Premalignant Research Articles

Updates in Human Papillomavirus Vaccination to Improve Medical Readiness in Active Duty Service Members

ABSTRACT Human papillomavirus (HPV) is the most common sexually transmitted infection in the United States and is associated with the development of genital warts, precancerous lesions, and cancers of the oropharynx, anus, penis, vulva, vagina, and cervix. HPV-associated diseases are preventable through vaccination. An Australian nationwide vaccination program will effectively eliminate cervical cancer as a public health concern by the year 2035. Despite safety and efficacy data, vaccination against HPV is not mandatory for military service members. Active duty service women are more likely to test positive for HPV when compared to dependents, and veterans diagnosed with HPV-associated cancers have higher health care resource utilization and costs. Even if a patient has a prior HPV infection, vaccination at the time of HPV diagnosis or following treatment can reduce recurrence of HPV and dysplasia. In addition, patients with a history of cancer have a higher incidence of secondary HPV-associated malignancies and should be offered vaccination or re-vaccination if they received a hematopoietic stem-cell transplant. Improving HPV vaccination rates within the military health system requires a multidisciplinary approach. Providers should use every opportunity to recommend HPV vaccination, including recommending HPV vaccines for individuals who do not have a record of HPV vaccination. The electronic health record can be utilized to generate prompts for vaccine reminders, and clinics should stock HPV vaccines to allow for easy administration. Additionally, adoption of an opt-out policy for HPV vaccines rather than mandatory vaccination may be an acceptable alternative to increase HPV vaccination while allowing active duty service members to retain autonomy.

Multiomics Approach Identifies Key Proteins and Regulatory Pathways in Colorectal Cancer.

The prevalence rate of colorectal cancer (CRC) has dramatically increased in recent decades. However, robust CRC biomarkers with therapeutic value for early diagnosis are still lacking. To comprehensively reveal the molecular characteristics of CRC development, we employed a multiomics strategy to investigate eight different types of CRC samples. Proteomic analysis revealed 2022 and 599 differentially expressed tissue proteins between CRC and control groups in CRC patients and CRC mice, respectively. In patients with colorectal precancerous lesions, 25 and 34 significantly changed proteins were found between patients and healthy controls in plasma and white blood cells, respectively. Notably, vesicle-associated membrane protein-associated protein A (VAPA) was found to be consistently and significantly decreased in most types of CRC samples, and its level was also significantly correlated with increased overall survival of CRC patients. Furthermore, 37 significantly enriched pathways in CRC were further validated via metabolomics analysis. Ten VAPA-related pathways were found to be significantly enriched in CRC samples, among which PI3K-Akt signaling, central carbon metabolism in cancer, cholesterol metabolism, and ABC transporter pathways were also enriched in the premalignant stage. Our study identified VAPA and its associated pathways as key regulators, suggesting their potential applications in the early diagnosis and prognosis of CRC.

Intestinal metaplasia key molecules and UPP1 activation via Helicobacter pylori /NF-kB: drivers of malignant progression in gastric cancer

Gastric cancer (GC) remains a significant global health challenge due to its high morbidity and mortality rates. The development of GC is a multi-hit process and the exploration of precancerous lesions is crucial. To elucidate the molecular and cellular dynamics underlying gastric carcinogenesis, we conducted an integrative single-cell RNA sequencing analysis of 26,028 high-quality cells from gastric antral mucosa biopsies across various stages, including non-atrophic gastritis, chronic atrophic gastritis, intestinal metaplasia, and early gastric cancer. By constructing a detailed single-cell atlas, we identified distinct epithelial cell subpopulations and their corresponding molecular signatures. We focused on the biological link between gastric epithelial cells and cancer cells. Notably, we observed that gland mucous cells acquired an intestinal-like stem cell phenotype during metaplasia, with MUC6, MUC2 and OLFM4 emerging as the specific markers for unique endocrine cells in early malignant lesions. Additionally, our analysis highlighted UPP1 as a key oncogene, with its expression progressively increasing from normal epithelial cells to malignant cells. UPP1 upregulation was shown to promote GC cell proliferation and migration, implicating it in the oncogenic process. Further, we explored the impact of Helicobacter pylori infection on gene expression, revealing that Helicobacter pylori infection upregulates UPP1 via the NF-κB pathway. Our cell-cell communication analysis underscored the significant role of the Macrophage migration inhibitory factor pathway in the tumor microenvironment, contributing to GC progression. Various key molecules involved in intestinal metaplasia, along with UPP1 and the Macrophage migration inhibitory factor pathway, collectively illustrate the multifaceted nature and complexity of gastric cancer evolution, highlighting the cumulative impacts that drive tumorigenesis.

