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J Transl MedJournal of Translational Medicine1479-5876BioMed Central London 1479-5876-3-111574828510.1186/1479-5876-3-11ResearchHLA class I and II genotype of the NCI-60 cell lines Adams Sharon 1sadams1@mail.cc.nih.govRobbins Fu-Meei 1FRobbins@mail.cc.nih.govChen Deborah 1dschen@mail.cc.nih.govWagage Devika 1DWagage@mail.cc.nih.govHolbeck Susan L 2holbecks@mail.nih.govMorse Herbert C III3hmorse@niaid.nih.govStroncek David 1DStroncek@mail.cc.nih.govMarincola Francesco M 1fmarincola@mail.cc.nih.gov1 Immunogenetics Section, Department of Transfusion Medicine, Clinical Center, National Institutes of Health, Bethesda, Maryland, 20892, USA2 Developmental Therapeutics Program, Information Technology Branch, National Cancer Institute, Bethesda, Maryland, 20892, USA3 Laboratory of Immunopathology, National Institute of Allergy and Infectious Diseases, Rockville, USA2005 4 3 2005 3 11 11 24 1 2005 4 3 2005 Copyright © 2005 Adams et al; licensee BioMed Central Ltd.2005Adams et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Sixty cancer cell lines have been extensively characterized and used by the National Cancer Institute's Developmental Therapeutics Program (NCI-60) since the early 90's as screening tools for anti-cancer drug development. An extensive database has been accumulated that could be used to select individual cells lines for specific experimental designs based on their global genetic and biological profile. However, information on the human leukocyte antigen (HLA) genotype of these cell lines is scant and mostly antiquated since it was derived from serological typing. We, therefore, re-typed the NCI-60 panel of cell lines by high-resolution sequence-based typing. This information may be used to: 1) identify and verify the identity of the same cell lines at various institutions; 2) check for possible contaminant cell lines in culture; 3) adopt individual cell lines for experiments in which knowledge of HLA molecule expression is relevant. Since genome-based typing does not guarantee actual surface protein expression, further characterization of relevant cell lines should be entertained to verify surface expression in experiments requiring correct antigen presentation.
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Background
A panel of sixty cancer cell lines of diverse lineage (lung, renal, colorectal, ovarian, breast, prostate, central nervous system, melanoma and hematological malignancies) was developed, characterized and extensively used by the National Cancer Institute's Developmental Therapeutics Program (NCI-60) since the early 90's as a screening tool for anti-cancer drug development [1]. This strategy [2-9]. yielded data about drug-related cytotoxicity for about 100,000 compounds. In addition, extensive functional characterization of the NCI-60 response to diverse biological or chemical stimulation has been accumulated [10-15]. Although originally developed for chemo-sensitivity testing, with the development of high-throughput analyses the NCI-60 panel has been broadly characterized for other biological applications [16-25]. Thus, patterns incidentally identified provided platforms for further investigations of mechanisms of tumorigenesis and cancer progression [5,6,26-30]. More recently, genomic DNA [24] and proteomics analyses have further characterized the profile of these cell lines [31]. The combined database provides the most comprehensive phenotyping of commonly accessible cancer cell lines offering correlative information about genetic, transcriptional and post-translational qualities. With growing interest in the identification of novel tumor antigens recognized by T cells as targets for antigen-specific immunization ([32], the NCI-60 could become an ideal tool for in silico discovery [33] ([34] and for tumor cell-specific T-cell reactivity testing [35]. For this purpose, accurate information about the extended human leukocyte antigen (HLA) phenotype of each cell line is necessary for the definition and validation of specific HLA/epitope combinations. Although antiquated and partial information about the HLA phenotype of some of the NCI-60 cell lines is available through the American Type Culture Collection (ATCC), Rockville, MD, no high-resolution information obtained by definitive sequence-based typing (SBT) has ever been published. Since T cell recognition of HLA-epitope complexes is narrowly restricted to unique combinations [36], this information is critical to select reasonable candidates for antigen-discovery choosing cell lines bearing HLA phenotypes most relevant to the disease population studied [37]. Accurate information about the HLA genotype of each cell line may, in addition, help their identification, validation and qualification among different laboratories excluding possible errors related to switching of cell lines or culture contamination. Therefore, we provide high-resolution SBT of the complete NCI-60 panel obtained from their original source: the National Cancer Institute's Developmental Therapeutics Program.

