TM II HPV28  to detect the high-risk (HR)-HPV genome in the distant lesions in these patients. Results: Eight cases of cervical cancer with a new secondary lesion were identified. In seven, HR-HPV DNA was detected in the biopsy of the distant lesion, which confirmed the diagnosis of cervical cancer metastasis. In the remaining case, no HPV was detected in the secondary lung biopsy, confirming the diagnosis of new primary lung cancer. Conclusion: Our results pave the way for HPV molecular genotyping use in cases of newly diagnosed distant lesions in patients with a history of HPV cervical neoplasia by using a routine diagnosis process to complete the clinical and histologic differential diagnosis when confronted with ambiguous situations.Background: Differentiating metastatic cervical cancer from another primary tumor can be difficult in patients with a history of cervical cancer and a distant lesion. The use of routine HPV molecular detection and genotyping tests could help in these cases. The objective of this study was to identify if an easy-to-use HPV molecular genotyping assay would allow differentiating between HPV tumor metastasis and a new independent primary non-HPV-induced tumor. Materials and Methods: Between 2010 and 2020, we identified patients with a primary cervical carcinoma who also had another secondary lesion. This identification included a clinical and histologic differential diagnosis of metastatic cervical cancer versus a new primary cancer or metastatic cancer from another site. We used a routine multiplex real-time PCR (rt-PCR) Anyplex Cervical cancer induced by high risk (HR)-human papilloma virus (HPV) infection is the fourth most frequent cancer in women worldwide with 569,000 new cases each year . Even inLesions of unknown origin out of the genital tract could occur in cervical cancer patients. Differential diagnosis between these lesions in the pathological analysis can be difficult, even with the help of immunohistochemistry techniques (IHC). However, the right identification of their origin will be essential for patients’ clinical care, treatment and prognosis.TM II HPV28  to study the HR-HPV genome in new distant lesions in cervical cancer patients. The objective of this study was to identify if such an easy-to-use HPV molecular genotyping assay would allow differentiating HPV tumor metastasis from a new independent primary non-HPV-induced tumor to improve care management of patients.Since HPV presence is required to maintain the tumoral phenotype,  HPV DNA We retrospectively included women with a histologically confirmed primary cervical carcinoma treated with surgery and/or chemoradiotherapy who also had another distant lesion. Patients were treated in two French medical centers  between 2010 and 2020. The lesion was suspected based on clinical symptoms or the results from imaging methods, namely magnetic resonance imaging (MRI), positron emission tomography (PET) or computed tomography scan (CT). All new lesions were biopsied and assessed by specialized pathologists in gynecological tumors.Clinical and histologic differential diagnoses for the distant lesion were performed in order to differentiate whether the lesion was a metastasis from either the known cervical cancer or caused by another primary/metastatic cancer. Classical HE (Hematoxylin Eosin Safran) and IHC analyses were performed. In most cases, the histological type and IHC were compatible with different origins (metastasis or other primary cancer), allowing for multiple diagnostic options.The diagnosis of any secondary lesions occurred at the same time as that for primary cervical cancer (synchronous), or later on during follow-up. For any secondary lesion, we recorded the year of diagnosis, location of the lesion, histology type and the p16 expression status .TM real-time PCR instrument  [TM and MuDTTM technologies [TM II HPV28 assay was shown to be suitable for an extended genotyping approach with a high sensitivity in FFPE specimens [Formalin-fixed, paraffin-embedded (FFPE) biopsies of distant lesions and the correspondent available tumor (only available for 4/8 patients) were sent to the ISO 15189-accredited virology laboratory of the Georges Pompidou European Hospital  for DNA extraction as described by Steinau et al.  prior to France) ,11. Basenologies , this aspecimens .Under our inclusion criteria, we identified eight women treated in two French comprehensive cancer centers between 2010 and 2020 .In these eight cases, the secondary lesion was difficult to identify as a cervical tumor metastasis or second primary tumor.The clinical and histological features of these women are described in Two cases were synchronous with primary cervical cancer. The other cases were diagnosed later (the time of recurrence/metastasis was between one and three years). Moreover, except for the synchronous cases that were treated with surgery, all other patients received chemoradiotherapy as initial treatment after the tumors were biopsied. The most common site of the second tumor was the lung (four cases).For the results interpretation, the quality of extracted DNA was confirmed by the systematic positivity of the internal control β-globin for all samples. Therefore, in the distant lesion, results from RT-PCR showed HPV-16 DNA in six cases and HPV-18 DNA in one case, thus completing the histological results and confirming the cervical origin of the new lesion . Lastly,In this study, eight documented cases of women with cervical cancer showed that the use of routine HPV molecular detection and genotyping allowed the differentiation between metastatic cervical cancer disease and a new primary tumor.We found HPV in all distant lesions except one case. This case, with negative HPV in the lesion, was considered as primary lung cancer and was treated with Navelbine with a fractionated dose of cisplatin (CDDP), unlike the cases diagnosed as metastatic cervical cancer, which were treated with carboplatin and taxol.The prognosis of patients with metastatic cervical cancer is poor . There are two types of metastases in cervical cancer patients: hematogenous and lymphatic. Patients with hematogenous metastasis have a higher risk of mortality than those with lymphatic metastasis. In addition, the management of metastasis may be different depending on its origin and location. Surgery, radiotherapy, chemotherapy or combinations of several treatments are options .In patients with HPV-related cancer and a distant lesion, the histologic differentiation of metastatic cervical cancer versus another primary tumor or metastasis from another cancer can be difficult, especially if the distant lesion shows similar histologic findings as the cervical cancer . However(INK4a) is a well-established surrogate marker in HPV-related malignancies [Overexpression of the cyclin-dependent kinase inhibitor gene p16gnancies . p16 expgnancies . This maTechniques such as in situ reverse transcriptase polymerase chain reaction (RT-PCR) for human papillomavirus RNA and in situ hybridization have allowed the differentiation of metastatic cervical carcinoma from either a new primary tumor or metastasis from another primary tumor . AlthougArfi et al. recently used the identification of the same HPV integration site in the paired DNA of endo-cervical and ovarian tumors as a proof of cervical metastasis in the ovaries . NeverthConversely, in the literature, we did not find any cases of HPV detection in distant lesions that were unrelated to cervical cancer, so the possibility of a false positive from this technique seems low.In our cases, we only identified HPV 16 or 18 in tumors and secondary lesions; however, other studies have detected other types, most frequently HPV 45 .As our study was a retrospective analysis, the sample size presented is low and represents a clear limitation of our study. Indeed, we only succeeded in achieving HPV genotyping in half of the initial cervical tumors, which allowed us to match the HPV genotype from both the initial and the distant lesion, due, in some cases, to the poor preservation of DNA in old FFPE samples. It also highlighted the interest of systematically performing HPV molecular genotyping of cervical cancer to optimize post-therapeutic medical care and monitoring of such patients. In view of our results, we suggest that HPV molecular genotyping of newly diagnosed distant lesions in patients with a history of cervical neoplasia could be helpful to improve the diagnosis and treatment of such new lesions in patients with doubtful distant tumors.


HPV+ and HPV- HNSCC share distinct epigenetic characteristics and clinicopathological features. The aim of our study was to assess whether DNA methylation plays a role in the progression of HPV+ HNSCC. We used a HumanMethylation450 BeadChip array (Illumina) to identify PRKCZ genes exhibiting different levels of DNA methylation between HPV+ and HPV- HNSCC. PRKCZ acts as a potent tumor promoter in HPV+ HNSCC. These findings may provide a possible explanation for the differences in the clinicopathological characteristics between HPV+ and HPV- HNSCC and promising ideas for the treatment of HPV+ HNSCC.Purpose: To study the role of target genes with aberrant DNA methylation in HPV+ HNSCC. Methods: A HumanMethylation450 BeadChip array (Illumina) was used to identify differentially methylated genes. CCK-8, flow cytometry, wound healing, and cell invasion assays were conducted to analyze the biological roles of PRKCZ. Western blot, qRT-PCR, immunohistochemistry, and animal studies were performed to explore the mechanisms underlying the functions of PRKCZ. Results: We selected PRKCZ, which is associated with HPV infection, as our target gene. PRKCZ was hypermethylated in HPV+ HNSCC patients, and PRKCZ methylation status was negatively related to the pathological grading of HNSCC patients. Silencing PRKCZ inhibited the malignant capacity of HPV+ HNSCC cells. Mechanistically, HPV might promote DNMT1 expression via E6 to increase PRKCZ methylation. Cdc42 was required for the PRKCZ-mediated mechanism of action, contributing to the occurrence of epithelial-mesenchymal transition (EMT) in HPV+ HNSCC cells. In addition, blocking PRKCZ delayed tumor growth in HPV16-E6/E7 transgenic mice. Cdc42 expression was decreased, whereas E-cadherin levels increased. Conclusion: We suggest that PRKCZ hypermethylation induces EMT via Cdc42 to act as a potent tumor promoter in HPV+ HNSCC. Head and neck squamous cell carcinoma (HNSCC) is the most common malignant tumor stemming from the oral cavity, with approximately 890,000 new cases and 450,000 deaths occurring worldwide in 2018 ,4. In adCDKN2A, DAPK, MGMT, and E-cadherin is crucial in HNSCC progression [The accumulation of epigenetic alterations, including aberrant DNA methylation, is an important carcinogenic feature of HNSCC . DNA metgression .PRKCZ gene is located on human chromosome 1 (1p36.33–p36.2) and encodes the following two proteins: Protein kinase M zeta (PKM-ζ) and Protein kinase C zeta . PKM-ζ is a constitutively active form of atypical PKC that is expressed only in the nervous system and is involved in long-term memory maintenance [The ntenance . PKC-ζ cntenance , which mntenance . PRKCZ hntenance . HoweverPRKCZ gene with different degrees of DNA methylation between HPV+ and HPV- HNSCC for the first time. Our data further illustrated that blocking PRKCZ expression could inhibit the proliferation, migration, and