Neural network-derived multivariate index assay demonstrates effective clinical performance in longitudinal monitoring of ovarian cancer risk.

OBJECTIVE: We recently characterized the clinical performance of a multivariate index assay (MIA3G) to assess ovarian cancer risk for adnexal masses at initial presentation. This study evaluated how MIA3G varies when applied longitudinally to monitor risk during clinical follow-up. METHOD: The study evaluated women presenting with adnexal masses from eleven centers across the US. Patients received an initial blood draw at enrollment and at the standard-of-care follow-up visits. MIA3G was determined for all visits but physicians did not have access to MIA3G scores to determine clinical management. The primary outcome was the relative change value (RCV) of MIA3G over the period of clinical observation. RESULTS: A total of 510 patients of 785 enrolled met study criteria. Of these, 30.8% had a second, 25.4% a third and 22.2% a fourth blood draw following initial collection. The median duration from initial draw was 131 d to second draw, 301.5 d to the third draw and 365.5 d to the fourth draw. MIA3G RCV of >50% was observed in 22-26% patients, whereas 70-75% patients had MIA3G RCV >5%. An empirical baseline RCV of 56% - transformed to 1 in logarithmic scale - was calculated from averaging RCVs of all patients who had no malignancy risk after 210 days. RCV > 1 log was associated with higher incidence of surgical intervention (29.6%) compared to RCV < 1 log (16.9%). CONCLUSIONS: Variation in MI3AG does not change the accuracy of the test for excluding malignancy, while marked changes may be associated with a slightly higher likelihood of surgical intervention. In addition to MIA3G score itself, the MIA3G RCV may be important for clinical management.

Ovarian Cancer surgical consideration is markedly improved by the neural network powered-MIA3G multivariate index assay.

Background: Surgery remains the main treatment option for an adnexal mass suspicious of ovarian cancer. The malignancy rate is, however, only 10-15% in women undergoing surgery. This results in a high number of unnecessary surgeries. A surveillance-based approach is recommended to form the basis for surgical referrals. We have previously reported the clinical performance of MIA3G, a deep neural network-based algorithm, for assessing ovarian cancer risk. In this study, we show that MIA3G markedly improves the surgical selection for women presenting with adnexal masses. Methods: MIA3G employs seven serum biomarkers, patient age, and menopausal status. Serum samples were collected from 785 women (IQR: 39-55 years) across 12 centers that presented with adnexal masses. MIA3G risk scores were calculated for all subjects in this cohort. Physicians had no access to the MIA3G risk score when deciding upon a surgical referral. The performance of MIA3G for surgery referral was compared to clinical and surgical outcomes. MIA3G was also tested in an independent cohort comprising 29 women across 14 study sites, in which the physicians had access to and utilized MIA3G prior to surgical consideration. Results: When compared to the actual number of surgeries (n = 207), referrals based on the MIA3G score would have reduced surgeries by 62% (n = 79). The reduction was higher in premenopausal patients (77%) and in patients ≤55 years old (70%). In addition, a 431% improvement in malignancy prediction would have been observed if physicians had utilized MIA3G scores for surgery selection. The accuracy of MIA3G referral was 90.00% (CI 87.89-92.11), while only 9.18% accuracy was observed when the MIA3G score was not used. These results were corroborated in an independent multi-site study of 29 patients in which the physicians utilized MIA3G in surgical consideration. The surgery reduction was 87% in this cohort. Moreover, the accuracy and concordance of MIA3G in this independent cohort were each 96.55%. Conclusion: These findings demonstrate that MIA3G markedly augments the physician's decisions for surgical intervention and improves malignancy prediction in women presenting with adnexal masses. MIA3G utilization as a clinical diagnostic tool might help reduce unnecessary surgeries.

Validation of a deep neural network-based algorithm supporting clinical management of adnexal mass.