Results and Discussion
Previous knowledge of the HLA phenotype of NCI-60 cell lines
We reviewed and collected available information about the HLA phenotype of the NCI-60 cell lines, performed according to serological testing before submission to the ATCC (Table 1). The information was collected through the ATCC website: . Most cell lines had not been previously typed; the large majority of the cell lines from which such information is available had been developed from Caucasian patients. HLA typing was reported according to the old serologic nomenclature at a very low level of resolution. In addition, several reported typings did not match the present typing as shown in Table 2 and 3. This was the case for the colon carcinoma cell line HT29 that maintained a correct haplotype (with the exclusion of the HLA-Cw locus) but had a completely different second haplotype. The melanoma cell line SK-MEL-5 had an almost identical haplotype with the exception of one HLA-B allele originally typed as Bw16 (inclusive of the molecularly-defined alleles: B*38 and B*39), while the present typing was HLA-B*07. Another melanoma cell line SK-MEL-28 maintained a haplotype similar to the previously reported HLA-A11, -B40 but appeared to have lost an HLA-A allele (HLA-A26) compared with the original ATCC description. Finally, the multiple myeloma cell line RPMI 8226 was matched at one haplotype (HLA-A19, -B15 and -Cw2) but was totally discrepant at the second haplotype (HLA-A*6802, -B*1510 and -Cw*0304). The HLA typing of the other two previously typed cell lines was confirmed in the present study. Overall, in spite of the discrepancies in HLA typing observed between the previous and the present analyses, a resemblance was noted in the cell line genotype suggesting that mis-typing related to the low accuracy of serological methods might have been at the basis of the discrepancy rather than contamination or switching of the cell lines.

Table 1 Available information from the ATCC about the NCI-60 panel

Name	ATCC no.	Sex	Race	Tumor Type	ATCC HLA typing	Discrepant	
BT-549	HTB-122	F	C	Breast CA			
HS 578T	HTB-126	F	C	Breast CA			
MCF7	HTB-22	F	C	Breast CA			
MDA-MB-231	HTB-26	F	C	Breast CA			
MDA-MB-435	HTB-129	F	C	Breast CA			
T-47D	HTB-133	F		Breast CA			
SF-268				CNS CA			
SF-295				CNS CA			
SF-539				CNS CA			
SNB-19				CNS CA			
SNB-75				CNS CA			
U251				CNS CA			
COLO 205	CCL-222	M	C	Colon CA			
HCC-2998				Colon CA			
HCT-116	CCL-247	M		Colon CA			
HCT-15	CCL-225	M		Colon CA			
HT29	HTB-38	F	C	Colon CA	A1,3,B12,17 Cw5	Yes	
KM12				Colon CA			
SW-620	CCL-227	M		Colon CA			
MOLT-4	CRL-1582	M		Leukemia, ALL			
CCRF-CEM	CCL-119	F	C	Leukemia, ALL			
HL-60	CCL-240	F	C	Leukemia, APL			
K-562	CCL-243	F		Leukemia, CML			
SR	CRL-2262	M	C	Leukemia, LCIL			
LOX IMVI				Melanoma			
M 14				Melanoma			
SK-MEL-2	HTB-68	M	C	Melanoma			
SK-MEL-5	HTB-70	F	C	Melanoma	A2,11, B40,Bw16	Yes	
SK-MEL-28	HTB-72	M		Melanoma	A11,26, B40,DRw4	Yes	
UACC-62				Melanoma			
UACC-257				Melanoma			
RPMI 8226	CCL-155	M		MM	Aw19, B15,37, Cw2	Yes	
A549/ATCC	CCL-185	M	C	NSCLC			
EKVX				NSCLC			
HOP-62				NSCLC			
HOP-92				NSCLC			
NCI-H23	CRL-5800	M	AA	NSCLC			
NCI-H226	CRL-5826	M		NSCLC			
NCI-H322M				NSCLC			
NCI-H460	HTB-177	M		NSCLC			
NCI-H522	CRL-5810	M	C	NSCLC			
IGROV1				Ovarian CA			
OVCAR-3	HTB-161			Ovarian CA			
OVCAR-4				Ovarian CA			
OVCAR-5				Ovarian CA			
OVCAR-8				Ovarian CA			
NCI/ADR-RES				Ovarian CA			
SK-OV-3	HTB-77	F	C	Ovarian CA			
DU-145	HTB-81	M	C	Prostate CA			
PC-3	CRL-1435	M	C	Prostate CA	A1,9	No	
786-O				Renal CA			
A498	HTB-44	F		Renal CA			
ACHN	CRL-1611	M	C	Renal CA			
CAK1-1	HTB-46	M	C	Renal CA	A9,B12,35	No	
SN12C				Renal CA			
TK-10				Renal CA			
UO-31				Renal CA			
RXF-393				Renal CA			
AA = African American; ALL = Acute Lymphoblastic Leukemia; APL = Acute promyelocytic leukemia; C = Caucasian; CA = Carcinoma; CML = Chronic Myelogenous Leukemia; CNS = Central Nervous System; F = Female; LCIL = Large Cell Immunoblastic Lymphoma; M = Male; MM = Multiple Myeloma; NA = Not Available; NSCLC = Non Small Cell Lung Cancer.

