Michael Dean

Cell‐free DNA from ascites identifies clinically relevant variants and tumour evolution in patients with advanced ovarian cancer

The emergence of targeted therapies has transformed ovarian cancer treatment. However, biomarker profiling for precision medicine is limited by access to quality, tumour‐enriched tissue samples. The use of cell‐free DNA (cfDNA) in ascites presents a potential solution to this challenge. In this study, next‐generation sequencing was performed on ascites‐derived cfDNA samples (26 samples from 15 human participants with ovarian cancer), with matched DNA from ascites‐derived tumour cells ( n  = 5) and archived formalin‐fixed paraffin‐embedded (FFPE) tissue ( n  = 5). Similar tumour purity and variant detection were achieved with cfDNA compared to FFPE and ascites cell DNA. Analysis of large‐scale genomic alterations, loss of heterozygosity and tumour mutation burden identified six cases of high genomic instability (including four with pathogenic BRCA1 and BRCA2 mutations). Copy number profiles and subclone prevalence changed between sequential ascites samples, particularly in a case where deletions and chromothripsis in Chr17p13.1 and Chr8q resulted in changes in clinically relevant TP53 and MYC variants over time. Ascites cfDNA identified clinically actionable information, concordant to tissue biopsies, enabling opportunistic molecular profiling. This advocates for analysis of ascites cfDNA in lieu of accessing tumour tissue via biopsy.

Whole‐genome sequencing of 1,083 HPV45 cases and controls identifies genetic variants associated with glandular cervical lesions

AbstractHuman papillomavirus type 45 (HPV45) causes ~6% of all cervical cancers and an even greater proportion of adenocarcinomas, the latter of which are challenging to detect using current cervical cancer screening. Little is known about how HPV45 genetic variation is related to the risk of cervical precancer/cancer. To investigate this, we whole‐genome sequenced a total of 1,083 HPV45‐positive samples from two large studies. We evaluated associations of HPV45 genetic variation (sublineages, subclades, and SNPs) with histology‐specific precancer/cancer risk using logistic regression and evaluated risk modification by self‐reported race/ethnicity. Compared to the common A1 sublineage, A2 and B1 were associated with increased precancer/cancer (A2, OR = 3.9, 95% CI = 1.9–8.5; B1, OR = 2.7, 95% CI = 1.3–5.8; B2, OR = 3.3, 95% CI = 1.6–7.3), and most strongly with the glandular precancers/cancers (AIS/ADC; A2, OR = 6.9, 95% CI = 1.0–184; B1, OR = 6.2, 95% CI = 1.1–159). The A2 sublineage was most prevalent in women in East Asia and women who self‐reported as Asian/Pacific Islander (PI) in the U.S.; East Asian and Asian/PI women had the greatest precancer/cancer risk associated with A2 infections (OR = 5.8, 95% CI = 1.3–37.4) compared to all other sublineages among these women. We further evaluated precancer/cancer risk associations for 262 individual HPV45 SNPs and identified four SNPs significantly associated with only glandular precancers/cancers after correction for multiple tests (ORs ranged 7.8–20.7). One of these SNPs was a nonsynonymous variant in both overlapping viral E2/E4 ORFs. In summary, we show that HPV45 genetic variation influences the risk of precancer/cancer, specifically glandular precancer/cancer. Further studies of these genetic variants may improve our understanding of glandular lesions.

Somatic mutations in 3929 HPV positive cervical cells associated with infection outcome and HPV type

AbstractInvasive cervical cancers (ICC), caused by HPV infections, have a heterogeneous molecular landscape. We investigate the detection, timing, and HPV type specificity of somatic mutations in 3929 HPV-positive exfoliated cervical cell samples from individuals undergoing cervical screening in the U.S. using deep targeted sequencing in ICC cases, precancers, and HPV-positive controls. We discover a subset of hotspot mutations rare in controls (2.6%) but significantly more prevalent in precancers, particularly glandular precancer lesions (10.2%), and cancers (25.7%), supporting their involvement in ICC carcinogenesis. Hotspot mutations differ by HPV type, and HPV18/45-positive ICC are more likely to have multiple hotspot mutations compared to HPV16-positive ICC. The proportion of cells containing hotspot mutations is higher (i.e., higher variant allele fraction) in ICC and mutations are detectable up to 6 years prior to cancer diagnosis. Our findings demonstrate the feasibility of using exfoliated cervical cells for detection of somatic mutations as potential diagnostic biomarkers.

