Validation of a HLA-A2 tetramer flow cytometric method, IFNgamma real time RT-PCR, and IFNgamma ELISPOT for detection of immunologic response to gp100 and MelanA/MART-1 in melanoma patients

Table 10 Immunologic response was detected in melanoma patients after vaccination

(A) IFNγ real time RT-PCR
Patient	gp100 ₂₀₉	gp100 _209/210M	gp100 _pool	MART-1	HIV
1	76.9	138.1	26.2	2.5	1
2	3.2	3.4	6.1	0.8	1
3	8.5	12.5	4.5	4.5	1
HLA-A2 PBMC from three melanoma patients known to have a positive clinical response was analyzed for IFNγ response by real time RT-PCR. IFNγ response fold increase over HIV, (IFNγ _peptide/CD8)/(IFNγ _HIV/CD8), is shown.
(B) IFNγ ELISPOT
Patient	gp100 ₂₀₉	gp100 _209/210M	gp100 _pool	MART-1	HIV
1	56	65	26	0	2
2	50	62	39	0	16
3	11	16	5	0	1
HLA-A2 PBMC from three melanoma patients known to have a positive clinical response was analyzed for IFNγ response by ELISPOT. IFNγ secreting cells (per well, average value from triplicate wells) are shown.
(C) Positive MART-1 response was seen in PBMC from a melanoma patient evaluated in all three validated assays.
Method	ELISPOT	Real Time RT-PCR	Tetramer Assay
IFNγ secreting cells	IFNγ copy number fold increase	% MART-1 tetramer positive cells
Baseline (Pre, 11/29/00)	0	ND	0.5
Post-Vaccine (Post-1 ^st dose, 12/20/00)	14	5.7	1.2
Study Completion (Post 6 ^th dose, 4/18/01)	7	4.2	2.4
Follow Up (8/15/01)	73	57.6	4.1

ND, not determined (insufficient cells for this analysis).
ELISPOT data is presented as the number of secreting cells after HIV background subtraction. Real time RT-PCR data is shown as copy number fold increase (over HIV) with CD8 as internal controls. Tetramer data is presented as % positive cells with negative tetramer background subtracted.

ISSN: 1479-5876