T cell receptors employ diverse strategies to target a p53 cancer neoantigen

Abstract

Adoptive cell therapy with tumor-specific T cells can mediate durable cancer regression. The prime target of tumor-specific T cells are neoantigens arising from mutations in self-proteins during malignant transformation. To understand T cell recognition of cancer neoantigens at the atomic level, we studied oligoclonal T cell receptors (TCRs) that recognize a neoepitope arising from a driver mutation in the p53 oncogene (p53R175H) presented by the major histocompatibility complex class I molecule HLA-A2. We previously reported the structures of three p53R175H-specific TCRs (38-10, 12-6, and 1a2) bound to p53R175H and HLA-A2. The structures showed that these TCRs discriminate between WT and mutant p53 by forming extensive interactions with the R175H mutation. Here, we report the structure of a fourth p53R175H-specific TCR (6-11) in complex with p53R175H and HLA-A2. In contrast to 38-10, 12-6, and 1a2, TCR 6-11 makes no direct contacts with the R175H mutation, yet is still able to distinguish mutant from WT p53. Structure-based in silico mutagenesis revealed that the 60-fold loss in 6-11 binding affinity for WT p53 compared to p53R175H is mainly due to the higher energetic cost of desolvating R175 in the WT p53 peptide during complex formation than H175 in the mutant. This indirect strategy for preferential neoantigen recognition by 6-11 is fundamentally different from the direct strategies employed by other TCRs and highlights the multiplicity of solutions to recognizing p53R175H with sufficient selectivity to mediate T cell killing of tumor but not normal cells. Adoptive cell therapy with tumor-specific T cells can mediate durable cancer regression. The prime target of tumor-specific T cells are neoantigens arising from mutations in self-proteins during malignant transformation. To understand T cell recognition of cancer neoantigens at the atomic level, we studied oligoclonal T cell receptors (TCRs) that recognize a neoepitope arising from a driver mutation in the p53 oncogene (p53R175H) presented by the major histocompatibility complex class I molecule HLA-A2. We previously reported the structures of three p53R175H-specific TCRs (38-10, 12-6, and 1a2) bound to p53R175H and HLA-A2. The structures showed that these TCRs discriminate between WT and mutant p53 by forming extensive interactions with the R175H mutation. Here, we report the structure of a fourth p53R175H-specific TCR (6-11) in complex with p53R175H and HLA-A2. In contrast to 38-10, 12-6, and 1a2, TCR 6-11 makes no direct contacts with the R175H mutation, yet is still able to distinguish mutant from WT p53. Structure-based in silico mutagenesis revealed that the 60-fold loss in 6-11 binding affinity for WT p53 compared to p53R175H is mainly due to the higher energetic cost of desolvating R175 in the WT p53 peptide during complex formation than H175 in the mutant. This indirect strategy for preferential neoantigen recognition by 6-11 is fundamentally different from the direct strategies employed by other TCRs and highlights the multiplicity of solutions to recognizing p53R175H with sufficient selectivity to mediate T cell killing of tumor but not normal cells. Adoptive cell therapy (ACT) with tumor-specific T cells can promote durable regression of diverse cancers, including metastatic melanoma, colon, bile duct, cervix, and breast cancers (1Rosenberg S.A. Restifo N.P. Adoptive cell transfer as personalized immunotherapy for human cancer.Science. 2015; 348: 62-68Google Scholar, 2Tran E. Robbins P.F. Lu Y.C. Prickett T.D. Gartner J.J. Jia L. Pasetto A. Zheng Z. Ray S. Groh E.M. Kriley I.R. Rosenberg S.A. T-cell transfer therapy targeting mutant KRAS in cancer.N. Engl. J. Med. 2016; 375: 2255-2262Google Scholar, 3Zacharakis N. Chinnasamy H. Black M. Xu H. Lu Y.C. Zheng Z. Pasetto A. Langhan M. Shelton T. Prickett T. Gartner J. Jia L. Trebska-McGowan K. Somerville R.P. Robbins P.F. et al.Immune recognition of somatic mutations leading to complete durable regression in metastatic breast cancer.Nat. Med. 2018; 24: 724-730Google Scholar, 4Stevanović S. Pasetto A. Helman S.R. Gartner J.J. Prickett T.D. Howie B. Robins H.S. Robbins P.F. Klebanoff C.A. Rosenberg S.A. Hinrichs C.S. Landscape of immunogenic tumor antigens in successful immunotherapy of virally induced epithelial cancer.Science. 2017; 356: 200-205Google Scholar, 5Tran E. Turcotte S. Gros A. Robbins P.F. Lu Y.C. Dudley M.E. Wunderlich J.R. Somerville R.P. Hogan K. Hinrichs C.S. Parkhurst M.R. Yang J.C. Rosenberg S.A. Cancer immunotherapy based on mutation-specific CD4+ T cells in a patient with epithelial cancer.Science. 2014; 344: 641-645Google Scholar). The therapeutic effect of these tumor-infiltrating lymphocytes (TILs) is mediated primarily by cytotoxic CD8+ T cells (6Dudley M.E. Gross C.A. Somerville R.P. Hong Y. Schaub N.P. Rosati S.F. White D.E. Nathan D. Restifo N.P. Steinberg S.M. Wunderlich J.R. Kammula U.S. Sherry R.M. Yang J.C. Phan G.Q. et al.Randomized selection design trial evaluating CD8+-enriched versus unselected tumor-infiltrating lymphocytes for adoptive cell therapy for patients with melanoma.J. Clin. Oncol. 2013; 31: 2152-2159Google Scholar). The main target of tumor-specific T cells are neoantigens that result from DNA alterations during malignant transformation (7Schumacher T.N. Scheper W. Kvistborg P. Cancer neoantigens.Annu. Rev. Immunol. 2019; 37: 173-200Google Scholar). Of special interest are neoantigens derived from oncogenes bearing driver mutations because these mutations are tumor-specific, important for tumor progression, and generally expressed by all tumor cells (8McGranahan N. Swanton C. Neoantigen quality, not quantity.Sci. Transl. Med. 2019; 11eaax7918Google Scholar). In a pioneering study of ACT, a patient with metastatic colorectal cancer was treated successfully with four ex vivo-expanded CD8+ T cell clones specific for a neoepitope arising from the G12D driver mutation in the KRAS oncogene (2Tran E. Robbins P.F. Lu Y.C. Prickett T.D. Gartner J.J. Jia L. Pasetto A. Zheng Z. Ray S. Groh E.M. Kriley I.R. Rosenberg S.A. T-cell transfer therapy targeting mutant KRAS in cancer.N. Engl. J. Med. 2016; 375: 2255-2262Google Scholar, 9Sim M.J.W. Lu J. Spencer M. Hopkins F. Tran E. Rosenberg S.A. Long E.O. Sun P.D. High-affinity oligoclonal TCRs define effective adoptive T cell therapy targeting mutant KRAS-G12D.Proc. Natl. Acad. Sci. U. S. A. 2020; 117: 12826-12835Google Scholar). TP53 (tumor protein p53) was the first tumor suppressor gene identified and is inactivated in the large majority of human cancers (10Garraway L.A. Lander E.S. Lessons from the cancer genome.Cell. 2013; 153: 17-37Google Scholar, 11Gerstung M. Jolly C. Leshchiner I. Dentro S.C. Gonzalez S. Rosebrock D. Mitchell T.J. Rubanova Y. Anur P. Yu K. Tarabichi M. Deshwar A. Wintersinger J. Kleinheinz K. Vázquez-García I. et al.The evolutionary history of 2,658 cancers.Nature. 2020; 578: 122-128Google Scholar). Mutations in TP53 effect most of the hallmarks of cancer cells, including proliferation, genomic instability, and metastasis (12Gao J. Aksoy B.A. Dogrusoz U. Dresdner G. Gross B. Sumer S.O. Sun Y. Jacobsen A. Sinha R. Larsson E. Cerami E. Sander C. Schultz N. Integrative analysis of complex cancer genomics and clinical profiles using the cBioPortal.Sci. Signal. 2013; 6pl1Google Scholar, 13Zehir A. Benayed R. Shah R.H. Syed A. Middha S. Kim H.R. Srinivasan P. Gao J. Chakravarty D. Devlin S.M. Hellmann M.D. Barron D.A. Schram A.M. Hameed M. Dogan S. et al.Mutational landscape of metastatic cancer revealed from prospective clinical sequencing of 10,000 patients.Nat. Med. 2017; 23: 703-713Google Scholar). Hotspot positions include R175, G245, R248, R273, and R282, which cluster in the central DNA-binding domain of p53 and alter its DNA-binding properties (14Sabapathy K. Lane D.P. Therapeutic targeting of p53: All mutants are equal, but some mutants are more equal than others.Nat. Rev. Clin. Oncol. 