Analysis of the progression of cervical cancer in a low-and-middle-income country: From pre-malignancy to invasive disease

To better understand cervical cancer progression, we analyzed RNA from 262 biopsies from women referred for colposcopy. We determined the HPV type and analyzed the expression of 51 genes. HPV31 was significantly more prevalent in precancer than stage 1 cancer and invasive cancer (p < 0.0001), and HPV16 increased in invasive disease (p < 0.0001). CCNE1, MELTF, and ULBP2 were significantly increased in HPV16-positive compared to HPV31 precancers, while NECTIN2 and HLA-E expression decreased. Markers of the innate immune system, DNA repair genes, and cell cycle genes are significantly increased during cancer progression (p = 0.0001). In contrast, the TP53 and RB1 tumor suppressor gene expression is significantly decreased in cancer cells. The T cell markers CD28 and FLT3LG expression decreased in cancer while FOXP3, IDO1, and ULBP2 expression increased. There is a significantly higher survival rate in individuals with increased expression of CD28 (p = 0.0005), FOXP3 (p = 0.0002), IDO1 (p = 0.038), FLT3LG (p = 0.026), APOBEC3B (p = 0.0011), and RUNX3 (p = 0.019), and a significantly lower survival rate in individuals with increased expression of ULBP2 (p = 0.035). These results will help us elucidate the molecular factors influencing the progression of cervical precancer to cancer. Understanding the risk of progression of specific HPV types and sublineages may aid in the triage of positive patients, and better knowledge of the immune response may aid in developing and applying immunotherapies.

Comparison of Hybribio-H13 and Hybrid Capture® 2 human papillomavirus tests for detection of CIN2+ and CIN3+

Introduction. Low-cost, accurate high-risk HPV tests are needed for cervical cancer screening in limited-resource settings.Objective. To compare the performance of the low-cost Hybribio-H13 test with the Hybrid Capture® 2 to detect cervical intraepithelial neoplasia grade 2 or 3 (CIN2 and CIN3).Materials and methods. Archived baseline samples tested by the Hybrid Capture® 2 from women of the ASCUS-COL trial, aged 20 to 69 years, with biopsy-colposcopy directed diagnosis of CIN2+ (n = 143), CIN3+ (n = 51), and &lt; CIN2 (n = 632) were blindly tested by the Hybribio-H13 test.Results. The relative sensitivity of the Hybribio-H13 test versus the Hybrid Capture® 2 for detecting CIN2+ was 0.89 (90% CI = 0,80-0,98; NIT = 0,66), and for CIN3+ was 0,92 (90% CI = 0,85-0,98; NIT = 0,35). Relative specificity was 1.19 (90% CI = 1.05-1.33; NIT &lt;0.00001). In the analysis restricted to women older than 30 years, the relative sensitivity of the Hybribio-H13 for CIN3+ was marginally below unity (ratio = 0.97; 90% CI = 0.95-0.99), and the specificity remained higher than the Hybrid Capture® 2 test.Conclusion. The Hybribio-H13 test was as specific as the Hybrid Capture® 2 for detecting CIN2+ or CIN3+ but less sensitive. Considering these results and the young age of the population recruited for screening because of ASCUS cytology, we suggest our results warrant the evaluation of the Hybribio-H13 for screening cervical cancer, especially in the evaluated population.

