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Background: It is possible that the relative lack of progress in treatment outcomes among adolescent and young adult (AYA) patients with cancer is caused by a difference in disease biology compared with the corresponding diseases in younger and older individuals. There is evidence that colon cancer is more aggressive and has a poorer prognosis in AYA patients than in older adult patients., Methods: To further understand the molecular basis for this difference, whole-exome sequencing was conducted on a cohort of 30 adult, 30 AYA, and 2 pediatric colon cancers., Results: A statistically significant difference in mutational frequency was observed between AYA and adult samples in 43 genes, including ROBO1, MYC binding protein 2 (MYCBP2), breast cancer 2 (early onset) (BRCA2), MAP3K3, MCPH1, RASGRP3, PTCH1, RAD9B, CTNND1, ATM, NF1; KIT, PTEN, and FBXW7. Many of these mutations were nonsynonymous, missense, stop-gain, or frameshift mutations that were damaging. Next, RNA sequencing was performed on a subset of the samples to confirm the mutations identified by exome sequencing. This confirmation study verified the presence of a significantly greater frequency of damaging mutations in AYA compared with adult colon cancers for 5 of the 43 genes (MYCBP2, BRCA2, PHLPP1, TOPORS, and ATR)., Conclusions: The current results provide the rationale for a more comprehensive study with a larger sample set and experimental validation of the functional impact of the identified variants along with their contribution to the biologic and clinical characteristics of AYA colon cancer. Cancer 2018;124:1070-82. © 2017 American Cancer Society., (© 2017 American Cancer Society.)

Robust and analytically validated assays are essential for clinical studies. We outline an analytical validation study of a targeted next-generation sequencing mutation-detection assay used for patient selection in the National Cancer Institute Molecular Profiling-Based Assignment of Cancer Therapy (NCI-MPACT) trial (NCT01827384). Using DNA samples from normal or tumor cell lines and xenografts with known variants, we assessed the sensitivity, specificity, and reproducibility of the NCI-MPACT assay in five variant types: single-nucleotide variants (SNVs), SNVs at homopolymeric (HP) regions (≥3 identical bases), small insertions/deletions (indels), large indels (gap ≥4 bp), and indels at HP regions. The assay achieved sensitivities of 100% for 64 SNVs, nine SNVs at HP regions, and 11 large indels, 83.33% for six indels, and 93.33% for 15 indels at HP regions. Zero false positives (100% specificity) were found in 380 actionable mutation loci in 96 runs of haplotype map cells. Reproducibility analysis showed 96.3% to 100% intraoperator and 98.1% to 100% interoperator mean concordance in detected variants and 100% reproducibility in treatment selection. To date, 38 tumors have been screened, 34 passed preanalytical quality control, and 18 had actionable mutations for treatment assignment. The NCI-MPACT assay is well suited for its intended investigational use and can serve as a template for developing next-generation sequencing assays for other cancer clinical trial applications., (Published by Elsevier Inc.)

Next Generation Sequencing (NGS) technologies are used to detect somatic mutations in tumors and study germ line variation. Most NGS studies use DNA isolated from whole blood or fresh frozen tissue. However, formalin-fixed paraffin-embedded (FFPE) tissues are one of the most widely available clinical specimens. Their potential utility as a source of DNA for NGS would greatly enhance population-based cancer studies. While preliminary studies suggest FFPE tissue may be used for NGS, the feasibility of using archived FFPE specimens in population based studies and the effect of storage time on these specimens needs to be determined. We conducted a study to determine whether DNA in archived FFPE high-grade ovarian serous adenocarcinomas from Surveillance, Epidemiology and End Results (SEER) registries Residual Tissue Repositories (RTR) was present in sufficient quantity and quality for NGS assays. Fifty-nine FFPE tissues, stored from 3 to 32 years, were obtained from three SEER RTR sites. DNA was extracted, quantified, quality assessed, and subjected to whole exome sequencing (WES). Following DNA extraction, 58 of 59 specimens (98%) yielded DNA and moved on to the library generation step followed by WES. Specimens stored for longer periods of time had significantly lower coverage of the target region (6% lower per 10 years, 95% CI: 3-10%) and lower average read depth (40x lower per 10 years, 95% CI: 18-60), although sufficient quality and quantity of WES data was obtained for data mining. Overall, 90% (53/59) of specimens provided usable NGS data regardless of storage time. This feasibility study demonstrates FFPE specimens acquired from SEER registries after varying lengths of storage time and under varying storage conditions are a promising source of DNA for NGS.

