Stratum spinosum). The microbial microenvironment of the cervical tissue determines the tissue homeostasis and the integrity of the protective mucous layer through the maintenance of a healthy immune and metabolic signalling. Pathological microorganisms and the resulting dysbiosis disturb this signalling. Thus, pathological inflammatory reactions occur, which manifest the HPV infection. About 90% of all women contract an HPV infection in the course of their lives. In about 10% of cases, the virus persists and cervical intra-epithelial neoplasia (CIN) develops. Approximately 1% of women with a high-risk HPV infection incur a cervical carcinoma after 10 to 20 years. In this non-systematic review article, we summarise how the sexually and microbial mediated pathogenesis of the cervix proceeds through aberrant immune and metabolism signalling via CIN to cervical carcinoma. We show how both the virus and the cancer benefit from the same changes in the immune and metabolic environment.Cervical cancer is responsible for around 5% of all human cancers worldwide. It develops almost exclusively from an unsolved, persistent infection of the squamocolumnar transformation zone between the endo- and ecto-cervix with various high-risk (HR) human papillomaviruses (HPVs). The decisive turning point on the way to persistent HPV infection and malignant transformation is an immune system weakened by pathobionts and oxidative stress and an injury to the cervical mucosa, often caused by sexual activities. Through these injury and healing processes, HPV viruses, hijacking activated keratinocytes, move into the basal layers of the cervical epithelium and then continue their development towards the distal prickle cell layer ( The microbiota is the community of microorganisms in a specific environment. It determines the metabolism and the immune environment of the host tissue through its microcosm and its own demands on metabolism and immune signalling. It has a decisive influence on homeostasis and the health of the organism. In the microbiome of the female lower reproductive tract (vagina and cervix), especially in the part of the cervix reviewed here, the microenvironment of the mucous membranes is colonised by a mixture of microorganisms. These mostly belong to the lactobacilli family . The uppThe female reproductive tract (FRT) can be divided into the upper and lower tracts. The upper tract includes the uterus, fallopian tubes, and ovaries; the lower tract the vagina, cervix, and vulva. The cervix, in turn, can be divided into two zones: (1) the endo-cervix or cervical canal leading into the uterus and (2) the ecto-cervix. At the border between both parts lies the transformation zone (TZ). The cervical canal is narrow and with its cervical mucus, which is produced by the mucus-forming glandular tissue, ensures that no germs and, in the sterile phase of the menstrual cycle, no sperm can rise into it. During pregnancy, it protects the developing life from rising germs by means of a tough plug of mucus. The ecto-cervix forms a stratified squamous epithelium, in contrast to the columnar endo-cervical epithelium. When malignant transformation of the cervix occurs, it mainly takes place in the squamocolumnar TZ ,9. MaligWith around 570,000 cases and 311,000 deaths in 2018, cervical cancer is the fourth most common cancer worldwide and the most common malignant transformation caused by HPV infection ,20. ThesThe community of symbiotic and pathogenic microorganisms and their metagenome  determine the tissue homeostasis and the pathogenesis of certain diseases, including cancer . The micEven if the term “pathobiont” as a symbiont that can trigger pathological processes is incorrect and does not represent the true occurrences in the microenvironment of the diseases , we haveFusobacterium nucleatum as well as Atopium parvulum and Actinomyces odontolyticus increased towards the advanced stages of colon cancer, respectively in multiple polyploid adenomas and/or intramucosal carcinomas. On the other hand, there were changes in the metabolite concentrations, such as increased levels of branched-chain amino acids and phenylalanines in intramucosal carcinomas, and respectively, increased bile acids such as deoxycholate in multiple polyploid adenomas and/or intramucosal carcinomas [One study showed that changes in the microbiome and metabolome occurred in the early stages of colon cancer development. On the one hand, the relative frequency of rcinomas .Lactobacillus dominant population altered the metabolism of amino acids and nucleotides. The Lactobacillus frequency was related to increased adenosine and cytosine levels and reduced inflammation parameters, the non-Lactobacillus frequency, via the glycochenodeoxycholate (GCDC) and carnitine metabolism, on the other hand, was related to the inflammation.In another study , three dLactobacillus crispatus. The Lactobacillus iners CST III group showed higher levels of biogenic amines, phospholipid and glycogen derivatives in HPV-positive women than in HPV-negative women. The low-Lactobacillus group CST IV, which stands for molecular bacterial vaginosis, had lower levels of glutathione, glycogen, and phospholipid derivatives than in HPV-negative women. Based on the relationship between HPV infection and glutathione levels, the authors conclude that antioxidant therapy could be used in the future.Another study  compared+) and their four corresponding enzymes, threonyl-tRNA synthetase, prolyl aminopeptidase, prolyl-tRNA synthetase, and acyl phosphatase. An increased succinate excretion, on the other hand, stood for the healthy, HPV-negative woman. HPV infection increased the diversity of microbial species and their ability to produce or use certain metabolites. This resulted in an increased incidence of Lactobacillus sp. and a decreased incidence of Lactobacillus iners and Shuttleworthia, which affected bacterial homeostasis [Others analysed the metabolome of 19 women from Puerto Rico using a model-based integration of metabolite observations and species abundance 2 (MIMOSA2) approach and found a connection between the three metabolites excreted in the urine, threonine, proline, acetate  thepsittaci ,41, (4) psittaci ,43, (5) psittaci , (6) thepsittaci , and (7)psittaci ,47,48, wpsittaci ,50. Bact thepsittpsittaci ; or the psittaci ,53.Anaerococcus, Atopium, Fusobacterium, Gardnerella, Gemella, Parvimonas, Peptostreptococcus, Prevotella, Sneathia, Shuttleworthia, and others, which displace the probiotic bacteria, influence the natural inflammatory reaction (anti-inflammatory cytokines such as the interleukin-1 receptor antagonists (IL-1RA) and antimicrobial peptides) [Helicobacter pylori, stimulate the typical characteristics of cancer, such as proliferation, survival, migration, and angiogenesis via the Wnt–ß-catenin signalling pathway [Chlamydia trachomatis, in turn, is able to carry out the epithelial–mesenchymal transition (EMT) via reduced E-cadherin and increased N-cadherin levels and the expression of SNAIL1, and the associated loss of cell adhesion between the epithelial cells. Together with the blockade of the DNA damage response, this leads to the promotion of tumour development [The effects of probiotic microorganisms on tissue health or possible disease progression through dysbiotic bacteria are largely determined by the integrity of the mucosal epithelium ,51,54,55eptides) ,57,58 toeptides) ,59. The eptides)  causes geptides) ,51. Bacteptides) . The chaeptides) ,3. Overaeptides) . Pathogeeptides) ,59,61. O pathway ,63. Chlaelopment .The human papillomavirus is associated with about 5% of all human cancer developments worldwide and in the majority of cases (99.7%) with cervical cancer . In addiIt is known that the altered glucose metabolism covers the changing need for energy and metabolic building blocks in the course of the changing microenvironment during cancer progression, the dissemination of cancer stem cells, and metastasis ,77,78,79Otto Warburg postulated aerobic glycolysis, also referred to as the “Warburg effect”, in the early 1920s as a characteristic feature in cancer tissue, in contrast to non-malignant tissue ,81,82. HThe mechanisms of the effects of the HPV oncoproteins on the metabolism are described in more detail under point 4.4.In order to understand why the HPV virus specifically affects the squamocolumnar transition zone (TZ) between the endo- and ecto-cervix and under what conditions a chronic infection leads to a malignant event, one must understand how the TZ is structured and the genome of the virus is organized, as it determines the individual phases of virus replication and the further fate towards cancer progression.In addition to the special location of the cervix in the woman’s vagina and the associated reaction mechanisms of the cells after injury and healing, when activated keratinocytes carry the virus into the wounded area , it is tThe genetic structure of the HPV genome in its common organisation determines the precise sequence of replication events of all (more than 200) HPV subtypes . The difThe prerequisite for the initiation of cervical carcinogenesis is the persistence of the HPV infection in the undifferentiated basal cells. For this purpose, the virus must ensure that not only extrachromosomal gene copies are made, but that the virus genome is bound to the host genome, since extrachromosomal copies of the virus DNA would be lost during mitotic cell division. The E2 protein seems to be responsible for this, as it is able to bind to the host chromatin during mitosis; and by binding to the promoter, that regulates the expression levels of E1, E2, E6, and E7, it has the ability to self-regulate even its own gene expression ,106. In Stratum spinosum) [During the transition from the undifferentiated to the differentiated part of the TZ, an increased viral protein biosynthesis only occurs in the direction of the most highly differentiated cell layers. This shift of the increased gene expression into the outer  boundary of the TZ has the strategic advantage that it is shifted to zones of the tissue that are largely beyond immune surveillance. The late L1 and L2 proteins are only expressed in the spinous layer  ,109,110.However, the HPV virus has to overcome one hurdle on the way to the completion of its replication cycle. It must break the cell cycle arrest that is usually present in the differentiating host cells and induce the host tissue to start replication again, since it is dependent on the host’s replication enzymes. The early E7 virus protein, by binding to members of the retinoblastoma (pRb) family, inactivates