invasion abilities and increase the apoptosis ratio of HPV+ HNSCC cells. In addition, downregulated PRKCZ expression could damage the process of epithelial-mesenchymal transition (EMT). These findings may provide a possible explanation for the differences in the clinicopathological characteristics between HPV+ and HPV- HNSCC, and promising ideas for the treatment of HPV+ HNSCC. In this study, we used the HumanMethylation450 BeadChip array (Illumina) to identify the Patients and clinical specimens 40 primary HNSCC tissue specimens, including 12 paraffin-embedded tissue specimens and 28 fresh tissue specimens, were derived from patients without history of previous treatments or autoimmune diseases who had accepted radical surgery at West China Hospital of Stomatology, Sichuan University. All patients gave informed consent. These specimens have been pathologically identified as HNSCC and divided into different grades on the basis of the International Union for Cancer Control’s latest standards. The clinicopathological characteristics of these patients are provided in An HumanMethylation450 BeadChip array  of clinical specimens from HNSCC patients who had accepted radical surgery at West China Hospital of Stomatology, Sichuan University, including 3 HPV-positive cases and 3 HPV-negative cases, was provided by oeBiotech Limited Company. The clinicopathological characteristics of these 6 patients were provided in Genomic DNA from 12 paraffin-embedded tissue specimens and 28 fresh tissue specimens was isolated using DNA Extraction Kit . Bisulfite conversion of DNA was carried out using the EZ DNA Methylation Kit  following the manufacturer’s procedure. Gene methylation was analyzed by Methylamp MS-qRT-PCT Fast Kit , which was performed on ABI PRISM 7900 sequence detection system  at 95 °C 7 min → (95 °C 10 s → 55 °C 10 s → 72 °C 8 s) × 40 cycles → 72 °C 1 min. The β-actin (ACTB) was used as an endogenous control. The primers for qMSP are as follows: PRKCZ-F: TTTTTTTATAGGGGATTTTGGATTC; PRKCZ-R: CACACTTACGAACTAAAACTACGAA;ACTB-F: TGGTGATGGAGGAGGTTTAGTAAGT;ACTB-R: AACCAATAAAACCTACTCCTCCCTTAA−(ΔCt PRKCZ–ΔCt ACTB) method [All experiments were repeated three times at least and the methylation degree of PRKCZ was analyzed using 2) method .® Green qPCR mix 2.0 (GeneCopoeia) on ABI PRISM 7900 sequence detection system. Reaction system: 12 μL DEPC water, 4 μL 5 × BlazeTaq qPCR Mix, 1 μL Forward Primer (10 μM), 1 μL Reverse Primer (10 μM), 2 μL template cDNA, final volume was 20 μL. Reaction conditions: 95 °C 30 s → (95 °C 10 s → 60 °C 30 s) × 40 cycles. Specific primers of each gene for qRT-PCR were shown in −△△CT method [Total RNA of tissue specimens was extracted according to Trizol instructions. The reverse transcription was carried out using SureScript™ First-Strand cDNA Synthesis Kit  was carried out for reverse transcription reaction. Then qRT-PCR was performed with BlazeTaq™ SYBRT method .Total proteins of tissue specimens were prepared via RIPA lysis buffer. Next, proteins were separated through sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Then, gel was transferred onto a PVDF membrane . After that, membranes with target protein were blocked in 5% defatted milk and incubated with specific primary antibody overnight at 4 °C. Primary antibodies were listed as follows: anti-PKC-ζ , anti-Cdc42 , anti-E-cadherin , anti-N-cadherin , anti-Vimentin , anti-HPV16 E6 , anti-HPV16 E7 , anti-DNMT1 , anti-DNMT3a , anti-DNMT3b . After incubating goat anti-rabbit or goat anti-mouse secondary antibody , membranes with target protein incubated with the chemiluminescence reagent  were visualized via ChemiDoc XRS+ System . All original Western blots can be found in Formalin-fixed paraffin-embedded sections were firstly dewaxed in xylene and rehydrated in graded alcohol solutions. Then antigen retrieval was performed by boiling for 3 min in 0.01 M citrate buffer (pH = 6.0), and sections were immersed in 3% hydrogen peroxide for 10 min and in PBS for 5 min twice. After incubating with 5% goat serum for antigen blocking, the sections were incubated with the primary antibody at 4 °C overnight in a moist chamber. Additionally, the following primary antibodies were listed: anti-PKC-ζ , anti-P16 , anti-Cdc42 , anti-E-cadherin , anti-N-cadherin , anti-Vimentin . After washing with PBS twice, the sections were incubated with biotinylated anti-mouse/rabbit IgG and sequent with streptavidin-biotin peroxidase both for 15 min. DAB was added to detect the primary antibody, and hematoxylin was used to stain the nucleus. As negative controls, sections were analyzed in parallel except incubating with isotype-specific immunoglobulin (IgG) instead of the primary antibody. Quantification was evaluated by three independent investigators who were blinded to patient characteristics. According to the degree of staining in ten randomly selected fields at 400× magnification, the level of target protein was assigned as 0 (negative), 1 (weak), 2 (moderate), and 3 (strong).2.HPV16-positive human HNSCC cell lines SCC47 and SCC090, HPV-negative human HNSCC cell lines Cal27, SCC25, and SCC9 were obtained from State Key Laboratory of Oral Disease, Sichuan University. SCC47, SCC090, Cal27, and SCC9 were cultured in DMEM  containing 10% fetal bovine serum . SCC25 was cultured in DMEM-F12 (Gibco) containing 10% fetal bovine serum . All of them were maintained at 37 °C in a humidified atmosphere with 5% COTM (GeneCopoeia) was used for transfections according to the manufacturer’s instructions. Further experiments were conducted 48 h after transfection. The plasmids for HPV16 E6 and E7 overexpression and negative control plasmid were designed by GenePharma Co., Ltd., China. Stably transfected Cal27 with E6 and E7 overexpression were derived from the parental cells by a fluorescence microscope  detection and puromycin  selection. The sequences of siRNAs are listed in GenePharma Co., Ltd., Suzhou, China provided the design and composition services of small-interfering RNAs (siRNAs) targeting E6, E7, and PRKCZ. EndoFectin3 (cells/well) and cultured at 37 °C in a humidified atmosphere with 5% CO2 for 1–5 days. Subsequently, cells were treated with 10 μL CCK-8 reaction solution  and incubated for 1 h in cell constant temperature incubator. Then, the light absorption value  at 450 nm was measured. Each assay was carried out in triplicate.CCK-8 assay was used to test cell proliferation capacity. Treated cells were seeded in 96-well plates at a density of 1 × 10Flow cytometry using the Annexin V-FITC Apoptosis Detection Kit (Invitrogen) was for the test of cell apoptosis. Treated cells were seeded in 6-well plates and digested after 48 h. Then, cells were resuspended with 1X Binding Buffer. Stained with 5 μL of Annexin V-FITC Conjugate and 5 μL of Propidium Iodide Solution, cells were incubated for 30 min at room temperature under dark conditions. After washing and resuspending, the percentage of stained cells was analyzed using a FACScalibur (Becton-Dickinson). Experiments were conducted in triplicate.Appropriate cells were seeded in 6-well plates to obtain 100% confluence next day. After 24 h of starvation in serum-free medium, a pipette tip was used to scratch the cells and form the clearance space with suitable width. After washing with PSC to clear out the dead cells, photomicrographs of clearance space were obtained at the timepoint of 0 and 48 h. Closed scratch areas were calculated by ImageJ software. Each experiment was conducted in triplicate.4 cells were seeded into the upper chamber of 24-well transwell plates  with Matrigel-coated membrane (BD Bioscience). After incubation for 24 h in cell constant temperature incubator, the medium in upper chamber was changed into serum-free, while that in lower chamber contained 10% FBS. Then, after 24 h incubation, cells that invaded through the Matrigel-coated membrane were fixed in 4% paraformaldehyde for 30 min and stained with 0.1% crystal violet and counted under a microscope. Five randomly selected fields were used to count the number of stained cells. Assays were conducted in triplicate.5 × 10n = 5), 4-NQO + PRKCZ siRNA group (n = 5) and 4-NQO group (n= 5). Mice were fed with distilled drinking water containing a concentration of 100 μg/mL 4-NQO (Sigma-Aldrich) solution for 10 weeks, then distilled water for another 10 weeks to generate HNSCC. After that, PKC-ζ pseudosubstrate inhibitor (Cayman) was injected near the tumor every three days at 100 mg/kg in 4-NQO + PKC-ζ pseudosubstrate inhibitor group. The 4-NQO + PRKCZ siRNA group was injected with PRKCZ siRNA according to the same concentration and method, while 4-NQO group was injected with DEPC water. Mice were killed by cervical dislocation after 3 weeks, and tongues were collected. Specimens bisected longitudinally were fixed with 10% formalin, paraffin-embedded, or frozen at −80℃. The sequence of PRKCZ siRNA was GCCATGAAGGTGGTAAAGAAG.For 4-nitroquinoline 1-oxide (4-NQO)-indued HNSCC model of transgenic mice, a total of fifteen 7-week-old HPV16-E6/E7 transgenic mice after one week of acclimation  were used and divided randomly into the following three groups: 4-NQO + PKC-ζ pseudosubstrate inhibitor group (t-tests or one-way ANOVA. The association between PRKCZ methylation and mRNA expression was assessed via 2-tailed Pearson’s statistics. All cellular experiments were conducted independently at least three times and in triplicate each time. GraphPad Prism 7.0 (GraphPad Software) was used for processing all data, and values were presented as means ± SD. p < 0.05 was considered to be statistically significant.Means comparisons were performed with Student To identify the differentially methylated genes between HPV+ and HPV- HNSCC, a HumanMethylation450K BeadChip array (Illumina) was performed. According to the analyzed data, 1344 genes were hypermethylated and 754 genes were hypomethylated in HPV+ HNSCC, compared with HPV- HNSCC A. We colp = 0.0469)  G. This d 0.0469) H,I.Furthermore, we detected the differences in PRKCZ methylation and mRNA levels between HPV+ HNSCC cells (SCC47 and SCC090) and HPV- HNSCC cells . Consistent with the results above, HPV+ HNSCC cells had increased methylation degrees and decreased mRNA and protein expressions A,B. To fp = 0.3138, p = 0.4679, p = 0.2365, p = 0.5322, and p = 0.5827, respectively), whereas PRKCZ methylation status was significantly related to the pathological grading of HNSCC patients (p = 0.0386). Patients with PRKCZ hypermethylation seemed to have worse pathological manifestations  with siRNA1, we found that the OD values on Days 3, 4, and 5 were significantly lower in the transfected cells compared with two cells transfected with control siRNA. This finding suggests that silencing PRKCZ impairs the proliferation abilities of HPV+ HNSCC cells C. We appIn addition, we used a specific inhibitor of PRKCZ, a PKC-ζ pseudosubstrate inhibitor, which decreased PRKCZ mRNA level in SCC47 cells to 10% G. SimilaE6 and E7, two main well-known carcinogens of HPV, target host tumor suppressor genes or proteins to drive carcinogenesis and progression . Thus, wThe DNMT family has been demonstrated to act as irreplaceable and crucial enzymes involved in DNA methylation ,34. ThusTo investigate the internal mechanisms by which PRKCZ influences HPV+ HNSCC cell biological behaviors, we performed a KEGG enrichment analysis of PRKCZ. The results suggested that among the pathways in which PRKCZ is involved, the Rap signaling pathway was ranked 7th among the top 20 KEGG pathways for abnormal gene methylation enrichment . In addiFirst, we detected the expression changes of several key genes involved in the Rap signaling pathway  in HPV+ HNSCC cells after PRKCZ expression was blocked. The qPCR results suggested that after PRKCZ silencing, Epac1, Rac1, Rap1A, Rap1B, Cdc42, and RhoB mRNA expression decreased, whereas the change in RhoA was not significantly different. In addition, Cdc42 mRNA levels changed most dramatically B. Cdc42 3 on average in inhibitor-treated mice and (1.799 ± 0.630) mm3 in siRNA-treated mice, whereas that in DEPC-treated mice was (5.108 ± 1.962) mm3  in rodents due to the similar histological and molecular changes observed in human oral carcinogenesis . In our 962) mm3 C. These Then, IHC staining demonstrated that Cdc42 expression decreased in tumors from the inhibitor-treated and siRNA-treated groups, whereas E-cadherin levels increased D. These The importance of DNA methylation in host–viral interactions and cancer development is self-evident ,41. The HPV has a specific effect on shaping the DNA methylome of HNSCC . In addiPRKCZ was the only gene associated with HPV infection among 50 selected genes with the most obvious methylation differences between HPV+ HNSCC and HPV- HNSCC according to array data. This finding was further supported by data obtained from TCGA, clinical specimens, and cell lines, revealing that PRKCZ was hypermethylated in HPV+ HNSCC. However, the difference in the level of methylation observed in TCGA data was not significant, which may be due to a discrepancy in the PRKCZ methylation degree and HPV status. PRKCZ hypermethylation plays a vital role in several diseases, such as type 2 diabetes mellitus , CTCF deHPV-mediated cancer development is inseparable from the continuous expression of the viral E6 and E7 oncogenes . In addiIn our study, we used two methods to inhibit PRKCZ expression, including siRNA and a pseudosubstrate inhibitor, which is a specific and efficient inhibitor of PKC-ζ . HoweverCdc42 is a Rho GTPase responsible for the dynamic reorganization of the actin filament system . Cdc42 pIn our HPV16-E6/E7 transgenic mouse model of tongue tumors, we found that tumor lesions in the tongue were much smaller in the inhibitor-treated and siRNA-treated groups compared with the DEPC-treated group, which might be due to decreased Cdc42 expression and increased E-cad expression. In our animal experiments, we did not demonstrate whether demethylation therapy with PRKCZ was effective in suppressing tumor growth because this function had been reported by Biktasova A et al. These researchers proposed that the application of 5-aza could inhibit HPV+ HNSCC tumor growth and mouse blood vessel invasion by repressing the expression and activity of matrix metalloproteinases . TherefoOverall, we illustrated for the first time that PRKCZ was hypermethylated in HPV+ HNSCC. In addition, PRKCZ acts as a tumor promotor that induces HPV+ HNSCC cell proliferation, invasion, and migration and prevents apoptosis. Mechanistically, HPV E6 might promote the expression of DNMT1 to induce PRKCZ hypermethylation, and PRKCZ increased Cdc42 levels to promote EMT in HPV+ HNSCC cells. These results indicated that PRKCZ hypermethylation might represent one possible reason why HPV+ HNSCC has a better prognosis and that blocking PRKCZ might be a new strategy for HPV+ HNSCC.


Second primary cancer (SPC) is the second most common cause of death among patients diagnosed with head and neck cancer. This study examined the risk of SPC following oropharyngeal squamous cell carcinoma (OPSCC) and the impact of human papillomavirus (HPV) on survival following SPC. The study was a population-based, retrospective study including all patients diagnosed with OPSCC in eastern Denmark from 2000–2020 who received curative intended treatment. The incidence rate ratio (IRR), age-adjusted incidence rates (AAIR), and hazard ratios (HR) were calculated. A total of 2584 patients with primary OPSCC were included (median follow-up time: 3.1 years), with 317 patients (12.3%) diagnosed with SPC. The risk of SPC was approximately five times the occurrence of cancer in the general population (IRR: 4.96). The median time to SPC after a primary OPSCC was 2.0 years (interquartile range (IQR) = 0.6–4.2 years). HPV-positive (HPV+) patients had a significantly longer median time to SPC, and a significant better survival compared to HPV-negative (HPV-) patients. SPC was most frequently found in lungs, head, and neck (LHN) for HPV- OPSCC patients and lungs followed by gender-specific  for HPV+ OPSCC. There was a significant difference between the two groups when distributed between “within” or “outside” LHN. Patients with SPC outside LHN had a significant better overall survival. This knowledge should be considered during post-treatment surveillance and might guide targeted imaging. The global incidence of oropharyngeal squamous cell carcinoma (OPSCC) is estimated to be approximately 93,000 new patients per year . During HPV+ OPSCC patients have a better prognosis and demonstrate distinct clinical, histopathological, and genetic characteristics compared to HPV-negative (HPV-) OPSCC patients ,12,13,14Secondary primary cancer (SPC) is the second most common cause of death among patients with head and neck squamous cell carcinomas (HNSCCs), including OPSCC ,17,18. SAlcohol consumption and tobacco smoking are notable risk factors for developing SPC, especially regarding cancers localized in the upper aerodigestive tract, lungs, and oesophagus . StudiesIt is important to better characterize the risk of SPC in OPSCC and the relation to HPV status to improve screening, treatment, and follow-up strategies and hence oncologic outcomes.To our knowledge, the incidence and pattern of SPC in patients with OPSCC have not been evaluated in a population-based study in a high-HPV-prevalence area in patients with known HPV DNA and p16 status. The aim of this study was to examine the risk of SPC in patients with OPSCC in eastern Denmark from 2000–2020. Further, we evaluated the survival following SPC in relation to HPV status.® , 52.3% [95%CI: 43.9–62.2%], and 45.1% [95%CI: 35.8–57.0%]. For patients with SPC within LHN, the one-, three-, and five-year overall survival was 64.3% [95%CI: 56.6–73.1%], 28.6% [95%CI: 20.3–40.4%], and 20.8% [95%CI: 12.6–34.3%], respectively.To our knowledge, this is the largest consecutive, population-based retrospective study including 2584 patients diagnosed with OPSCC that all were HPV- and p16-tested. In this cohort from eastern Denmark from years 2000–2020, we found a high risk of SPC following a primary OPSCC. The risk was nearly five times higher (IRR = 4.96) compared to the risk of any cancer  in the general population in Denmark . It is eWe found an increased risk of SPC in older patients, which is not surprising since age is a well-established risk factor in all types of cancer. We also saw a higher risk of SPC in patients with a UICC8 stage II OPSCC at the time of diagnosis, which calls for further investigation since we cannot describe a clear explanation. Patients with a N2 classification at the time of diagnosis of OPSCC were shown to have a significantly lower risk of SPC in our analysis. This might be because HPV+ patients more often have lymph node involvement at the time of diagnosis. It would be of interest to examine baseline characteristics stratified by HPV status to further investigate the observed association. The risk of SPC was not associated with T-stage OPSCC or sublocation. HPV-positivity showed to be a significant protecting factor, which is in line with the before-mentioned differences in baseline characteristics between the subgroups of HPV+ and HPV- OPSCC patients. Further, Bosshart, S.L. et al.  also fouMost patients with OPSCC received primary radiation therapy with or without chemotherapy, which is a well-known risk factor for developing SPC, but according to Hashibe, M. et al. , the earp = 0.003), at 37.6% and 55.0%, respectively. We also found that patients with SPC outside the LHN area had a significant and better survival compared to patients with SPC within the LHN (p = 0.01), which probably reflects the inferior survival of lung cancer and the challenges of treating a second cancer in the head and neck area. Holstead, R. et al. [p = 0.001) and have a worse survival, which is in line with other studies [The overall predominant sites of SPC were the lungs, head, and neck, and the gastrointestinal tract. The SPC localizations differed between patients with HPV+ and HPV- OPSCC index tumours and head and neck were only the third most frequent site for HPV+ OPSCC compared to the second most frequent site in HPV- OPSCC. In contrast to patients with HPV- OPSCC, gender-specific SPCs were more common for patients with HPV+ OPSCC than head and neck malignancies. Further, HPV+ patients had a better overall survival than HPV- patients, reflecting the well-established clinical differences between patients with HPV+ and HPV- OPSCC. Since the lungs are the most predominant site for both HPV+ and HPV- and since the lungs are a common site for distant metastasis from OPSCC, we find it relevant, in the future, to further studies to investigate new malignancies in the lungs following OPSCC to better differentiate between SPC and recurrence . Further. et al.  also fou studies ,24. Addip = 0.02) and loco-regional (p = 0.001) recurrence following OPSCC. In this study, we found a significantly better overall survival following SPC for HPV+ OPSCC compared to HPV- (p = 0.003). These findings could be due to the patients diagnosed with HPV+ OPSCC being younger, demonstrating less comorbidity, and having a more favourable smoking history, as shown earlier [According to HPV status and the overall risk of SPC, we found a significantly lower risk of SPC in patients with HPV+ OPSCC index tumours. Other studies also found a lower risk of SPC in patients with p16+ OPSCC ,32 but d earlier . FurtherThe current study was restricted by its retrospective design. Further, there is no international consensus on how to define an SPC, and hence, the definition differs between studies ,21,31,38The strength of this study was the large size of the population-based, non-selected, consecutive cohort of all patients diagnosed with OPSCC in eastern Denmark with a high number of events, appropriate follow-up time, and a broad knowledge of clinical data and tumour characteristics, including both HPV DNA and p16 status. Further, it was a strength that all patients received a standardized treatment through the whole period, following the same guidelines.This study identified a high incidence of SPC (12.3%) among 2584 patients following a primary OPSCC in eastern Denmark from 2000–2020. The risk of SPC in patients diagnosed with OPSCC was nearly five times higher (IRR = 4.96) compared to the risk of cancer in the general population in Denmark. The most frequent localization of SPC was the LHN, but a different distribution was found in HPV+ and HPV- OPSCC, with SPC within the LHN being significantly lower in HPV+ OPSCC. The survival after SPC was significant better in HPV+ OPSCC and patients with SPC outside the LHN area.This knowledge should be considered during post-treatment surveillance and might guide targeted imaging for early detection and thereby improve prognostication.


