NSD1) gene have been observed in head and neck squamous cell carcinomas (HNSCC). NSD1 encodes a histone 3 lysine-36 methyltransferase. NSD1 mutations are correlated with improved clinical outcomes and increased sensitivity to platinum-based chemotherapy agents in human papillomavirus-negative (HPV-) tumors, despite weak T-cell infiltration. However, the role of NSD1 and related family members NSD2 and NSD3 in human papillomavirus-positive (HPV+) HNSCC is unclear.Frequent mutations in the nuclear receptor binding SET domain protein 1 , we compared the relative level of mRNA expression of NSD1, NSD2 or NSD3 were not correlated with altered lymphocyte infiltration in HPV+ HNSCC. More importantly, low expression of NSD1, NSD2, or NSD3 correlated with significantly reduced overall patient survival in HPV+, but not HPV- HNSCC.Expression levels of NSD1, NSD2, and NSD3 expression levels should be further investigated as novel clinical metrics for improved prognostication and patient stratification in HPV+ HNSCC.These results starkly illustrate the contrast in molecular features between HPV+ and HPV- HNSCC tumors and suggest that The online version contains supplementary material available at 10.1186/s13027-021-00347-6. E6 and E7 oncogenes that deregulate cell growth and gene expression [Head and neck squamous cell carcinomas (HNSCC) comprise a group of heterogeneous cancers that arise from multiple anatomical subsites in the head and neck region. Collectively, they represent the 7th most common human cancer type  and are pression , 9. Impopression , 11, allpression .Histone methylation plays a critical role in the epigenetic control of gene expression. Specialized enzymes methylate/de-methylate individual amino acids that are found on the histone tails H1, H2A, H2B, H3, and H4. The nuclear receptor binding SET domain protein (NSD) family of histone-lysine N-methyltransferases are composed of three paralogous proteins: NSD1, NSD2 (WHSC1), and NSD3 (WHSC1L1). NSD1 mediates the transfer of a methyl group onto H3 lysine-36 (H3K36) and H4 lysine-20 (H4K20) . NSD2 meNSD1 H3K36 methyltransferase [NSD1 that contain a substitution of K36 to methionine in H3. Thus, mutation of the substrate of NSD1 phenocopies the methylation signature of direct mutational activation of this enzyme. This signature represents approximately 13% of HPV- HNSCC and identifies NSD1 inactivation as a mechanism of epigenome deregulation [NSD1 gene alterations exhibited improved survival compared to patients with wild-type NSD1 tumors [NSD1 in HPV- HNSCC has been reported to confer increased sensitivity to platinum-based chemotherapy agents in vitro [NSD1 or NSD2 leads to significantly better clinical outcomes in HPV- HNSCC of the larynx [NSD1, or its paralogs NSD2 and NSD3 in HPV+ HNSCC, except that mutation of these genes appears to occur at a reduced frequency as compared to HPV- HNSCC [NSD1 mutation is correlated with reduced survival [We and others used hierarchical clustering of DNA methylation data to identify a subset of HPV- HNSCC tumors enriched for mutations in the nsferase , 9. Thesgulation . Studies1 tumors , 22. Furin vitro , despitein vitro . Anothere larynx , furtherV- HNSCC  and NSD1NSD1, NSD2, and NSD3 between HPV+ HNSCC, HPV- HNSCC, or normal control tissues. We also investigated the relationship between the levels of expression of NSD1, NSD2, or NSD3 with tumor-infiltrating lymphocytes (TILs) in either the HPV+ or HPV- HNSCC samples and whether expression of NSD1, NSD2, or NSD3 correlated with overall survival. Our aim was to determine if there were differences in these molecular features that could serve as novel clinical metrics for improved prognostication and patient stratification in HNSCC.In this study, we used data from over 500 HNSCC patients from the Cancer Genome Atlas (TCGA) to compare the expression levels of mRNA for https://gdac.broadinstitute.org/). RNA-seq viral read counts for HPV E6 and E7 was extracted from the supplementary data files from Chakravarthy et al. [Patient data from the Cancer Genome Atlas (TCGA), including the Merged Clinical data and Level 3 RNA-Seq by Expectation-Maximization (RSEM) normalized Illumina HiSeq RNA expression data for the HNSCC cohort, was downloaded from the Broad Genome Data Analysis Centers Firehose server  utilizing the ggplot2 package [Level 3 RSEM normalized RNA-seq data for y et al. . A pairw package . The