The information about the ATCC cell lines (Cell Lines with ATCC no.) was obtained accessing the following URL: . Additional information was obtained through the National Cancer Institute's Developmental Therapeutics Program URL: .

Table 2 Sequence-based typing of NCI-60 HLA class I Loci

Cell Line	ID	Tissue	A locus	B Locus	Cw Locus	
BT-549	41292-D	Breast CA	N.R.	151701, 5501	030301, 07a	
HS 578T	41293-D	Breast CA	03a, 24a	35a, 40a	030401, 04a	
MCF7	41294-D	Breast CA	020101	18a, 44a	05a	
MDA-MB-231	41296-D	Breast CA	0201, 0217	4002, 4101	020202, 17a	
MDA-MB435	41297-D	Breast CA	110101, 240201	15a, 35a	030301, 04a	
T47D	41298-D	Breast CA	3301	1402	0802	
SF-268	41286-D	CNS CA	010101, 3201	0801, 4002	020202, 07a	
SF-295	41287-D	CNS CA	010101, 2601	070201, 5501	03a, 07a	
SF-539	41288-D	CNS CA	020101	08a, 35a	04a, 07a	
SNB-19	41289-D	CNS CA	020101	18a	05a	
SNB-75	41290-D	CNS CA	020101, 110101	35a, 39a	04a, 120301	
U251	41291-D	CNS CA	020101	18a	05a	
COLO 205	41299-D	Colon CA	01a, 02a	07a, 08a	070201, 07a	
HCC-2998	41300-D	Colon CA	02a, 24a	3701, 400601	04a, 0602	
HCT-116	41301-D	Colon CA	01a, 02a	18a, 4501	05a, 07a	
HCT-15	41302-D	Colon CA	02a, 24a	08new, 350101	04a, 07a	
HT29	41303-D	Colon CA	01a, 24a	35a, 440301	04a	
KM12	41304-D	Colon CA	02new	70201	70201	
SW-620	41305-D	Colon CA	02a, 24a	07a, 15a	070201, 07a	
MOLT 4	41281-D	Leukemia, ALL	010101, 2501	18a, 570101	0602, 120301	
CCRF-CEM	41282-D	Leukemia, ALL	N.R.	08a, 40a	030401, 07a	
HL-60	41284-D	Leukemia, APL	10101	570101	0602	
K-562	41280-D	Leukemia, CML	110101, 310102	18a, 40a	03a, N.R.	
SR	41285-D	Leukemia, LCIL	02a, 03a	3701, 3901	0602, 120301	
LOX IMVI	41315-D	Melanoma	110101, 2902	070201, 440301	070201, 1601	
M 14	41316-D	Melanoma	110101, 240201	15a, 35a	030301, 04a	
SK-MEL-2	41317-D	Melanoma	03a, 26a	35a, 38a	04a, 120301	
SK-MEL-5	41319-D	Melanoma	020101, 110101	07a, 40a	030401, 070201	
SK-MEL-28	41318-D	Melanoma	110101	4001	030401	
UACC-62	41321-D	Melanoma	02a, 32a	39a, 44a	05a, 12a	
UACC-257	41320-D	Melanoma	020101	18a, 44a	05a, 07a	
RPMI-8226	41283-D	MM	3001, 6802	1503, 1510	020204, 030402	
A549/ATCC	41306-D	NSCLC	2501, 3001	18a, 440301	120301, 1601	
EKVX	41307-D	NSCLC	010101	3701	0602	
HOP-62	41308-D	NSCLC	030101	07a, 44a	05a, 070201	
HOP-92	41309-D	NSCLC	03a, 24a	27a, 470101	01a, 06a	
NCI-H23	41312-D	NSCLC	8001	5001	0602	
NCI-H226	41311-D	NSCLC	010101, 240201	07a, 39a	070201, 120301	
NCI-H322M	41310-D	NSCLC	2902	440301	1601	
NCI-H460	41313-D	NSCLC	24a, 68a	35a, 51a	03a, 15a	
NCI-H522	41314-D	NSCLC	020101	44a, 5501	030301, 05a	
IGROV1	41322-D	Ovarian CA	240201, 3301	4901	07a	
OVCAR-3	41323-D	Ovarian CA	020101, 2902	070201, 5801	070201, 07a	
OVCAR-4	41324-D	Ovarian CA	010101, 3201	0801, 4002	07a, 15a	
OVCAR-5	41325-D	Ovarian CA	01a, 02a	08a, 44a	05a, 07a	
OVCAR-8	41326-D	Ovarian CA	010101, 2501	570101	0602	
NCI/ADR-RES	41295-D	Ovarian CA	010101, 2501	570101	0602	
SK-OV-3	41327-D	Ovarian CA	03a, 68a	18a, 35a	04a, 05a	
DU-145	41328-D	Prostate CA	030101, 3303	5001, 570101	0602	
PC-3	41329-D	Prostate CA	010101, 240201	1302, 5501	01a, 06a	
786-O	41330-D	Renal CA	030101	07a, 44a	05a, 070201	
A498	41331-D	Renal CA	020101	0801	07a	
ACHN	41332-D	Renal CA	2601	4901	07a	
CAKI-1	41333-D	Renal CA	2301, 240201	3502, 440301	04a, 04new	
SN12C	41334-D	Renal CA	03, 24new	07a, 44a	05a, 070201	
TK-10	41335-D	Renal CA	3301	1402	0802	
UO-31	41336-D	Renal CA	010101, 030101	07a, 14a	07a, 08a	
RXF-393	41337-D	Renal CA	02a, 24a	1401, 44a	05a, 0802	
Sequence-based typing for the HLA class I loci are reported with the highest degree of resolution. Non-resolved ambiguities are reported as two digit denominations with a superscript a as previously described 43. HLA typings divergent from those originally described in the ATCC database are reported in red. ID# refers to the HLA laboratory reference number. New alleles are indicated by the suffix new following the allele. N.R. – Ambiguity not resolved at the lower level of resolution.