The 5-year CSS rates of GNEC and GAC were 57.4% and 50.1%, respectively (p = 0.012). Besides, multivariable cox regression analyses showed that GNEC was an independent predictor of improved OS  and CSS  in the matched data. The prognosis of GNEC was better than GAC in Whites, showing significant ethnic differences. Appropriate treatments and follow-up strategies for GNEC in Whites are probably different from East Asians. The potential genetic and molecular mechanisms need to be further explored.It was generally believed that the prognosis of gastric neuroendocrine carcinoma (GNEC) was worse than gastric adenocarcinoma (GAC). However, almost all previous studies compared the prognosis of GNEC and GAC based on East Asians. In this study, we evaluated the clinicopathological features and prognosis of GNEC and GAC in Whites. Patients with GNEC and GAC were identified from 2000 to 2018 in the Surveillance, Epidemiology, and End Results (SEER) database. We used propensity score matching (PSM) analysis to match the age, sex, TNM stage, and treatments received between GNEC and GAC, then compared the overall survival (OS) and cancer-specific survival (CSS) in the two types. A total of 392 cases of GNEC and 12,835 cases of GAC in Whites were recognized. After PSM, the 5-year OS rates of GNEC and GAC were 50.3% and 43.0%, respectively ( Gastric cancer is the fifth most common malignant tumor all over the world with the fourth highest mortality . Among ap < 0.001). Although the threshold was controversial, containing neuroendocrine components was regarded as a symbol of poor prognosis in gastric cancer ) .p < 0.001, CSS p = 0.013). In addition, after PSM . Furthermore, GNEC was an independent prognostic factor for the distant metastatic stage gastric cancer in the multivariable analysis   .p value was 0.019 in the unmatched data and the p value was 0.052 in the matched data. In terms of CSS, there was no statistically significant difference in prognosis between the two groups. The p value was 0.104 in the unmatched data and 0.229 in the matched data. Besides, we showed the survival curves of GNEC and GAC among different races in the unmatched data . The prognosis of GAC in East Asians was better than in Whites .The baseline characteristics of patients with GNEC and GAC in East Asians are shown in hed data . The proIn this study, we evaluated the clinicopathological features and prognosis of GNEC in comparison to GAC in Whites. To our knowledge, this study is the first to compare the prognosis of GNEC and GAC in Whites. Inconsistent with the results of previous research studies focused on East Asians ,15,16,17It was believed that the prognosis of GNEC was worse than that of GAC. Containing neuroendocrine components was regarded as an indicator of poor prognosis in GAC. In 2013, Ishida et al.  comparedp = 0.010) or CSS (p = 0.012). On top of that, GNEC remained an independent prognostic factor by multivariable analysis . However, there is no statically significant difference for CSS in the two groups (CSS p = 0.103). The reason might be the small number of East Asian patients with GNEC, and this conclusion needs further validation. Dasari et al. [The second finding in this study was that race might be an important prognostic factor for patients with GNEC . In the i et al.  found thi et al.  did not i et al. ,13,14,15i et al. ,25,26.Besides, we found that most of the clinicopathological features of GNEC in Whites were similar to the previous study except for sex. In our review of 392 patients with GNEC, the median age of the patients was 63.5 years, which was similar to the previous study . GNEC waOur study has numerous strengths. Firstly, a large population database was utilized to compare the prognosis of GNEC and GAC in Whites, and a relatively great quantity of patients with GNEC, an “orphan” disease, were identified, leading to an adequately powered study. As far as I know, this is the first study focused on Whites, because all of the relevant previous research studies have been based on East Asians. Secondly, PSM was conducted to eliminate the observed bias in baseline covariates between the GNEC and GAC groups. We further confirmed our conclusion in different stages of GNEC. Lastly, we validated the previous conclusion that the prognosis of GNEC was worse than GAC in East Asians and the prognosis of GAC was worse in Whites than in East Asians. That could prove the reliability of our data and conclusions. There were also some limitations to our study. Firstly, specific pathological information and treatment details, such as the expression of Ki67 and chemotherapy regimen, were unavailable in the SEER database, impeding the opportunity to further eliminate the imbalance between GNEC and GAC. Moreover, because of the lack of information in several cases, we had to give up some patients, resulting in a further reduction of samples. Besides, the GAC group consists of a heterogeneous group of subtypes. The prognostic curves on GAC may not be representative for all of the subtypes within the group. Finally, socioeconomic- or treating facility-related factors were not included. Nevertheless, in consideration of the rarity of GNEC, our study has included a relatively large sample size, which could suggest the need for paying attention to racial disparities in the prognosis of GNEC.The results and conclusions of the present study were very meaningful. Treatment and follow-up patterns are determined according to the disease’s prognosis. In the past, GNEC was regarded as a malignant pathological type and treated with more radical treatment, such as salvage surgery after endoscopic resection for early GNEC, additional close follow-up strategies, and more aggressive adjuvant therapies . HoweverWe first discovered that the prognosis of GNEC was better than GAC in Whites. This will help us to further understand GNEC, especially its racial difference. A better understanding could lead to more appropriate treatment patterns and reduce unnecessary medical costs and psychological burden. The treatments and follow-up duration of GNEC might be adjusted according to different races. Further validation and potential genetic and molecular mechanisms among different racial groups should be investigated in the future, so as to further improve the prognosis of patients with GNEC.


Globally, cervical cancer is the fourth most common cancer among females and a major public health problem in low- and middle-income countries (LMICs). There are several screening tests available for cervical cancer screening; however, due to a lack of organised screening facilities as well as factors such as low participation rates in screening programmes, many women die due to cervical cancer. To reach out to a large number of women, an easy, non-invasive and time-saving screening method is required. Evidence supports that cervical cancer screening with human papillomavirus deoxyribonucleic acid (HPV DNA) testing is the most effective technique for lowering the incidence and mortality associated with cervical cancer when compared to other screening methods. Furthermore, a small number of studies have reported that menstrual blood can be used as an alternative sample for HPV detection for cervical cancer screening. We have done a systematic review of the studies that have reported the diagnostic accuracy of menstrual blood to detect HPV. We found five studies in our literature search. The studies showed the diagnostic accuracy of menstrual blood in terms of sensitivity ranging from 82.8% to 97.7% and specificity ranging from 50.0% to 98.0% in cervical intraepithelial neoplasia or HPV infection detection. This review supports the use of menstrual blood as a screening tool for cervical cancer especially in LMICs where women are reluctant to participate in cervical cancer screening due to issues such as embarrassment and discomfort to test procedures as well as busy schedules. However, further studies are required to compare the diagnostic accuracy of menstrual blood in detecting HPV compared to other self-sampled HPV detection methods. This is one of the methods that can be explored further for use as a cervical cancer screening test. Globally, cervical cancer is the fourth leading cancer in terms of incidence and mortality among females . Of the Human papillomavirus (HPV) testing, cytology, visual inspection with acetic acid (VIA) and visual inspection with lugol’s iodine are well-known standard screening tests for cervical cancer. There have been significant advancements in cervical cancer prevention techniques. Automated visual evaluation using Artificial Intelligence (AI) is gaining momentum for screening. Also, self-collected cervical samples for HPV deoxyribonucleic acid (HPV DNA) is an upcoming method and undergoing evaluation as a screening test –10. FurtHPV detection on menstrual blood, although not studied extensively, can be used for HPV based screening. In LMICs, the use of a menstrual cloth/pad is a common menstrual hygiene practice among women and the use of menstrual blood collected on a menstrual pad/cloth as a sample for HPV testing can be an alternative. In most of the African countries as well as countries like India, it has been reported that most of the women use cloth as a menstrual blood absorbent –18. We wStudies on the use of menstrual blood to detect HPV were included.Studies indicating the use of non-invasive menstrual hygiene practices such as menstrual pad/cloth/sanitary napkins to collect menstrual blood were included; studies were excluded if the menstrual hygiene practices included use of tampons or other invasive methods.Studies showing the diagnostic accuracy results of menstrual blood in HPV or high-risk HPV (HR-HPV) or low-risk HPV (LR-HPV) or cervical intraepithelial neoplasia (CIN) lesions were included. Studies were included if the results were reported in terms of sensitivity and/or specificity.No restrictions were applied in terms of study designs and study setting due to limited literature search results. We have included studies published up until December 2021. English literature full-text articles published in peer-reviewed journals were included.We conducted a thorough search using electronic databases including PubMed, Web of Science and academic search engines like Google Scholar. The potential eligible articles available in the English language were considered. The articles were searched using controlled vocabulary [For PubMed: Medical Subject Headings (MeSH) Terms] as well as keywords including ‘Human Papillomavirus testing’, ‘Menstrual pad’, ‘Cervical cancer screening’, ‘Menstrual Hygiene Products’. The reference list of each eligible article was carefully examined to capture more articles. The search strategy for PubMed is described in The number of included and excluded studies at each step is presented in a Preferred Reporting Items for Systematic Review and Meta-Analysis (PRISMA) flow chart .We identified a total of five studies that met the inclusion criteria of the review –22. The et al [et al [Of these five studies, four studies included women who had a pathological diagnosis of premalignant cervical lesions or positive with HR-HPV infection on cervical sample –22; wheret al  was donel [et al  used a sl [et al –22.et al [et al [et al [et al [et al [In these five studies, the day on which the menstrual blood was collected ranged from 1 to 4. Budukh et al  collectel [et al  collectel [et al  encouragl [et al , 21 did l [et al –22 excepl [et al  where woet al [et al [In the laboratory, a small piece of menstrual pad/cloth was cut by punching  or by uset al  used attet al , 21, 22 et al . The detet al –21 excepet al , 21, L1,et al . Moreoveet al , 21 185 et al ; whereaset al [Wong et al  includedet al [Lee et al  conducteA population-based study  conducteet al [A study by Wong et al  recruiteet al [Zhang et al  evaluateOverall, these five studies showed diagnostic accuracy of menstrual blood in terms of sensitivity ranging from 82.8% to 97.7% and specificity ranging from 50% to 98%. The screening for precancerous cervical lesions is one of the three objectives set by the World Health Organization (WHO) to achieve the global goal of eliminating cervical cancer by the year 2030. Cervical cancer screening with HPV DNA testing is a proven method. Recent evidence from the International Agency for Research on Cancer suggests that HPV DNA testing is the most effective technique for lowering the cervical cancer incidence and mortality when compared to other screening methods such as cytologic analysis and VIA. It has been observed that the probability of CIN3+ is lower following a negative HPV DNA test result than after a negative cytologic test result over a period of 3–10 years .For self-sampled HPV testing, a variety of samples such as cervico-vaginal fluid