them, forcing the keratinocytes of the distal epithelial layers back into the cell cycle. This strongly stimulates the transcription of the virus genes E1, E2, E1^E4 (connection of E1 amino acids with the open reading frame of E4), and E5 ,112,113 Both the innate and adaptive immune systems are cooperatively suppressed by several mechanisms that result from the effects of the early HR E5, E6, and E7 virus proteins and the late L2 protein on the host’s immune responses. The HPV infection usually leads to a corresponding immune response, which in the best case eliminates the virus via regression of the lesions . In caseThe interferon response is one of the innate immune response pathways ,122. The+ and CD4+ lymphocytes, Langerhans cells (LCs), and other dendritic cells (DCs) [An infection is usually associated with increased inflammatory cytokine production . In HPV-ls (DCs) ,136. It ls (DCs) ,137 , which controls the MHC pathway via the antigen presentation and is expressed in almost all cells on their surface. The HR HPV oncoproteins E5, E6, and E7 are responsible for down-regulating MHC I expression and probably cause the suppressed or delayed T cell reaction ,139. E7 If HPV infection persists, the benign or malignant lesions that it causes also grow over time. There is competition for nutrients and oxygen in the centre of the proliferating lesions. Normally, this leads to cell cycle arrest, which can lead to cell death. Angiogenesis, the formation of new blood vessels through sprouting and splitting reactions from existing blood vessels, counteracts cell death ,140,141.In particular, the two oncoproteins E6 and E7 occur individually or in combination to ensure that the HPV infection is maintained in the cervix and other tissues. Only the high-risk types of E6 and E7 cause the transformation to malignant progression. The targets of these two oncogenic proteins originate from the various regulatory mechanisms of the cell, such as the cell cycle, gene expression, replication, and signalling. We want to discuss these in detail now.An important building block in maintaining replication in differentiating cells of the TZ of the cervix is the binding to members of the retinoblastoma (pRb) protein family. These are considered tumour suppressors. They include p105 (pRb), p107 and p130 B. NormalAn accompanying reaction to the abrogation of the pRb tumour suppressor-functionality is the compensatory stabilisation of the tumour suppressor-protein p53, which negatively influences virus replication and can trigger apoptosis. In terms of developmental biology, the HR E6 oncoproteins have therefore evolved to prevent apoptosis via the degradation of p53 and the immortalisation of the keratinocytes  Figure B. With tAs long as the replication cycle of the virus is complete, and its DNA—the tandem of E6 and E7—does not integrate into the host genome, the HPV infection, and this also applies to the HR HPV variants, will be completely eliminated by 90% of cases from the host over the course of about 2 years, mediated by a potent immune system . In thisThe previously described cooperative repression of the tumour suppressor proteins pRb and p53 and the associated apoptosis suppression by HR HPV E6 and E7 leads, via the E6-mediated blockade of the p53-activated regulator of glycolysis and apoptosis TIGAR (TP53-induced glycolysis and apoptosis regulator), to the activation of glycolysis . In addi+/glucose co-transporters [+/glucose co-transporter and increases the influx of glucose [Xenopus laevis oocytes also led to a significantly increased distribution of SGLT1 in the cell membrane [HR HPV16 E7 also seems to activate HIF1, at least in oral squamous cell carcinoma  Figure C. HR HPVsporters ,175,176,sporters . EGFR is glucose ,173,178. glucose . HPV16 E glucose ,181. HPVmembrane .Overall, the HPV E6 and E7 oncoproteins intervene at various points in the glycolytic pathway, but also in the Krebs cycle. For example, by inactivating p53, as the antagonist of G6PDH, the key enzyme in the PPP, increases and, with it, the nucleotide levels. The HPV E6 oncoprotein also positively regulates pyruvate dehydrogenase kinase 2 (PDK2) and subsequently negatively regulates pyruvate dehydrogenase (PDH), thereby lowering the acetyl CoA level and shifting the metabolism towards lactate production, another hallmark of cancer. Furthermore, the p53 degradation causes a reduced glutaminolysis via the down-regulation of glutaminase 2 (GLS2), which, via the reduction in the α-ketoglutarate level, attenuates the function of the Krebs cycle, which is also one of the hallmarks of cancer. The enzymatic activity of the foetal form (M2) of pyruvate kinase (PK), called PKM2 or tumour-M2-PK, is more enzymatically active in glycolytic metabolism than the adult form PKM1 ,182. It 0F1-ATPase), regulates crystal morphology [P53, which is degraded by the viral oncoprotein E6 via the proteasome, induces the protein expression of mitochondrial proteins, e.g., cytochrome C oxidase 2 (SCO2) , apoptosrphology . The ROSrphology . Variousrphology . The HPVrphology .ras lead to further DNA mutations that drive the transformation [Cervical cancer, but also other cancers caused by human papillomaviruses, show properties of immortalised, undifferentiated cells. For its development, however, the HPV virus absolutely needs differentiating cell types, such as those found in the transformation zone (TZ) between the endo- and ecto-cervix. For virus replication, it is imperative that the virus produce extrachromosomal episomes. In cancer cells, however, the virus genomes are integrated into the host genome in the form of the E6 and E7 DNA segments. This is probably another reason why cancer cells do not produce virions. The virus replication cycle ends with the incorporation of its DNA into the host DNA. It is in fact the case that nude mice, which are immortalised through the incorporation of E6 and E7 oncoproteins do not automatically show tumourigenicity. HPV infection is necessary for this, but in itself is not sufficient to initiate the malignant transformation. The prolonged passage during cell cultivation and a second oncogene such as ormation . The E6 ormation . The proormation . In addilapatinib in therapy leads to treatment resistance in a model of HER2/HPV-positive SiHa cervical carcinoma cells; however, by the combinational treatment with glycolysis inhibitors such as 2-deoxy-D-glucose (2-DG), a glucose analogue that is not metabolised by the cells, resistance can be abolished [Resistance to chemotherapy and radiation therapy is an obstacle that is difficult to overcome on the way to healing the patient. The hypoxic microenvironment of the tumour and the aberrant metabolism via aerobic glycolysis (Warburg effect) are mainly responsible for this ,196. In bolished ,200.Akkermansia muciniphila had a positive correlation with the clinical effectiveness of the therapy, which restored the effectiveness of the PD-1 blockade via interleukin-12 through the accumulation of CCR9+CXCR3+CD4+ T lymphocytes in the tumour beds [Finally, we would like to recommend a few review articles to the reader, which report on the positive effects of the microbiota on patient health and the alleviation of side effects from chemo-, radiation-, and immunotherapy ,6,7. ThrLactobacillus crispatus, and the drug Lactobacillus crispatus CTV-05 (LACTIN-V) derived from it, on vaginal health; and the authors seek to restore women’s vaginal health by replenishing the dysbiotic vaginal microbiota (VMB) with this hydrogen peroxide (H2O2)-producing bacterium, thereby preventing recurrent inflammation and bacterial vaginosis (BV).Another publication  presentsSneathia and the reduction in the number of symbiotic species of Lactobacillus, as well as an increase in the pH value of the vagina. Nevertheless, the knowledge about the mechanisms of the crosstalk between host and microorganism is still limited and needs further clarification. The influence of the microbiota on tissue homeostasis and the positive effects of a healthy probiotic epithelial landscape certainly deserve our special attention, especially in the prophylaxis of urogenital diseases, in order to maintain and promote the quality of life and health of women. In order to get back to our provocative title, and to the question of which of the many microorganisms is good and which is bad, one must surely come to the conclusion that this depends on the respective species, the tissue context and the respective pathological situation. The answer to our question is therefore in the grey area and should still take some effort if it can ever be answered.The influence of the vaginal microbiota and its interactions with the microbiota of other epithelial locations in women in diseases of the urogenital tract, including malignant transformations, has only recently been investigated in more detail. Clinical studies with a larger number of patients must follow, in order to recognise their value for a putative clinical treatment and to make valid statements about the effectiveness of probiotic treatment methods. Since there are also, probably, differences in the epidemiological development of cervical cancer between women of different origins, the ethnic and socio-economic origins of the patients must also be taken into account. In the future, questions about the age of sexual maturity, the number of children, the type of contraceptive, and possible influences from the effects of hormone preparations or the question about accompanying sexually transmitted diseases, triggered by pathological microorganisms should be raised. Interestingly, in women of Spanish origin, for example, the occurrence of cervical cancer was associated with the increased incidence of the pathological species