Table 3 Sequence-based typing of NCI-60 HLA class II Loci

Cell Line	ID	Tissue	DRβ1 Locus	DQB1 Locus	DPB1 Locus	
BT-549	41292-D	Breast CA	11a, 13a	030101, 060401	020102, 0401	
HS 578T	41293-D	Breast CA	01a, 150101	050101, 0602	0401, 7801	
MCF7	41294-D	Breast CA	03a, 15a	0201, 0602	020102, 0401	
MDA-MB-231	41296-D	Breast CA	0701, 1305	0202, 030101	020102, 1701	
MDA-MB435	41297-D	Breast CA	040501, 130101	0302, 0603	1301, 1901	
T47D	41298-D	Breast CA	010201	050101	020102, 0401	
SF-268	41286-D	CNS CA	03a, 04a	0201, 0302	0401, 0601	
SF-295	41287-D	CNS CA	14a, 15a	050301, 0602	0401	
SF-539	41288-D	CNS CA	030101, 12a	0201, 030101	010101, 0401	
SNB-19	41289-D	CNS CA	030101	0201	0402	
SNB-75	41290-D	CNS CA	0103, 11a	03a, 050101	0401, 0402	
U251	41291-D	CNS CA	030101	0201	0402	
COLO 205	41299-D	Colon CA	040101, 130101	0603	0401	
HCC-2998	41300-D	Colon CA	11a, 16a	030101, 050201	0401	
HCT-116	41301-D	Colon CA	N.R.	02new, 03new	030101, 0402	
HCT-15	41302-D	Colon CA	03a, 14a	02a, 050301	010101, 0401	
HT29	41303-D	Colon CA	0402, 0701	02a, 0302	0401	
KM12	41304-D	Colon CA	040101	0302	1301	
SW-620	41305-D	Colon CA	0103, 130101	050101, 0603	010101, 0401	
MOLT 4	41281-D	Leukemia, ALL	07new, 12new	0202, 030101	20102	
CCRF-CEM	41282-D	Leukemia, ALL	030101, 0701	0201, 0202	0401, 1301	
HL-60	41284-D	Leukemia, APL	N.R.	030302	0401, 1301	
K-562	41280-D	Leukemia, CML	03a, 04a	0201, 0302	0401, 0402	
SR	41285-D	Leukemia, LCIL	01a, 160101	050101, 050201	0401	
LOX IMVI	41315-D	Melanoma	0701, 150101	0202, 0602	0401, 110101	
M 14	41316-D	Melanoma	040501, 130101	0302, 0603	1301, 1901	
SK-MEL-2	41317-D	Melanoma	0402, 130101	030101, 0603	020102, 0401	
SK-MEL-5	41319-D	Melanoma	040101, 130101	0302, 0603	030101, 1601	
SK-MEL-28	41318-D	Melanoma	0404	0302	030101	
UACC-62	41321-D	Melanoma	12a, 130101	030101, 0603	0401, 1401	
UACC-257	41320-D	Melanoma	040101	030101, 0302	0401	
RPMI-8226	41283-D	MM	030101, 0701	0201, 0202	010102, 1301	
A549/ATCC	41306-D	NSCLC	0701, 110401	0202, 030101	N.R.	
EKVX	41307-D	NSCLC	150101	0602	0401	
HOP-62	41308-D	NSCLC	13a, 15a	06a, 06a	0402	
HOP-92	41309-D	NSCLC	01a, 150101	050101, 0602	0401, 0402	
NCI-H23	41312-D	NSCLC	130101	0603	1901	
NCI-H226	41311-D	NSCLC	150101, 160101	050201, 0602	020102, 0401	
NCI-H322M	41310-D	NSCLC	0701	0202	0401	
NCI-H460	41313-D	NSCLC	01a, 04a	030101, 050101	N.R.	
NCI-H522	41314-D	NSCLC	040101, 150101	03a, 0602	0401	
IGROV1	41322-D	Ovarian CA	11a, 11a	03new	new, 0501	
OVCAR-3	41323-D	Ovarian CA	080101, 080401	0402	020102, 0401	
OVCAR-4	41324-D	Ovarian CA	030101, 040101	0201, 030101	0401, 1301	
OVCAR-5	41325-D	Ovarian CA	030101, 040101	0201, 030101	0401	
OVCAR-8	41326-D	Ovarian CA	0701, 150101	030302, 0602	020102, 1301	
NCI/ADR-RES	41295-D	Ovarian CA	0701, 150101	030302, 0602	020102, 1301	
SK-OV-3	41327-D	Ovarian CA	01a, 030101	0201, 050101	020102, 0401	
DU-145	41328-D	Prostate CA	N.R.	030302, 050101	0401	
PC-3	41329-D	Prostate CA	0701, 130101	0202, 0603	0401	
786-O	41330-D	Renal CA	13a, 15a	06a, 06a	0402	
A498	41331-D	Renal CA	030101	0201	010101	
ACHN	41332-D	Renal CA	160101	050201	020102	
CAKI-1	41333-D	Renal CA	0701, 110401	0202, 03a	020102, 1001	
SN12C	41334-D	Renal CA	040101, 150101	03a, 0602	N.R.	
TK-10	41335-D	Renal CA	010201	050101	0402	
UO-31	41336-D	Renal CA	130201, 150101	0602, 0609	0402, 0501	
RXF-393	41337-D	Renal CA	110101, 150101	030101, 0602	010101, 0401	
Sequence-based typing for the HLA class II loci are reported with the highest degree of resolution. Non-resolved ambiguities are reported as two digit denominations with a superscript a as previously described [43]. HLA typings divergent from those originally described in the ATCC database are reported in red. ID# refers to the HLA laboratory reference number. New alleles are indicated by the suffix new following the allele. N.R. = Ambiguity not resolved at the lower level of resolution.