or lavages or urine and sample-collection methods including Dacron swabs, cotton-tipped swabs, tampon and cytobrushes have been used , 24. AltHPV detection using menstrual blood has advantages over other patient-performed HPV screening tests. Patient-performed HPV screening tests are not only inconvenient to collect, but also necessitate the storage and delivery of specimens in liquid media; whereas menstrual blood collected on a menstrual pad can simply be placed in a zip-lock bag and mailed to the laboratory. All the studies included in the review used zip-lock plastic bags to store the sample. Menstrual blood samples are both clinically and socially relevant to the ultimate success of primary screening for cervical cancer –21.et al [et al [Menstrual blood used for HPV detection may increase women’s participation in cervical cancer screening as the procedure is non-invasive when using a menstrual pad/cloth, time-saving and convenient. Wong et al  evaluatel [et al  also supl [et al .et al [et al [It is interesting to note that the delivery of sample by a health worker from participants to the laboratory can be beneficial. It has been reported that the uptake of the screening is increased when samples are collected by a familiar face, such as a health worker , 27. Wonet al  reportedl [et al  found thThe day of menstrual blood sample collection was documented in three out of five studies and this may also have an impact on the outcome. Based on the results, the authors noted that the viral levels of HR-HPV infections may decrease with the course of menstruation. The authors observed that when menstrual blood HR-HPV tests were performed during MCD 1 in women with CIN 3 or worse, the sensitivity and NPV of the tests, as well as the agreement rates for detecting HR-HPVs using cervical and menstrual blood samples, were at their highest . HoweverCost effectiveness is one of the most important factors while organising screening especially in LMICs. Organising cervical cancer screening requires a lot of effort as it involves a spacious screening clinic, trained human resources , mobile van and utilisation of the services of village administrative offices. It has been reported that the organising screening clinic costs 20%–40% of the total cost. Menstrual pads used as a cervical cancer screening tool can reduce the cost of organising a screening clinic , 28.Cervical cancer can be prevented easily if we provide easy and comfortable access of diagnosis and treatment to the participants. The major load of this cancer is in LMICs because of the difficulty of organising population based cervical cancer screening programmes. As per World Bank data, the majority of the LMICs population live in rural areas – on average 60%  . In cervThe studies showed diagnostic accuracy of menstrual pad in terms of sensitivity in the range of 82.8%–97.7% and specificity 50.0%–98.0%. The diagnostic accuracy values across the studies were comparable. It is interesting to note that all studies have been conducted in Asian countries and the diagnostic performance of this method may vary in different regions of the world. Further studies are required in different parts of the world with social and cultural differences.The WHO has declared that through cost-effective, evidence-based interventions, we can eliminate cervical cancer by the turn of the century if we achieve the following three goals by 2030 – 90% of girls to receive HPV vaccination by the age of 15 years, 70% of women to undergo screening at least twice in a lifetime and 90% of women with pre-cancer and invasive cancer to receive treatment . HoweverThe limitation of this review includes the overall smaller sample size as there are only five studies which show low reliability of the results. Furthermore, there is a heterogeneity in the studies such as the sensitivity was checked based on HPV positivity and/or on varying level of cervical neoplasia (from CIN1 to CIN3). Type of menstrual hygiene device used for sample collection may also influence the outcomes in detecting HPV. However, there is no supportive evidence to validate the type of menstrual hygiene devices that should be used for which further research is required. Apart from these limitations, there is a need to compare the menstrual blood HPV detection with other self-sampled HPV detection techniques to validate the method on a large sample size with a standardised method for menstrual blood sample collection, including type of HPV DNA test and the DNA extraction method. Few other factors that require exploration are type of device for menstrual blood collection (pad/cloth) and contamination of sample. Furthermore, HPV testing should be type specific as well as viral region specific , as per evidence. L1 expression is sometimes lost after HPV integration into the human genome, while E6/E7 expression is always present, which explains why there are no E6- or E7-negative cancers. A test designed solely for L1 will miss around 10% of all invasive tumours .Overall, the use of menstrual blood for HPV detection shows promising results and can be a potential tool for HPV detection, especially in places where cultural and social barriers are more prevalent. This method also holds promise for improved participation of women.To conclude, menstrual blood is a potential screening method for under-served women. However, to consider menstrual blood as a sample for HPV detection for a cervical cancer screening strategy by the public health departments of LMICs, further research is required. Initial studies from Asian countries are encouraging; however, larger population-based studies from different parts of the world with social and cultural differences are required.None.None.Priyal Chakravarti: Literature search, writing manuscript, quality control.Amita Maheshwari: Conceptualisation, reviewing the draft and providing critical inputs.Shweta Tahlan: Conceptualisation, reviewing the draft and providing critical inputs.Prithviraj Kadam: Literature search, assistance in writing the draft, Quality control.Sonali Bagal: Literature search, assistance in writing the draft, Quality control.Suvarna Gore: Literature search, assistance in writing the draft, Quality control.Nandkumar Panse: Reviewing the draft and providing critical inputs.Kedar Deodhar: Reviewing the draft and providing critical inputs.Pankaj Chaturvedi: Reviewing the draft and providing critical inputs.Rajesh Dikshit: Conceptualisation, reviewing the draft and providing critical inputs.Atul Budukh: Conceptualisation, overall supervision, reviewing the draft and providing critical inputs.


Circulating cells in the blood of cancer patients with solid tumors are known to provide clinically relevant information about treatment response, tumor progression, metastasis, and the development of drug resistance. We identify for the first time the distinctive morphological characterization of endometrial giant macrophage-like cells, called CAMLs, in the context of the presence of circulating tumor cells in patients. Two distinct size-based subtypes of CAMLs, <20 µm (tiny) and >20 µm (giant), were demonstrated. The giant CAMLs have distinctive polymorphic morphologies with mononuclear or fused polynuclear structures, including (1) apoptotic CAMLs; (2) CAML–WBC doublets; (3) conjoined CAMLs; (4) CAML–WBC clusters; and (5) circulating tumor cell–CAML–WBC clusters. In enumerating CAMLs and circulating tumor cells simultaneously, we observed that all circulating tumor cell-positive patients are also positive for CAMLs, in contrast to 55% of patients out of all CAML-positive patients that were found positive for circulating tumor cells.The blood of patients with solid tumors contains circulating tumor-associated cells, including epithelial cells originating from the tumor mass, such as circulating tumor cells (CTCs), or phagocytic myeloid cells (differentiated monocytes), such as circulating cancer-associated macrophage-like cells (CAMLs). We report for the first time the identification and in-depth morphologic characterization of CAMLs in patients with endometrial cancers. We isolated CAMLs by size-based filtration on lithographically fabricated membranes followed by immunofluorescence, using a CD45+/CK 8,18,19+/EpCAM+/CD31+/macrophage-like nuclear morphology, from > 70 patients. Irrespective of the histological and pathological parameters, 98% of patients were positive for CAMLs. Two size-based subtypes of CAMLs, <20 µm (tiny) and >20 µm (giant) CAMLs, of distinctive polymorphic morphologies with mononuclear or fused polynuclear structures in several morphological states were observed, including apoptotic CAMLs, CAML–WBC doublets, conjoined CAMLs, CAML–WBC clusters, and CTC–CAML–WBC clusters. In contrast, CAMLs were absent in patients with non-neoplastic/benign tumors, healthy donors, and leucopaks. Enumerating CTCs simultaneously from the same patient, we observed that CTC-positive patients are positive for CAMLs, while 55% out of all CAML-positive patients were found positive for CTCs. Our study demonstrated for the first time the distinctive morphological characteristics of endometrial CAMLs in the context of the presence of CTCs in patients. Distant metastasis in solid tumors requires viable tumor cells to travel to the secondary site physically. To form a secondary metastatic colony, epithelial tumor cells of solid tumors undertake a journey that, among many functions, demands the display of specific properties not characteristic of classical epithelial cells. The epithelial tumor cells lose their inherent property of attachment to the ECM  prior to the process of intravasation to enter blood/lymphoid circulation and survive in the circulation. Finally, they extravasate to get seeded and colonized, aided by regaining their embryonic epithelioid property by the process of MEC . The entire event of the metastasis-associated tumor progression has been postulated to involve various cell types other than tumor cells, which include endothelial progenitor cells, cancer mesenchymal stem cells, hybrid cancer cells, and circulating monocytic cells ,2,3. ForCancer-associated macrophage-like cells (CAMLs) are one of the cellular elements of liquid biopsy in solid tumors. As defined by Adams et al., circulating CAMLs are giant, fused hybrids of multi-nucleated stromal macrophages  with a sizeable atypical nucleus and a diffuse cytokeratin-positive cytoplasm found in the peripheral blood of patients with solid tumors ,7. CAMLsAlthough CAMLs are identified in patients with several solid tumors ,8, detaiHere, we report the first in-depth characterization of CAMLs in endometrial cancers. In our cohort, 98% of patients with endometrial cancers were positive for some type of CAMLs in their blood at the time of surgery. Identifying CAMLs from more than 70 patients with endometrial cancers of various histopathological parameters, we identified two distinct size-based types of CAMLs, tiny CAMLs (<20 µM) and giant CAMLs (>20 µM). These two types of CAMLs are characterized by their distinct shapes, nuclear morphologies, expression of epithelial/myeloid markers, and their presence in patients with different histopathological parameters. We also identified several morphological states of CAMLs, including apoptotic CAMLs, CAML–WBC doublets, conjoined CAMLs, CAML–WBC clusters, and CTC–CAML–WBC clusters.Cell lines from endometrial and lung cancers , as well as HUVEC cells, were procured from ATCC and were cultured according to the standard cell culture procedures as per ATCC recommendations. Leucopak and PBMC  were procured from Lonza . The CellSieve enumeration kit with either DAPI/CK-FITC/EpCAM-PE/CD45-Cy5 or DAPI/CK-FITC/CD31-PE/CD45-Cy5 was procured from Creatv Microtech .Anonymized peripheral blood samples were collected for enumeration of CAMLs and CTC at surgery from patients with endometrial cancers. Informed (IRB approved: Protocol Number Study: 2017.053-100399_ExVivo001) consent for receiving the peripheral blood was obtained from 72 enrolled patients with endometrial cancers. Blood samples were collected in commercially available CellSave collection tubes . We included samples from patients with solid tumors at any stage/grade of the disease undergoing surgery/biopsy with or without pre-treatment/history of any previous carcinoma. We did not include any bone-marrow transplant patients or patients with liquid tumors. All experimental protocols were approved by the institutional and/or licensing committee. All methods were carried out in accordance with the relevant guidelines and regulations.