Purpose: Veliparib, a PARP inhibitor, demonstrated clinical activity in combination with oral cyclophosphamide in patients with BRCA-mutant solid tumors in a phase I trial. To define the relative contribution of PARP inhibition to the observed clinical activity, we conducted a randomized phase II trial to determine the response rate of veliparib in combination with cyclophosphamide compared with cyclophosphamide alone in patients with pretreated BRCA-mutant ovarian cancer or in patients with pretreated primary peritoneal, fallopian tube, or high-grade serous ovarian cancers (HGSOC)., Experimental Design: Adult patients were randomized to receive cyclophosphamide alone (50 mg orally once daily) or with veliparib (60 mg orally once daily) in 21-day cycles. Crossover to the combination was allowed at disease progression., Results: Seventy-five patients were enrolled and 72 were evaluable for response; 38 received cyclophosphamide alone and 37 the combination as their initial treatment regimen. Treatment was well tolerated. One complete response was observed in each arm, with three partial responses (PR) in the combination arm and six PRs in the cyclophosphamide alone arm. Genetic sequence and expression analyses were performed for 211 genes involved in DNA repair; none of the detected genetic alterations were significantly associated with treatment benefit., Conclusion: This is the first trial that evaluated single-agent, low-dose cyclophosphamide in HGSOC, peritoneal, fallopian tube, and BRCA-mutant ovarian cancers. It was well tolerated and clinical activity was observed; the addition of veliparib at 60 mg daily did not improve either the response rate or the median progression-free survival., (©2015 American Association for Cancer Research.)

In two experiments, the interaction of location and distance cues in the recall of pre-selected movements was investigated. In Expt 1, separate groups of subjects were required to remember either ther terminal location of, or the distance moved during, a criterion movement pre-selected within a 30 cm response region. Following either a 5 s or 30 s unfilled retention interval, subjects were required to recall the criterion movement using the particular movement cue (i.e. location or distance) in question. In Expt 2, a similar procedure was used, except that recall of the criterion movement followed either a 5 s or a 20 s unfilled, or a 20 s filled (backward counting) retention interval. Systematic manipulation of both the direction and magnitude of the starting position for recall movements revealed the subjects were unable to make the movement uninfluenced by the "unattended' movement cue. The interfering effect of this irrelevant cue was independent of the ongoing activity during the retention interval. The results suggest that memory for preselected movements is based on a combination of the two movement cues generated during production of the criterion movement.

A murine monoclonal antibody, 12.7G3, directed against an Ia antigen encoded by genes in the I-Ab subregion of the H-2 genetic complex, was found to be cytotoxic against human B lymphocytes. When tested against a random panel of normal human donors, the reactivity of 12.7G3 exhibited a correlation coefficient of 0.58-0.68 with cells expressing HLA-DR2. Antibody reactivity segregated with HLA-DR2 in two families studied. Binding of 12.7G3, as detected by immunofluorescence using flow microfluorometry, was positive for two human cell lines, GM 3161 and HFB-1, both expressing HLA-DR2, and negative for two other cell lines, GM 3104 (DR1,1) and GM 3164 (DR4,4).

The immune response to beef insulin in mice is controlled by genes in the IA subregion. We have previously shown that B6.C-H-2bm12 (bm12) mice, an A beta gene mutation of B6, have a selective loss of responsiveness to beef insulin, whereas other IAb controlled responses such as (TG)AL and collagen are unchanged. F1 hybrid mice between two nonresponder genotypes Ik and Ibm12 were found to be good responders to beef insulin suggesting functional complementation. In this report, we define the cellular and molecular basis of this complementation by investigating the determinants on Ia molecules and nominal antigen that are recognized by (B10.A X bm12)F1 proliferating T cells. Genetic analyses demonstrated that the Ik region was the only nonresponder genotype that complemented Ibm12, thus restoring responsiveness to beef insulin. More precisely an IAk and not an IEk gene product was found to be responsible for this complementation. Antibody blocking studies furthermore showed that the A alpha b:A beta k hybrid Ia mediated the response to beef insulin in (B10.A X bm12)F1 mice. Clonal analyses of the response to beef insulin in these F1 mice confirmed these conclusions, because the insulin-specific response in all 21 F1-T cell clones studied thus far was found to be dependent upon presentation via the A alpha b:A beta k hybrid Ia molecule. Dissection of the antigenic specificity of the F1-T cell clones demonstrated recognition of at least two insulin determinants, one A-loop (A8-A10) associated and the other non-loop (A4 or B chain) associated. Therefore these studies identify the molecular and antigenic basis of the Ir gene complementation seen in the response to beef insulin of (B10.A X bm12)F1 hybrids.

