FLOW CYTOMETRY ANALYSIS OF NEURAL DIFFERENTIATION MARKERS EXPRESSION IN HUMAN GLIOBLASTOMAS MAY PREDICT THEIR RESPONSE TO CHEMOTHERAPY

Background

• Glioblastoma multiforme (GBM) represents the most malignant and chemoresistant form of central nervous system tumors of the astrocytic origin.
• Despite the associated unfavorable prognosis (life expectancy of 62 weeks with radiation/chemotherapy), a small number of patients display prolonged survival times. It is not known, however, what genetic and phenotypic characteristics allow a distinction between long-term survivors who respond to radiation/chemotherapy and other patients with GBM.
• Differences in chemosensitivity exist between histological glioma tumor types, as well as histopathologically identical neoplasms. In studies examining genetic factors that may result in these differences, certain chromosomal abnormalities have been linked with longer recurrence-free survival after chemotherapy. Likewise, expression of certain markers (eg, nestin, vimentin, A2B5) has been correlated with tumor malignancy, proliferative activity, invasive potential, and patient prognosis.
• However, knowledge regarding associations between tumor markers and chemosensitivity is limited. In vitro chemosensitivity assays also have limitations such as heterogenicity of biopsy specimen, in vitro drifting of cultured cells from homogenous heteroclonal culture, heterogeneity of chemosensitivity, and possible cytotoxic effects of metabolites as opposed to parent compounds.
• Flow cytometry has the potential to overcome issues related to in vitro chemosensitivity assays, therefore, it appears reasonable to utilize this strategy to define potential relationships between the lineage of glioma tumor cells (defined by immunophenotype) and their responsiveness to chemotherapy.

Objective

• To evaluate the expression of selected neural, as well as non-neural, differentiation markers in GBM tumors using multiparametric flow cytometry.
• To correlate the observed immunophenotype with in vitro chemosensitivity using methyl thiazolyl tetrazolium (MTT) assay.

Title/Citation

Balik V, Mirossay P, Bohus P, et al. Flow cytometry analysis of neural differentiation markers expression in human glioblastomas may predict their response to chemotherapy. Cell Mol Neurobiol. 2009;29:845-858. Epub 2009 Mar 14.

Funding

The work was supported by Slovak Research and Development Agency, P.J. Safarik University, and League Against Cancer.

Trial design

Prospective study at a single center in Slovakia.

Enrollment

11 patients diagnosed with GBM between 2002 and 2004.

Inclusion criteria

Patients diagnosed with grade IV GBM between 2002 and 2004, who provided informed consent prior to surgery.

Exclusion criteria

Exclusion criteria were not provided.

Study groups

Patients diagnosed with GBM.

End points

• Expression of selected neural and non-neural differentiation markers (A2B5, CD34, CD45, CD56, CD117, CD133, EGFR, GFAP, Her-2/neu, LIFR, nestin, NGFR, Pgp, vimentin) on GBM tumor specimens.
• Relationship between immunophenotype and chemosensitivity
• Sensitivity of tumor cells to panel of chemotherapeutic agents, including:
• Carmustine (BCNU)
• Cisplatin (CDDP)
• Dacarbazine (DTIC)
• Daunorubicin (DAU)
• Etoposide (VP16)
• Lomustine (CCNU)
• Paclitaxel (TAX)
• Topotecan (TOPO)
• Vincristine (VCR)

Statistical analysis

• SPSS Base version 13.0 software package for calculation of mean, median, and range values for each variable.
• Two-tailed Spearman’s correlation test for calculation of bivariate correlations between selected variables.

Baseline characteristics

• Mean age was 50 (range 36–62).
• 4 patients were female, 7 were male.

End points

• Expression of selected neural and non-neural differentiation markers on GBM tumor specimens:
• All tumors were positive for A2B5, CD56, nestin, and vimentin; the strongest activity was found for nestin and vimentin.
• CD133, EGFR, LIFR, NGFR, and Pgp were expressed only by minor tumor cell subpopulations.
• CD34, CD45, CD117, and Her-2/neu were constantly negative.
• Expression of GFAP was only weak or undetectable.
• Relationship between immunophenotype and chemosensitivity:
• Direct correlations were found between immunophenotypic markers and chemosensitivity: A2B5 vs CCNU, CD56 vs CDDP, %Pgp vs VCR, %NGFR vs DAU and TOPO.
• Inverse correlations were observed between EGFR vs TAX, CD133 vs DTIC, and LIFR vs DAU.
• Sensitivity of tumor cells to panel of chemotherapeutic agents, including:
• The most frequent in vitro responses were observed for TAX, followed by DTIC and TOPO.
• Approximately 50% of the tumors displayed high or partial chemosensitivity to CCNU or CDDP.
• All but 1 tumor were resistant to VP16 and only 2 displayed sensitivity to BCNU or VCR.
• Direct correlations were found between chemosensitivity to BCNU, CCNU, CDDP, DTIC, DAU, and TAX.
• Chemosensitivity/chemoresistance to CCNU correlated significantly with that to CDDP, DTIC, and TAX.
• Association was found between resistance to CDDP and TAX.
• Resistance to BCNU showed a correlation with resistance to DTIC and DAU.
• No significant association was found between chemosensitivities to BCNU and CCNU.

Conclusions

• Histopathologically identical GBM tumors displayed marked immunophenotypic heterogeneity.
• The expression of A2B5, CD56, NGFR, and Pgp appeared to be associated with chemoresistance, whereas, CD133, EGFR, and LIFR expression was characteristic of chemosensitive tumors.

Study strengths/ limitations

Strengths:

• Several observations are consistent with those of previous studies. For example, abundant glioma expression of nestin, vimentin, A2B5, CD56, as opposed to GFAP (absent from most gliomas) was found in several studies, including this one.
• The growth factor receptors (specifically EGFR, HER-2/neu, LIFR, NGFR) that were selected for analysis are consistent with those that have been documented in the literature to be present in a considerable percentage of glioma cases.
• Associations between immunophenotypic characteristics and chemoresistance observed in this study are more likely to reflect clinical reality because the MTT assay (as opposed to an in vitro chemosensitivity assay) was employed, which underestimates rather than overestimates chemosensitivity and, thus, may yield more consistent and reliable chemoresistance estimates.

Limitations:

• As a result of varying patient characteristics, expression of neural and non-neural differentiation markers among patients in Slovakia may differ from those found in the United States, which may limit applicability of results.
• Lack of information regarding how patients were selected and excluded.
• Objective did not clearly identify all end points of the study.
• Results are limited by the small number of patients included in the study.
• It remains to be clarified whether findings are applicable under in vivo conditions (whether tumor immunophenotype would correlate with in vivo response to chemotherapy).

Implications

• Flow cytometric analysis enables detection of multiple phenotypic markers on a single cell; therefore it allows for more complex analysis than traditional methods (eg, immunohistochemistry).
• Flow cytometric immunophenotypic analysis of GBM may predict chemoresponsiveness and help to identify patients who may potentially benefit from chemotherapy.