−/CK8,18,19+/EpCAM+/DAPI+ for determining the CTCs (FDA-approved definition of CTC) and CD45+/CK8,18,19+/EpCAM+/DAPI+ for determining CAMLs by the immunofluorescence method. DAPI was used for the evaluation of the nuclear size and morphology. The measurement of the cell and nuclear diameters are presented in the photomicrographs of CTC was determined by triple-immunofluorescence  following isolation using lithographic microfilters as presented elsewhere . In shor−/CK8,18,19−/CD31+/DAPI+-stained HUVEC cells and CD45−/CK8,18,19+/CD31−/DAPI+-stained tumor cells.The detection of CAMLs in patients’ blood was validated using CD31 staining. Blood samples were processed, and cells were stained using a kit containing a cocktail of CD45-CK8,18,19-CD31 antibodies. Parallel blood samples were spiked with CD31-positive HUVEC cells. CAMLs were identified as CD45+/CK8,18,19+/CD31+/DAPI+-stained cells in contrast to CD45Informed consent was obtained from 72 patients with endometrial cancers whose blood samples were received for standardization and detection of CAMLs. −/EpCAM−/DAPI+-stained 10.38 µM WBC from blood of a patient with a Grade 1, Stage IA endometrial endometrioid adenocarcinoma tumor. We tested the CD31 positivity of CAMLs with a parallel validation by CD31-positive HUVEC cells  of our tested blood samples in patients with endometrial cancers. EC cells A. FigureThe nuclear morphology of leukocytes, including monocytes of the immune system, is associated with multiple functions ,17. The Since it is reported that M2 polarization correlates with an increased degree of cell elongation , we quanIn addition to different shapes of endometrial CAMLs, we recorded CAMLs in the blood in various states. CAMLs (either tiny or giant) were identified in close to 100% of the tested blood samples from patients. CAMLs are one of the cells that can be identified in liquid biopsies in solid tumors . In our We identified CTC in the same patient by triple immunofluorescence A,B,D andWe evaluated the co-expression of CAMLs and CTCs in our patient cohort. A pictorial representation of grades, stages, MMR status in the context of the presence of CTC, and types of CAMLs in The distribution of giant CAMLs, when coupled with the presence of CTC in our study, showed no association with pathological parameters such as grade, stage, LVI, or % of myometrial invasion. The 79.77 µM elongated giant CAML with more than 1 DAPI+ fused nuclei from the blood sample of the CTC-positive patient with endometrioid adenocarcinoma A was preCAMLs are specialized polyploid myeloid cells transiting in the circulation of patients with various types of solid malignancies ,19. We r− with a DAPI+ ≥8 µM “salt–pepper” nucleus); (2) CTC clusters ; (3) apoptotic CTCs ; (4) CTC debris ; (5) EMT CTCs ; (6) stem-cell CTCs ; (7) PD-L1-CTCs ; and (8) macrophage–tumor fusion cells . CAMLs belong to the category of cancer-associated circulating extra-CTC cells characterized by distinctly polymorphic, mononuclear/polynuclear, and polymorphonuclear (syn- or heterokaryon) features with dual epithelial and macrophage/myeloid marker phenotypes. They collectively comprise the components of blood-based biopsies, i.e., "liquid" biopsies. Thus, CAMLs are components of "liquid" biopsies. In our study, we identified a novel subset of CAML, tiny CAMLs (15–20 µM), for the first time in the blood of endometrial patients. We also observed that the distribution of tiny CAMLs is not specific for any stage/grade/histological type of the disease. These tiny CAMLs can be accompanied by the classical giant (≥30 µM) CAMLs. Although it has been reported that macrophages adopt different geometries in vivo; how these changes in cell shape might feed back into regulating their functional phenotype has not been delineated [Circulating cells in the peripheral blood of patients with solid tumors can be of several types, including circulating tumor cells, modified blood cells, plasma cells, fused cancer cells, or giant macrophage-like cells (CAMLs). Circulating cancer-associated cell subtypes includes (1) traditional CTCs  or multi-lobed nucleus. Interestingly, nuclear morphology determines two important functions of a cell, including transcriptional activity and flexibility of movement. Indeed, leukocytes of the immune system have lobed nuclei, which are known to impart their flexibility and migration . CellulaIn the organ-type of solid tumors where CAMLs have been identified so far, their presence has been reported in every stage of the disease ,19. In lMacrophages contribute to the various stages of tumor progression, from initiation to formation of distant metastasis . The relConsidering their size, particularly giant CAMLs, if they originate from tumor tissue, then what is their mode and route from a distant primary tumor? Expressing markers for both CTCs and macrophages, how close are CAMLs, both tiny and giant, to TAM or M2? Are they a product of a fusion of a cancer cell/CTC and macrophage (M2)? What is their contextual relation with WBCs/T-cells in circulation? Although our study cannot answer such questions at present, interrogations will be crucial in determining the functions and clinical relevance of CAMLs in endometrial cancers. Macrophage fusion hybridization with tumor cells [Fusion cells hold a critical role in being a key component in cancer progression, metastasis, and drug resistance ; indeed,or cells ,32 is reor cells ,33. Indeor cells . M2 macror cells ,36. Sincor cells , it can or cells . Future Our data confirm the presence of CAMLs in the peripheral blood of patients with endometrial tumors. The CAMLs present in the blood of these patients are morphologically classified into tiny and giant based on their size. Giant CAMLs present a wide range of morphologies and forms, including apoptotic and in doublet/clusters with WBC or CTCs. In enumerating CTCs and CAMLs simultaneously from the blood of the same patient, we observed that all CTC-positive patients are positive for CAMLs in contrast to 55% out of all CAML-positive patients who were found positive for CTCs. Our study demonstrates for the first time the distinctive morphological characteristics of endometrial CAMLs in the context of the presence of CTC in the patients. Our data indicate a need for an in-depth study on the clinical relevance of CAMLs in the context of CTC in endometrial cancers.The study presented in the MS is part of a patent application .


Cartilage oligomeric matrix protein (COMP) is an extracellular matrix (ECM) glycoprotein that is critical for collagen assembly and ECM stability. Mutations of COMP cause endoplasmic reticulum stress and chondrocyte apoptosis, resulting in rare skeleton diseases. The bouquet-like structure of COMP allows it to act as a bridging molecule that regulates cellular phenotype and function. COMP is able to interact with many other ECM components and binds directly to a variety of cellular receptors and growth factors. The roles of COMP in other skeleton diseases, such as osteoarthritis, have been implied. As a well-established biochemical marker, COMP indicates cartilage turnover associated with destruction. Recent exciting achievements indicate its involvement in other diseases, such as malignancy, cardiovascular diseases, and tissue fibrosis. Here, we review the basic concepts of COMP and summarize its novel functions in the regulation of signaling events. These findings renew our understanding that COMP has a notable function in cell behavior and disease progression as a signaling regulator. Interestingly, COMP shows distinct functions in different diseases. Targeting COMP in malignancy may withdraw its beneficial effects on the vascular system and induce or aggravate cardiovascular diseases. COMP supplementation is a promising treatment for OA and aortic aneurysms while it may induce tissue fibrosis or cancer metastasis.  Articular cartilage consists of dense extracellular matrix (ECM) and sparsely distributed chondrocytes ,2. ECM iCOMP promotes the secretion and assembly of collagens and maintains the stability of ECM . MutatioDue to the protective effects of COMP on cartilage, a thorough understanding of COMP functions and the related signaling pathways is beneficial for novel therapeutic strategies for cartilage degradation diseases. Recent exciting achievements have implied that COMP is an important gene contributing to other diseases, such as cancer/malignancy ,23,24, ctrans retinol [COMP is a secretory pentamer multidomain protein that belongs to TSP family subgroup B . Remarka retinol , which m retinol   progression , suggestCOMP expression levels in cancer tissues are significantly higher than in neighboring normal tissues . Increas2+ release from ER [+ human breast cancer cell line, MCF7 cells) [Serum COMP is elevated in the advanced breast cancer patients with metastasis and significantly associated with histological subtype, estrogen receptor positivity, and metastasis at diagnosis . Moreove from ER ,104. COM from ER . Multipl from ER . It seem from ER     mice, while there is no difference in pulmonary hemodynamics between them [−/− mice display elevated blood pressure and impaired endothelium-dependent relaxation induced by acetylcholine [2+ influx, thus increasing endothelial nitric oxide synthase activity and nitric oxide generation [COMP is a protective factor for cardiovascular system . Patienteen them . Hypoxiaeen them ,113. COMeen them . Comparelcholine . Mechanineration   receptor. COMP deficiency activates the AT1 receptor/β-arrestin-2 signal , which e of COMP . Human s of COMP . These f−/−COMP−/− mice to ApoE−/− mice increases the formation of atherosclerotic plaques, suggesting that bone marrow-derived COMP may play a crucial role in preventing atherosclerosis [COMP deficiency causes aging-related vascular dysfunction, implying that COMP is also critical in preventing vascular aging and the senescence of vascular smooth muscle cells (VSMC) . COMP isclerosis . Mechaniclerosis . Moreoveclerosis . COMP biclerosis   than in smaller keloids, implying that COMP expression levels were related to disease progression and might be used as a biomarker to identify disease severity [COMP is a fibrillar collagen assembly regulator , which iseverity . Indeed,severity ,127. Up-severity ,123,128 severity  . COMP was secreted mainly by mesenchymal cells and most probably fibroblasts in lung . Serum CSerum COMP can be served as a useful biomarker for screening bone and cartilage involvement in psoriasis patients  and helpWith a long-term effort of more than 20 years, notable understandings of the pathophysiology of COMP-related genetic diseases have been achieved, and novel mutations in COMP gene continue to be discovered ,137. ProThe potential of serum COMP as a biomarker is well-characterized, at least, in OA. However, the broad expression location and complex connections with multiple diseases impair the specificity of COMP. To date, COMP is a non-specific biomarker, such as erythrocyte sedimentation rate or C-reactive protein, serving as clinical alarm signals. More studies are required to define the gold criteria that can be used for diagnosis and differential diagnosis. Combination with other biomarkers may help to improve the specificity of COMP. Recent studies have implied that tumor expression of COMP can be a good diagnostic and prognostic indicator in human cancers. More importantly, COMP seems to be more relevant to metastasis than malignancy onset, suggesting that it has a critical role in cancer cell behaviors, such as migration and invasion. Indeed, accumulating findings have indicated that COMP promotes the aggressive behaviors and stemness of cancer cells. However, the expression of COMP in stroma and cancer cells is rarely distinguished. Moreover, the immune response and immune cell infiltration have not been assessed in the high COMP-expressing tumors. It is not surprising that COMP may modify the interaction between cancer cells and immune cells. As previous studies indicate, COMP is a potential target for cancer, giving promise to the anti-COMP therapy. However, COMP is widely expressed in many kinds of tissues and shows distinct functions in different diseases. Systematic administration of COMP-targeting regent in cancer patients may withdraw its beneficial effects to the vascular system and induce or aggravate cardiovascular diseases. It may be limited to cancer patients without basic cardiovascular diseases. In this case, local targeted therapy is needed. Recent achievements of nanoparticle-based RNA indicate that RNA-based therapy delivered by nanotechnology may be a promising way to go. Since COMP interacts with other proteins to facilitate its functions, targeting the interactions rather than COMP expression may be a promising approach for preventing tumor progression. Due to its protective effects on cartilage and vascular system, COMP supplementation is a potential treatment for OA and cardiovascular diseases. However, potential tissue fibrosis or cancer metastasis needs to be considered because of its notable functions in these diseases.