Endometrial hyperplasia, benign endometrial tumors and endometrial carcinoma: A review study

Background. During the reproductive phase, the endometrium undergoes cyclical structural changes. Endometrial hyperplasia (EH) is a prevalent condition that arises from a combination of estrogen exposure and insufficient progesterone levels. Endometrial cancer (EC) is the second most common gynecologic malignancy, with the likelihood of EH progressing to EC ranging from 1% to 29%. The objective of this study is to conduct a comprehensive assessment of various forms of endometrial hyperplasia, benign endometrial tumors, and endometrial cancer (EC). Methods. A review of published papers from major databases, including PubMed, Scopus, and Google Scholar, was conducted. The search included original research articles and reviews on EH, benign endometrial tumors, and EC. Case reports, series, and clinical images were excluded. Results. Information on the selected topics was summarized, including epidemiology, etiology, histopathological classification, anatomy, biological and molecular characteristics, detection and prevention, clinical manifestations, staging, diagnostic workup, prognostic factors, and management. Conclusion. Endometrial hyperplasia and endometrial cancer are two of the most common problems in women. EH is a major precancerous condition for EC, and EC is associated with a poor prognosis if diagnosed late. The epidemiology and outcomes of both conditions vary according to several factors, including ethnicity, environment, socioeconomic status, heredity, drugs, hormones, and the aging process.

Breaking Barriers: Empowering Cervical Cancer Screening with HPV Self-Sampling for Sex Workers and Formerly Incarcerated Women in Toronto

Background: Although cervical cancer (CC) is highly preventable through appropriate screening methods like the Papanicolaou (Pap) test, which enables early detection of malignant and precancerous lesions, access to such screening has not been equitable across social groups. Sex workers and people with records of incarceration are among the most under-screened populations in Ontario. Little is known about the acceptability and feasibility of HPV self-sampling (HPV-SS) as an alternative cervical cancer screening method for these groups. This online, community-based mixed-methods pilot study aimed to address this knowledge gap. Methods: Eighty-four under- and never-screened sex workers and ex-prisoners aged 25–69 years and residing in the Greater Toronto Area, were recruited by community peer associates. Participants completed an online survey and viewed short videos about CC and screening with Pap and HPV-SS. Those who opted for HPV-SS conducted the test at one of two collaborating organizations. Results: The median age of participants was 36.5 years. Most had limited knowledge about CC and screening. Approximately 13% identified as non-binary, and 5% as two-spirit or trans men, with the majority having completed secondary education. Of the participants, 88% chose HPV-SS, and one-third tested positive for high-risk HPV types. The ability to self-sample without judgment from healthcare providers was noted as a key advantage. However, there was a need for training on proper HPV-SS techniques. Conclusions: To improve cervical cancer screening among sex workers, increasing awareness through participatory community co-creation of sexual health education is essential. Additionally, offering HPV-SS as a screening option is crucial, given its demonstrated acceptability and feasibility within this population, many of whom lack a primary care provider and face discriminatory attitudes in healthcare settings.

Curcumin Alleviates Arecoline-induced Oral Submucous Fibrosis via the FOSL1/MAPK8 Axis.

