Jatinderpal Kalsi

Ovarian cancer screening and mortality in the UK Collaborative Trial of Ovarian Cancer Screening (UKCTOCS): a randomised controlled trial

Summary Background Ovarian cancer has a poor prognosis, with just 40% of patients surviving 5 years. We designed this trial to establish the effect of early detection by screening on ovarian cancer mortality. Methods In this randomised controlled trial, we recruited postmenopausal women aged 50–74 years from 13 centres in National Health Service Trusts in England, Wales, and Northern Ireland. Exclusion criteria were previous bilateral oophorectomy or ovarian malignancy, increased risk of familial ovarian cancer, and active non-ovarian malignancy. The trial management system confirmed eligibility and randomly allocated participants in blocks of 32 using computer-generated random numbers to annual multimodal screening (MMS) with serum CA125 interpreted with use of the risk of ovarian cancer algorithm, annual transvaginal ultrasound screening (USS), or no screening, in a 1:1:2 ratio. The primary outcome was death due to ovarian cancer by Dec 31, 2014, comparing MMS and USS separately with no screening, ascertained by an outcomes committee masked to randomisation group. All analyses were by modified intention to screen, excluding the small number of women we discovered after randomisation to have a bilateral oophorectomy, have ovarian cancer, or had exited the registry before recruitment. Investigators and participants were aware of screening type. This trial is registered with ClinicalTrials.gov, number NCT00058032. Findings Between June 1, 2001, and Oct 21, 2005, we randomly allocated 202 638 women: 50 640 (25·0%) to MMS, 50 639 (25·0%) to USS, and 101 359 (50·0%) to no screening. 202 546 (>99·9%) women were eligible for analysis: 50 624 (>99·9%) women in the MMS group, 50 623 (>99·9%) in the USS group, and 101 299 (>99·9%) in the no screening group. Screening ended on Dec 31, 2011, and included 345 570 MMS and 327 775 USS annual screening episodes. At a median follow-up of 11·1 years (IQR 10·0–12·0), we diagnosed ovarian cancer in 1282 (0·6%) women: 338 (0·7%) in the MMS group, 314 (0·6%) in the USS group, and 630 (0·6%) in the no screening group. Of these women, 148 (0·29%) women in the MMS group, 154 (0·30%) in the USS group, and 347 (0·34%) in the no screening group had died of ovarian cancer. The primary analysis using a Cox proportional hazards model gave a mortality reduction over years 0–14 of 15% (95% CI −3 to 30; p=0·10) with MMS and 11% (−7 to 27; p=0·21) with USS. The Royston-Parmar flexible parametric model showed that in the MMS group, this mortality effect was made up of 8% (−20 to 31) in years 0–7 and 23% (1–46) in years 7–14, and in the USS group, of 2% (−27 to 26) in years 0–7 and 21% (−2 to 42) in years 7–14. A prespecified analysis of death from ovarian cancer of MMS versus no screening with exclusion of prevalent cases showed significantly different death rates (p=0·021), with an overall average mortality reduction of 20% (−2 to 40) and a reduction of 8% (−27 to 43) in years 0–7 and 28% (−3 to 49) in years 7–14 in favour of MMS. Interpretation Although the mortality reduction was not significant in the primary analysis, we noted a significant mortality reduction with MMS when prevalent cases were excluded. We noted encouraging evidence of a mortality reduction in years 7–14, but further follow-up is needed before firm conclusions can be reached on the efficacy and cost-effectiveness of ovarian cancer screening. Funding Medical Research Council, Cancer Research UK, Department of Health, The Eve Appeal.

Genetic, structural and functional properties of an IgG DNA-binding monoclonal antibody from a lupus patient with nephritis

Antibodies binding to double‐stranded (ds) DNA are strongly associated with renal involvement in patients with systemic lupus erythematosus (SLE). We have generated two new IgG DNA‐binding monoclonal antibodies (mAb), RH‐14 and DIL‐6, from the peripheral blood lymphocytes of two SLE patients with glomerulonephritis using the heteromyeloma cell line CB‐F7. RH‐14 is an IgG1 λ antibody which also bound to single‐stranded (ss)DNA, histones and nucleosomes. DIL‐6 is an IgG3 λ antibody with restricted antigen binding specificity. cDNA encoding the variable regions of the heavy (VH) and light (VL) chains of RH‐14 was sequenced and the antigen binding site of this mAb was computer modelled. Sequence analysis of VH and VL regions of RH‐14 showed that VH is derived from germ‐line gene V3‐7, a member of the VH3 family, and VL is derived from DPL 11, a member of the Vλ2 family. Somatic mutations and basic amino acid residues are identified in the complementarity‐ determining regions of both VH and VL regions. The nephritogenic properties of these mAb were analyzed by implanting and growing the hybridoma cells secreting the mAb in the peri toneum of SCID mice. The animals that received the RH‐14 hybridoma produced higher levels of proteinuria (3 to ≥ 4) (p < 0.001) compared to the groups that received DIL‐6 (trace to ≥ 1) or CB‐F7 (trace). Electron microscopy of kidney sections from all the RH‐14‐ implanted animals showed granular immunoglobulin deposition in the renal glomerular capil laries and mesangium. In this study we have shown for the first time using electron micros copy that a human IgG anti‐dsDNA mAb, RH‐14, is nephritogenic and that deposition of such an antibody alone is sufficient to induce renal damage.