Papillomaviruses dysregulate the G1/S cell cycle transition in order to promote DNA synthesis in S phase, which is a requirement for viral replication. The human papillomaviruses (HPV) E6 and E7 oncoproteins mediate degradation of the cell cycle regulators p53 and Rb, which are two of the most universally disrupted tumor-suppressor genes in all of cancer. The G1/S checkpoint is activated in normal cells to allow sufficient time for DNA repair in G1 before proceeding to replicate DNA and risk propagating unrepaired errors. The TP53 pathway suppresses a variety of such errors, including translocation, copy number alterations, and aneuploidy, which are thus found in HPV-associated tumors similarly to HPV-negative tumors with other mechanisms of TP53 disruption. However, E6 and E7 maintain a variety of other virus–host interactions that directly disrupt a growing list of other DNA repair and chromatin remodeling factors, implying HPV-specific repair deficiencies. In addition, HPV-associated squamous cell carcinomas tumors clinically respond differently to DNA damaging agents compared to their HPV negative counterparts. The focus of this review is to integrate three categories of observations: (1) pre-clinical understanding as to the effect of HPV on DNA repair, (2) genomic signatures of DNA repair in HPV-associated tumor genomes, and (3) clinical responses of HPV-associated tumors to DNA damaging agents. The goals are to try to explain why HPV-associated tumors respond so well to DNA damaging agents, identify missing pieces, and suggest clinical strategies could be used to further improve treatment of these cancers. The high-risk human papillomaviruses (HR-HPV) infect the basal layer of epithelium in the anogenital region and the oropharynx. While most infections are cleared, in some individuals, HR-HPV infections and the introduction of oncoproteins E6 and E7 is the founding carcinogenic event for the development of squamous cell carcinomas arising from these anatomic locations. While sufficient for the immortalization of keratinocytes, E6 and E7 are insufficient for transformation into malignancy, and the acquisition of additional driver alterations is required. In murine models, the expression of HPV18 E6/E7 alone does not induce malignancy, which also requires an additional oncogene (ras or fos) ,3,4. In Locoregionally advanced head and neck squamous cell carcinomas are often best managed with concurrent chemoradiation, consisting of 70 Gy in 2 Gy fractions with concurrent cisplatin either weekly or in three bolus doses over the course of radiotherapy . The valIn 2008, a landmark analysis of a prospective Eastern Cooperative Oncology Group (ECOG) trial of concurrent chemoradiation for head and neck cancers established superior outcomes for HPV-associated squamous cell carcinomas in terms of local control following radiotherapy as well as overall survival . The supPre-clinical models of HPV-associated HNSCC are few, but multiple groups have replicated the demonstrated increased radiation sensitivity in collections of HPV+ HNSCC cell lines compared to HPV− HNSCC cell lines using gold standard clonogenic survival assays ,35. In pThe cell-intrinsic radiosensitivity found in clonogenic survival assays has been linked to a DNA double-strand break repair defect. Again using a comparison of cell line collections in HPV+ and HPV− categories, the resolution of γH2AX foci after radiation is delayed in HPV+ cell lines, suggesting a defect in DSBR (double strand break repair) ,35. IsogBoth E6 or E7 individually and joint E6/E7 expression have been linked to DSBR defects and multiple DSBR pathways are proposed to be abrogated, including both homologous recombination ,41 and nFollowing a double-strand break created by irradiation, an estimated 70% of breaks are repaired through nonhomologous end joining, a rapidly completed pathway consisting of end recognition by the XRCC5/6 heterodimer and DNA-PK, which forms a synaptic complex across the break site. Compatible ends can be directly ligated through Ligase IV, or limited end processing can occur to remove oxidatively damaged residues before subsequent ligation. In S and G2 cells, CTIP and the MRN complex can initiate end-resection, leaving a single strand 3′ prime end, which can invade a sister chromatid strand, find homology, and complete homologous recombination. A less well-characterized pathway, alternative end joining (Alt-EJ), also makes use of the exposed single stranded 3′ end from each side of the break. Small stretches of homology between opposite strands of the break, termed ‘microhomology’, can be annealed, initiating repair through polymerization and the clipping of non-complementary tails.HPV E6/E7 expression has been linked to homologous recombination deficiency. Liu et al. have described an abrogation of TGF-β signaling in HPV+ HNSCCs and a subsequent de-repression of miR-182, which in turn inhibits BRCA1 and FOXO3 expression, thereby inhibiting homologous recombination directly through BRCA1 and indirectly through FOXO3 effects on ATM activity . InhibitIn contrast, when comparing panels of HPV+ and HPV− cell lines that did exhibit separable radiation sensitivities in the hands of multiple groups ,35, no sThe E6/E7 oncoproteins have also been proposed to influence repair through end joining. Using three different methods, our group measured the impact of E7 on DSBR and found an E7-mediated suppression of NHEJ, with a concomitant increase in Alt-EJ and HR, similar to the phenotype of NHEJ-inhibiting drugs ,54. The While many studies have focused on how E6 and E7 modulate DSBR, viral replication itself requires DDR activation , includiIn addition to the DNA repair related, cell-intrinsic radiosensitivity mechanisms, other possibilities include incomplete TP53 inactivation , expressA complementary approach to cell-based analyses of DSBR is to analyze cancer genomes to measure characteristics DNA repair scars (“signatures”) that can reflect the underlying DNA repair capacity of the tumor. Examples include cancers defective in mismatch repair exhibiting microsatellite instability in the genome and cancers defective in homologous recombination exhibiting a variety of associated genomic alterations. What can the underlying genomics of HPV+ cancers teach us about DNA repair in these cancers?p = 0.026) and a higher percentage of HPV+ HNSCC genomes are classified as “M” type, driven by more mutations in cancer drivers than copy number changes (58% vs. 27%) [First, we know that the expression of E6/E7 into cells can cause genomic instability, which is reflected by rearrangements, translocations, and amplifications ,78, and Second, the overall frequency of mutations does not differ between HPV+ and HPV− HNSCC genomes. However, the mode of acquisition of single base substitutions does differ substantially. HPV− genomes are dominated by C>A substitutions induced by smoking, whereas HPV+ genomes exhibit dominance of the APOBEC signature involving C>T and C>G mutations in TpCpN trinucleotides. The APOBEC cytidine deaminases mediate anti-viral activity through deamination of T to U in single-stranded viral DNA. As a harmful by-product, single-stranded DNA occurring in the genome during replication can similarly be affected by APOBEC enzymes, inducing base substitutions.Third, when focusing on genomic signatures present in cancer deficient in homologous recombination, HPV+ tumors do not exhibit SBS3, the single base substitution pattern found in BRCA1/2 biallelic mutated cancers defective in HR , and if p = 0.04) [What is different between HPV+ and HPV− tumors is the frequency of deletions associated with microhomology-based repair used in alternative end joining . Those H = 0.04) . Subsequ = 0.04) . To reco = 0.04) ,85 and t = 0.04) .Another means of assessing real-world homologous recombination deficiency is to compare the clinical response rates of recurrent and metastatic HPV+ tumors to platinum salts and PARP inhibitors to those of HPV− tumors. Platinum and PARP inhibitor sensitivity are largely determined by HR capacity. For instance, the overall response rates to platinum in germline bi-allelic mutant BRCA1/2 cancers range from 65 to 95% ,90,91,92The radiosensitivity of HPV+ HNSCC, anal and vulvar cancers is a clinically important topic, as few other biomarkers of radiation sensitivity exist. In pre-clinical models, there is a considerable body of work supporting both HR and NHEJ deficiency induced by E6/E7. HPV+ tumor genomes demonstrate an absence of canonical HR deficiency signatures, such as SBS3 and LST, but they do exhibit increased Alt-EJ scars relative to their HPV− counterparts, possibly supporting increased Alt-EJ in response to a partial NHEJ deficiency. Finally, available clinical data strongly support a role for cisplatin in concurrent chemoradiotherapy, but as a stand-alone agent, it does not result in the kind of response rates common in true HR-deficient cancers. Opportunities for improvement in this field include additional prospective validation of Alt-EJ signatures and clinical investigation of DNA damage response inhibitors that can best take advantage of HPV-associated DSBR defects.JEL and DSH are listed inventors on a patent filed by Memorial Sloan Kettering Cancer Center regarding the use of alternative end-joining genomic signatures to predict radiation sensitivity in connection with prior work . There a


Advanced Driver Assistance Systems (ADAS) are being developed and installed in increasing numbers. Some of the most popular ADAS include blind spot monitoring and cruise control which are fitted in the majority of new vehicles sold in high-income countries. With more drivers having access to these technologies, it is imperative to develop policy and strategies to guarantee the safe uptake of ADAS. One key issue is that ADAS education has been primarily centred on the user manual which are not widely utilised. Moreover, it is unclear if user manuals are an adequate source of education in terms of content and readability. To address this research gap, a content analysis was used to assess the differences in ADAS-related content and readability among the manuals of the highest selling vehicles in Australia. The qualitative findings showed that there are seven themes in the user manuals: differences between driving with and without ADAS, familiarisation requirements, operational limits of the ADAS, potential ADAS errors, behaviour adaptation warnings, confusion warnings, and malfunction warnings. The quantitative analysis found that some of the manuals require several years of education above the recommended for a universal audience (>8 years) to be understood. Additionally, there is a notable number of text diversions and infographics which could make comprehension of the user manual difficult. This investigation shows that there is a lack of standardisation of ADAS user manuals (in both content and delivery of information) which requires regulatory oversight. Driver ADAS education needs to be prioritised by policymakers and practitioners as smart technology continues to increase across the transport system. It seems that current strategies based on user manuals are insufficient to achieve successful adoption and safe use of these technologies. A wide range of Advanced Driver Assistance Systems (ADAS) have been developed and installed in vehicles within the past two decades. These technologies are designed to assist