In the Netherlands, the Health Council has advised that the human papillomavirus (HPV) vaccination should be offered to both boys and girls. Additionally, boys and men up to the age of 26 years should be included in a catch-up program. In this study, we examine the cost-effectiveness of this HPV catch-up program.We used a static Markov model to estimate the amount of cancers prevented and the incremental cost-effectiveness ratio (ICER) for different scenarios.Vaccinating men from 12 until the age of 26 years would result in an average of 48 cancer cases prevented in every cohort , with an average ICER of €32,256.We found that the catch-up vaccination program results in a relevant number prevented cases against an acceptable cost-effectiveness ratio. Policymakers should take these findings into account when evaluating a gender-neutral HPV vaccination program in the Netherlands. In addition to vaccinating 12-year-old girls, all girls aged 13–16 years were invited to receive the HPV vaccine in a catch-up program .Since the introduction of the program in 2009, vaccination coverage in girls has been suboptimal reaching less than half the girls that are invited. Thus, the government decided to further expand the HPV vaccination program to boys, to further reduce the burden of HPV-related disease in the Netherlands .Fig. 1In 2022, boys are planned to receive HPV-vaccination in the Netherlands . The HPVThere is still debate around whether men until the age of 26 should have the opportunity to receive the HPV-vaccine.Previously, we showed that the vaccination of 12-year-old boys can be considered cost-effective . Here, w22.1The developed model reflects a lifetime multi-stage static Markov approach with time cycles of one year, comparing HPV vaccination of one single cohort of 100,000 boys or men with the current situation of the female-only strategy. In this model, one cohort is vaccinated and followed until the age of 95. A full overview of the model, all of its assumptions and input parameters, have been previously described, and has been added in the The age of vaccination was varied between 12 and 26 years old based on the recent Dutch Health council advice . We asseAdditionally, multiple scenarios combining different age groups were assessed. These analyses estimate to what degree a catch-up program could be considered as (cost-)effective in preventing HPV-related cancers, and which age groups should be included.We performed a multi-cohort analysis to assess the total cost of implementing a catch-up program.We assessed three different scenarios. In the first scenario, boys aged 12–14 years are vaccinated. In the second scenario, boys aged 12–16 years are vaccinated, the same age range previously established in the girl’s vaccination scheme. Finally, we assessed the scenario in which boys aged 12–26 years are vaccinated. In all scenarios, boys aged 12–14 years receive two doses, and those aged 15 and older receive three doses. We assumed a vaccine coverage of 30% with a vaccine price of €50 per dose, which is 50% lower than the current listed price , since vFinally, sensitivity analyses were performed to assess the impact of key parameters on the outcome of the model. Three deterministic sensitivity analyses (DSA) were performed at different ages  to assess the impact of different variables, for example: female vaccination coverage, infection rate, vaccine cost and vaccine efficacy. An overview of the values used for the deterministic sensitivity analysis is shown in the 3In the base case, vaccinating 12-year-old boys prevented 56 cases of HPV-induced cancer, with a corresponding ICER of €17,907. When increasing the vaccination age, the number of prevented cancers declined, and conversely, the ICER increased. At age 26 years, the number of HPV-induced cancers that were prevented over the remaining lifetime decreased of approximately 40%, to 32 cases, with a corresponding ICER of €53,173.3.1In the first scenario, the model predicted that vaccinating boys aged 12 to 14 years would prevent 166 cancer cases. On average, this resulted in 55 cases per vaccinated cohort and an ICER of €18,197.In the second scenario, vaccinating boys aged 12 to 16 years prevented 275 cancer cases with an average ICER of €22,109.In the final scenario, boys and men until the age of 26 years were vaccinated in the model. This resulted in a total of 720 cases of HPV-induced cancer prevented and an average ICER of €32,256. An overview of all scenarios is shown in 3.23.2.1In the DSA, the ICER was found to be most sensitive to the degree of herd immunity from the female population in all three age groups, see 3.2.2The scatterplot with the simulations from the PSA is shown in 4plus girls’ vaccination program. The most favorable ICER, €18,197, is achieved by vaccinating all boys until, and including, the age of 14 years. Implementing an HPV vaccination program for all boys 12 to 16 years of age (the same age cohort used in the previous vaccination program for girls) would result in an ICER of €22,109, which is also considered to be a nearly cost-effective strategy, based on a willingness to pay (WTP) threshold of €20,000. Including HPV vaccination until the age of 26 years, would result in an ICER of €32,256. However, the projected ICER only included HPV-related cancer prevention in males. Including female cancer prevention due to herd immunity would improve the ICER significantly, as was seen in previous published studies  and from PAG Ltd (100%). M.J. Postma declares outside of the submitted work financial support from Asc Academics as an adviser for this company. J.J.M. Simons, T.A. Westra and M.J. Postma declare no other financial and non-financial relationships and activities.


P value of less than .05. Men who have sex with men had a statistically significantly higher prevalence of oral HPV , genital HPV , and concordant oral and genital HPV  infections compared with heterosexual men. Improved HPV prevention among men is needed.The objective of our study was to describe oral and genital human papillomavirus (HPV) infection prevalence and concordance by sexual orientation among US men using a nationally representative sample. We conducted a retrospective cross-sectional analysis of the 2013-2016 National Health and Nutrition Examination Survey. The survey conducts a physical examination and collects oral rinse and genital swab specimens; demographic and health behaviors are self-reported. We used descriptive statistics and multivariate regression models to estimate HPV infection prevalence and the likelihood of HPV infection, respectively. All analyses were adjusted for National Health and Nutrition Examination Survey design and weights, and statistical significance was tested at a 2-sided  The incidence of human papillomavirus (HPV)–associated cancers, particularly oropharyngeal and anal cancers, is rising rapidly among US men . Gay, biWe conducted a retrospective cross-sectional analysis of the 2013-2016 National Health and Nutrition Examination Survey (NHANES), a nationally representative survey of noninstitutionalized individuals in the United States. A clustered multistage probability sample of participants is identified, and information on sociodemographic and health and sexual behaviors is collected by trained interviewers during the home interview or in mobile examination centers. The survey also conducts a physical examination and collects oral rinse and genital swab specimens in the mobile examination centers. The oral rinse sample  and self-collected external genital swabs are refrigerated at 4°C and shipped by the NHANES personnel to a laboratory to perform polymerase chain reaction assay for genotyping. Details regarding the survey are available on the NHANES website .2 test. Multivariable logistic regression models, adjusted for age, race, and ethnicity ; cigarette use; lifetime number of sex partners; history of sexually transmitted infections; age at first sex; and circumcision, estimated the odds ratio for HPV infection and concordant infection. Statistical significance was tested at a 2-sided P value less than .05. All analyses were performed with SAS software version 9.4  using SAS PROC SURVEY procedures to incorporate sampling weights and to adjust for the complex survey design. The study was deemed exempt from review by the institutional review board committee as it utilizes deidentified publicly available dataset.In the current study, men aged 18-59 years of age with nonmissing data on sexual orientation and oral and/or genital HPV tests were identified. Sexual orientation was self-reported . The HPV genotypes were identified from assay results and classified (as per prior studies) as type 16, type 18, 4-valent vaccine types ; 9-valent vaccine types ; and any high-risk types  . ConcordP = .009), 9-valent types , 4-valent , and type 16 and 18  . Prevalence of genital 9-valent , 4-valent , and type 16 and 18  HPV types was also statistically significantly higher among MSM compared with heterosexual men. Concordant oral and genital infection prevalence of any high-risk  and 9-valent  HPV types was more common among MSM compared with heterosexual males. The likelihood of oral and genital any high-risk, 9-valent, 4-valent, and types 16 and 18 HPV infection was statistically significantly higher for MSM compared with heterosexual men  men with information on oral HPV infection, 2954  men with data on genital HPV infection, and 2883  men with information on both infections ( < .001) . PrevaleThis study documents a higher prevalence of oral and genital HPV infection among MSM compared with heterosexual men. Our study further reports for the first time that concordant oral and genital HPV  infection prevalence is also greater among MSM compared with heterosexual men. A previous study reported higher genital HPV prevalence among men having sex with men and women than among MSM or heterosexual men . A priorOur study has important cancer prevention and policy implications. The higher prevalence of oral and genital HPV among MSM underscores the importance of intensive efforts for HPV prevention. Unlike heterosexual males, the MSM population may not benefit from herd protection through female HPV vaccination, which increases the importance of achieving high vaccination coverage among males. Studies have documented that HPV vaccination is effective for protection against anogenital and oral HPV among MSM, particularly if initiated at younger ages ,14. Unfopkac088_Supplementary_DataClick here for additional data file.