finNSD1, NSD2, and NSD3 were converted into a standard score (zscore) and grouped into high, mid, and low expression based on the following criteria: high = z-score > 0.5, mid = − 0.5 < z-score < 0.5, and low = z-score < − 0.5. Five-year overall survival outcomes were compared in both HPV+ and HPV- subsets of patients grouped by either high, mid, or low expression of NSD1, NSD2, or NSD3. Log-rank statistical analysis was performed using GraphPad Prism v7.0 . Furthermore, log-rank p values were assessed for significance after correcting for false discovery rate (FDR) using the Benjamini–Hochberg method with an FDR threshold of 10%. Figures were assembled into final form using Adobe Illustrator . Univariate analysis was performed through RStudio (version 1.2.1335) based on a Cox Proportional-Hazards Model with the survival package (version 2.41–3). Finally, stepwise bidirectional multivariate analysis was performed with the grouped expression of the NSD paralogs and the following clinical variables: sex, age, subsite, T stage, N stage, overall stage, HPV type, and smoking history. The smoking history clinical variable for the HPV+ cohort was stratified as heavy smokers (> 20 pack year history) or non-smokers based on our previous study that employed a similar stratification approach [p values were derived from the Wald test on survival coefficients.RSEM normalized RNA-seq data for each of approach . MoreoveNSD1, NSD2, and NSD3 relative to that of the T-cell infiltration signature were compared in a pairwise fashion and concordance calculated by Spearman’s Rho analysis.To estimate T-cell infiltration, we used a previously generated T-cell signature based on mean expression of 13 transcripts  as utiliNSD1 and its two paralogs NSD2 and NSD3 in HPV+ HNSCC. Previous work has suggested that NSD1 RNA expression can serve as a measure of NSD1 proficiency in HNSCC [Given that NSD1 and NSD2 have been linked to various aspects of HPV- HNSCC, including epigenetic alterations, immune status of the tumor, and predicting patient outcome at specific subsites , 21–26, in HNSCC . As suchNSD paralogs in the HPV+ cohort was due to the constitutive expression of viral oncogenes, we correlated HPV E6 and E7 mRNA expression levels with those of NSD1, NSD2, or NSD3. Our analysis revealed a statistically significant positive Spearman correlation coefficient between NSD2 expression and either E6 or E7 , NSD2 (low vs high expression), and NSD3 (low vs high expression and low vs mid expression) were statistically significant, with NSD2 (low vs mid expression) trending towards significance. Since expression of these genes might not be independent predictors of survival, we analyzed the relationship between survival and our gene expression groups  for NSD1, NSD2, and NSD3 concurrently by multivariate analysis  and NSD3 (low vs high expression and low vs mid expression) remained significant  = 3.23–333.33, p = 0.003; HR = 103.1, 95% CI = 8.55–1.24e03, p = 0.0003; HR = 135.3, 95% CI = 6.62–2.76e03, p = 0.001, respectively), indicating that low expression of NSD1 and NSD3 is a significant, and potentially independent, contributor to overall survival in the HPV+ HNSCC cohort. The final multivariate model also included age , overall stage , and HPV type  as statistically significant contributing factors to survival.To determine the extent that each of the NSD family members could influence patient outcomes in the HPV+ HNSCC cohort, we generated a hazard ratio (HR) for each gene by univariate analysis Table . As expeIn general, the clinical management of HNSCC is complex, often associated with significant treatment induced morbidities and associated with unacceptably low clinical outcomes. HPV-dependent tumors of the oropharynx are a notable exception, as this subset of HNSCC exhibits dramatically better clinical outcomes. As a result, there is an effort to de-intensify treatment for patients with HPV+ disease in an effort to reduce the acute and chronic toxicities associated with the aggressive treatment protocols necessary for treatment of HPV- HNSCC . NeverthNSD1 methyltransferase gene and its paralogs are actively involved in the development of a subset of HNSCC [NSD1, NSD2, and NSD3 compared to their HPV- counterparts and normal control tissues (Fig. E6 and E7 oncogenes. Correlation of the expression of E6 and E7 mRNA with those of NSD1, NSD2, or NSD3 in a pairwise fashion