2018; 15: 13-30Google Scholar). Mutations at these sites are attractive candidates for targeted immunotherapy because they confer a growth advantage to tumor cells and are associated with malignant progression. The immunogenicity of p53 mutations in cancer patients has been demonstrated by the detection of T cell responses against several p53 neoantigens, most notably R175H in which arginine at position 175 is replaced by histidine (15Malekzadeh P. Pasetto A. Robbins P.F. Parkhurst M.R. Paria B.C. Jia L. Gartner J.J. Hill V. Yu Z. Restifo N.P. Sachs A. Tran E. Lo W. Somerville R.P. Rosenberg S.A. et al.Neoantigen screening identifies broad TP53 mutant immunogenicity in patients with epithelial cancers.J. Clin. Invest. 2019; 129: 1109-1114Google Scholar, 16Lo W. Parkhurst M. Robbins P.F. Tran E. Lu Y.C. Jia L. Gartner J.J. Pasetto A. Deniger D. Malekzadeh P. Shelton T.E. Prickett T. Ray S. Kivitz S. Paria B.C. et al.Immunologic recognition of a shared p53 mutated neoantigen in a patient with metastatic colorectal cancer.Cancer Immunol. Res. 2019; 7: 534-543Google Scholar). This driver mutation is the most frequently observed mutation in TP53 as well as the most common mutation in any tumor suppressor gene (17Grossman R.L. Heath A.P. Ferretti V. Varmus H.E. Lowy D.R. Kibbe W.A. Staudt L.M. Toward a shared vision for cancer genomic data.N. Engl. J. Med. 2016; 375: 1109-1112Google Scholar). A number of T cell receptors (TCRs) have been isolated from TILs of epithelial cancer patients that target a neoepitope corresponding to residues 168 to 176 of p53R175H (HMTEVVRHC; mutant amino acid in bold) (15Malekzadeh P. Pasetto A. Robbins P.F. Parkhurst M.R. Paria B.C. Jia L. Gartner J.J. Hill V. Yu Z. Restifo N.P. Sachs A. Tran E. Lo W. Somerville R.P. Rosenberg S.A. et al.Neoantigen screening identifies broad TP53 mutant immunogenicity in patients with epithelial cancers.J. Clin. Invest. 2019; 129: 1109-1114Google Scholar, 16Lo W. Parkhurst M. Robbins P.F. Tran E. Lu Y.C. Jia L. Gartner J.J. Pasetto A. Deniger D. Malekzadeh P. Shelton T.E. Prickett T. Ray S. Kivitz S. Paria B.C. et al.Immunologic recognition of a shared p53 mutated neoantigen in a patient with metastatic colorectal cancer.Cancer Immunol. Res. 2019; 7: 534-543Google Scholar). The TCRs are restricted by HLA-A∗02:01, which is the most frequent major histocompatibility complex (MHC) class I allele in the U.S. population (18González-Galarza F.F. Takeshita L.Y. Santos E.J. Kempson F. Maia M.H. da Silva A.L. Teles e Silva A.L. Ghattaoraya G.S. Alfirevic A. Jones A.R. Middleton D. Allele frequency net 2015 update: New for and and Res. 2015; Scholar). TCRs effective in and the p53R175H mutation a lymphocytes for (15Malekzadeh P. Pasetto A. Robbins P.F. Parkhurst M.R. Paria B.C. Jia L. Gartner J.J. Hill V. Yu Z. Restifo N.P. Sachs A. Tran E. Lo W. Somerville R.P. Rosenberg S.A. et al.Neoantigen screening identifies broad TP53 mutant immunogenicity in patients with epithelial cancers.J. Clin. Invest. 2019; 129: 1109-1114Google Scholar, 16Lo W. Parkhurst M. Robbins P.F. Tran E. Lu Y.C. Jia L. Gartner J.J. Pasetto A. Deniger D. Malekzadeh P. Shelton T.E. Prickett T. Ray S. Kivitz S. Paria B.C. et al.Immunologic recognition of a shared p53 mutated neoantigen in a patient with metastatic colorectal cancer.Cancer Immunol. Res. 2019; 7: 534-543Google Scholar). the of TCR recognition of cancer neoantigens at the atomic level, we previously structures of three p53R175H-specific TCRs 38-10, and 1a2) in complex with and the neoepitope p53R175H D. R. for oligoclonal T cell recognition of a shared p53 cancer 2020; Scholar). The structures revealed that these TCRs discriminate between WT and mutated p53 by on the R175H mutation, with which they extensive Here, we report the structure of a fourth p53R175H-specific TCR (6-11) bound to the p53R175H peptide and In contrast to 12-6, 38-10, and 1a2, TCR 6-11 makes no contacts with the R175H mutation, yet is able to distinguish mutant from WT p53. these structures that are solutions to recognizing the p53R175H neoepitope with sufficient affinity and to mediate the killing of tumor cells mutant p53 normal cells WT a for clinical in due to TCR recognition S.R. recognition strategies to T-cell receptors for