To further delineate the functional defects of bm12 mice in antigen presentation, we analyzed antigen-specific T lymphocyte clones derived from B6 or bm12 for their ability to recognize antigens presented on either B6 or bm12 APC. Both complex antigens, such as PPD and GAT, and restricted antigens, such as beef insulin, were used. Our results indicate that for complex antigens, more than 50% of the B6 T lymphocyte clones recognized antigens only if presented by B6 APC, whereas the rest could not discriminate B6 from bm12 APC. Similarly, bm12 T lymphocyte clones responding to complex antigens could be divided into two groups depending on the sources of the APC. We have also isolated B6 T lymphocyte clones specific for the more restricted antigen, beef insulin, to which bm12 failed to respond. All B6 T lymphocyte clones could be stimulated only with B6 APC and not with bm12 APC. These data are consistent with the notion that there are antigen-specific association sites on the Ia molecule, and that complex antigens have more than one such association site. Furthermore, these studies demonstrate that both the gain and loss determinants associated with the bm12 mutation are recognized by a significant number of bm12 and B6 antigen-specific T lymphocyte clones, respectively, thus defining the importance of this region of the A beta polypeptide chain in antigen presentation.

To characterize the T cell receptor repertoire in an immune response in which the Ia and nominal antigenic determinants are defined and limited, we have cloned and sequenced the expressed receptors from four independent, beef insulin-specific T cell lines from C57BL mice. Each of these lines responded to beef but not to the pork insulin, thus defining the nominal antigenic determinant recognized. Furthermore, each of these lines could only be presented antigen by B6 but not mutant B6.C-H-2bm12 antigen-presenting cells, thus defining the requisite Ia recognition or antigen-association site. In spite of this functional similarity in ligand specificity, each of these T cell lines was found to use different V alpha and V beta gene segments. Moreover, structural comparisons of implied protein sequences of each of these receptors showed no stretches of conserved amino acid residues that could be implicated in ligand interaction. However, the V alpha genes used by these four clones appeared considerably more homologous to each other than were their V beta genes.

These experiments assessed the interrelationship between location and distance cues in the coding of movements. In separate experiments subjects recalled either the terminal location or the distance of constrained (Experiment 1) or preselected (Experiment 2) movements following a 15-sec retention interval. Changes in direction amd amplitude of starting position were used to ascertain whether recall errors were related to these changes. The findings of both experiments indicated that location and distance were recalled with similar accuracy when the starting position was identical for the criterion and recall movement. However, analysis of constant errors when the recall starting position was varied in either direction clearly indicated neither terminal location nor distance are coded independently, and memory for movement is based on an interaction between these cues.

The structure of the highly polymorphic Ia dimer is the genetically determined factor that controls the immune response to foreign antigens, albeit the mechanism remains unresolved. However, it is clear that, in diverse immune responses, effector T lymphocytes require recognition of self-Ia and foreign antigenic determinants on the surface of an antigen-presenting cell or an antibody-secreting B cell. Furthermore, a single Ia molecule has been found to possess several independently acting functional domains. In this report T-cell recognition of Ia was limited to a single, defined structure by using the Ia mutant mouse strain B6.C-H-2bm12 (bm12). The Ia determinant being recognized is the site of the mutation that represents a difference in three of five amino acid residues in a hypervariable region of its beta chain. This mutation has been proposed to have resulted from a gene conversion-like event and is known to have functional importance. Recognition of the bm12 mutation site was studied here in in vitro cultures of T cells generated against Iabm12 antigens. The specificity of these alloreactive T cells was tested by using stimulator cells expressing various Ia alloantigens of known structure. Our findings provide direct genetic evidence that T cells recognize predominantly conformational determinants on Ia molecules and not their primary structure. The implications of these findings on our understanding of the genetic control of the immune response and the potential to modulate these responses in an antigen-specific way are discussed.