drivers with key driving functions including speed management via intelligent speed adaptation systems, and timely braking via autonomous braking systems. Previous studies have found ADAS to increase safety , 2. A reThe surge of smart vehicle technologies appears to lack any standardisation, as many automakers use this technology to create competitive advantages. As a result, road users now have a variety of options to choose from when selecting a vehicle. While some newer vehicle models contain similar technologies, such as blind-spot monitoring, many of these technologies differ between vehicle brands and models. For example, collision avoidance technology (CAT) varies significantly across vehicle models. CAT systems can use either radar, lidar, a camera, or a combination of these to detect an object or obstacle that the vehicle may collide with . AdditioFamiliarisation with novel ADAS technology is not the only challenge that users face when operating a new vehicle. Labelling irregularities within the vehicle industry introduce confusion into the learning process, whereby users must recognise that different manufacturers often label technology differently. For example, the vehicle’s ability to regulate a consistent speed autonomously while driving may be named ‘Adaptive Cruise Control’ by one company and ‘Distance Assist’ by another. The American Automobile Association  examinedOne key issue is that education and training about ADAS has mainly been the responsibility of the driver. Generally, drivers are required to read the user manuals and complement this with other sources of information, such as internet research. However, reports show that the majority of drivers resort to inaccurate learning methods, rather than utilising user manuals or receiving instructions from the dealer . Indeed,A further complication associated with ADAS implementation is that, even if drivers know they possess a system, they often do not understand the functioning or limitations of their ADAS technology. For example, a national cross-sectional study of drivers in Spain found that not knowing how to use the system was one of the main reasons for not using ADAS, including fatigue detection systems and adaptive cruise control . More coThe present study aims to analyse a variety of vehicle operations manuals from different brands and to develop a descriptive model of the information presented. User manuals have been used in previous research as effective sources of educational information for operators across a range of industries , 14, 15.A content analysis was used to assess the differences in ADAS-related content and readability among the manuals of the highest selling vehicles in Australia. This process comprised two sections; the first of which was the search strategy used to locate vehicles that would be eligible for use in the study, and their corresponding user manuals. The second was the data processing used to analyse the content of each user manual.A search on the Google search engine was performed to identify vehicles equipped with advanced driver assistance systems. The search was designed to identify the most purchased vehicle models in Australia during 2018, as it was surmised that these vehicles would be equipped with more advanced driving systems. The vehicles examined were limited to those listed as popular in Australia, as it has been previously found that vehicles in different countries differ, despite being branded as the same make and model . Therefoth vehicle with which to replace the missing manual, the Mitsubishi Triton was omitted from the study, and the total number of vehicles examined was nine.It was also important that the vehicles used were representative of the vehicles owned by the general population, rather than top-of-the-range models. For example, a recent Tesla model may be fitted with more advanced driver assistance systems than a recent Toyota Hilux, however a brand-new Tesla model would be unaffordable for much of the population. The ten most purchased vehicles in 2018 were considered : Toyota Despite differences between vehicle models, a vehicle owner will receive a manual that covers all models of a vehicle for that year. For example, while a Toyota Hilux TGN1*6 and a Toyota Hilux KUN1*5 will have different engines, the owner will be provided with the same vehicle manual that will include information on both models. and be labelled as the operations manuals for Toyota Hilux. The present study has focused only on these broad, all-inclusive vehicle manuals.The data processing stage comprised a two-part process. The themes were first determined through a deductive approach, wherein the sections pertaining to smart vehicle technology were read by two members of the research team, and relevant sections were noted. These sections were; intelligent speed adaptation/ speed limit warning/adaptive cruise control and blind spot monitoring/warning with lane change assist. From these two sections, broad parent themes were created from the content found across all manuals. The second part of the process utilised an inductive approach, wherein the content of each theme was examined and analysed. Themes that related to driver safety and safe usage of ADAS technologies was retained.For ease of analysis, Intelligent speed adaptation/Speed limit warning/Adaptive Cruise control and any other advanced driver system that moderates and adapts the speed of the vehicle when in use has been named ‘Cruise Control’. Similarly, any ADAS that monitors the driver’s blind spot or assists the driver in lane changing by checking the surrounding road environment has been named “Blind Spot Monitoring”. Each car manual was assessed on these two applications: cruise control and blind spot monitoring. Within each of these applications, each manual was coded according to seven different themes. The data was coded by three members of the research team. After the qualitative analysis was completed, we conducted the quantitative analysis of the manuals. The results are presented in the order in which the analyses were conducted.The content of the manuals was coded according to seven different themes based on the manual content. The themes included:informing differences” considers when the user manual describes the differences between driving with and without ADAS. This includes when the manual specifies the difference between driving with ADAS in an operational mode or driving with the system disengaged. For example, manually adjusting the vehicle speed rather than using the cruise control technology. Manuals were coded with either ‘Yes’ (present) or ‘No’ (absent). A manual that is coded as ’No’ indicates that it did not specify any difference between driving with or without the technology engaged.The theme “familiarisation” refers to a notice within the manual itself that advises the owner to become familiar with the manual and the smart technology. This is categorised as any time that any form of advice is given in regard to becoming familiar with any of the specified technology. Manuals will be coded for familiarisation as either ‘Yes’ (present) or ‘No’ (absent). A manual that is coded as ’No’ indicates that no message or warning indicates that the user should become familiar with the instructions for the smart technology and/or the user manual itself.The theme ““operational limits” considers when the user manual explains the operational limitations of the ADAS, including the warnings messages that indicate when the ADAS should not be used because of its own limitations. This warning could reference the road environment and highlight situations such as rain or snow. Manuals will be coded in regard to application warnings as either ‘Yes’ (present) or ‘No’ (absent). Manuals that are coded as ’Yes’ must have clearly signed warnings that are in bold text and draw the attention of the reader. They must also clearly highlight when the application should and should not be used.The theme “potential ADAS errors” refers to the potential situations when the ADAS may present an error when the system is operating. Compared to the operational limits, errors are issues that occur when the ADAS is operating in situation where typically should operate normally. This can take the form of an explanation that highlights an action that may cause an unexpected error and cause the ADAS to cease normal function, or an action that will prevent the application from being activated. Manuals will be coded for potential error warning as either ‘Yes’ (present) or ‘No’ (absent). Manuals that are coded as ’No’ will demonstrate no warnings of potential errors within the system, nor any actions that can cause or remedy a potential error.The theme behaviour adaptation warnings” considers warnings regarding behaviour change as a result of using ADAS. This can take the form of a warning that explains how the technology could change their behaviour, and what behaviours the driver will need to retain to maintain safe driving behaviour. Manuals will be coded for behaviour adaptation warnings as either ‘Yes’ (present) or ‘No’ (absent). Manuals that are coded as ’Yes’ display warnings in clearly bolded and noticeable text, and clearly indicate the effect that using the smart technology may have on the driver, and how the driver should behave accordingly.The theme ““Confusion warnings” describes a notification within the manual that indicates that there is the possibility that the user may not be aware of a normal function. This can be in the form of a delay, or a sound that the operator may not be aware of, however is part of the normal function of the system. Manuals will be coded for confusion warnings as either ‘Yes’ (present) or ‘No’ (absent). Manuals that are coded as ’No’ indicate that there are no notifications about potential confusion regarding the use of the smart technology within the vehicle.The theme “Malfunction warning” includes instructions of how to respond to a malfunction, e.g. vehicle should be returned to the dealer or manufacturer due to an error. This may also refer to the need to take the vehicle to a repair shop that is licensed to deal with the specific vehicle brand. This theme goes above and beyond the ‘potential errors’ theme, in that these malfunctions are more serious in nature and may endanger the driver. User manuals were coded for malfunction warnings as either ‘Yes’ (present) or ‘No’ (absent). User manuals that are coded as ’No’ indicate that there are no warnings that the vehicle may need to be returned to a dealer or taken to a specialised repairer due to an error or malfunction.The