clinical 2019; Scholar). T cell 6-11 was isolated by screening TILs from patients with metastatic colorectal cancer for the mutated p53R175H neoantigen W. Parkhurst M. Robbins P.F. Tran E. Lu Y.C. Jia L. Gartner J.J. Pasetto A. Deniger D. Malekzadeh P. Shelton T.E. Prickett T. Ray S. Kivitz S. Paria B.C. et al.Immunologic recognition of a shared p53 mutated neoantigen in a patient with metastatic colorectal cancer.Cancer Immunol. Res. 2019; 7: 534-543Google Scholar). This TCR the p53R175H neoepitope using and for the and and for the gene are different from by TCRs 12-6, 38-10, and 1a2, which recognize the as 6-11 We to the affinity of TCR 6-11 for with mutant WT p53 peptide TCR 6-11 and by in from was to a and of 6-11 the T cell 6-11 bound with a of This affinity is to of TCRs and D. R. for oligoclonal T cell recognition of a shared p53 cancer 2020; Scholar). and for the binding of 6-11 to and corresponding to a of in with the from analysis T cell 6-11 bound WT with which is affinity than for mutant no between TCRs 12-6, 38-10, and WT D. R. for oligoclonal T cell recognition of a shared p53 cancer 2020; Scholar). based on 6-11 is not as specific for mutant as these other In T cells with TCR 6-11 by cells with of mutant p53R175H but they not to WT p53 at higher W. Parkhurst M. Robbins P.F. Tran E. Lu Y.C. Jia L. Gartner J.J. Pasetto A. Deniger D. Malekzadeh P. Shelton T.E. Prickett T. Ray S. Kivitz S. Paria B.C. et al.Immunologic recognition of a shared p53 mutated neoantigen in a patient with metastatic colorectal cancer.Cancer Immunol. Res. 2019; 7: 534-543Google Scholar). a of for 6-11 binding to by is the affinity for the of 6-11 T TCR and P. Pasetto A. Robbins P.F. Parkhurst M.R. Paria B.C. Jia L. Gartner J.J. Hill V. Yu Z. Restifo N.P. Sachs A. Tran E. Lo W. Somerville R.P. Rosenberg S.A. et al.Neoantigen screening identifies broad TP53 mutant immunogenicity in patients with epithelial cancers.J. Clin. Invest. 2019; 129: 1109-1114Google P. Pasetto A. Robbins P.F. Parkhurst M.R. Paria B.C. Jia L. Gartner J.J. Hill V. Yu Z. Restifo N.P. Sachs A. Tran E. Lo W. Somerville R.P. Rosenberg S.A. et al.Neoantigen screening identifies broad TP53 mutant immunogenicity in patients with epithelial cancers.J. Clin. Invest. 2019; 129: 1109-1114Google P. Pasetto A. Robbins P.F. Parkhurst M.R. Paria B.C. Jia L. Gartner J.J. Hill V. Yu Z. Restifo N.P. Sachs A. Tran E. Lo W. Somerville R.P. Rosenberg S.A. et al.Neoantigen screening identifies broad TP53 mutant immunogenicity in patients with epithelial cancers.J. Clin. Invest. 2019; 129: 1109-1114Google K. Lane D.P. Therapeutic targeting of p53: All mutants are equal, but some mutants are more equal than others.Nat. Rev. Clin. Oncol. 2018; 15: 13-30Google in a To understand TCR 6-11 between WT and mutant p53 and to by 6-11 with that by 12-6, 38-10, and 1a2, we the structure of the complex to The between TCR and was in for of the four complex in the of the The in positions for the TCR and including the p53R175H from to for the four the of interactions to all in the of the T cell 6-11 in a with the of and the The of TCR to R.L. TCRs and Rev. Immunol. 24: is which is to the of and but more than that of The Z. A for of T cell Sci. 2013; which to the of of TCR is for compared to for 12-6, for 38-10, and for the 6-11 complex is most the complex with to and most the complex with to T cell TCRs 12-6, 38-10, and D. R. for oligoclonal T cell recognition of a shared p53 cancer 2020; is the of the p53R175H which is the of the driver mutation at To the we the positions of the TCR the the is with the peptide and a more a Of 6-11 the reported which is as as but more than The of these TCRs is the to to discriminate between WT and mutant p53 by the of TCR 6-11 on the 6-11 contacts with the p53R175H peptide mainly the are no contacts with the between the mutant and WT T. Kim D.E. D. of Scholar). In contrast to TCRs 12-6, 38-10, and all and 1a2) the of 6-11 on that of and in with the and of these the of T cell 6-11 interactions from of 12-6, 38-10, but with some broad Of the number of contacts that 6-11 makes