Risk Algorithm Using Serial Biomarker Measurements Doubles the Number of Screen-Detected Cancers Compared With a Single-Threshold Rule in the United Kingdom Collaborative Trial of Ovarian Cancer Screening

Purpose Cancer screening strategies have commonly adopted single-biomarker thresholds to identify abnormality. We investigated the impact of serial biomarker change interpreted through a risk algorithm on cancer detection rates. Patients and Methods In the United Kingdom Collaborative Trial of Ovarian Cancer Screening, 46,237 women, age 50 years or older underwent incidence screening by using the multimodal strategy (MMS) in which annual serum cancer antigen 125 (CA-125) was interpreted with the risk of ovarian cancer algorithm (ROCA). Women were triaged by the ROCA: normal risk, returned to annual screening; intermediate risk, repeat CA-125; and elevated risk, repeat CA-125 and transvaginal ultrasound. Women with persistently increased risk were clinically evaluated. All participants were followed through national cancer and/or death registries. Performance characteristics of a single-threshold rule and the ROCA were compared by using receiver operating characteristic curves. Results After 296,911 women-years of annual incidence screening, 640 women underwent surgery. Of those, 133 had primary invasive epithelial ovarian or tubal cancers (iEOCs). In all, 22 interval iEOCs occurred within 1 year of screening, of which one was detected by ROCA but was managed conservatively after clinical assessment. The sensitivity and specificity of MMS for detection of iEOCs were 85.8% (95% CI, 79.3% to 90.9%) and 99.8% (95% CI, 99.8% to 99.8%), respectively, with 4.8 surgeries per iEOC. ROCA alone detected 87.1% (135 of 155) of the iEOCs. Using fixed CA-125 cutoffs at the last annual screen of more than 35, more than 30, and more than 22 U/mL would have identified 41.3% (64 of 155), 48.4% (75 of 155), and 66.5% (103 of 155), respectively. The area under the curve for ROCA (0.915) was significantly (P = .0027) higher than that for a single-threshold rule (0.869). Conclusion Screening by using ROCA doubled the number of screen-detected iEOCs compared with a fixed cutoff. In the context of cancer screening, reliance on predefined single-threshold rules may result in biomarkers of value being discarded.

The role of antibodies to DNA in systemic lupus erythematosus— A review and introduction to an international workshop on DNA antibodies held in London, May 1996

The term ’lupus erythematosus’ was first used approximately 150 y ago and its potential systemic involvement has been widely acknowledged during the past 100 y. In contrast the major serological marker of systemic lupus erythematosus (SLE), antibodies to DNA, were distinguished by four different groups as late as 1957.1--4 During the next 20 y a large panoply of antibodies were identified in the serum of lupus patients (see Table 1 and Reference

Functional and modelling studies of the binding of human monoclonal anti-DNA antibodies to DNA.

The relationships between the antigen-binding specificities of four human monoclonal anti-DNA antibodies and the structural aspects of the combining sites of two of these were examined. Competition ELISAs were used to examine the reactivities of two IgM MAbs (WRI-176 and RT-79) and two IgG mAbs (D5 and B3) to a wide range of polynucleotides. The mAbs WRI-176 and RT-79 were found to bind predominantly ssDNA, with a preference for poly (dT), whilst D5 and B3 bound components of both ss- and dsDNA, and Z-DNA. The mAb B3 also exhibited a preference for A(T) rich nucleotides. Computer models were generated for the Fv regions of WRI-176 and B3. Models for RT-79 and D5 were not generated as the structure of the long CDR-H3 loops in these mAbs could not be predicted. The B3 combining site contains a groove flanked by three arginines at positions CDR-L1-27A, CDR-L2-54 and CDR-H2-53. Using interactive molecular graphics, B-DNA was docked into the B3 antigen combining site along the plane of the VH/VL interface, whilst Z-DNA was best-fitted at approximately 90 degrees to this direction. The models provide a hypothesis to explain the ability of a single autoantibody to bind two different antigens. In addition, aspects of the base specificity of B3 may be explained. The model of the WRI-176 Fv region revealed a relatively flat surface, on which a large number of hydrophobic and aromatic residues were present. Trp-H52, in particular, is prominent on the surface. This may participate in ssDNA binding through base stacking interactions. The models allow identification of potential targets for site-directed mutagenesis.