Oral submucous fibrosis (OSF) is a precancerous lesion of the oral cavity. Areca nut consumption can cause OSF through sustained activation of buccal mucosal fibroblasts (BMFs). This study explored the effect of curcumin on arecoline-induced BMF activation and its mechanism of action. BMFs were isolated and identified by immunofluorescence detection of fibroblast surface markers vimentin and S100A4. After transfection with FOSL1- or MAPK8-related vectors, BMFs were activated by arecoline and treated with curcumin. Scratch and transwell assays were performed to detect cell migration. ChIP and luciferase reporter assays were conducted to detect the binding of FOSL1 to the MAPK8 promoter. RT-qPCR was used to detect FOSL1 and MAPK8 mRNA expression. Western blotting was used to detect FOSL1, MAPK8, COL1A1, α-SMA, Smad2, and p-Smad2 proteins. Curcumin treatment inhibited arecoline-induced fibroblast migration, reduced the expression of myofibroblast markers COL1A1, α-SMA, and p-Smad2, and downregulated the expression of FOSL1 and MAPK8. FOSL1 or MAPK8 overexpression enhanced migration and increased COL1A1, α-SMA, and p-Smad2 expression in curcumin-treated cells. FOSL1 bound to the MAPK8 promoter and promoted MAPK8 expression. Simultaneous FOSL1 overexpression and MAPK8 knockdown, compared to FOSL1 overexpression, reduced cell migration and inhibited COL1A1, α-SMA, and p-Smad2 expression. In conclusion, curcumin targets FOSL1 to reduce MAPK8 expression, thereby suppressing arecoline-induced fibroblast activation.

High Risk of HPV Related Preneoplastic and Neoplastic Vulvar Lesions in Women Living With HIV.

We aimed to investigate the epidemiology of human papilloma virus (HPV)-related preneoplastic and neoplastic vulvar lesions in a large cohort of women living with HIV (WLWH). We retrospectively selected 1,796 WLWH who had a gynecological examination, cervical cytology, high-risk (HR-) HPV test, vulvoscopy, and colposcopy with targeted biopsies when necessary between 1987 and 2020 at 2 Italian institutions. Univariable and multivariable regression analyses were carried out to test the association of the anamnestic and clinical data with the development of precancerous and cancerous lesions. At baseline, 348 (19.4%) of 1,796 WLWH had genital warts, 30 (1.7%) had vulvar high-grade intraepithelial neoplasia (VHSIL), and 2 (0.1%) had squamous cell carcinoma of the vulva. Among 895 WLWH who had more than 1 year of follow-up, we found 40 (4.5%) new cases of VHSIL and 7 (0.8%) cases of vulvar cancer. The cumulative incidence of VHSIL and vulvar cancer was respectively 0.56 and 0.10 per 100 person-years. Risk factors independently associated with the development of vulvar HSIL and cancer included history of injection drug use (p < .01), genital warts at baseline (p < .001), HR-HPV test positivity at diagnosis (p < .001), and severe immunodepression (CD4 cell count <200 cells/mL) at diagnosis (p < .01). WLWH are at high risk of vulvar high-grade intraepithelial neoplasia and cancer, especially those with severe immunodepression. A careful inspection of vulva, perineum and anus, possibly with the aid of colposcopy, should become part of the surveillance protocol of HIV-infected women.

Upregulation of HPV16E1 and E7 expression and FOXO3a mRNA downregulation in high-grade cervical neoplasia.

Cervical cancer remains a significant global health concern, ranking as the fourth most prevalent cancer among women worldwide. Human papillomaviruses (HPV) transcribe many genes that might be responsible for cervical cancer development. This study aims to investigate the correlation between the expression of HPV16 early genes and the mRNA expression of human FOXO3a, a tumor suppressor gene, in association with various stages of cervical precancerous lesions. Eighty-five positive HPV16 DNA cervical swab samples were recruited and categorized based on cytology stages, i.e., negative for intraepithelial lesion or malignancy (NILM), atypical squamous cells of undetermined significance (ASC-US), low-grade squamous intraepithelial lesion (LSIL), atypical squamous cell cannot exclude HSIL (ASC-H), high-grade squamous intraepithelial lesion (HSIL). RT-qPCR was performed to amplify HPV16E1, E4, E6, E6*I, E7, and human FOXO3a mRNA expression in all samples. The relative expression of those genes was calculated using GAPDH as a control. Detection of FOXO3a mRNA expression in the cervical cancer cell line by RT-qPCR and meta-analysis of FOXO3a expression using the RNA-Seq dataset by GEPIA2 were analyzed to support the conclusions. Among the cervical samples, HPV16E1 and E7 were significantly increased expression correlating to disease severity. HPV16E4 mRNA expression was 100% detected in all LSIL samples, with a significant increase observed from normal to LSIL stages. Conversely, FOXO3a mRNA expression decreased with disease severity, and the lowest expression was observed in HSIL/squamous cell carcinoma (SCC) samples. In addition, similar results of FOXO3a downregulation were also found in the cervical cancer cell line and RNA-Seq dataset of cervical cancer samples. HPV16 early mRNA levels, including E1 and E7, increase during cancer progression, and downregulation of FOXO3a mRNA is a characteristic of cervical cancer cells and HSIL/SCC. Additionally, HPV16E4 mRNA expression was consistently detected in all LSIL samples, suggesting the presence of active viral replication. These findings might lead to further investigation into the interplay between HPV gene expression and host cell factors for targeted therapeutic strategies in cervical cancer management.