Splenic B cells and B cell blasts from the I-A mutant mouse strain B6.C-H-2bm12 were tested by serology with a series of new monoclonal anti-Iab antibodies. Four out of 5 of those monoclonal antibody-defined specificities that are determined by wild-type I-Ab antigens were undetectable on B6.C-H-2bm12 cells. Specificities both present and absent on mutant cells appear to be determinants on the same wild-type molecule, as indicated by sequential precipitation experiments with soluble H-2b antigens. The lack of expression of certain Ia specificities on mutant cells was found not to be the result of disparate control by the Xid gene, which was previously shown to control the expression of Ia.W39, another specificity absent in B6.C-H-2bm12 mice. Serologic testing of Ia specificities on cells and blasts from F1-hybrid mice suggested that the Iabm12 antigens are codominantly expressed, indicating a failure to detect trans regulation or complementation of the mutant phenotype. Another monoclonal antibody-defined Ia specificity dependent on the expression of the E beta polypeptide was normally expressed in B6.C-H-2bm12 mice. These data thus suggest that the lesion of these mutant mice occurred in the A alpha and/or A beta structural gene, resulting in the loss of several Ia specificities.

The IA mutant mouse strain, B6.C-H-2bm12 (bm12) has been used to address several important questions for the role of Ia molecules in immune responses to foreign antigens. Numerous publications using bm12 mice have led to conclusions concerning (1) the number and relative importance of functional sites on Ia molecules; (2) the effects of qualitative versus quantitative differences in Ia; (3) whether T cells recognize Ia sequence or conformation; and (4) if gene conversion events, such as the one that putatively occurred in bm12, transfer functional Ir gene epitopes. Because of the importance of these conclusions, as well as their controversial nature, we have undertaken a comprehensive and systematic analysis of the aberrant immune response of bm12 mice to heterologous insulin. Responses to beef, horse, and sheep insulin were compared in B6 and bm12 mice by T cell proliferation, enumeration of plaque-forming cells, and quantitation of serum antibody levels. Various doses of antigen were administered and the kinetics of each response was monitored at various times. The findings of these studies suggest (1) B6 and bm12 mice both mount comparably high levels of response to sheep and horse insulins; (2) in contrast to the good response of B6 mice to beef insulin, bm12 mice showed dramatically impaired responses, as evident from both the lower magnitude of the response in all three assays as well as the difference in the kinetics of the response in B6 and bm12 mice; and (3) the response to sheep insulin is controlled by IA and IE encoded genes. These new findings differ from and extend previously published reports using bm12 mice, and therefore have substantive implications on the above stated conclusions regarding recognition of Ia. One such implication is that the bm12 gene conversion did not result in the transfer of a functional epitope for sheep insulin, but rather resulted in the creation of a functionally unique Ia molecule. Furthermore, this critical definition of the Ir gene lesion in bm12 permits us to address mechanistic questions regarding the nature of its Ir gene defect to beef insulin.

Detailed serologic and biochemical characterization of D region products of the wild-derived mouse strain B10.GAA37 (Dw16) were performed and compared with previous studies of the D region products of the H-2d,b, and q haplotypes. Serologic analysis revealed that the antigens encoded by the Dw16 region express a unique combination of specificities defined by monoclonal antibodies (mAb) with established activity for the Ld and Dd molecules. Two out of five anti-Ld-reactive mAb reacted with B10.GAA37 cells, whereas one of three anti-Dd mAb showed B10.GAA37 reactivity. Sequential immunoprecipitation of B10.GAA37 antigens demonstrated the existence of at least two antigenically distinct molecules (designated Dw16 and Lw16) encoded by genes associated with the Dw16 region. Peptide map comparisons of the Dw16 and Lw16 molecules defined multiple differences in their primary protein structure, suggesting they are products of separate genes. Structural comparisons of the Lw16 and Dw16 molecules with the Ld and Dd molecules implied a) that the Dw16 and Dd regions did not result from a recent evolutionary divergence of a common primordial haplotype, and b) that the Lw16 and Dw16 molecules are more structurally homologous to each other than the Ld and Dd molecules are. Comparison of these findings with our previous studies of antigens encoded by the D regions suggest that each of these haplotypes has unique properties in terms of the number of gene products expressed and/or the structural relatedness of products of the same region.