indicated that only NSD2 had a statistically significant correlation with either E6 or E7 (Fig. NSD paralog on a mutually exclusive basis, or if their expression might be coordinately regulated. Our pairwise analysis indicated that tumors expressing high levels of one of these methyltransferases expressed high levels of the other two paralogs (Fig. NSD paralogs does not appear to be a specific consequence of HPV oncogene expression, as it is observed in both HPV+ and HPV- tumors.A variety of evidence indicates that the of HNSCC  and thatof HNSCC , 25. UsiNSD1, or low NSD1 expression leads to an immune “cold” phenotype in HPV- HNSCC [NSD paralogs was also linked to TIL levels in HPV+ samples. Despite confirming a weak correlation between NSD1 RNA expression and TIL signature in HPV- HNSCC (Fig. NSD1, NSD2, or NSD3 in HPV+ HNSCC (Fig. NSD paralogs is not correlated with the infiltrating T-cell component of the tumor microenvironment in HPV+ HNSCC. This illustrates yet another difference between HPV+ and HPV- HNSCC. Interestingly, no significant relationship between NSD2 or NSD3 expression with the TIL signature was detected in the HPV- HNSCC cohort. This was unexpected, given the positive correlation with NSD1, and the observation that NSD paralogs appear to be expressed in a coordinate fashion regardless of HPV status (Fig. One study reported that mutations in V- HNSCC . This waV- HNSCC . As HPV+V- HNSCC , we testSCC Fig. b, and coSCC Fig. , no signSCC Fig. a. Thus, NSD1 and NSD2 were reported to define a subset of stage 3 and 4 laryngeal tumors with favorable prognosis [NSD1 are also present more frequently in heavy smokers and correlated with improved overall survival [NSD paralogs other than NSD1 are relatively infrequent in the HPV+ TCGA HNSCC cohort [NSD1, NSD2, or NSD3 RNA expression was related to overall survival (Fig. NSD1 transcript expression levels are a reasonable estimate of NSD1 proficiency [NSD1, NSD2, or NSD3 expression in HPV+ samples (Fig. NSD1 and NSD3 analyses were independent predictors of survival based on multivariate analysis, with very large effects on relative risk of death (Table NSD expression predict poor outcome. In stark contrast, damaging mutations in NSD1 and NSD2 predict a favourable prognosis in HPV- HNSCC. Thus, NSDs could potentially serve as prognostic tools in completely different ways in different HNSCC types. Importantly, despite the obvious potential for using NSD gene expression levels as a tool to identify those HPV+ patients with poor prognosis, NSD levels could also be used as a guide to select those patients that could benefit from treatment de-intensification. For example, those patients in the upper two-thirds of NSD expression exhibit excellent outcomes that may allow less aggressive treatment, while those in the lower third exhibit poor outcomes that may warrant more aggressive treatment.Damaging mutations in rognosis . In addisurvival . As mutaC cohort , we instval Fig. . A previficiency . Therefoles Fig. a. These NSD paralogs all function as H3K36 methyltransferases, it would seem likely that those HPV+ tumors expressing high NSD levels might exhibit methylation and/or gene expression profiles that differ from those expressing low NSD levels. However, intensive efforts based on hierarchical clustering did not identify HPV+ subsets with different methylomes that predict clinical outcomes [NSD2 knockout impaired the ability of type-I interferon to induce expression of an antiviral gene response [NSD levels in HPV+ HNSCC are not correlated with an increased TIL score, suggesting that any potential effects on innate immunity do not correlate with increased infiltration of these already highly infiltrated tumors.Given that the outcomes . Intriguoutcomes , 36 and outcomes . As bothresponse . HoweverNSD members predicts improved clinical outcome in HPV- HNSCC, low level expression of these genes is a very strong predictor of poor outcome in HPV+ HNSCC. Thus, further investigation of the expression levels of NSD1, NSD2, and NSD3 is warranted, as these could serve as rapidly exploitable prognostic biomarkers in HPV+ HNSCC, providing a metric for appropriate treatment de-intensification.Taken together, this study provides strong evidence that the NSD methyltransferases play opposite roles within HPV+ and HPV- HNSCC. Most importantly and paradoxically, while mutation/loss of function of Additional file 1.