Compression optical coherence elastography as a new tool for non-invasive identification of precancerous lesions of the oral mucosa

Compression optical coherence elastography as a new tool for non-invasive identification of precancerous lesions of the oral mucosa

Perinatal outcomes after surgical treatment of cervical LSIL and conservative treatment of endometrial cancer. A clinical case analysis

This article describes a clinical case of pregnancy management in a patient after surgical treatment of low-grade squamous intraepithelial lesions (LSIL) of the cervix and endometrial cancer. In the structure of female oncological morbidity, precancerous conditions and malignant diseases of the female genital organs occupy leading positions and require the most sparing treatment in women who have not realized reproductive function. The frequency of miscarriage in early pregnancy associated with a history of endometrial cancer, requiring repeated invasive manipulations to assess the effectiveness of treatment, reaches 36–40%, depending on the age of the woman. After surgical interventions on the cervix, regardless of the method of exposure, the frequency of miscarriage significantly increases due to the formation of an organic form of isthmic-cervical insufficiency, which dictates the need for preventive cervical circumcision in a regulated time. In addition, the results of numerous studies also indicate an infectious cause as the main etiological factor in the development and progression of isthmic-cervical insufficiency. Management tactics, obstetric risks, as well as the possibilities of choosing a treatment method are discussed on the basis of the presented clinical case.

The HPV101 E7 protein shares host cellular targets and biological activities with high-risk HPV16 E7

Human papillomaviruses (HPVs) are a diverse family of viruses with over 450 members that have been identified and fully sequenced. They are classified into five phylogenetic genera: alpha, beta, gamma, mu, and nu. The high-risk alpha HPVs, such as HPV16, have been studied the most extensively due to their medical significance as cancer-causing agents. However, while nearly 70% of all HPVs are members of the gamma genus, they are almost entirely unstudied. This is because gamma HPVs have been considered medically irrelevant commensals as most of them infect the skin and are not known to cause significant clinical lesions in immunocompetent individuals. Members of the gamma 6 HPVs, however, have been detected in the anogenital tract mucosa and HPV101 has been isolated from a premalignant cervical lesion. Moreover, gamma 6 HPVs have a unique genome structure. They lack E6 proteins but in place of E6, they encode unique, tiny hydrophobic proteins without any close viral or cellular homologs that have been termed E10. Here we report that HPV101 E7 shares biochemical activities with the high-risk alpha HPV16 E7, including the ability to target the pRB and PTPN tumor suppressor for degradation. This study underscores the importance of further characterizing HPV101 and other unstudied HPV species.

The association between oral microbiome and gastric precancerous lesions.

Gastric precancerous lesions are thought to be precursors in the occurrence and development of gastric cancer through Correa's cascade. Recent studies have investigated the association between the oral microbiome and gastric precancerous lesions. However, there has yet to be a comprehensive synthesis review of the existing literature on the relationship between oral microbiome and gastric precancerous lesions. A systematic review was conducted to characterize the literature on the association between oral microbiome and gastric precancerous lesions. The studies show that oral microbiome is dynamic in individuals with gastric precancerous lesions. Oral-derived microorganisms were colonized in the gastric precancerous lesions. Interactions between oral and gastric microbiomes affect the response of the host immunity. The abnormal proliferation of oral-associated microorganisms may be linked to the reduction of gastric acid. The present review supports the potential association between oral microbiome and gastric precancerous lesions. However, the interactions are complex and multifaceted, which require further investigation.