theme User manuals were also coded for attributes that could facilitate or make their use and understanding difficult. Three main attributes were identified: diversions, use of infographics, and readability.Diversion involves asking users to refer to another section of the manual for further information. Manuals include diversions that can be categorised as ‘outside the section’ and ‘inside the section’. Diversions that are coded as ‘outside the section’ indicate that a message has led the reader to another paragraph or message that is outside the section or chapter that they are currently reading. For example, within the manual for the Mazda CX5, in chapter 4, page 170, there is the message “Refer to Message Indicated on Display on page 7–65” which leads the reader to page 65 of chapter 7 and would therefore be found to be ‘outside the section’. In contrast, on page 172 of chapter 4, the message ‘Refer to Cruise Control Function on page 4–180’ would be found to be ‘within the same section’, as the diversion leads the reader to a part within the same chapter. The measure of section diversion will comprise of the sum total of ‘outside the section’ diversions.In the context of driving manuals, there are three main types of infographics see : illustrhttps://www.online-utility.org/english/readability_test_and_improve.jsp). The online calculator measures text based on the Gunning Fog index, which indicates the number of years of formal education required to easily read and understand the text on the first read through. Texts intended for a near universal audience, such as vehicle owners, the ideal score is reported to be six to eight. Texts intended for a wide audience generally require a Gunning Fog index of 12 at maximum. Scores above 12 are reported to be too complex for wider audiences to understand with ease. For both cruise control and blind spot monitoring, each section was copied into the readability software in the entirety and the Gunning Fog index was recorded. The entire chapters of cruise control and blind spot monitoring were analysed. Prior to entering the text, each chapter was edited to remove bullet points or any other formatting anomalies that may influence the software’s ability to analyse the text. If there was no formatting, the text was entered into the program without any changes being made. In addition to the overall score, readability analyses were conducted from samples from the beginning, middle and end of the chapters.Readability is the level of ease or difficulty with which text material can be understood by the reader. An online readability calculator was used to determine how easily the manuals can be read by those that purchase the vehicle , are critical to warrant safe use of the ADAS, i.e., operational limits, potential ADAS errors, and behaviour adaptation warnings. For example, previous research has shown that drivers can misuse ADAS systems and reduce protective behaviours as a consequence of the ADAS , 21, 22,The findings from the present research show that user manuals have safety-critical information about using ADAS, which is consistent with earlier research . Whilst Manuals also include several human factors issues regarding the number of times a reader must divert around the manual to find information and the wide range of infographics that must be interpreted and understood. Diversions in a user manual can create challenges effecting the text flow as well as distractions from the ADAS. In this study, we found that in some vehicles the number of diversions was high, which decreases readability. If the automobile industry expects that education about their systems be solely or primarily based on user manuals, they should try to improve them. Similarly, we identified a wide range of imagery used in manuals which can also significantly erode a reader’s efforts to understand and put into practice a manual’s content. Relevant to this, Thompson et al.  found amA key finding of this study is that to be able to both read and comprehend the manuals a high number of years of education is required. Some of the vehicles and ADAS require several years of education above the recommended for a universal audience (>8). This creates limitations in the understanding of the manuals particularly among young drivers, people with disability, people with limited English proficiency, or those who have not completed the assumed level of education required. Existing literature has examined the difficulty faced by users to read and understand manuals based on the language and grammar used to relay important information. Smart et al.  found thth vehicle from the list used to select the vehicles. Also, in the quantitative analysis, the Gunning Fog Index is based on a formula which determines the complexity of a word based on the number of syllables contained within the said word. As a result, commonly used words that contain three syllables are valued as ‘more complex’ than a word with two syllables that is less well known or has a definition that is more difficult to understand. This index may not precisely represent the complexity of the text. With this in mind, it is important to recognise that this investigation did not analyse perceptions of users directly. By obtaining direct information from human subjects about their experience using the manuals, we may gain additional understanding of readability and usability of the manuals. In addition, studies using human subjects can help to understand the actual impact that interactions with manuals have on road user behaviour. Also, the present study only analysed the technology component of user manuals. This means that we cannot conclude whether the presence of this technology is the reason for why the manuals are complex to read or not. Future research should seek to compare vehicle manuals with and without ADAS to understand the impact that the technology has on readability of the manuals. Generally, additional studies are needed to broaden our understanding of the gaps and best practice of ADAS education policy.The present manuscript has a number of limitations. Firstly, car models considered in this study were top sellers in Australia, which may not represent the features available in other jurisdictions. Next, we only analysed the content of user manuals of vehicles, but the users can also obtain information from the internet, or from friends and family. That information can have varying levels of accuracy, and it is unclear if it can correctly complement, or indeed undermine, the user manuals. Similarly, only nine of the ten vehicles listed were able to be examined, due to the unavailability of the Mitsubishi Triton manual online, and the lack of an 11To conclude, the information presented in the user manuals of vehicles with ADAS is safety-critical for driving. However, the large variability in the content of the manuals highlights the need for benchmarks of the minimum education needed to drive with ADAS safely. Another critical issue is that the manuals present several potential usability issues such as large numbers of text diversions and infographics. It was also found that these manuals are generally written to be understood by a highly educated audience. These issues likely decrease the usability of the manuals, which could potentially contribute to low readership. This investigation shows that there is a lack of standardisation of ADAS user manuals (in both content and delivery of information) which requires regulatory oversight.S1 Appendix(DOCX)Click here for additional data file.


Apart from tobacco and alcohol, viral infections are proposed as risk factors for laryngeal cancer. The occurrence of oncogenic viruses including human papilloma virus (HPV) and Epstein–Barr virus (EBV), in laryngeal squamous cell carcinoma (LSCC) varies in the world. Carcinogenesis is a multi-step process, and the role of viruses in LSCC progression has not been clarified. We aimed to analyze the presence and co-expression of HPV, EBV, human cytomegalovirus (HCMV) and human adenovirus (HAdV) in LSCC. We also investigated if p16 can act as surrogate marker for HPV in LSCC.Combined PCR/microarrays (PapilloCheck®) were used for detection and genotyping of HPV DNA, real-time PCR for EBV, HCMV and HAdV DNA detection, and EBER in situ hybridization (EBER-ISH) for EBV detection in tissue from 78 LSCC patients. Additionally, we analyzed p16 expression with immunohistochemistry.Thirty-three percent (26/78) of LSCC tumor samples were EBV positive, 9% (7/78) HCMV positive and 4% (3/78) HAdV positive. Due to DNA fragmentation, 45 samples could not be analyzed with PapilloCheck®; 9% of the remaining (3/33) were high-risk HPV16 positive and also over-expressed p16. A total of 14% (11/78) of the samples over-expressed p16.These findings present a mapping of HPV, EBV, HCMV and HAdV, including the HPV surrogate marker p16, in LSCC in this cohort. Except for EBV, which was detected in a third of the samples, data show viral infection to be uncommon, and that p16 does not appear to be a specific surrogate marker for high-risk HPV infection in LSCC.The online version contains supplementary material available at 10.1007/s12672-022-00475-4. Laryngeal cancer is detrimental for voice and airway, leading to life-changing consequences.The worldwide annual incidence of newly detected laryngeal cancer is 210,000 cases/year. The estimated prevalence is ∼1,000,000 cases/year and 126,000 deaths/year [Laryngeal squamous cell carcinoma (LSCC) can develop from several causes, with tobacco and alcohol use being recognized risk factors . Other pEBV and HPV can each lead to cancer development independently, but these viruses can also co-infect and enhance cancer progression through interactions with other mechanisms .Cancer in the tonsils and base of tongue is associated with high-risk HPV genotypes HPV16 and HPV18. These types of cancer differ from non-HPV associated malignancies both biologically and clinically , which hp16 is the second most common tumor suppressor gene , and itsThe presence of high-risk HPV types in LSCC has been reported in 5–24% of cases . VariatiEBV is reported in 60% of laryngeal cancers . EBV is EBV, HPV, human cytomegalovirus (HCMV), and human adenovirus (HAdV) all replicate in the airway epithelium. They are transmitted via saliva or inhalation of droplets from infected individuals and can establish persistent infections in their host in part through evading host immune surveillance.HCMV and HAdV infections can be devastating in immunocompromised patients. HCMV is not a typical human tumor virus, but is thought to have