Insights into the mechanisms and structure of breakage-fusion-bridge cycles in cervical cancer using long-read sequencing

Cervical cancer is caused by human papillomavirus (HPV) infection, has few approved targeted therapeutics, and is the most common cause of cancer death in low-resource countries. We characterized 19 cervical and four head and neck cancer cell lines using long-read DNA and RNA sequencing and identified the HPV types, HPV integration sites, chromosomal alterations, and cancer driver mutations. Structural variation analysis revealed telomeric deletions associated with DNA inversions resulting from breakage-fusion-bridge (BFB) cycles. BFB is a common mechanism of chromosomal alterations in cancer, and our study applies long-read sequencing to this important chromosomal rearrangement type. Analysis of the inversion sites revealed staggered ends consistent with exonuclease digestion of the DNA after breakage. Some BFB events are complex, involving inter- or intra-chromosomal insertions or rearrangements. None of the BFB breakpoints had telomere sequences added to resolve the dicentric chromosomes, and only one BFB breakpoint showed chromothripsis. Five cell lines have a chromosomal region 11q BFB event, with YAP1-BIRC3-BIRC2 amplification. Indeed, YAP1 amplification is associated with a 10-year-earlier age of diagnosis of cervical cancer and is three times more common in African American women. This suggests that individuals with cervical cancer and YAP1-BIRC3-BIRC2 amplification, especially those of African ancestry, might benefit from targeted therapy. In summary, we uncovered valuable insights into the mechanisms and consequences of BFB cycles in cervical cancer using long-read sequencing.

The D2 and D3 Sublineages of Human Papilloma Virus 16–Positive Cervical Cancer in Guatemala Differ in Integration Rate and Age of Diagnosis

Abstract Human papillomavirus (HPV) 16 displays substantial sequence variation; four HPV16 lineages (A, B, C, and D) have been described as well as multiple sublineages. To identify molecular events associated with HPV16 carcinogenesis, we evaluated viral variation, the integration of HPV16, and somatic mutation in 96 cervical cancer samples from Guatemala. A total of 65% (62/96) of the samples had integrated HPV16 sequences and integration was associated with an earlier age of diagnosis and premenopausal disease. HPV16 integration sites were broadly distributed in the genome, but in one tumor, HPV16 integrated into the promoter of the IFN regulatory factor 4 (IRF4) gene, which plays an important role in the regulation of the IFN response to viral infection. The HPV16 D2 and D3 sublineages were found in 23% and 30% of the tumors, respectively, and were significantly associated with adenocarcinoma. D2-positive tumors had a higher rate of integration, earlier age of diagnosis, and a lower rate of somatic mutation, whereas D3-positive tumors were less likely to integrate, had later age of diagnosis, and exhibited a higher rate of somatic mutation. In conclusion, Guatemalan cervical tumors have a high frequency of very high-risk HPV16 D2 and D3 sublineages harboring distinct histology, which may help guide future therapeutic strategies to target the tumor and reduce recurrence. Significance: This study details the biological and molecular properties of the most pathogenic forms of HPV16, the cause of the majority of cervical cancers.

Cervical Visual Inspection with Acetic Acid (VIA) and Oncogenic Human Papillomavirus Screening in Rural Indigenous Guatemalan Women: Time to Rethink VIA

Single-visit “screen-and-treat” strategies using visual inspection with acetic acid (VIA) and cryotherapy (liquid nitrous oxide ablation) in low-resource settings are commonly used to detect and treat precancerous lesions for cervical cancer prevention. This study compared VIA sensitivity and specificity in rural indigenous Guatemalan communities, to that of oncogenic human papillomavirus (HPV) testing for detection of precancerous changes, using cytology as the reference standard. Between 3–8 September 2017, trained nurses examined 222 women aged 23–58 years with VIA. Specimens for liquid-based cytology and HPV testing were obtained prior to VIA with a cytobrush and transported in PreservCyt to a US clinical laboratory. VIA and HPV test sensitivities were assessed as proportions of women with abnormal cytology that had abnormal VIA or HPV results, respectively, and specificities, as proportions with normal cytology with normal VIA or negative HPV tests. Of 222 women, 18 (8.1%) had abnormal cytology (1 carcinoma in a participant who received VIA-based cryotherapy in 2015, 4 high- and 5 low-grade squamous intraepithelial lesions, and 8 atypical squamous cells of undetermined significance (ASCUS)). Excluding ASCUS, sensitivities of VIA and HPV were 20.0% and 100%, respectively. VIA-based screening may not be acceptable for detecting precancerous lesions, and field cryotherapy for preventing malignancy. The World Health Organization recommended in 2021 “…using HPV DNA detection as the primary screening test rather than VIA or cytology”.