Artificial Intelligence–Assisted Colonoscopy for Detecting Polyps, Adenomas, Precancerous Lesions, and Colorectal Cancer

What Is the Issue? Colorectal cancer is 1 of the most common cancers globally. In Canada, it is estimated that more than 25,000 people will be diagnosed with colorectal cancer in 2024 and that more than 9,000 people will die of it. To reduce the incidence and mortality of colorectal cancer, screening programs across various jurisdictions in Canada have implemented screening strategies involving routine fecal testing and colonoscopy. While colonoscopy is often considered the gold standard for colorectal cancer screening, missed polyps remain a challenge with this modality. In recent years, many artificial intelligence (AI)–enabled polyp detection systems have been developed for use during colonoscopy. A review of the clinical and cost-effectiveness of these systems could help clarify their potential role in clinical practice. What Did We Do? To inform decisions regarding the use of AI-assisted colonoscopy, we conducted a rapid review to identify and summarize evidence that compared the clinical and cost-effectiveness of AI-assisted colonoscopy to conventional colonoscopy and among different AI-assisted colonoscopy systems. We searched key resources, including journal citation databases, and conducted a focused internet search for relevant evidence published since 2019. One reviewer screened articles for inclusion based on predefined criteria, critically appraised the included studies, and narratively summarized the findings. What Did We Find? We found 1 health technology assessment (HTA), 3 systematic reviews (SRs), and 1 randomized controlled trial (RCT) that evaluated the clinical and cost-effectiveness of AI-assisted colonoscopy for detecting polyps, adenomas, precancerous lesions, and colorectal cancer. AI-assisted colonoscopy may improve clinical outcomes compared to conventional colonoscopy, including adenoma detection rates, the number of adenomas detected per procedure, and adenoma miss rates, although it may also lead to longer withdrawal times. However, not all studies included in this report demonstrated statistically significant differences between the groups for each of these outcomes. The included RCT allocated participants to receive AI-assisted colonoscopy with either Deep-GI or CAD EYE, but the authors did not perform statistical testing to compare the outcomes between these 2 treatment groups. Findings from 6 economic evaluations summarized in the HTA suggest that AI-assisted colonoscopy is likely to be cost-effective or dominant — meaning it is less costly and more effective — compared to conventional colonoscopy. We did not find any studies on the relative cost-effectiveness of different AI-assisted colonoscopy systems that met our selection criteria for this review. None of the included studies reported long-term outcomes, such as colorectal cancer incidence and mortality; therefore, the impact of AI-assisted colonoscopy on these outcomes is unknown. What Does This Mean? AI-assisted colonoscopy may improve clinical outcomes and be more cost-effective than conventional colonoscopy for detecting polyps, adenomas, precancerous lesions, and colorectal cancer. The clinical and cost-effectiveness of different types of AI-assisted colonoscopy systems compared to each other is unknown. Clinicians and decision-makers can use the evidence summarized in this review to inform decisions regarding the implementation of AI-assisted colonoscopy.

The Undetermined Significance of Screening for Monoclonal Gammopathy of Undetermined Significance

AbstractThe identification of premalignant lesions in solid tumors such as colorectal and cervical cancer has led to the development of highly effective population-wide screening programs for these malignancies. Although precursor clinical states are not well-established for most hematologic malignancies, multiple myeloma is a notable exception. Virtually all patients with multiple myeloma first develop monoclonal gammopathy of undetermined significance (MGUS), an asymptomatic but easily detectable condition. However, broad screening for MGUS has not been adopted primarily because there is no proven intervention to delay or prevent the progression of MGUS to multiple myeloma. Furthermore, the incidence of MGUS is poorly understood; a better understanding of its epidemiology will be critical in designing appropriate screening guidelines once an effective treatment has been developed. In the current issue of Cancer Epidemiology, Biomarkers &amp; Prevention, Ji and colleagues provide important epidemiologic insights on MGUS, including a critical discussion on how its incidence and preclinical duration vary by age and race, acknowledging that both MGUS and multiple myeloma are more common in Black than White populations.See related article by Ji et al., p. 1690

Dental pulp stem cells promote malignant transformation of oral epithelial cells through mitochondrial transfer.

Oral epithelial dysplasia includes a range of clinical oral mucosal diseases with potentially malignant traits. Dental pulp stem cells (DPSCs) are potential candidates for cell-based therapies targeting various diseases. However, the effect of DPSCs on the progression of oral mucosal precancerous lesions remains unclear. Animal experiments were conducted to assess the effect of human DPSCs (hDPSCs). We measured the proliferation, motility and mitochondrial respiratory function of the human dysplastic oral keratinocyte (DOK) cells cocultured with hDPSCs. Mitochondrial transfer experiments were performed to determine the role mitochondria from hDPSCs in the malignant transformation of DOK cells. hDPSCs injection accelerated carcinogenesis in 4NQO-induced oral epithelial dysplasia in mice. Coculture with hDPSCs increased the proliferation, migration, invasion and mitochondrial respiratory function of DOK cells. Mitochondria from hDPSCs could be transferred to DOK cells, and activated mTOR signaling pathway in DOK cells. Our study demonstrates that hDPSCs activate the mTOR signaling pathway through mitochondrial transfer, promoting the malignant transformation of oral precancerous epithelial lesions.