Overall, there was no evidence of contamination among the cell lines tested with clean homozygous or heterozygous combinations observed in all loci analyzed. SBT of HLA class I and HLA class II loci are reported in Table 2 and 3 respectively. Information about the HLA typing of the cell lines is also available through the Molecular Targets URL: . Approximately 17% of the cell lines (10 out of 58 including: T47D, SNB-19, U251, KM12, RPMI-8226, EKVX, NCI-H23, NCI-H322M, A498, ACHN and TK-10) exhibited a pseudo-homozygous pattern suggestive of complete loss of heterozygosity encompassing the HLA class I and HLA class II regions. This frequency is close to the loss of haplotype that we originally described for melanoma cell lines generated at the National Cancer Institute (Bethesda, MD) [38,39] and subsequently observed in other cancers [40,41]. We conclude that this is an unlikely representative of patients' homozygosity because complete HLA class I and II homozygosity is exceedingly rare in the population at large. To corroborate this statement, we analyzed 554 genomic DNA specimens from normal donors recently typed with the same technology in our laboratory. Genomic DNA for the normal donors was obtained from whole blood samples. Only 5 individuals were found to be truly homozygous for all HLA class I and class II loci for a frequency of 0.9%.

Overall, discrepancies between ATCC typings and the present typing or the unbalanced frequency of homozygosity could be related to accumulated genetic alterations between the cell lines since the time of their original expansion from the patient and should not be surprising.

A particular case was represented by the NCI/ADR-RES cell line which was previously believed to be an adriamycin derivative of the breast cancer cell line MCF-7. Subsequently, it was discovered not to be related to MCF-7, but it's derivation was unclear [42]. Karyotyping analysis suggested it was related to the ovarian cell line OVCAR-8. Subsequent DNA fingerprinting confirmed that both cell lines were generated from the same individual. HLA genotyping confirms this since the cell lines are indeed identical.