Molecular cloning and expression of the fabs of human autoantibodies in Escherichia coli - Determination of the heavy or light chain contribution to the anti-DNA/-cardiolipin activity of the Fab

The Fabs of three human autoantibodies (B3/33H11, anti-DNA; UK4, anti-phospholipid) and six related hybrids have been cloned, expressed in Escherichia coli, and purified to homogeneity. SDS-polyacrylamide gel electrophoresis and Western blot analysis of the recombinant Fab demonstrated the purified Fab to be of correct size and in assembled form. Protein expression levels of up to 5–9 mg per liter of culture were achievable. A sensitive and reliable comparative anti-DNA enzyme-linked immunosorbent assay, involving a defined biotinylated 35-mer oligonucleotide in its single- or double-stranded form, is also described. Crithidia assay and anti-DNA or anti-cardiolipin antibody enzyme-linked immunosorbent assay analyses demonstrated convincing binding of the recombinant Fab proteins to DNA/cardiolipin, confirming the expression of functional molecule. The comparative DNA/cardiolipin binding analyses of the nine Fabs revealed that the anti-DNA (light, B3/33H11) or anti-cardiolipin (heavy, UK4) activity lies predominantly on one of the two chains. However, a compatible partner chain is necessary for optimum antigen binding activity of the antibody.

The UKCTOCS experience--reasons for hope?

Abstract There has been concern about current ovarian cancer screening strategies after the publication showing a lack of benefit from screening in the ovarian part of the Prostate, Lung, Colorectal and Ovarian Cancer Screening Trial. The ongoing United Kingdom Collaborative Trial of Ovarian Cancer Screening involves 202,638 low-risk postmenopausal women. The performance characteristics on prevalence screen in sensitivity, specificity, and stage distribution have been encouraging. Screening differs from the Prostate, Lung, Colorectal and Ovarian Cancer Screening Trial in the use of algorithms to interpret CA125 and ultrasound imaging and well-defined, centrally coordinated management of screen-detected abnormalities with protocols for intervention based on screening findings. There is a possibility that these essential differences may be sufficient to alter the natural history of ovarian cancer and ultimately lead to demonstration of a mortality benefit from screening when the trial reports in 2014/2015.

IgG Subclasses in Systemic Lupus Erythematosus and Other Autoimmune Rheumatic Diseases

In this study the concentration of the different subclasses of IgG in sera from patients with a range of autoimmune rheumatic diseases (ARD) was detected by radial immunodiffusion. In the second part the IgG subclasses of autoantibodies that recognize single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), Ro, La, Sm and RNP in patients with ARD were measured by enzyme-linked immunosorbent assay. We studied 15 patients with lupus, 20 patients each with primary and secondary Sjögrens syndrome (SS) and 10 each with rheumatoid arthritis (RA), scleroderma and myositis. Twenty healthy controls were also measured. The serum concentration of IgG2 in ARD patients was generally reduced. In contrast, the concentrations of IgG1, IgG3 and IgG4 subclasses were normal or raised. A high degree of correspondence in the IgG1, IgG2 and IgG3 responses to dsDNA and ssDNA in SLE was found. Notable differences in the IgG1 anti-Ro and ssDNA responses compared to the other subclasses were seen in 1° and 2° SS. In addition, an unexpected high level of IgG4 antibodies to ssDNA in 1° SS (65%) and IgG4 antibodies to Sm/RNP in RA was observed.

Anti-DNA antibodies: from gene usage to crystal structures

Abstract Serum anti-DNA antibodies are a key feature of systemic lupus erythematosus. Gene usage, structural details of their binding to antigens and their role in the disease process were the main subjects discussed at a recent meeting ∗ ∗The meeting DNA Antibody Workshop was held at London, UK, on 11–12 May 1996..

Protein Z: A putative novel biomarker for early detection of ovarian cancer

Ovarian cancer (OC) has the highest mortality of all gynaecological cancers. Early diagnosis offers an approach to achieving better outcomes. We conducted a blinded‐evaluation of prospectively collected preclinical serum from participants in the multimodal group of the United Kingdom Collaborative Trial of Ovarian Cancer Screening. Using isobaric tags (iTRAQ) we identified 90 proteins differentially expressed between OC cases and controls. A second targeted mass spectrometry analysis of twenty of these candidates identified Protein Z as a potential early detection biomarker for OC. This was further validated by ELISA analysis in 482 serial serum samples, from 80 individuals, 49 OC cases and 31 controls, spanning up to 7 years prior to diagnosis. Protein Z was significantly down‐regulated up to 2 years pre‐diagnosis (p = 0.000000411) in 8 of 19 Type I patients whilst in 5 Type II individuals, it was significantly up‐regulated up to 4 years before diagnosis (p = 0.01). ROC curve analysis for CA‐125 and CA‐125 combined with Protein Z showed a statistically significant (p= 0.00033) increase in the AUC from 77 to 81% for Type I and a statistically significant (p= 0.00003) increase in the AUC from 76 to 82% for Type II. Protein Z is a novel independent early detection biomarker for Type I and Type II ovarian cancer; which can discriminate between both types. Protein Z also adds to CA‐125 and potentially the Risk of Ovarian Cancer algorithm in the detection of both subtypes.