B lymphoblasts, F. T lymphoblasts, G. B lymphoblasts, H. Mature T cells. Samples were stored for up to 72 hours in preservation media prior to flow cytometric acquisition. Red = abnormal populations. 

Should flow cytometry by standard of care in CSF staging and monitoring for leukemia? 
There are a number of clinical and technical considerations that necessitate caution given current data. First, existing COG thresholds and associated prognoses were established based on cytospin morphology assessment. Flow cytometry is known to be more sensitive than cytomorphology for lymphoblast identification,30,32,33 such that its use under the current staging system would be expected to lead to a subset of low cellularity, morphologically negative diagnostic staging samples (CNS1) being upstaged to positive (CNS2) and leading to unnecessary overtreatment.34-36 Second, cells in paucicellular samples, including CSF, tend to degenerate rapidly. Flow cytometry, which relies on intact cells for cell surface marker identification, is optimally performed on paucicellular samples shortly after sample procurement and/or following addition of cell preservation media.37-39 A prospective clinical trial utilizing sample stabilization and central flow cytometry review is needed to answer this question. If feasible, flow cytometry may provide a more reproducible method for ALL assessment in CSF compared with traditional cytomorphology.  

What about molecular assays? 
In primary large B-cell lymphoma of the central nervous system (primary CNS lymphoma), for example, polymerase chain reaction (PCR) for MYD88 gene mutation in CSF is a sensitive and specific marker,40 and multiplex genomic assays incorporating MYD88 have recently been proposed.41,42 In addition to flow cytometry, PCR and next-generation sequencing (NGS) assays for clonal immunoglobulin heavy chain (IgH) and/or T-cell receptor (TCR) gene sequences, are increasingly being used for minimal/measurable residual disease monitoring of leukemia in bone marrow and peripheral blood.43 For CSF, however, IGH and/or TCR sequencing could be non-specific, showing so-called “pseudo-clonal” sequences based on amplification of rare sequences in paucicellular samples. As with applying flow cytometry to CSF samples from children who would otherwise be staged as CNS1 and do well with current therapeutic protocols, the sensitivity of molecular techniques may be inappropriately high for the clinical question at hand. Despite the available technologies, we must “first do no harm.” 

In summary, CSF evaluation in the staging, monitoring, and treatment of standard treatment of childhood leukemia is a modern medical success story highly dependent on laboratory medical professionals. Best practices for use of technologies including flow cytometry and molecular assays on CSF assessment in leukemia remain to be determined over the next, likely fewer than 100, years. 


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