To avoid possible misinterpretations, a large number of alleles are not presented here with their definitive nomenclature but rather at a two digits level of resolution because some of the ambiguities could not be completely resolved by SBT as previously described [43]. However, more detailed information about individual cell lines can be obtained by contacting Sharon Adams directly at the HLA laboratory, Department of Transfusion Medicine, Bethesda, MD. As previously described [43], it is possible to resolve most of these ambiguities using various methods including sequence-specific primer PCR or pyro-sequencing [44]. If necessary in the future, the NIH HLA laboratory may assist in further characterization of individual HLA alleles. Another caveat is that the identification of HLA alleles at the genomic level does not necessarily correspond to surface expression of their protein products since various abnormalities in transcription, translation and assembling could influence the surface expression of HLA molecules [39,45,46].

Finally, several new alleles were identified (referred to in the tables as new, for which a nomenclature is pending; in detail KM12 HLA-A*02new = Genebank Accession # AY918166; SN12C HLA-A*24new = # AY918167; CAKI-1 HLA-Cw04new = # AY918170). Information regarding the sequence of these alleles could be obtained by directly contacting the HLA laboratory, Department of Transfusion Medicine, Bethesda, MD.

Materials and Methods
Cell Lines
Genomic DNA from the NCI-60 cell line anticancer drug discovery panel was obtained from SH of the National Cancer Institute Developmental Therapeutics Program (Bethesda, MD). Cells were grown in RPMI 1640 supplemented with 10% fetal bovine serum and 5 mM L-glutamine.

DNA Isolation
Genomic DNA was isolated from peripheral blood using the Gentra PUREGENE isolation kit (Gentra Systems, Minneapolis, MN, USA). The DNA was re-suspended in Tris HCl buffer (pH 8.5) and the concentration was measured using a Pharmacia Gene Quant II Spectrophotometer. The DNA was then stored at -70°C until testing.