438 The association of multiple cutaneous squamous cell carcinomas with premalignant lesions and basal cell carcinomas

438 The association of multiple cutaneous squamous cell carcinomas with premalignant lesions and basal cell carcinomas

Diagnostic Performance of Cytocolposcopy versus Biopsy for Premalignant and Malignant Lesions in a Women's Hospital Dysplasia Clinic

Background: Cervical cancer (CRC) is a public health problem because it is the fourth most common gynecologic neoplasm worldwide. The screening tests used to diagnose this pathology are cervical cytology, which in suspected malignancy or with malignancy requires colposcopy to identify the affected area and thus guide the biopsy, which is the gold standard for diagnosis. Therefore, these tests are complementary, and a high diagnostic concordance is required to make a confident diagnosis. Subjects and Methods: A retrospective, cross-sectional, observational, and analytical study was performed. A total of 1470 medical records were analyzed, of which 175 patients met the inclusion criteria. The cyto-colposcopic diagnostic yield was compared with the histopathologic yield. The concordance between screening tests and the gold standard was calculated using Cohen's kappa coefficient Results: The sample comprised 175 subjects who met the selection criteria (11.9%). The mean age was 34.59 + 11.01 years, ranging from 17 to 65 years. The mean sexual debut was 16.6 years, with a mean of 3.1 ± 2 sexual partners. When patients were classified according to lesion type, the highest percentages were found in low-grade squamous intraepithelial lesions (LSIL). With 45.71, 61.14, and 49.14% for cytologic, colposcopic, and histopathologic examination, respectively. The highest concordance between histopathology and cytology was found in the high-grade squamous intraepithelial lesion (HSIL) with 0.41, and the concordance between histopathology and colposcopy in HSIL and cancer was 0.55 and 0.74, respectively. Conclusions: Papanicolaou tests and colposcopy showed moderate concordance with histopathologic findings; the diagnostic accuracy of colposcopy is superior to that of cytology.

Robust Assessment of Cervical Precancerous Lesions from Pre- and Post-Acetic Acid Cervicography by Combining Deep Learning and Medical Guidelines

Robust Assessment of Cervical Precancerous Lesions from Pre- and Post-Acetic Acid Cervicography by Combining Deep Learning and Medical Guidelines

Chemoprophylaxis for precancerous lesions of the large intestine

Chemoprophylaxis for precancerous lesions of the large intestine

Exploring thenew Kyoto guidelines for managing pancreatic cysts: an overview and comparison with previous guidelines.

The rising use of diagnostic imaging has led to an increase in the incidental detection of pancreatic cysts, with reported incidences of 1.2-2.6% on Computed Tomography and 2.4-49.1% on Magnetic Resonance Imaging. While many of these cysts are asymptomatic and benign, the enhanced imaging techniques have also revealed malignant and premalignant lesions. Mucinous neoplasms, including intraductal papillary mucinous neoplasms (IPMNs) and mucinous cystic neoplasms, are particularly concerning due to their potential for malignant transformation. Recent studies highlight significant variations in dysplasia across IPMN types, with main duct IPMNs showing a higher likelihood of high-grade dysplasia or invasive carcinoma compared to branch duct IPMNs. Management and follow-up of these lesions remain controversial due to inconsistent guidelines. This article reviews and compares six major guidelines: the 2015 American Gastroenterological Association guidelines, the 2017 International Association of Pancreatology (IAP/Fukuoka) guidelines, the 2017 American College of Radiology guidelines, the 2018 American College of Gastroenterology guidelines, the 2018 European Study Group guidelines, and the newly released, 2024 Kyoto guidelines. We summarize key differences in risk factors, surveillance protocols, and surgical referral criteria, with a focus on the updated 2024 Kyoto guidelines and the implications of recent research advancements.