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The 2017 11th Workshop on Recent Issues in Bioanalysis took place in Los Angeles/Universal City, California, on 3-7 April 2017 with participation of close to 750 professionals from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was once again a 5-day, week-long event - a full immersion week of bioanalysis, biomarkers and immunogenicity. As usual, it was specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small- and large-molecule analysis involving LC-MS, hybrid ligand-binding assay (LBA)/LC-MS and LBA approaches. This 2017 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2017 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers the recommendations for large-molecule bioanalysis, biomarkers and immunogenicity using LBA. Part 1 (LC-MS for small molecules, peptides and small molecule biomarkers) and Part 2 (hybrid LBA/LC-MS for biotherapeutics and regulatory agencies' inputs) are published in volume 9 of Bioanalysis, issues 22 and 23 (2017), respectively.

Objective: To determine whether polymorphism in the manganese superoxide dismutase (MnSOD) gene is associated with susceptibility or disease outcome in rheumatoid arthritis (RA)., Methods: We used a case-control approach with 153 RA patients and 218 control subjects to examine for any associations between MnSOD genotypes and susceptibility to RA. We also investigated the influence of genotypes on radiologic outcome, as measured using the Larsen score for radiographs of the hands and feet, and on functional outcome, as assessed by the Health Assessment Questionnaire. MnSOD typing was carried out using polymerase chain reaction-based methods. Results were analyzed using multiple regression analysis, with adjustment for age, sex, and disease duration. In separate analyses, we corrected for rheumatoid factor (RF) status and/or the presence of the HLA-DRB1 "shared epitope" (SE). We also examined whether radiologic outcome was influenced by interactions between MnSOD and glutathione S-transferase (GST) genes., Results: No association between MnSOD genotype and development of RA was found. The MnSOD VV genotype was associated with a significantly higher (P = 0.04) Larsen score (104.4) than MnSOD AA (83.0), while MnSOD AV was associated with an intermediate score (91.8). Correction for RF status had no significant effect on the results of the analysis, but significance was lost (P = 0.09) after correction for the presence of the SE. There was evidence of interaction between the GSTT1 and MnSOD genotypes, with the MnSOD VV/GSTT1-null combination being associated with the highest Larsen score (142.1; P = 0.007 after correction for the SE)., Conclusion: Polymorphism in the MnSOD gene is not associated with susceptibility to RA. Our data suggest that MnSOD VV is associated with more severe radiologic outcome, although this relationship may not be independent of the effect of the SE. However, interaction between MnSOD and GST genes appears to influence radiologic outcome independently of the SE.

Allelic variation at the melanocyte stimulating hormone receptor (MC1R) gene has been linked with sun-sensitive skin types, suggesting it is a susceptibility candidate for melanoma. We determined the frequency of the val92met, asp294his, and asp84glu MC1R alleles in 190 Caucasian controls and 306 melanoma cases and studied their association with skin type and hair color. The percentage of controls with at least one val92met, asp294his, or asp84glu allele was 17.3%, 6.8%, and 3.5%, respectively. Individually, frequencies of the val92met, asp294his, or asp84glu alleles in the controls with skin types 3 and 4 were similar to those with skin types 1 and 2. Trend analysis, however, did identify an association (exact p = 0.048, two-sided test) between skin type and MC1R variants in the group comprising all controls with any one or more of these alleles. There was no association between MC1R alleles and hair color. Allele frequencies were not different in melanoma cases and controls. There were no associations between skin types and the proportion of cases with the asp294his or asp84glu alleles, though the association between skin type and the val92met allele approached significance (exact p = 0.09, two-sided test). Unexpectedly, in the group comprising all cases with one or more variant alleles, the proportion of subjects with variant alleles increased with skin types associated with tanning rather than burning, although trend analysis showed that this association did not quite reach statistical significance (exact p = 0.08, two-sided test). Asp84glu (but not val92met or asp294his) variant alleles were more common in subjects with blonde hair, although the relationship between the asp84glu allele and hair color did not achieve statistical significance (chi(2)3 = 6.16, exact p = 0.10). We interpret the data presented as indicating that polymorphism at MC1R does not appear a major determinant of skin type, at least in terms of these allelic variants. Furthermore, considered alone, these alleles are not susceptibility candidates for malignant melanoma.