oncomodulatory activity . HCMV anCarcinogenesis is a multistep process, and the role of viruses in this progression in LSCC has not been fully identified. If viruses turn out to be important in development of LSCC, anti-viral vaccines could potentially be used for prevention, and possibly even treatment of this disease. Vaccination holds a promise for a vast reduction of HPV-related cancer in tonsils and base of tongue. Our hypothesis is that LSCC is associated with oncogenic/oncomodulatory virus. Therefore, our purpose is to map HPV, EBV, HCMV and HAdV in a group of cases with LSCC, and study co-infection. In addition, we aim to analyze if p16 is a reliable surrogate marker for HPV in LSCC.We identified 236 consecutive LSCC samples from cases between 2000 and 2017 in SymPathy . Slides were histopathologically re-examined (by KN and KO) for correct diagnosis and classification of cancer site.Exclusion criteria for analysis were: (1) repeated biopsies from the same case (112 samples), (2) previous radiotherapy to the neck (39 samples), (3) classification errors (5 samples) and (4) too little tumor tissue for analysis (2 samples).SymPathy data provided access to clinical chart databases to supplement patient characteristics.Descriptive data on 78 LSCC samples included in the PCR analysis of EBV, HCMV, HAdV and p16  are presented. Due to HPV DNA fragmentation, 45 LSCC samples could not be analyzed, and were therefore drop-outs in the HPV PCR results  LSCC samples were assessed for eligibility. The case ages ranged from 41 to 86 with a mean of 68.1 years (± 10.3). The sex distribution was 15 (19%) women and 63 (81%) men, and other clinical characteristics are shown in Table Thirty-three LSCC samples underwent HPV genotyping, with an age range of 46–83, mean of 67.2 years (± 10.2). The sex distribution was 6 (18%) women and 27 (82%) men.Combined PCR/microarrays  were used for detection and genotyping of HPV and real-time PCR for EBV, HCMV and HAdV DNA detection. EBER in situ hybridization (EBER-ISH) was used for EBV detection in slides from LSCC tumors. In addition, we used immunohistochemistry (IHC) for mapping of p16 . Quality and quantity of total DNA was measured spectrophotometrically.HPV detection by PapilloCheck® requires a DNA fragment of 350 bp for successful PCR performance. To assess the degree of DNA fragmentation prior to detection and typing using the PapilloCheck®, we performed three different PCR assays amplifying amplicon lengths of 536, 268 or 100 bp of the ß-globin housekeeping genes, and one real-time PCR assay detecting 60 bp of the RNasP housekeeping gene.The PapilloCheck® is a multiplex PCR method with fluorescent primers, for genotyping of 24 HPV-types including 15 high‐risk HPV types; HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73 and 82, two probably high or intermediate risk types; HPV 53 and 66, and seven low‐risk HPV types; HPV 6, 11, 40, 42, 43, 44 and 70.BamHI-W region of the EBV genome was used. The sequences of the forward and reverse primers were 5′-GCATAATGGCGGACCTAGG-3′ and 5′-AAGATAGCAGCAGCGCAGC-3′, respectively, flanking the dual-labelled probe 5′-(FAM)TAAAACCCCCAGGAAGCGGGTCTATG(TAMRA)-3′. Real-time PCR detection towards the polymerase gene of HCMV consisted of the amplification primers 5′-TGDGTCACGCCGTTCTCTAG-3′ and a 50:50 mix of 5′-ATCATCATGGCCCACAACCT-3′ and 5′-ATCATCATGGCTCACAACCT-3′, flanking the dual-labelled fluorescent probe 5′-(FAM)ACTCGCCACCCGGCACCAAC(TAMRA)-3′.For EBV detection, a real-time PCR assay of the For detection of HAdV DNA a real-time PCR assay of the hexon region was used. The sequences of the degenerated forward and reverse primers were 5-CWTACATGCACATCKCSGG-3′ and 5′-CRCGGGCRAAYTGCACCAG-3′, respectively, flanking the dual-labelled probe 5′-(FAM)CCGGGCTCAGGTACTCCGAGGCGTCCT (TAMRA)-3′.The EBER-ISH is an in-situ hybridization kit for EBER transcripts (Epstein–Barr Encoding Region), and this was applied for EBV mapping in tumor and connective tissue.Two tissue sections were cut from each LSCC biopsy, one was incubated with an RNA-positive control probe . The second section was tested for EBER-1 and EBER-2 by the Inform EBER Probe . NBT- BCIP Detection System  was employed for visualization. We used a Ventana BenchMark ULTRA system  for staining.EBER-positive cells were counted in tumor and connective tissue by three of the authors . Consensus was reached after comparison of individual results.EBER was graded as positive when there were ≥ 2 positive cells and graded as negative when there were one or no positive cells.To detect the p16 protein, five µm sections were cut from paraffin blocks. The staining system  was applied, and slides pretreated in CC1  for 48 min. For detection of p16, CINtec® p16 Histology  was used. To visualize the antibody, the Opti View DAB IHC Detection Kit  was employed.p16 expression was evaluated by three members of the group  using a light microscope  and a semiquantitative Quickscore system by Detre et al. . Using tDescriptive statistics  were presented regarding age, gender, smoking, cancer site and presence of virus and p16. Cross tabulations were used to describe numbers and proportions of virus and p16. The association between virus and p16 were analyzed using Chi-square test and Fisher’s exact test. A probability value of equal or less than 0.05 (2-sided) was considered statistically significant.HPV was assessed in a two-step fashion. First, 45 samples were excluded due to DNA fragmentation, exhibiting fragments smaller than the 350 bp needed for successful detection and typing. To ensure correct sorting, an HPV analysis with PapilloCheck® was performed on these excluded samples, without adding new results. Real-time PCR of the endogenous target gene RNAsP indicated that all samples contained DNA of at least 60 bp.Secondly, we performed PapilloCheck® on a sub-group of 33 samples. Nine percent (3/33) were high-risk HPV16 positive Fig. .Fig. 2EBTwo of three HPV16 positive cancers were in the true vocal cords, both patients were females and one also a smoker. The third patient was a non-smoking male with subglottic cancer. We did not identify any imbalance comparing the sub-group (n = 33) to the group in total (n = 78) regarding age, sex, site, or smoking status.One patient was positive for both HPV and EBV 1/33)  and 18 (2).We detected EBV by PCR in 33% (26/78), comprising tumor and connective tissue. Thereafter, EBER-ISH revealed distribution of EBER-positive cells in the tissue Fig. .Fig. 4EBThe amount of positive cells/slide ranged from 2 to 10, with a mean of 5 cells in tumor tissue and 4.6 cells in connective tissue. Five percent (4/78) of samples were EBER-positive in tumor tissue, and 6% (5/78) were EBER-positive in connective tissue. Two of these samples were EBER-positive in both tumor and connective tissue, thus 9% (7/78) of LSCC samples were EBER-positive (Table Nine percent (7/78) were HCMV positive and 4% (3/78) of samples were HAdV positive Fig. .Six percent (5/78) of samples were positive for both EBV (PCR) and HCMV Fig. .One percent (1/78) of the samples were positive for both EBV and HAdV Fig. .One percent (1/78) of the samples were positive for both HCMV and HAdV Fig. .The analysis of p16 in tumor tissue for intensity and proportion showed a Quickscore (QS) ranging from 0 to 18 Fig. .Fig. 5ReWe ranked our Quickscore results in three groups: negative QS (QS = 0), low QS (QS = 1 and 2) and high QS (QS = 3–18). The results were: negative QS = 42% (33) of samples, low QS = 44% (34) and high QS 14% (11) of samples Fig. . Nine peWe performed a complete mapping of four different viruses and p16 in the same group of cases with LSCC, which showed that 33% (26/78) of samples were EBV infected and 9% (3/33) were high-risk HPV16 positive. These oncogenic viruses were expressed individually; in one case only (of 33) co-expression was noted.Three out of 11 p16 high QS samples correlated with high-risk HPV16 positive samples, indicating that p16 was not a very sensitive surrogate marker for HPV16 in LSCC.The common upper airway viruses HCMV and HAdV, were present in low amounts; 9% (7/78) were HCMV positive and 4% (3/78) HAdV positive. It would be relevant to map the same viruses in non-tumorous laryngeal mucosa, but ethical consideration makes it impossible to collect samples from a healthy control group, as the true vocal cords are susceptible to injuries in this type of sampling process.We searched for high- and low risk HPV types with PapilloCheck® in 33 LSCC samples. Nine percent (3/33) were high-risk HPV16 positive. Remaining 45 samples could not be analyzed due to random fragmentation of DNA caused by aging. Comparison of the groups did not show any differences regarding age, sex, site, or smoking status.PapilloCheck® is a readily available, sensitive, and robust technique, used routinely in the clinical setting for screening of high and low- risk HPV types in the cervix. E6/E7 mRNA technique is by some considered a reference diagnostic test of active HPV infection , 13. HowRegardless of the detection method, HPV positivity appears to be low in LSCC. E6/E7 mRNA expression is reported between 2 and 8.6% , 14, andUsing multidimensional outcome measures, we first found EBV by PCR in 33% of the tumor samples. Secondly, mapping of EBER-stained cells revealed roughly equal distribution in tumor (5%) and adjacent connective tissue (6%). EBER-ISH is not a quantitative measure, but it can map location of the EBV-infected cells in one slice of the total tissue sample. Epstein–Barr Encoded Regions 1 and 2 (EBERs) are good markers of EBV infection and present in numerous copies in latently EBV infected cells. They are easily identified by in situ hybridization (ISH) . EBERs aEBV PCR, on the contrary, was used to assess EBV presence or absence in a bigger piece (2 × 2 mm) of the tumor. This explains the different numeric results between PCR and EBER-ISH.It is important to distinguish EBV in actual tumor epithelial cells vs non-tumor cells, since EBV infection of epithelial cells is considered pathological . It is uWe found one patient (1/33) positive for both HPV and EBV in LSCC samples. EBV is known to interact with HPV in adult-onset recurrent respiratory papillomatosis, a benign lesion on the vocal folds with a risk of malignant conversion . In LSCCOne of the target viruses in the analysis, HCMV, might have “oncomodulatory” activities, resulting in secretion of cellular and viral growth factors and cytokines in tumor microenvironment . Nine peNone of the LSCC samples was HCMV and HPV positive. Although implicated as a cofactor in HPV-mediated carcinogenesis, an