Background: Conservative management of adnexal mass is warranted when there is imaging-based and clinical evidence of benign characteristics. Malignancy risk is, however, a concern due to the mortality rate of ovarian cancer. Malignancy occurs in 10-15% of adnexal masses that go to surgery, whereas the rate of malignancy is much lower in masses clinically characterized as benign or indeterminate. Additional diagnostic tests could assist conservative management of these patients. Here we report the clinical validation of OvaWatch, a multivariate index assay, with real-world evidence of performance that supports conservative management of adnexal masses. Methods: OvaWatch utilizes a previously characterized neural network-based algorithm combining serum biomarkers and clinical covariates and was used to examine malignancy risk in prospective and retrospective samples of patients with an adnexal mass. Retrospective data sets were assembled from previous studies using patients who had adnexal mass and were scheduled for surgery. The prospective study was a multi-center trial of women with adnexal mass as identified on clinical examination and indeterminate or asymptomatic by imaging. The performance to detect ovarian malignancy was evaluated at a previously validated score threshold. Results: In retrospective, low prevalence (N = 1,453, 1.5% malignancy rate) data from patients that received an independent physician assessment of benign, OvaWatch has a sensitivity of 81.8% [95% confidence interval (CI) 65.1-92.7] for identifying a histologically confirmed malignancy, and a negative predictive value (NPV) of 99.7%. OvaWatch identified 18/22 malignancies missed by physician assessment. A prospective data set had 501 patients where 106 patients with adnexal mass went for surgery. The prevalence was 2% (10 malignancies). The sensitivity of OvaWatch for malignancy was 40% (95% CI: 16.8-68.7%), and the specificity was 87% (95% CI: 83.7-89.7) when patients were included in the analysis who did not go to surgery and were evaluated as benign. The NPV remained 98.6% (95% CI: 97.0-99.4%). An independent analysis set with a high prevalence (45.8%) the NPV value was 87.8% (95% CI: 95% CI: 75.8-94.3%). Conclusion: OvaWatch demonstrated high NPV across diverse data sets and promises utility as an effective diagnostic test supporting management of suspected benign or indeterminate mass to safely decrease or delay unnecessary surgeries.

Validation of a gene expression signature to measure immune quiescence in kidney transplant recipients in the CLIA setting.

Aim: Allograft rejection remains a major cause of graft failure in kidney transplantation. Here the authors report the validation of a non-invasive molecular diagnostic assay, AlloMap Kidney, using peripheral blood. Methods: The AlloMap Kidney test is a gene expression profile utilizing the RNA-seq platform to measure immune quiescence in kidney transplant patients. Results/Conclusions: Analytical validation showed robust performance characteristics with an accuracy correlation coefficient of 0.997 and a precision coefficient of variation of 0.049 across testing. Clinical validation from the prospective, multi-center studies of 235 samples (66 rejection and 169 quiescence specimens) demonstrated the sensitivity of 70% and specificity of 66% for allograft rejection, while the negative predictive value was 95% to discriminate rejection from quiescence at 10% prevalence of rejection.

Gene expression and splicing alterations analyzed by high throughput RNA sequencing of chronic lymphocytic leukemia specimens.

BACKGROUND: To determine differentially expressed and spliced RNA transcripts in chronic lymphocytic leukemia specimens a high throughput RNA-sequencing (HTS RNA-seq) analysis was performed. METHODS: Ten CLL specimens and five normal peripheral blood CD19+ B cells were analyzed by HTS RNA-seq. The library preparation was performed with Illumina TrueSeq RNA kit and analyzed by Illumina HiSeq 2000 sequencing system. RESULTS: An average of 48.5 million reads for B cells, and 50.6 million reads for CLL specimens were obtained with 10396 and 10448 assembled transcripts for normal B cells and primary CLL specimens respectively. With the Cuffdiff analysis, 2091 differentially expressed genes (DEG) between B cells and CLL specimens based on FPKM (fragments per kilobase of transcript per million reads and false discovery rate, FDR q < 0.05, fold change >2) were identified. Expression of selected DEGs (n = 32) with up regulated and down regulated expression in CLL from RNA-seq data were also analyzed by qRT-PCR in a test cohort of CLL specimens. Even though there was a variation in fold expression of DEG genes between RNA-seq and qRT-PCR; more than 90 % of analyzed genes were validated by qRT-PCR analysis. Analysis of RNA-seq data for splicing alterations in CLL and B cells was performed by Multivariate Analysis of Transcript Splicing (MATS analysis). Skipped exon was the most frequent splicing alteration in CLL specimens with 128 significant events (P-value <0.05, minimum inclusion level difference >0.1). CONCLUSION: The RNA-seq analysis of CLL specimens identifies novel DEG and alternatively spliced genes that are potential prognostic markers and therapeutic targets. High level of validation by qRT-PCR for a number of DEG genes supports the accuracy of this analysis. Global comparison of transcriptomes of B cells, IGVH non-mutated CLL (U-CLL) and mutated CLL specimens (M-CLL) with multidimensional scaling analysis was able to segregate CLL and B cell transcriptomes but the M-CLL and U-CLL transcriptomes were indistinguishable. The analysis of HTS RNA-seq data to identify alternative splicing events and other genetic abnormalities specific to CLL is an added advantage of RNA-seq that is not feasible with other genome wide analysis.