The influence of polymorphism in the glutathione S-transferase, GSTM3 gene on susceptibility to cutaneous basal cell carcinoma (BCC) has been investigated. We have reported previously two GSTM3 alleles, GSTM3*A and GSTM3*B, distinguished by a recognition motif for the YY1 transcription factor in GSTM3*B. In this study, immunohistochemistry was used to identify GSTM3 expression in the epidermis of skin samples from 11 controls and 9 patients with BCC. A PCR method was used to identify GSTM3*A and GSTM3*B and thereby the GSTM3 AA, GSTM3 AB, and GSTM3 BB genotypes in 300 controls and 286 Caucasians with 1-35 primary BCCs. Genotypes at GSTM1, GSTT1, and the cytochrome P450 CYP1A1 and CYP2D6 loci were also determined. Frequencies of GSTM3, GSTM1, GSTT1, CYP2D6, and CYP1A1 genotypes in the cases and controls were not different. Dividing the BCC cases into groups of 92 patients with 1 lesion and 194 patients with 2-35 lesions showed that the frequencies of GSTM3 BB (2.6%) and GSTM1 A/B (1.3%) in the group with 2-35 tumors were almost significantly lower than in the group with 1 lesion (7.6%, exact P = 0.0601, chi 2(1) = 3.390; 6.5%, exact P = 0.055, chi 2(1) = 4.946, respectively). Within the cases with 2-35 tumors, a Poisson regression model was used to identify genotypes, characteristics such as skin type, and interactions between genotypes and characteristics associated with increasing numbers of tumors. This showed, after correction for male gender and age, that GSTM3 AA was not associated with risk of increased numbers of tumors, although in combination with skin type 1, GSTM1 null, and CYP1A1 m1m1, the genotype did confer increased risk (P < 0.001, rate ratio, 2.058; P < 0.001, rate ratio, 1.606; P < 0.001, rate ratio, 1.470 respectively). The data suggest that, like other allelic GST, GSTM3 influences cancer risk. As GSTM3 AA was associated with increased tumor numbers, it appears that YY1 acts as an activator of the recognition motif in GSTM3*B.

The factors that determine progression of cervical intra-epithelial neoplasia (CIN) to squamous cell carcinoma (SCC) are unknown. Cigarette smoking is a risk factor, suggesting polymorphism at loci that encode carcinogen-metabolizing enzymes such as glutathione S-transferase (GSTT1, GSTM1) and cytochrome P450 (CYP2D6) may determine susceptibility to these cancers. We have studied the frequency of the null genotype at the theta class GSTT1 locus in women with low-grade CIN, high-grade CIN and SCC. The control group comprised women with normal cervical pathology suffering menorrhagia. We found the frequency of GSTT1 null in the control and case groups was not significantly different, though frequency distributions of combinations of the genotype with smoking in mutually exclusive groups in the high-grade CIN group and the other case groups were significantly different. Interactive effects of GSTT1 null with the GSTM1 null and CYP2D6 EM genotypes, and cigarette smoking were also studied by comparing the multinomial frequency distributions of these factors over mutually exclusive categories. These showed no significant differences between the controls and SCC or low-grade CIN. Frequency distributions in high-grade CIN, however, were significantly different to the controls, and both SCC and low-grade CIN; frequency distributions of GSTT1 null with smoking and CYP2D6 EM, individually and in combination, were significantly different. However, inspection of our data does not indicate that GSTT1 null is a major factor mediating risk. Thus, comparison of chi 2 values for the differences between frequency distributions in high-grade CIN and other groups shows that values for combinations of GSTT1 null with other factors are lower than those for equivalent combinations with smoking and CYP2D6 EM. Interestingly, the combination GSTT1 null/GSTM1 null did not appear to influence susceptibility to CIN or SCC.