Imagine this clinical scenario: a recently diagnosed six-year-old German Shepherd Dog with B-cell lymphoma. With the increasing availability of flow cytometers and antibodies against species-specific cellular markers in the veterinary sector, you wonder whether flow cytometric analysis can provide useful prognostic information on your patient.
Twelve studies were critically appraised, all of which were observational cohort studies. Eight studies identified an association between specific cellular markers and prognosis in B-cell lymphoma, which may be useful in predicting survival after treatment with cytotoxic drugs.
Patients with a high percentage of CD25+ B-cells in peripheral lymph nodes had a significantly decreased progression-free survival (Mizutani et al., 2016). This correlation was also found by Wolf-Ringwall et al. (2019) who evaluated CD25 expression in dogs with multicentric B-cell lymphoma. CD25 binds the growth factor interleukin-2 which stimulates clonal expansion and maturation of activated T or B lymphocytes (Lowenthal et al., 1985).
Dogs with high-grade B-cell lymphoma were found to have upregulated expression of programmed death-1 (PD-1) on peripheral and tumour infiltrating lymphocytes, and upregulated expression of cytotoxic T-lymphocyte associated antigen-4 (CTLA-4) on peripheral CD4+ T-cells (Tagawa et al., 2018). PD-1 and CTLA-4 are immune checkpoint molecules believed to help evasion of the host immune system. Dogs with CTLA-4 expression below the established cut-off value had significantly longer survival times compared to those above cut-off value. Although increased in dogs with B-cell lymphoma, the expression of PD-1 was not found to reliably correlate with survival times.
Class II major histocompatibility complex (MHC) is a cellular marker primarily expressed on professional antigen-presenting cells. Dogs with low class II MHC expression were 2.9 times more likely to die in any period compared to high expression (Rao et al., 2011). The study by Pinheiro et al. (2014) supported this, finding decreased class II MHC expression to be associated with increased time to remission. However, Wolf-Ringwall et al. (2019) did not find a correlation between class II MHC expression and outcome in dogs with diffuse large B-cell lymphoma. The authors suggest that class II MHC levels may not be important for survival in some histopathological subtypes; however, as diffuse large B-cell lymphoma is the most common type of B-cell lymphoma in dogs, it would be expected that these dogs made up a large proportion of the population in the study by Rao et al. (2011).
Ki-67 is a marker of cellular proliferation and is frequently used to assess the growth fraction of neoplastic populations (Schlüter et al., 1993). Poggi et al. (2015) assessed the percentage of Ki-67+ cells in lymph node samples in dogs with lymphoma (65 of which had B-cell lymphoma) and confirmed increased Ki-67 percentage in high-grade lymphoma samples compared to low grade – although only three dogs with low-grade lymphoma were included in the study. The authors established a suggested cut-off value for Ki-67 of 12.2 percent to detect high-grade lymphoma.
The authors suggested that Ki-67 could be a useful prognostic indicator to include in the routine panel of labelling for high-grade B-cell lymphoma
Poggi et al. (2017) found Ki-67 percentage significantly influenced lymphoma-specific survival; dogs with intermediate Ki-67 percentage, compared to a high or low Ki-67 percentage, had a significantly longer relapse-free interval and lymphoma-specific survival. Ki-67 percentage was a significant predictor for one- and two-year survival. The authors suggested that Ki-67 could be a useful prognostic indicator to include in the routine panel of labelling for high-grade B-cell lymphoma.
Large B lymphocytes may correlate to more immature and aggressive neoplastic cells, which are negatively associated with survival. Rao et al. (2011) classified cells taken from samples from peripheral lymph nodes of dogs with B-cell lymphoma, based on size evaluated by flow cytometry. Patients in the large-cell group, which had medium forward scatter of more than 720U, were found to be 2.8 times more likely to die in any period compared to those in the small-cell group.
In contrast, Wolf-Ringwall et al. (2019) found no correlation between cell size with survival. However, the large-cell group only comprised 3.1 percent of patients (2 out of 64), which may explain the lack of statistical correlation. As far fewer patients were assigned to the large-cell group compared to the small-cell group in both studies, cell size measured by this basis may not provide a widely applicable prognostic test.
Bone marrow infiltration
Marconato et al. (2013) used flow cytometry in a novel way, to correlate bone marrow infiltration by multicentric B-cell lymphoma with time to progression and lymphoma-specific survival. Bone marrow infiltration was reported as percentage of large CD21+ cells out of total CD45+ cells in the sample. Dogs with bone marrow infiltration of 3.0 percent or more had a 3.3 times higher probability of progression, and a 3.6 times higher probability of death when compared to dogs with less than 3.0 percent bone marrow infiltration.
Joetzke et al. (2012) used the ratio between T and B lymphocytes (log [T:B]), established by flow cytometry, to differentiate between lymphoma-infiltrated and healthy samples from blood, bone marrow, liver and spleen. Mean log (T:B) values in all samples were significantly lower in dogs with B-cell lymphoma compared to healthy control dogs. The authors suggest that the log (T:B) could be used as an indicator of the degree of lymphoma cell infiltration to predict prognosis; however, this would need to be investigated in further studies.
Peripheral lymphocyte/monocyte ratio
Marconato et al. (2015) found that the probability of lymphoma progression was 3.7 times higher in dogs with a peripheral lymphoctye/monocyte ratio (LMR) below 1.2, compared to dogs with an LMR greater than 1.2. This technique could be useful as it makes use of a peripheral blood sample rather than aspirates of lymph nodes, which may be less stressful for the patient.
Peripheral regulatory T-cell population
Baek et al. (2017) investigated the percentage of regulatory T-cells (also called Tregs) in total lymphocytes of the peripheral blood in dogs with B-cell lymphoma of different stages. The percentage of Tregs in peripheral blood was increased in dogs with lymphoma versus control dogs, and dogs with World Health Organization (WHO) stage V lymphoma had an increased percentage of Tregs compared to dogs with WHO stage IV lymphoma. A limitation was that an association of Treg percentage and survival was not assessed.
Pinheiro et al. (2014) found that increased forkhead box P3 protein (FOXP3) expression in patients was associated with poorer progression-free survival and overall survival times
In dogs with B-cell lymphoma, forkhead box P3 protein (FOXP3) and Helios expression is used as a surrogate for levels of regulatory T-cells in the affected lymph nodes. Pinheiro et al. (2014) found that increased FOXP3 expression in patients was associated with poorer progression-free survival and overall survival times. Patients with higher Helios expression also had a shorter progression-free survival time.
Overall, there is currently good evidence for the use of percentage expression of CD25 and Ki-67 cellular markers in providing prognostic information in canine B-cell lymphoma, with more than one good-quality study supporting this. Future studies should identify cut-off values for estimates of survival or response to treatment.
There is also evidence for the prognostic value of various other forms of flow cytometric analysis, as detailed above. However, these need to be further investigated with larger sample sizes, before introduction to clinical practice to confirm their value.