KRAS mutation status impacts diagnosis and treatment decision in a patient with two colon tumours: a case report.

KRAS mutation status predicts response to anti-EGFR therapy in colorectal cancer patients. Here we report an interesting case of discordant KRAS mutation status in a patient with two separate tumour foci. Tumour A in sigmoid colon invaded through muscularis propria into the subserosal fat with metastatic disease in regional lymph nodes (pT3N2b). Tumour B in ascending colon had a relatively lower stage and no metastasis (pT2N0). Both tumours showed similar morphology, immunohistochemical staining and microsatellite instability pattern. KRAS mutation, however, was detected only in tumour A. These findings indicate distinct clonal nature of these two tumours. The discordance of KRAS mutation status also suggests that a combination of anti-epidermal growth factor receptor and chemotherapy is likely the best treatment option for this patient. This case exemplifies a notion that comprehensive pathological work-up comprising molecular testing is critical to guide the diagnosis and treatment decisions for colorectal cancer patients with multiple tumours.

The proto-oncogene MYC is required for selection in the germinal center and cyclic reentry.

After antigenic challenge, B cells enter the dark zone (DZ) of germinal centers (GCs) to proliferate and hypermutate their immunoglobulin genes. Mutants with greater affinity for the antigen are positively selected in the light zone (LZ) to either differentiate into plasma and memory cells or reenter the DZ. The molecular circuits that govern positive selection in the GC are not known. We show here that the GC reaction required biphasic regulation of expression of the cell-cycle regulator c-Myc that involved its transient induction during early GC commitment, its repression by Bcl-6 in DZ B cells and its reinduction in B cells selected for reentry into the DZ. Inhibition of c-Myc in vivo led to GC collapse, which indicated an essential role for c-Myc in GCs. Our results have implications for the mechanism of GC selection and the role of c-Myc in lymphomagenesis.

Integrity of the AID serine-38 phosphorylation site is critical for class switch recombination and somatic hypermutation in mice.

Activation-induced cytidine deaminase (AID) is a single-stranded (ss) DNA-specific cytidine deaminase that initiates Ig heavy chain (IgH) class switch recombination (CSR) and Ig somatic hypermutation (SHM) by deaminating cytidines within, respectively, IgH switch (S) regions and Ig variable region (V) exons. AID that is phosphorylated on serine residue 38 interacts with replication protein A (RPA), a ssDNA binding protein, to promote deamination of transcribed double-stranded DNA in vitro, which, along with other evidence, suggests that AID may similarly gain access to transcribed S regions and V exons in vivo. However, the physiological role of AID phosphorylation at serine residue 38 (S38), and even the requirement for the S38 residue, with respect to CSR or SHM has been debated. To address this issue, we used gene targeting to generate an endogenous mouse AID locus that produces AID in which S38 is substituted with alanine (AID(S38A)), a mutant form of AID that retains similar catalytic activity on ssDNA as WT AID (AID(WT)). B cells homozygous for the AID(S38A) mutation show substantially impaired CSR and SHM, correlating with inability of AID(S38A) to interact with endogenous RPA. Moreover, mice haploinsufficient for AID(S38A) have even more severely impaired CSR when compared with mice haploinsufficient for AID(WT), with CSR levels reduced to nearly background levels. These results unequivocally demonstrate that integrity of the AID S38 phosphorylation site is required for normal CSR and SHM in mice and strongly support a role for AID phosphorylation at S38 and RPA interaction in regulating CSR and SHM.

Genotoxic stress regulates expression of the proto-oncogene Bcl6 in germinal center B cells.

Antigen-specific B cells are selected in germinal centers, the structure in which these cells proliferate while accomplishing genome-remodeling processes such as class-switch recombination and somatic hypermutation. These events are associated with considerable genotoxic stress, which cells tolerate through suppression of DNA-damage responses by Bcl-6, a transcription factor required for the formation of germinal centers. Here we show that the expression of Bcl-6 is regulated by DNA damage through a signaling pathway that promotes Bcl-6 degradation. After DNA damage accumulated, the kinase ATM promoted Bcl-6 phosphorylation, leading to its interaction with the isomerase Pin1 and its degradation by the ubiquitin-proteasome system. Because Bcl-6 is required for the maintenance of germinal centers, our findings suggest that the extent of genotoxic stress controls the fate of germinal center B cells by means of Bcl-6.