Sequence-Based Typing (SBT)
HLA class I loci sequence-based typing (SBT) was performed as previously described ([43]. The primary PCR amplification reaction produced a 1.5 kb amplicon encompassing exon 1 through intron 3 of the HLA class I locus. All reagents necessary for primary amplification and sequencing were included in the HLA-A, HLA-B and HLA-C alleleSEQR Sequenced Based Typing Kits (Atria Genetics, Hayward, CA, U.S.A.). The primary amplification PCR products were purified from excess primers, dNTPs and genomic DNA using ExoSAP-IT (American Life Science, Cleveland, OH, U.S.A.). Each template was sequenced in the forward and reverse sequence orientation for exon 2 and exon 3 according to protocols supplied with the SBT kits. Excess dye terminators were removed from the sequencing products utilizing an ethanol precipitation method with absolute ethanol. The reaction products were reconstituted with 15 μl of Hi-Di™ Formamide (PE Applied Biosystems / Perkin-Elmer, Foster City, CA, U.S.A.) and analyzed on the ABI Prism* 3700 DNA Analyzer with Dye Set file: Z and mobility file: DT3700POP6 [ET].
==== Refs
Shoemaker RH Monks A Alley MC Scudiero DA Fine DL McLemore TL  Development of human tumor cell line panels for use in disease-oriented drug screening Prog Clin Biol Res 1988 276 265 286 3051021 
Monks A Scudiero D Skehan P Shoemaker R Paull K Vistica D  Feasibility of a high-flux anticancer drug screen using a diverse panel of cultured human tumor cell lines J Natl Cancer Inst 1991 83 757 766 2041050 
Grever MR Schepartz SA Chabner BA  The National Cancer Institute: cancer drug discovery and development program Semin Oncol 1992 19 622 638 1462164 
Stinson SF Alley MC Kopp WC Fiebig HH Mullendore LA Pittman AF  Morphological and immunocytochemical characteristics of human tumor cell lines for use in a disease-oriented anticancer drug screen Anticancer Res 1992 12 1035 1053 1503399 
Monks A Scudiero DA Johnson GS Paull KD Sausville EA  The NCI anti-cancer drug screen: a smart screen to identify effectors of novel targets Anticancer Drug Des 1997 12 533 541 9365500 
Weinstein JN Myers TG O'Connor PM Friend SH Fornace AJ JrKohn KW  An information-intensive approach to the molecular pharmacology of cancer Science 1997 275 343 349 8994024 10.1126/science.275.5298.343 
Gmeiner WH Skradis A Pon RT Liu J  Cytotoxicity and in-vivo tolerance of FdUMP[10]: a novel pro-drug of the TS inhibitory nucleotide FdUMP Nucleosides Nucleotides 1999 18 1729 1730 10474257 
Wells G Seaton A Stevens MF  Structural studies on bioactive compounds. 32. Oxidation of tyrphostin protein tyrosine kinase inhibitors with hypervalent iodine reagents J Med Chem 2000 43 1550 1562 10780912 10.1021/jm990947f 
Voeller DM Grem JL Pommier Y Paull K Allegra CJ  Identification and proposed mechanism of action of thymidine kinase inhibition associated with cellular exposure to camptothecin analogs Cancer Chemother Pharmacol 2000 45 409 416 10803925 10.1007/s002800051010 
Fong WG Liston P Rajcan-Separovic E St Jean M Craig C Korneluk RG  Expression and genetic analysis of XIAP-associated factor 1 (XAF1) in cancer cell lines Genomics 2000 70 113 122 11087668 10.1006/geno.2000.6364 
Salomon AR Voehringer DW Herzenberg LA Khosla C  Apoptolidin, a selective cytotoxic agent, is an inhibitor of F0F1-ATPase Chem Biol 2001 8 71 80 11182320 10.1016/S1074-5521(00)00057-0 
Johnson JI Decker S Zaharevitz D Rubinstein LV Venditti JM Schepartz S  Relationships between drug activity in NCI preclinical in vitro and in vivo models and early clinical trials Br J Cancer 2001 84 1424 1431 11355958 10.1054/bjoc.2001.1796 
Segraves NL Robinson SJ Garcia D Said SA Fu X Schmitz FJ  Comparison of fascaplysin and related alkaloids: a study of structures, cytotoxicities, and sources J Nat Prod 2004 67 783 792 15165138 10.1021/np049935+ 
Ross DD Doyle LA  Mining our ABCs: pharmacogenomic approach for evaluating transporter function in cancer drug resistance Cancer Cell 2004 6 105 107 15324691 10.1016/j.ccr.2004.08.003 
Szakacs G Annereau JP Lababidi S Shankavaram U Arciello A Bussey KJ  Predicting drug sensitivity and resistance: profiling ABC transporter genes in cancer cells Cancer Cell 2004 6 129 137 15324696 10.1016/j.ccr.2004.06.026 
Staunton JE Slonim DK Coller HA Tamayo P Angelo MJ Park J  Chemosensitivity prediction by transcriptional profiling Proc Natl Acad Sci U S A 2001 98 10787 10792 11553813 10.1073/pnas.191368598 
Weinstein JN  Pharmacogenomics. Teaching old drugs new tricks N Engl J Med 2002 343 1408 1409 11070107 10.1056/NEJM200011093431910 
Weinstein JN  Searching for pharmacogenomics markers: the synergy between omic and hypothesis-driven research Dis Markers 2002 17 77 88 11673654 
Weinstein JN Scherf U Lee JK Nishizuka S Gwadry F Bussey AK  The bioinformatics of microarray gene expression profiling Cytometry 2002 47 46 49 11774349 10.1002/cyto.10041 
Kuo WP Jenssen TK Butte AJ Ohno-Machado L Kohane IS  Analysis of matched mRNA measurements from two different microarray technologies Bioinformatics 2002 18 405 412 11934739 10.1093/bioinformatics/18.3.405 