association between oral cancer and HCMV has not yet been identified .The second target virus, HAdV, confers mild and self-limiting infections such as the “common cold”. HAdV is oncogenic in rodents, but oncogenicity has not been observed in humans . Humans There is not a widely accepted specific p16 cut-off value for positivity in the literature. The College of American Pathologists recommends that for positive diagnosis, p16 shows strong, diffuse nuclear and cytoplasmic staining in 70% of tumor cells .We used the Quickscore (QS) method from Detre et al.  to assesThe tumor suppressor protein p16 was expressed with high intensity and quantity in the HPV16 positive cancer samples, whereas 8 samples over-expressed p16 without any correlation to HPV16. This supports the interpretation that p16 is not a reliable surrogate marker for HPV infection in LSCC.Whether HPV, EBV, HCMV and HAdV are just ‘passerby’ viruses in LSCC, or have oncogenic or oncomodulatory capacities, is a relevant clinical question. HPV and EBV have capabilities of virus-induced metabolic changes that share similarities with tumor associated metabolic reprogramming . MetabolThis is the first thorough report mapping HPV, EBV, HCMV and HAdV, including p16, in the same group of patients with LSCC. Except for EBV, which was detected in a third of the LSCC samples, viruses were not found in a large proportion of cases with this tumor type in this cohort. The findings also demonstrate that p16 may not be reliable as a surrogate marker for high-risk HPV infection in LSCC.Supplementary file 1 Quickscore system by Detre et al. Figure illustration by PhD Anna Holm (DOCX 28 KB)


P16 immunostaining is considered a useful surrogate of transcriptionally active high-risk (hr) HPV infection. Only strong and widespread “block-like” immunoreactivity is considered specific, whereas weak/focal p16 positive immunostaining is considered not specific, and follow-up and HPV molecular detection is not indicated. The aim of the study was to evaluate the presence of HPV DNA and Ki67 immunostaining in 40 cervical atypical biopsies (CALs) with mild and focal histological features suggestive of HPV infection—20 cases with weak/focal p16 positive immunoreactivity and 20 cases negative for p16 expression. In 16/20 weak/focal p16 positive CALs (80%), the INNO-LiPA HPV genotyping detected hrHPV genotypes . Co-infection of two or more hrHPV genotypes was often evidenced. HPV16 and 18 genotypes were never detected. Ki67 immunostaining was increased in 10/20 cases (50%). In 19/20 p16 negative CALs, hrHPV infection was absent and Ki67 was not increased. These results suggest that weak/focal p16 immunostaining represents the early stage of transcriptionally active infection, strongly related to the presence of less common hrHPV genotypes, probably with a slower transforming power, but with a potential risk of progression if the infection persists. HPV DNA genotyping and follow-up could be useful in these cases to verify if they are able to evolve into overt dysplastic changes and to improve knowledge of less common hrHPV genotypes. The etiological role of HPV infection in cervical cancer is well known. To date, more than 200 HPV types have been completely sequenced, characterized, and numbered. Among these, approximately 40 can infect the human mucosa, above all the anogenital and aerodigestive tracts .The International Agency for Research on Cancer (IARC) classified 12 HPV types as human carcinogens (Group 1), which are commonly referred to as “high risk-HPVs” .High-risk (hr) HPV types include HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59; probable or possible hrHPV types (IARC Group 2A and 2B) include HPV 26, 53, 66, 67, 68, 70, 73, and 82; and low-risk (lr) HPV types include HPV 6, 11 (IARC Group 3), 40, 42, 54, 55, 61, 62, 64, 69, 71, 72, 81, 83, 84, and 89 [Many women get cervical HPV infections, and most do not progress to cervical cancer because infections are transient and viral clearance by the immune system occurs spontaneously without ever causing lesions. High risk HPV infections that “persist” are more likely to evolve into high-grade squamous intraepithelial lesion (HSIL). The evolution to cervical carcinoma may take several years if left untreated .The integration of HPV into the cell genome is considered one of the main factors for HSIL and cervical carcinoma development ,7.The pathogenesis of HPV infection consists in an overexpression of E6 and E7 oncoproteins, which can interact with a variety of cellular proteins, inducing proliferation and, eventually, malignant transformation.The best-regarded interactions are with TP53 and pRB, both master regulators of the cell cycle and mutated in many human cancers.Degradation of p53 by E6 oncoprotein allows cells to continue replicating . BindingUp-regulation of CDKN2A leads to p16 expression, absent in the normal cervix, as the host’s response to deactivated pRB by HPV proteins. So, the immunohistochemical assessment of p16 is considered a surrogate for transcriptionally active hrHPV infection.The expression of this marker in the cervical epithelium is of diagnostic value only when a diffuse and intense positivity is observed, so called “block-like positivity” ,11,12.Weak and focal p16 positive (w/f p16+) immunostaining is considered not specific, because it has also been reported in reactive squamous epithelium .Ki67 is a nuclear antigen associated with cell proliferation. The protein is present during all active phases of the cell cycle, but is absent in resting cells (G0). In the normal exocervical epithelium, Ki67 is localized within the basal and suprabasal layers, whereas in squamous intraepithelial lesions it is increased ,13. So, In cervical biopsies lacking obvious dysplastic alterations, w/f p16+ immunostaining does not impose either HPV DNA testing or strict follow-up.Previous studies have demonstrated that, despite the use of strict histologic criteria for the diagnosis of CIN 1 lesions, there was a false positive rate, or an overinterpretation of CIN 1, in up to 34% of cervical atypical biopsies that, via molecular methods, were negative for HPV infection .In cervical biopsies lacking obvious dysplasia, defined as “cervical atypical lesions” (CALs), a significant statistical correlation between the Ki67 expression in the higher layers of cervical squamous epithelium and the presence of hrHPV was reported .As some CALs show w/f p16+ immunostaining, the aim of this study is to correlate w/f p16+ immunostaining with Ki67 immunoreactivity and to perform HPV DNA testing to investigate the presence of HPV DNA so as to better understand the real meaning of w/f p16+ in cervical biopsies.The study was carried out on 20 formalin fixed, paraffin embedded cervical biopsies with histological features of CALs showing weak and focal p16 immunostaining (w/f p16+ CALs), and 20 CALs negative for p16 immunostaining (p16- CALs), consecutively received between June and December 2020. Patients’ ages ranged from 27 to 53 (mean age 35).Histological features of HPV infection  were inconsistently present or only weakly/focally evident, raising the question of a differential diagnosis between HPV-associated changes and repair/reactive or metaplastic changes. Cases with obvious dysplasia and/or with “block-like” p16 expression were excluded.A “block-like” p16 pattern was considered when strong/diffuse nuclear and cytoplasmic immunoreactivity represented greater than or equal to 50% reactivity in all the epithelium layers. A weak and focal p16 expression was considered when weak/focal/scattered nuclear and cytoplasmic positivity was present in more than 10 basal/parabasal cells and in less than 50% of the epithelium thickness.Immunohistochemical stains were carried out with a BenchMark ULTRA automated slide staining system  according to the manufacturer’s instructions, using the following primary antibodies: p16  and Ki67 . For all the immunohistochemical stains, the 3,3-diaminobenzidine kit was used as chromogen. The slides were observed on a Leica DM2000 microscope . The microphotographs were obtained using a Leica DFC320 camera .For the HPV detection and genotyping of each biopsy, two or three 5 µm paraffin-embedded sections were placed in a microcentrifuge tube with 200 μL of Feoli-Fonseca buffer  containing 300 μg/mL of proteinase K. The mixture was incubated at 56 °C overnight. Proteinase K was then denatured at 95 °C for 10 min, and after centrifugation at 13,000 rpm for 5 min the supernatant was purified with QIAmp DNA mini kit (QIAgen), following protocol for DNA purification from blood or body fluids.The DNA quality was checked by β-globin amplification. The INNO-LiPA HPV Genotyping Extra II kit  was used, based on the combined use of PCR SPF10 and LiPA hybridization. SPF general primers detected at least 43 different HPV genotypes . The LiPAs negative controls, 10 p16 negative surgical cervical samples of uteri resected for the presence of leiomyoma were assessed with an HPV DNA test and Ki67 immunostaining. They appeared histologically normal.p values < 0.05.Statistical analysis was performed by using a Chi-square test with Yates correction. Statistical significance was assumed for HPV DNA test: 16/20 w/f p16+ CALs (80%) were positive for at least one genotype among hrHPV or probable hrHPV, one case (5%) was positive for lrHPV, and in three cases (15%) no HPV DNA was detected. Ten different HPV genotypes were identified: four hrHPV , five probable hrHPV  for Groups 1 and 2 of IARC classification, respectively, and only one lrHPV (genotype 54).In 11/20 cases (55%), at least one of the IARC Group 1 subtypes was identified. The most frequent HPV subtypes were 51, 31, and 73 (each one found in 4/20 cases (20%)). Multiple HPV genotype infections were found in 7/20 (35%) cases. The subtypes most often involved in co-infections were 51 (four cases), 66, 31, and 73 (three cases for each); HPV 51 and HPV 66 were present only in co-infection cases and never alone. Of note, the HPV16 and 18 genotypes were never found .Ki67 immunostaining: 10/20 (50%) w/f p16+ CALs showed Ki67 nuclear positivity limited to the basal/parabasal layer of the squamous epithelium.In addition, 10/20 cases (50%) showed nuclear positivity for Ki67 in all layers of the squamous epithelium.Among the latter, 9/10 (90%) showed hrHPV (or probable hrHPV) genotypes.Among all hrHPV positive cases, 9/16 (56.3%) showed a high proliferation rate with Ki67 positive immunostaining in all of the squamous epithelium layers . In 7/16In some cases, in the background of w/f p16+ immunostaining, small and circumscribed “block-like” p16 positive areas (less than 50% of the epithelium) with full thickness Ki67 immunostaining were present .HPV DNA test: 19/20 p16- CALs (95%) were negative for HPV-DNA. One case (5%) showed the presence of hrHPV DNA (genotype 16).Ki67 immunostaining: Ki67 expression was limited to the basal layer of the squamous epithelium in all cases.p values < 0.01).The statistical analysis, performed using a Chi-square test with Yates correction, showed a significant statistical difference for the presence of hrHPV (Groups 1 and 2 of the IARC classification) between the two groups  positivity is considered specific. Recently, Yuxin Liu and al.  emphasizNoteworthy, they did not investigate cases with weak, focal, and discontinuous p16 staining, which they considered as a “negative pattern”. To our knowledge, no systematic studies have been performed to assess the meaning of this w/f p16+ pattern in CALs.In this study, hrHPV (or possible hrHPV) genotypes were found in 16/20 (80%) w/f p16+ CALs, often as the co-infection of multiple genotypes. Moreover, 9/16 (56%) cases showed a high proliferation rate, with Ki67 nuclear immunostaining in all epithelial layers, suggesting that a transcriptionally active viral phase is ongoing.The remaining 7/16 (43.8%) w/f p16+ CALs with a low proliferative activity  probably represent latent, relatively harmless, HPV infection with less clinical consequences, if underdiagnosed.Of note, 19/20 p16- CALs were negative for HPV DNA presence, suggesting that they are metaplastic and reactive lesions unrelated to HPV, in keeping with previous studies [P16 immunohistochemistry was hypothesized to distinguish between transforming and non-transforming HPV infections, based on the concept that HPV-mediated transformation is triggered by the dysregulated expression of the viral oncogenes E6 and E7 in the basal and parabasal cells.However, although the basal and parabasal cells are considered to be the ones in which transformation begins, only the block positive p16 expression pattern was defined as a hallmark of HPV-dependent transformation, and thus considered as p16+ ,21,22.The results of the present study suggest that weak and focal p16 expression is not a negative or non-specific result. According to the hypothesis that transformation begins from basal and parabasal cells, it could be the early pattern of transforming HPV infection, as demonstrated by the strongly relation with the presence of rarer hrHPV genotypes and by the increase of ki67 immunoreactivity in half of the w/f p16+ cases.In fact, the w/f p16+ pattern showed 80% sensitivity, 95% specificity, 94.12% positive predictive value (PPV), and 82.61% negative predictive value (NPV) for hrHPV infection.The coexistence of areas with circumscribed “block-like” p16 immunoreactivity and the increase of Ki67, in a background of w/f p16+ expression, suggests that the w/f p16+ pattern could evolve in a “block-like” p16 immunoreactivity if the infection persists.If these results are confirmed, HPV DNA testing and a follow-up should be recommended, at least for cases with increased Ki67 expression.The study presents some limitations. The first limitation is the small number of cases, and the second is the short follow-up period. Nevertheless, this is a pilot study, and it has some strengths, as follows: the first is the high PPV and NPV for hrHPV infection. The second is the capability of highlighting the presence of rarer hrHPV genotypes at the very early stages of infection, allowing patients to be subjected to an accurate follow-up.This is in keeping with recent studies highlighting the importance of the use of p16 immunostaining, alone or in combination with extended HPV genotyping, to improve the screening programs and the subsequent management of the patients ,24,25.The results of long-time follow-up data will be reported in future studies.Remarkably, HPV16 or 18 genotypes were not detected in w/f p16+ cases, probably because HPV16 and 18 have a faster transforming power, quicky reaching the block p16+ stage, making hard to detect the w/f p16+ in the early stage. To confirm this hypothesis, further studies are necessary.Most low-grade lesions spontaneously regress in few months, probably due to immunological intervention. Morphological criteria alone are not sufficient to distinguish lesions that may regress from those that might persist and progress.Cases with a w/f p16+ expression may represent an early stage of transformation related to the less common hrHPV genotypes, probably with a slower transforming power and perhaps less prone to persist. Ki67 immunostaining, HPV DNA extended genotyping, and follow-up should be warranted to evaluate, in case of hrHPV positivity, the subsequent evolution , into overt dysplastic changes.The present study and future follow-up reports could highlight the benefits of a greater attention to weak/focal p16+ cases, improving the knowledge of rarer hrHPV genotypes, with a positive impact on cervical cancer screening programs.


Echinococcus multilocularis acquires energy. Fructose-1, 6-bisphosphate aldolase (FBA) plays an important role in this process, but it is not fully characterized in E. multilocularis yet. The results of genome-wide analysis showed that the Echinococcus species contained four fba genes (FBA1-4), all of which had the domain of FBA I and multiple conserved active sites. EmFBA1 was mainly located in the germinal layer and the posterior of the protoscolex. The enzyme activity of EmFBA1 was 67.42 U/mg with Km and Vmax of 1.75 mM and 0.5 mmol/min, respectively. EmFBA1 was only susceptible to Fe3+ but not to the other four ions , and its enzyme activity was remarkably lost in the presence of 0.5 mM Fe3+. The current study reveals the biochemical characters of EmFBA1 and is informative for further investigation of its role in the glycolysis in E. multilocularis.Glycolysis is one of the important ways by which  Echinococcus multilocularis, the causative agent of alveolar echinococcosis (AE), is widely distributed in the Northern hemisphere  is the substrate concentration, and mK is the Michaelis-Menten constant.Using the same reaction system, the enzyme kinetics were determined under the optimal conditions , and the experimental data were fitted into the formula through Origin 7.5 :V=Vmax[S2+, K+, Na+, Fe3+ and Mg2+) at a different final concentration were added to evaluate the effects on the enzyme activity of EmFBA1.Similarly, different metal ions (Can = 3). Origin Pro 7.5  was used to plot, analyze the kinetic data and estimate mK and maxV using the non-linear fit function.Data were presented as mean ± SD (E.multilocularis contained four potential fba genes, named as emfba1-4, and there were also four homologues in E.granulosus (fba had only one copy, and the four fba genes in each parasite were distributed on different chromosomes (fba genes, the overall amino acid similarity was 74.64%, whereas each fba shared >92% similarity with its corresponding ortholog. The phylogenetic tree showed that these fba genes formed four branches, each of which consisted one fba of E.multilocularis and its corresponding ortholog of E.granulosus (The genome-wide analysis revealed that anulosus . Each fbomosomes A. Among anulosus B. Moreovanulosus .E. granulosus, Taenia asiatica, and Taenia hydatigena, suggesting that FBA1 is widely expressed in cestodes. For the localization of EmFBA1, strong immunofluorescent signals were predominantly present along the germinal layer and in the posterior of unevaginated and evaginated protoscoleces (The purity of EmFBA1 was estimated to be more than 92% . Westernscoleces B. No sigscoleces B.mK and maxV of EmFBA1 were 1.75 mM and 0.5 mmol/min, respectively (Comparison of EmFBA1 enzyme activity demonstrated that the optimal reaction temperature and pH were 37 °C and 7.5, respectively A,B. Neveectively D.3+ significantly inhibited the EmFBA1 activity compared with other ions, including Na+, Ca2+, Mg2+ and K+ (3+ at a final concentration of 0.5 mM (It was further found that Fe+ and K+ E,F. Moref 0.5 mM F.E. multilocularis and E. granulosus can produce energy by glycolysis without a significant difference in the rate of glycogen use under aerobic and anaerobic conditions [Trypansoma brucei during its lifecycle, as it is the only source of ATP. Only 50% glycolysis inhibition is enough to kill T. brucei, which makes it a potent target for drugs [S. japonicum, FBA was potentially involved in the growth and development of sporocysts [E. multilocularis and E. granulosus, all eight FBA shared the same active site residues, which were also reported in the FBA isoenzymes in Clonorchis sinensis [E. granulosus. This may be explained by the fact that a set of emFBA genes might have been formed before speciation of both Echinococcus parasites.Glycolysis is a process of the decomposition of glucose or glycogen into lactic acid and the production of ATP in the presence or absence of oxygen, named as aerobic glycolysis or anaerobic glycolysis. Both nditions ,23. It hor drugs ,25. Theror drugs ,27,28. Iorocysts . In apicorocysts ,30. In bsinensis , suggestE. granulosus [Opisthorchis viverrini, which may provide energy to muscle tissues [Plasmodium spp., which may be involved in the movement and invasion of parasites [It was herein shown that EmFBA1 was widely localized along the germinal layer and in the posterior of the protoscolex. This distribution pattern is consistent with that of FBA1 in anulosus . As the anulosus , EmFBA1  tissues , and on arasites ,34. Of iS. japonicum that has eight different FBAs [E. multilocularis FBA proteins are functionally redundant. As expected, EmFBA1 show the highest enzymatic activity at 37 °C and pH 7.5. In the presence of four ions including Na+, Ca2+, Mg2+ and K+, EmFBA1 exhibits no significant alterations in enzymatic activity, suggesting ion independence. It was also shown that EmFBA1 was susceptible to Fe3+ ion, which has never been described for other FBAs. This adverse effect of Fe3+ is still unclear. Whether other emFBAs have similar traits or not needs to be investigated in future.FBA plays a key role in the anaerobic metabolism of glucose. The activity of EmFBA1 was 67.42U/mg, as three times lower as one of ent FBAs . This diE. multilocularis.We herein genetically and biochemically characterized FBA1. The results demonstrated that FBA1 was conserved in all cestodes investigated and EmFBA1 showed a relatively lower enzymatic activity. The current study reveals the discrepancy in the biochemical characters of EmFBA1 and is informative for further investigation of its role in the glycolysis in