Andreas Martin Kaufmann

PURPOSE: This study investigates the interplay between T-cell receptor (TCR) immune characteristics and microbiome profiles to explore the relationship between immune diversity and microbial composition in cervical samples from Ethiopia. METHODS: Cervical specimens were collected from patients at Tikur Anbessa Specialized Hospital in Addis Ababa, and rural Butajira, south-central Ethiopia. Patient data, including age, human papillomavirus status, pathology, and TCR immune characteristics, were analyzed with a focus on the interactions between TCR profiles and microbiome compositions in malignant samples. RESULTS: Three distinct TCR profiles were identified: Group 1 (TCR active) exhibited features of active immune engagement, including high diversity, clonal expansion, and repertoire richness. Group 2 (TCR restricted) showed reduced TCR diversity and expansion, suggesting a restricted repertoire. Group 3 (TCR balanced) had moderate diversity and clonal activity. TCR repertoire groups were linked with microbial diversity, with Group 1 (TCR active) showing the highest number of microbes (high operational taxonomic units and microbial diversity). Maximum TCR clonal expansion positivity associated with microbial richness, while Group 3 (TCR balanced) was linked to reduced microbial alpha diversity. Taxonomic analysis revealed specific organisms enriched in TCR repertoire group. CONCLUSION: Variations in TCR profiles are linked to distinct microbial environments in cervical cancer with greater microbial richness in patients with greater maximum productive frequency. These findings underscore the interplay between TCR diversity, microbiome composition, and malignancy, offering insights into the potential implications for microbiome-targeted therapies and prognostic biomarkers in cervical cancer.

Purpose: High-risk human papillomavirus (hr-HPV) types are the primary cause of cervical and anogenital cancers. Understanding patterns of persistence, clearance, and re-infection is essential, as persistent infections contribute to precancerous and invasive lesions. This study evaluated hr-HPV infection dynamics and genotype specific outcomes over two years among Ethiopian women. Patients and Methods: A cohort of 893 women aged 30– 49 years was followed for two years using self-collected samples for multiplexed hr-HPV genotyping by BSGP5+/6+ PCR. Women testing positive were re-evaluated at 6 and 24 months with HPV genotyping, Visual Inspection with Acetic Acid (VIA) and cytology. Persistent infection was defined as continuous HPV DNA presence over consecutive visits, while clearance indicated no detectable virus. Re-infection occurred if a cleared HPV type reappeared, or a new type was detected. Results: After six months, 26.3% had persistent infections, while 73.7% cleared the infection. After two years, 13.2% experienced persistence, and 86.8% cleared their infections. Among women who tested negative at baseline, 74 were re-tested and showed hr-HPV incidence of 4.05% after 24 months. Genotype persistence rates varied, with HPV68, 82, 53, 52, and 56 showing the highest persistence after six months. After 24 months, HPV59, 68, 66, 52, and 16 had the highest persistence. Additionally, 29.9% of women at six months had abnormal cytology, including Atypical squamous cells of undetermined significance (ASCUS) and High grade squamous intraepithelial lesion (HSIL), which was 10.3% of those tested. Conclusion: The findings show that most hr-HPV infections among rural Ethiopian women cleared within two years, with varying persistence and clearance rates across HPV types. This emphasizes the need for regular monitoring and targeted prevention in high HPV prevalence populations.