Evolution of the immunoglobulin heavy chain class switch recombination mechanism.

To mount an optimum immune response, mature B lymphocytes can change the class of expressed antibody from IgM to IgG, IgA, or IgE through a recombination/deletion process termed immunoglobulin heavy chain (IgH) class switch recombination (CSR). CSR requires the activation-induced cytidine deaminase (AID), which has been shown to employ single-stranded DNA as a substrate in vitro. IgH CSR occurs within and requires large, repetitive sequences, termed S regions, which are parts of germ line transcription units (termed "C(H) genes") that are composed of promoters, S regions, and individual IgH constant region exons. CSR requires and is directed by germ line transcription of participating C(H) genes prior to CSR. AID deamination of cytidines in S regions appears to lead to S region double-stranded breaks (DSBs) required to initiate CSR. Joining of two broken S regions to complete CSR exploits the activities of general DNA DSB repair mechanisms. In this chapter, we discuss our current knowledge of the function of S regions, germ line transcription, AID, and DNA repair in CSR. We present a model for CSR in which transcription through S regions provides DNA substrates on which AID can generate DSB-inducing lesions. We also discuss how phosphorylation of AID may mediate interactions with cofactors that facilitate access to transcribed S regions during CSR and transcribed variable regions during the related process of somatic hypermutation (SHM). Finally, in the context of this CSR model, we further discuss current findings that suggest synapsis and joining of S region DSBs during CSR have evolved to exploit general mechanisms that function to join widely separated chromosomal DSBs.

Regulation of activation induced deaminase via phosphorylation.

Immunoglobulin gene diversification by somatic hypermutation (SHM), class switch recombination (CSR), and gene conversion is dependent upon activation-induced cytidine deaminase (AID). AID is a single-stranded DNA specific cytidine deaminase that is expressed primarily in activated mature B lymphocytes. AID appears to catalyze DNA cytidine deamination of immunoglobulin heavy (IgH) and light chain (IgL) variable region (V) exons and IgH switch (S) region sequences to initiate, respectively, IgH and IgL somatic hypermutation (SHM) and IgH class switch recombination (CSR). Here, we will discuss the implications of recent studies that demonstrate the role of AID phosphorylation in augmenting AID activity with respect to these two processes.

BCL6 interacts with the transcription factor Miz-1 to suppress the cyclin-dependent kinase inhibitor p21 and cell cycle arrest in germinal center B cells.

The BCL6 proto-oncogene encodes a transcriptional repressor that is required for germinal center formation and has been linked to lymphomagenesis. BCL6 functions by directly binding to specific DNA sequences and suppressing the transcription of target genes. Here we report an alternative mechanism by which BCL6 controls the transcription of genes lacking a BCL6 binding site and show that this mechanism was required for the prevention of tumor suppressor p53-independent cell cycle arrest in germinal center B cells. BCL6 interacted with the transcriptional activator Miz-1 and, via Miz-1, bound to the promoter and suppressed transcription of the cell cycle arrest gene CDKN1A. Through this mechanism, BCL6 may facilitate the proliferative expansion of germinal centers during the normal immune response and, when deregulated, the pathological expansion of B cell lymphomas.

The BCL6 proto-oncogene suppresses p53 expression in germinal-centre B cells.

The human proto-oncogene BCL6 encodes a BTB/POZ-zinc-finger transcriptional repressor that is necessary for germinal-centre formation and is implicated in the pathogenesis of B-cell lymphoma. The precise function of BCL6 in germinal-centre development and lymphomagenesis is unclear because very few direct BCL6 target genes have been identified. Here we report that BCL6 suppresses the expression of the p53 (also known as tp53) tumour suppressor gene and modulates DNA damage-induced apoptotic responses in germinal-centre B cells. BCL6 represses p53 transcription by binding two specific DNA sites within the p53 promoter region and, accordingly, p53 expression is absent in germinal-centre B cells where BCL6 is highly expressed. Suppression of BCL6 expression via specific short interfering RNA leads to increased levels of p53 messenger RNA and protein both under basal conditions and in response to DNA damage. Most notably, constitutive expression of BCL6 protects B cell lines from apoptosis induced by DNA damage. These results suggest that an important function of BCL6 is to allow germinal-centre B cells to tolerate the physiological DNA breaks required for immunoglobulin class switch recombination and somatic hypermutation without inducing a p53-dependent apoptotic response. These findings also imply that deregulated BCL6 expression contributes to lymphomagenesis in part by functional inactivation of p53.