with and and compared with and by and more interactions with than of is not as in the complex as in the and for and of contacts with The than interactions in all four is mainly due to the of the TCRs the of the p53R175H peptide which from the and atomic contacts with peptide and of contacts the number of of between with a contacts the number of in a between with a T cell 6-11 makes more interactions with the than the A and as a of the binding of which is by of In 6-11 and which makes contacts the but from and and and which the and to 6-11 the using all three and and a cluster of with and 6-11 to are three between and and of of these including mediated by and is to the complex binding TCR 6-11 of the peptide compared to for 12-6, for 38-10, and for Of the number of contacts that 6-11 makes with the and and compared with and by and The of 6-11 in peptide recognition of that of and and is by all in the four We previously showed that the large majority of contacts between TCRs 38-10, 12-6, and and the p53R175H peptide residues and and that these contacts are between these residues D. R. for oligoclonal T cell recognition of a shared p53 cancer 2020; TCRs specific recognition of mutant p53 peptide to WT by interactions with the central and of which are in the WT and on the mutation at In contrast to 38-10, 12-6, and 1a2, 6-11 makes no interactions with the of TCR to discriminate between mutant and WT p53 A and The complex at The of at of the of 6-11 is on the with which four 6-11 6-11 6-11 and 6-11 in T. Kim D.E. D. of with the complex as the of in 6-11 TCR In 6-11 makes contacts with and and are and in structures of the WT and mutant p53R175H D. R. for oligoclonal T cell recognition of a shared p53 cancer 2020; the TCR 6-11 WT from mutant p53 is not 6-11 TCR binding affinity and for p53 peptide and WT p53 peptide 6-11 affinity by using the structure of the TCR complex as of the with of for all peptide are not The to attractive and of the with of for all peptide are not The to attractive and of the with of for all peptide are not The to attractive and to 6-11 affinity by using the structure of the TCR complex as of the with of for all peptide are not The to attractive and in a to To we the effect of by which to to the WT p53 we in silico mutagenesis using T. Kim D.E. D. of Scholar). The peptide was in the structure of the by and to The was to with the loss in 6-11 binding affinity for WT p53 peptide that we by To the for affinity the the This revealed that the energetic cost of desolvating during complex formation with TCR 6-11 the in binding of of the affinity the for at that position to interactions of the with the 6-11 TCR and its The effect of desolvating versus by TCR 6-11 is in with the to amino acid transfer of and V. of from the of J. Med. which between and as well as more amino acid C. G. C. of atomic from the structures of that showed for versus with the these versus amino acid are the 60-fold binding affinity loss to of observed for TCR 6-11 due to the to a analysis for TCRs 38-10, 12-6, and showed that of was mainly for affinity of and for WT loss of interactions for the affinity of D. R. for oligoclonal T cell recognition of a shared p53 cancer 2020; Scholar). We the energetic of of p53R175H to binding TCR 6-11 by to in was to have a effect on 6-11 TCR binding by T. Kim D.E. D. of which is the structure revealed no interactions between TCR and using of for contacts and for by 6-11 bound with which is affinity than for This is in with the in silico which 6-11 binding for to versus to In the of the the attractive the binding that TCR contacts of the are not in the other TCR contacts of the for 6-11 which is from are interactions that are To the TCR 6-11 binding of at we mutagenesis to the of all amino at that position amino acid residues 6-11 based on several and residues at and are to major in 6-11 binding in to and mutagenesis in has been in the of other B. Yu Y. W. M. Z. Structure-based design of a T-cell to in binding affinity for Scholar, L.M. M. Z. design of the affinity and of a therapeutic T cell 2014; due to of