Although the exact mechanisms have yet to be elucidated, it is clear that cellular immunity plays a role in clearance of human papillomavirus (HPV) infections as it relates to the development of warts. Patients with extensive, recalcitrant, or treatment‐refractory warts may have an underlying immune system impairment at the root of HPV susceptibility. Early recognition of genetic disorders associated with immunologic defects that allow for recalcitrant HPV infection may expedite appropriate treatment for patients. Early recognition is often pivotal in preventing subsequent morbidity and/or mortality that may arise from inborn errors of immunity, such as WHIM  syndrome. Among these, cervical cancer is one of the most common malignancies associated with HPV, can be fatal if not treated early, and is seen more frequently in patients with underlying immune deficiencies. A review of diseases with susceptibility to HPV provides clues to understanding the pathophysiology of warts. We also present diagnostic guidance to facilitate the recognition of inborn errors of immunity in patients with extensive and/or recalcitrant HPV infections.  Early diagnosis and treatment are important to limit progression to more serious morbidities, such as cancer. This review compiles information into one article to help specialists who may encounter patients with recalcitrant warts due to an underlying immunodeficiency.2Human papillomavirus (HPV) is a ubiquitous and diverse species of viruses comprising >200 subtypes3Herein, we aim to leverage the current literature and our clinical experience evaluating the underlying causes of HPV‐derived recurrent and recalcitrant warts. We conducted a PubMed search for articles containing the search terms ‘primary immunodeficiency’, ‘human papilloma virus (HPV)’ and ‘warts’. The search was performed in June 2021 and included English language results published at any time. Selection criteria for incorporation of the sources found in these searches consisted of relevance to the topic and primacy of the data. We then incorporated the published information on immunodeficiency in HPV infections and developed a proposed algorithm for evaluation and treatment of patients with severe or refractory HPV‐related infections.4n = 99), followed by alpha (n = 65), beta (n = 54), mu (n = 3) and nu (n = 1).HPV is divided into alpha, beta, gamma, mu and nu genera based on phylogenetic analysis.Unlike most viruses, multiple HPV strains may be present in a patientPerhaps the most consequential aspect of HPV physiology is its oncogenic potential. In women, high‐risk HPV is associated with cervical intraepithelial neoplasia (CIN), a precursor to invasive cervical cancer if not cleared by the host's immune system. In men, high‐risk HPV is associated with head and neck squamous cell carcinoma and penile cancer.5In immunocompetent hosts, HPV infection is eventually recognised and cleared. For subtypes causing genital infections, median HPV infection clearance is 5.9 months, regardless of whether low or high risk, and 75% of patients clear HPV infection within 12 months.Inability to clear HPV may predispose malignant transformation.In patients with immunodeficiencies, host recognition and clearance of HPV may be impaired and may perpetuate infection simultaneously with viral immune evasion strategies.6Noninherited causes of immunodeficiency, including Human Immunodeficiency Virus (HIV), malignancy, connective tissue disease, chemotherapy, biologic therapy, and immunosuppressive agents, can lead to symptomatic HPV infections via impairment of several immune system components Table .2, 31Many iatrogenic forms of immunosuppression increase HPV risk, including in patients receiving immunosuppression for solid organ transplants and patients receiving TNF inhibitors.DOCK8) mutations, GATA binding protein 2 (GATA2) mutations, interleukin 2 receptor subunit gamma (IL2RG) or Janus kinase 3 (JAK3) deficiency, ataxia telangiectasia (AT), and T‐cell CD28 deficiency can predispose patients to HPV infections in the form of warts and HPV‐related malignancies (Table ITGB2), WASP actin nucleation promoting factor (WAS), adenosine deaminase 2 (ADA2), and NFKB inhibitor alpha (NFKBIA) also affect broad elements of the immune system and bring occasional susceptibility to HPV warts.Inherited syndromes and/or immunodeficiencies may also carry greater risk of HPV infections. Diseases such as epidermodysplasia verruciformis (EV), WHIM syndrome, leucocyte adhesion deficiency 1 (LAD1), hyper IgE syndromes caused by dedicator of cytokinesis 8  can cause disease in patients with EV.8CXCR4) defect and experience recurrent bacterial infections due to neutropenia from impaired migration of polymorphonuclear cells to peripheral bloodCXCR4 mutations highlights the role of neutrophils in HPV immunity.CXCR4 gain‐of‐function mutation.CXCR4 increases cell proliferation and immortalisationCXCR4 antagonism reduced gamma HPV predominance in patients with WHIM syndrome over time in a clinical trial, a potential target for wart treatment.Individuals with WHIM syndrome are uniquely susceptible to cutaneous warts associated with gamma HPVs—which generally do not cause disease in immunocompetent individuals.9LAD1 results in frequent skin and mucosal surface infection beginning in infancy and can be fatal.10DOCK8, present with a defect in dendritic cell and T‐cell migration, resulting in immunodeficiency characterised by increased cutaneous viral infection susceptibility, including HPV, herpes simplex virus, molluscum contagiosum, and varicella zoster virus.Mutations underlying hyper IgE syndromes, such as 11DOCK8, missense or null mutations in GATA2, a transcription factor involved in haematopoiesis and stem cell maintenance, lead to a variety of presentations, with >75% of patients with GATA2 deficiency being infected with HPV.GATA2 mutations typically present in older children or even in adulthoodGATA2 haploinsufficiency can also be attributed to progressive multiple cytopenias, resulting in mycobacterial and fungal infections, and high risk of myelodysplastic syndrome, acute myeloid leukemia, lymphoedema, and pulmonary alveolar proteinosis. Around 50% of patients with GATA2 haploinsufficiency have increased susceptibility to recurrent warts.Similar to 12IL2RG or JAK3 deficiency have a reduced number of natural killer (NK) cells with decreased cytotoxic activity and, presumably, an impaired ability to eliminate virally infected cells.Patients with IL2RG mutation was shown to have a second, somatic mutation in IL2RG that reversed the T‐cell deficiency but left the NK cell population affected. The expansion of the HPV skin virome and recalcitrant HPV‐related skin and mucosal warts of this patient support a role for NK cells in HPV susceptibility and clearance. In this case, both benign and malignant HPV‐associated warts cleared with HSCT.One patient with a pathogenic germline X‐linked 13ATM gene, affects multiple systems and leads to cerebellar degeneration, telangiectasia, immunodeficiency, and susceptibility to cancer. These mutations result in low B‐cell and naïve CD4+ and CD8+ T‐cell counts and abnormal B‐cell and T‐cell receptor repertoires due to the role of ATM in double‐stranded DNA breaks in V(D)J recombination.ATM defect ultimately results in impaired antigen recognition, antibody production, and cytotoxic elimination of HPV‐infected cells, resulting in around 20% of patients developing persistent HPV warts.Ataxia telangiectasia (AT), which is caused by biallelic mutations of the 14A family with inherited hyperkeratotic cutaneous papillomatosis caused by HPV‐2 infection was identified to have a mutation in CD28. CD28, a costimulatory receptor expressed by T cells, is essential for interactions with antigen‐presenting cells. Patients carrying these mutations exhibit deficient CD4+ immune response against HPV, suggesting a dependence on HLA class II interactions for immunity against some HPV strains.GATA2, EV, and CD28 deficiency, while defects in antigen presentation are hallmarks of EV, CD28 deficiency, and DOCK8 mutations.IL2RG/JAK3 deficiency, and WHIM syndrome.The genetic pathologies listed above that are associated with susceptibility to HPV infection and warts all compromise essential elements of immune function, specifically viral immunity. Impaired T‐cell development, function, and/or mobilisation is present in This highlights the importance of recognising potential signs of underlying immunodeficiency, which may include recalcitrant warts. Indeed, genetic mosaicism and incomplete gene penetrance in some immunodeficiencies may complicate diagnosis.15With no universally accepted definition of treatment‐refractory, or recalcitrant, warts, a standard approach to evaluating and managing affected patients has been challenging. Leung suggested that warts are recalcitrant if not responding after five treatments over 6 months.16Prevalence of subclinical immune deficiencies is currently unknown. Nevertheless, in patients with severe, recurrent, or refractory warts, variations in aforementioned gene product expression and resulting immune deficiency may be considered possible components. Including immunodeficiencies in the differential diagnosis of recalcitrant warts may facilitate diagnosis in some patients. Considering current literature and direct experience in treating patients, we propose the following algorithm to aid clinicians in assessment and treatment of patients with recalcitrant warts in whom immunodeficiency should be suspected.Patients with recalcitrant or recurrent severe warts should be evaluated for evidence of other infections ; this may be the first indicator of underlying immunodeficiency. History of recurrent infection, frequent antibiotic use since childhood, or autoimmune disease may raise suspicions of underlying immunity impairment. Additionally, family history of immunodeficiency suggests an inherited cause. With personal or family history suggestive of immunodeficiency or in warts persisting for >2 years, consideration of laboratory testing to exclude acquired and inherited forms of immune deficiency should be performed. Testing should include a complete blood count, comprehensive metabolic panel, HIV test, antinuclear antibody, and quantitative immunoglobulin assessments. If suspicion remains high and diagnosis elusive, genetic screening  may confirm diagnosis Figure .17The HPV vaccine has led to substantial decrease in incidence and prevalence of HPV‐related cancers and genital warts; despite initial antibody increases, long‐term immunity is not always maintained in people with immunodeficiency.Many approaches exist to treat warts in healthy patients, although randomised controlled trials have mostly demonstrated equivalent efficacy, and no therapy will be effective in all patients.In conclusion, HPV is a common human‐associated virus typically cleared by the host's immune system. In cases of unusual wart presentation , there may be underlying immunodeficiency warranting evaluation that may affect wart prognosis and management. Patients with suspected immunodeficiencies should be referred to an immunologist for evaluation, including genetic testing, as appropriate, to confirm or exclude inborn errors of immunity. For patients affected by such inborn errors, early diagnosis and tailored treatment could profoundly impact outcome, particularly in those patients who may go on to develop HPV‐related malignancies that can be fatal without prompt, targeted intervention.Zampella reports receiving consulting fees from X4 Pharmaceuticals. Cohen has no conflict of interest to declare.John G Zampella: Conceptualization, Data curation‐Supporting, Formal analysis, Funding acquisition‐Supporting, Investigation, Methodology‐Supporting, Project administration‐Supporting, Resources, Validation, Visualization, Writing ‐ original draft‐Supporting, Writing ‐ review & editing‐Supporting. Bernard Cohen: Conceptualization, Data curation‐Supporting, Formal analysis, Funding acquisition‐Supporting, Investigation, Methodology‐Supporting, Project administration‐Supporting, Resources, Validation, Visualization, Writing ‐ original draft‐Supporting, Writing ‐ review & editing‐Supporting.