Blower PE Yang C Fligner MA Verducci JS Yu L Richman S  Pharmacogenomic analysis: correlating molecular substructure classes with microarray gene expression data Pharmacogenomics J 2002 2 259 271 12196914 10.1038/sj.tpj.6500116 
Le QT Sutphin PD Raychaudhuri S Yu SC Terris DJ Lin HS  Identification of osteopontin as a prognostic plasma marker for head and neck squamous cell carcinomas Clin Cancer Res 2003 9 59 67 12538452 
Lee JK Bussey KJ Gwadry FG Reinhold W Riddick G Pelletier SL  Comparing cDNA and oligonucleotide array data: concordance of gene expression across platforms for the NCI-60 cancer cells Genome Biol 2003 4 R82 14659019 10.1186/gb-2003-4-12-r82 
Roschke AV Tonon G Gehlhaus KS McTyre N Bussey KJ Lababidi S  Karyotypic complexity of the NCI-60 drug-screening panel Cancer Res 2003 63 8634 8647 14695175 
Weinstein JN Pommier Y  Transcriptomic analysis of the NCI-60 cancer cell lines C R Biol 2003 326 909 920 14744097 
Weinstein JN Kohn KW Grever MR Viswanadhan VN Rubinstein LV Monks AP  Neural computing in cancer drug development: predicting mechanism of action Science 1992 258 447 451 1411538 
Bates SE Fojo AT Weinstein JN Myers TG Alvarez M Pauli KD  Molecular targets in the National Cancer Institute drug screen J Cancer Res Clin Oncol 1995 121 495 500 7559726 10.1007/BF01197759 
O'Connor PM Jackman J Bae I Myers TG Fan S Mutoh M  Characterization of the p53 tumor suppressor pathway in cell lines of the National Cancer Institute anticancer drug screen and correlations with the growth-inhibitory potency of 123 anticancer agents Cancer Res 1997 57 4285 4300 9331090 
Ross DT Scherf U Eisen MB Perou CM Rees C Spellman P  Systematic variation in gene expression patterns in human cancer cell lines Nat Genet 2000 24 227 235 10700174 10.1038/73432 
Scherf U Ross DT Waltham M Smith LH Lee JK Tanabe L  A gene expression database for the molecular pharmacology of cancer Nat Genet 2000 24 236 244 10700175 10.1038/73439 
Nishizuka S Charboneau L Young L Major S Reinhold WC Waltham M  Proteomic profiling of the NCI-60 cancer cell lines using new high-density reverse-phase lysate microarrays Proc Natl Acad Sci U S A 2003 100 14229 14234 14623978 10.1073/pnas.2331323100 
Renkvist N Castelli C Robbins PF Parmiani G  A listing of human tumor antigens recognized by T cells Cancer Immunol Immunother 2001 50 3 15 11315507 10.1007/s002620000169 
Vinals C Gaulis S Coche T  Using in silico transcriptomics to search for tumor-associated antigens for immunotherapy Vaccine 2001 19 2607 2614 11257399 10.1016/S0264-410X(00)00500-4 
Inozume T Matsuzaki Y Kurihara S Fujita T Yamamoto A Aburatani H  Novel melanoma antigen, FCRL/FREB, identified by cDNA profile comparison using DNA chip are immunogenic in multiple melanoma patients Int J Cancer 2004 
Fang X Shao L Zhang H Wang S  Web-based tools for mining the NCI databases for anticancer drug discovery J Chem Inf Comput Sci 2004 44 249 257 14741034 10.1021/ci034209i 
Bettinotti M Kim CJ Lee K-H Roden M Cormier JN Panelli MC  Stringent allele/epitope requirements for MART-1/Melan A immunodominance: implications for peptide-based immunotherapy J Immunol 1998 161 877 889 9670966 
Kim CJ Parkinson DR Marincola FM  Immunodominance across the HLA polymorphism: implications for cancer immunotherapy J Immunother 1998 21 1 16 9456431 
Marincola FM Shamamian P Alexander RB Gnarra JR Turetskaya RL Nedospasov SA  Loss of HLA haplotype and B locus down-regulation in melanoma cell lines J Immunol 1994 153 1225 1237 8027550 
Ferrone S Marincola FM  Loss of HLA class I antigens by melanoma cells: molecular mechanisms, functional significance and clinical relevance Immunol Today 1995 16 487 494 7576053 10.1016/0167-5699(95)80033-6 
Garrido F Cabrera T Accola RS Bensa JC Bodmer WF Dohr G  Charron D  HLA and cancer: 12th International Histocompatibility Workshop study Genetic diversitity of HLA Functional and medical implications 1997 Sevres, France: EDK 445 452 
Koopman LA Corver WE van der Slik AR Giphart MJ Fleuren GJ  Multiple genetic alterations cause a frequent and heterogeneous human histocompatibility leukocyte antigen class I loss in cervical cancer J Exp Med 2000 191 961 976 10727458 10.1084/jem.191.6.961 
Scudiero DA Monks A Sausville EA  Cell line designation change: multidrug-resistant cell line in the NCI anticancer screen J Natl Cancer Inst 1998 90 862 9625176 
Adams SD Barracchini KC Chen D Robbins F Wang L Larsen P  Ambiguous allele combinations in HLA Class I and Class II sequence-based typing: when precise nucleotide sequencing leads to imprecise allele identification J Transl Med 2004 2 30 15363110 10.1186/1479-5876-2-30 
Ramon D Braden M Adams S Marincola FM Wang L  Pyrosequencing trade mark : A one-step method for high resolution HLA typing J Transl Med 2003 1 9 14641938 10.1186/1479-5876-1-9 
Hicklin DJ Marincola FM Ferrone S  HLA class I antigen downregulation in human cancers: T-cell immunotherapy revives an old story Mol Med Today 1999 5 178 186 10203751 10.1016/S1357-4310(99)01451-3 
Wang Z Marincola FM Rivoltini L Parmiani G Ferrone S  Selective human leukocyte antigen (HLA)-A2 loss caused by aberrant pre-mRNA splicing in 624MEL28 melanoma cells J Exp Med 1999 190 205 215 10432284 10.1084/jem.190.2.205