Head and neck squamous cell carcinoma (HNSCC) with discordant diagnostic patterns of HPV⁺/p16⁻ or HPV⁻/p16⁺ correlate with worse prognosis. This study aims to identify truly HPV-driven HNSCCs using a QuantiGene-Molecular-Profiling-Histology (QG-MPH) assay for identifying transcriptionally active HPV. Of 97 FFPE samples analyzed, 68 were valid for HPV DNA detection by PCR and quantification of HPV E7 and p16 ^INK4a mRNA by QG-MPH. p16 ^INK4a mRNA expression was compared with p16 protein expression via immunohistochemistry (p16 IHC). Among the 68 cases, 26 (38.2%) showed increased high-risk HPV E7 mRNA expression (hrHPV E7 mRNA⁺), while 37 (54.4%) were hrHPV DNA⁺. Concordance between HPV DNA and mRNA status was 70.1%. Notably, 79.2% of E7 mRNA⁺ cases were p16 IHC⁺, compared to 55.9% of DNA⁺ cases, demonstrating better concordance between HPV E7 mRNA⁺ status and p16 mRNA expression plus p16 IHC positivity. All patients (19/19) in the HPV E7 mRNA⁺/p16 IHC⁺ group survived the 5-year follow-up, compared to 59.5% (22/37) in the HPV E7 mRNA⁻/p16 IHC⁻ group (p = 0.001). Specifically, the OS rate was 57.1% (8/14) in the group with discordant HPV DNA and p16 IHC results, compared to 40% (3/5) in the group with discordant HPV E7 mRNA and p16 IHC results. These findings highlight the better outcomes for the transcriptionally active HPV cases and indicate the prognostic disadvantage for patients with discordant patterns and the advantages for incorporating the molecular mRNA profiling by QG-MPH to p16 IHC. In conclusion, QG-MPH quantification of E7 and p16 ^INK4a mRNA more precisely identifies truly HPV-driven from non-HPV-driven HNSCC, compared to HPV DNA testing alone or with p16 IHC, which reduces misclassification and provides valuable implications for improved prognosis prediction and therapeutic decision-making.

Background Head and neck squamous cell carcinoma (HNSCC) with discordant diagnostic pat-terns of HPV⁺/p16⁻ or HPV⁻/p16⁺ correlate with worse prognosis. This study aims to identify truly HPV-driven HNSCCs using mRNA profiling by QuantiGene Molecu-lar-Profiling-Histology (QG-MPH) assay for more precise etiological diagnosis. Methods Of 97 FFPE samples analyzed, 68 were valid for HPV DNA detection by PCR and quantification of HPV E7 and p16ᴵᴺᴷ⁴ᵃ mRNA using QG-MPH. p16ᴵᴺᴷ⁴ᵃ mRNA expression was then compared with p16 protein expression by immunohistochemistry (p16 IHC). Results Among the 68 cases, 26 (38.2%) showed increased high-risk HPV E7 mRNA expression (hrHPV E7 mRNA⁺), while 37 (54.4%) were hrHPV DNA⁺. The concordance between HPV DNA and mRNA status was 70.1%. Notably, 79.2% of E7 mRNA⁺ cases were p16 IHC⁺, compared to 55.9 % of DNA⁺ cases, demon-strating better concordance between HPV E7 mRNA⁺ status and p16 mRNA expression plus p16 IHC positivity. All patients (19/19) in the HPV E7 mRNA⁺/p16 IHC⁺ group survived the 5-year follow-up, compared to 59.5% (22/37) in the HPV E7 mRNA⁻/p16 IHC⁻ group (p = 0.001). Specifi-cally, the OS rate was 57.1 % (8/14) in the group with discordant HPV DNA and p16 IHC results, compared to 40% (3/5) in the group with discordant HPV E7 mRNA and p16 IHC results. These findings highlight the better outcome for the transcriptionally active HPV cases and indicate the prognostic disadvantage for patients with discordant categories and the advantages for incorpo-rating the molecular mRNA profiling by QG-MPH to p16 IHC in making therapeutic decision and predicting patient outcomes in HNSCC. Conclusion QG-MPH quantification of E7 and p16ᴵᴺᴷ⁴ᵃ mRNA is more precise in identifying truly HPV-driven HNSCC than HPV DNA testing alone or with p16 IHC, enabling improved prognosis prediction and therapeutic deci-sion-making.