the binding can these of affinity complex are from the between the The of a large that by attractive and in to a analysis of the structure revealed that the affinity of TCR 6-11 for WT than for mutant is primarily due to the higher energetic cost of desolvating in the WT p53 peptide than in the mutant. strategy for WT from mutant p53 not on direct contacts between TCR 6-11 and in contrast to the more strategies employed by TCRs 38-10, 12-6, and 1a2, which on direct we not direct contacts between TCR 6-11 and we indirect interactions mediated by bound The of the structure not the of with and in the in several structures have been to that interactions and G. N.P. for of the human T-cell to a 2017; Scholar). In to are for targeting of cancer A specific for the targeted by TCR was reported J. S. S.R. A. et a neoantigen derived from a common TP53 Scholar). In the structure of bound to the and a and in which the of is of a with and TCRs 38-10, 12-6, and 1a2, distinguish WT from mutant p53 direct contacts with which is fundamentally different from the indirect strategy by TCR The and of p53R175H-specific TCR 6-11 from patients with colorectal cancer was previously (14Sabapathy K. Lane D.P. Therapeutic targeting of p53: All mutants are equal, but some mutants are more equal than others.Nat. Rev. Clin. Oncol. 2018; 15: 13-30Google Scholar). TCR 6-11 for affinity and structure was by in from expressed in E. as previously for other p53R175H-specific TCRs D. R. for oligoclonal T cell recognition of a shared p53 cancer 2020; Scholar). the TCR and and the was to the of TCR TCR 6-11 was using and with WT p53 peptide mutant p53R175H peptide was by in of E. as D. R. for oligoclonal T cell recognition of a shared p53 cancer 2020; Scholar). and using and To a was to the was with of the TCR TCR 6-11 was with in a at a of at by in The complex in and the with and at of the The and using the Z. W. of in Scholar). are in was using the for protein Scholar). The TCR structure was by with the and by with N. J.J. M. A. P.D. structure with Scholar). The was by with P. B. K. and of based on and The of a TCR P. A. S. R. K. S.A. J. S.R. J. R. L. et TCR recognition of human Immunol. 2019; the of TCR A. K. S. C. H. E. W. A. T. P. W. S. P. et in a common CD8+ T cell Immunol. 2017; and D. R. for oligoclonal T cell recognition of a shared p53 cancer 2020; with the and peptide as to the and position of the are in residues identified with the for protein and as residues from a of the binding using The of TCR 6-11 with and was by using a was on a at cell was with to as a the analysis of TCR solutions different of 6-11 with the The the was as the by and to a binding using of and was as previously D. R. for oligoclonal T cell recognition of a shared p53 cancer 2020; Scholar). mutagenesis to 6-11 TCR binding affinity was using a in T. Kim D.E. D. of which was previously to affinity of a therapeutic L.M. M. Z. design of the affinity and of a therapeutic T cell 2014; and was to effective for and for R. G. R. I. J. and energetic of binding domain recognition and the of Scholar). was and in silico mutagenesis in as with a A of TCR positions as previously D. R. for oligoclonal T cell recognition of a shared p53 cancer 2020; Scholar). The complex was and a in study D. R. for oligoclonal T cell recognition of a shared p53 cancer 2020; with the to the This complex was to the TCR domain and its the its position and position the and in positions compared with for other class I in that from the R. The T cell 2019; all positions the 6-11 TCR position reported in study D. R. for oligoclonal T cell recognition of a shared p53 cancer 2020; Scholar). and structure for the TCR complex have been in the This for protein The no We for with We F. Robbins and A. Rosenberg Cancer for in report are based on at and at the of by for the U.S. of of and D. W. and R. G. D. W. and R. G. B. G. P. and R. A. M. B. G. P. and R. A. M. D. R. B. G. and R. A. M. This was by of B. G. and R. A. by of D. and by of D. The is the of the and not the of the of

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