Although cancer incidence and mortality are rising in Ethiopia, lay and health professional perceptions of the disease remain limited. To explore perceptions of cancer, including its causes, signs and symptoms, and transmission within a rural community in Ethiopia. We conducted a qualitative study in four rural neighbourhoods of Butajira in central Ethiopia. Seven Focus Group Discussions (FGDs) and six In-Depth Interviews (IDIs) were held with community members, women representatives, religious leaders and key informants using two interview guides (divided by method). Behaviour change theories and a community research framework were used to summarize the findings. Across respondent categories and data collection methods, respondents described cancer or nekersa, which translates as 'an illness that cannot be cured', as serious and fatal. Cancer was further viewed as becoming more common and as underpinning more deaths particularly among women. Causes of cancer largely focused on individual behaviours namely mitch (referring to exposure to sunlight), poor personal hygiene and urinating on the ground/dirty areas. Almost all participants strongly related cancer to a wound that does not heal and entails a foul-smelling discharge. Bleeding and weight loss were other commonly mentioned complaints of cancer. Although cancer is known among rural communities in this area, misconceptions about cancer aetiology and conflation of the signs and symptoms of cancer versus other diseases merit health messaging. Our study calls for design research to determine how to culturally tailor educational materials and deliver health campaigns regarding cancer causes, signs and symptoms within this context.

Background Intense efforts have focused on identifying heterogeneity of the cellular composition in ovarian cancer. However, tissue composition and physiological conditions of cancer cells in cisplatin-sensitive ovarian cancer remains largely unknown. Moreover, comparisons of different cellular states in normal tissue, in treatment naive ovarian cancer, and in cisplatin-treated tissue after adjuvant therapy of cisplatin-sensitive ovarian cancer at the single-cell level might offer clues for ovarian cancer treatment and prevention of cisplatin-resistance formation. Methods Single-cell transcriptome sequencing of a cisplatin-treated ovarian cancer was performed. Data sets of non-tumorous ovarian tissues and treatment-naive ovarian cancer were downloaded from the European Genome-phenome Archive (accession number EGAS00001004987). Quality control, batch effect correction, integration, and clustering analysis of the integrated single-cell transcriptome data was done. Cell subsets were annotated based on surface marker phenotypes. Finally, the proportions of subclusters, the immune cell population, and the potential biological processes among different cellular states were compared. Results Sixteen distinct cell subsets were identified from the integrated single-cell transcriptome sequencing data of a pool of all tissues. The composition of the three different tissue types was characterized. The proportion of fibroblasts in cisplatin-treated ovarian tumor was remarkably lower than in treatment-naive ovarian tumor (1.33% vs. 13.53%, p < 0.05). Moreover, each subject’s sample had differing relative proportions of the identified cell types. In primary untreated ovarian cancer, the prevalent immune cells were B cells and myeloid-related immunosuppressive M2 macrophages. However, there were less B cells and myeloid-related immunosuppressive M2 macrophages after cisplatin-treatment, while significantly more T cells were found. The physiological cellular state in primary untreated ovarian tumors was associated with dysfunctional gene expression and modulation of cellular homeostasis, while cells from cisplatin-treated tumor showed more activation of immune and inflammatory genes as compared to healthy ovarian tissue. Conclusion Our molecular gene expression analysis allowed for the separation and identification of differences in normal ovarian tissues, treatment-naive, and cisplatin-sensitive ovarian cancer cell populations at single-cell resolution. We identified different cell type composition and discriminative marker expression concerning specific cell subsets and identified differences among their physiological cell states. This knowledge may open new possibilities for elucidating important pathogenetic features and therapeutic strategies for treating ovarian cancer.

Cervical cancer has killed millions of women over the past decade. In 2019 the World Health Organization launched the Cervical Cancer Elimination Strategy, which included ambitious targets for vaccination, screening, and treatment. The COVID-19 pandemic disrupted progress on the strategy, but lessons learned during the pandemic – especially in vaccination, self-administered testing, and coordinated mobilization on a global scale – may help with efforts to achieve its targets. However, we must also learn from the failure of the COVID-19 response to include adequate representation of global voices. Efforts to eliminate cervical cancer will only succeed if those countries most affected are involved from the very start of planning. In this article we summarize innovations and highlight missed opportunities in the COVID response, and make recommendations to leverage the COVID experience to accelerate the elimination of cervical cancer globally.

Suppl. 1. Comparison of different storage and transportation conditions