Cutaneous squamous cell carcinoma (cSCC) is a common form of skin cancer with a complex but not fully understood pathogenesis. Recent research suggests the role of beta human papillomavirus (HPV) types and HPV-associated inflammatory processes in cSCC development. Beta HPV types are components of the normal flora; however, under the influence of certain cofactors, the virus may trigger a malignant process. Dysregulation of the immune system (chronic inflammation and immunosuppression), environmental factors (ultraviolet radiation), and genetic factors are the most important cofactors involved in beta HPV-related carcinogenesis. In addition, the oncoproteins E6 and E7 of beta HPV types differ biochemically from their counterparts in the structure of alpha HPV types, resulting in different mechanisms of action in carcinogenesis. The aim of our manuscript is to present an updated point of view on the involvement of beta HPV types in cSCC pathogenesis. Cutaneous squamous cell carcinoma (cSCC) is the second most common nonmelanoma skin cancer (NMSC) after basal cell carcinoma (BCC), originating from the keratinocytes located in the epidermis or adnexal structures. The tumour is mainly diagnosed in middle-aged and older adults, the young individuals being less frequently affected. In some cases, cSCC exhibits an aggressive behaviour with a significant risk of metastasis , 2. Its Chronic exposure to radiations, arsenic, or polycyclic hydrocarbons is also associated with the risk of developing cSCC . ImmunosIn this review, we summarize data on the incidence of beta HPV types among patients with cSCC and discuss the main mechanisms possibly involved in beta HPV-related skin carcinogenesis emphasizing the role of chronic inflammation.HPV genome comprises eight genes which are expressed in the early  and late (L1 and L2) stages of the viral cycle . HPV earHPV penetrates into the epidermis through microlesions. The replication cycle of the virus depends on the differentiation process of keratinocytes. The infection starts at the basal layer of the epidermis. HPV enters the cells through endocytosis, and the cell penetration facilitated by the viral proteins L1 and L2 is mediated by various components such as heparan sulfate, proteoglycans, and annexin A2; however, the cell receptor remains unknown , 12, 13.Later, the infected cells leave the basal layer and reach the upper layers of the epidermis. The viral genes are overexpressed, especially E6 and E7; and the number of copies per cell exceeds 1000. The expression of E6 and E7 genes is controlled by E2 . High-riPapillomaviridae family [HPV is a nonenveloped virus that belongs to the e family . HPV is e family .So far, over 200 types have been described and classified into 5 genera: alpha, beta, gamma, mu, and nu. In turn, each genus comprises several species and each species includes certain types. Thus, alpha papillomaviruses include species with cutaneous tropism  and with tropism for mucous membranes . In fact, the vast majority of alpha papillomaviruses exhibit mucosal tropism. The beta genus comprises 5 species with cutaneous tropism including HPV 5 and 8 also known as epidermodysplasia verruciformis (EV) types, being classically reported in patients with EV. The genera gamma, mu, and nu exhibit skin tropism and cause warts –22. ThusRecent research on skin cancer has suggested that beta HPV types may also be involved in carcinogenesis, in the emergence of NMSC . The haiCurrent studies that attempt to confirm or disprove the association between beta HPV types and cSCC use various methods such as the detection of viral DNA in the skin, in eyebrow hair follicles, or in tumour tissue as well as the detection of circulating antibodies against some viral antigens .Individuals are often infected with many beta HPV types. The presence of HPV DNA may represent a latent infection but the detection of antibodies denotes an active infection in the past which elicited an immune response resulting in the synthesis of antibodies . It seemProby et al.  have shoTo determine the role of HPV in the emergence of cSCC, Arron et al.  evaluateThe viral load is higher in actinic keratoses than in cSCC, which supports the hypothesis that beta HPV types play a role in the initiation of the carcinogenesis but not in maintaining it . Some auData on beta HPV seroreactivity are scarce; however, antibody detection is considered a marker of an infection with increased viral load . SeveralSerological tests have revealed an increased incidence of antibodies against L1 major capsid protein of beta HPV types in the general population. Thus, in the German population an incidence of 52% was reported, and in the Italian population of 67%. However, beta HPV DNA has been identified in over 90% of individuals. Low viral load and high keratinocyte turnover may explain the lower proportion of seroconversion . The stuThe meta-analysis by Bzhalava et al.  has reveIn the study by Farzan et al.  1.408 paHowever, there are studies that have shown no differences concerning HPV seropositivity between patients with cSCC and controls . PlasmeiIn cervical cancer, HPV infection is persistent and the viral genome is detected in all tumour cells. After the initiation of the malignant process, E6 and E7 continue to play an essential role in the propagation of the carcinogenic process . In contLike its counterpart in the structure of alpha HPV types, beta HPV E6 has the ability to bind to Bak, p300, as well as to cellular histone acetyltransferase. Beta HPV E6 does not bind to PDZ domains of proteins, having a different structure compared to alpha HPV E6, resulting in a lower degree of invasiveness. PDZ containing proteins are involved in cell cycle regulation. The main difference is that beta HPV E6 protein does not have the ability to degrade p53, an ability possessed by alpha HPV types, via the ubiquitin pathway. Beta HPV types does not bind to ubiquitin ligase E6AP, which is required for the degradation of p53. However, E6 proteins of HPV 5 and 8 (beta-1), most commonly incriminated in cSCC, participate in p300 degradation, blocking its phosphorylation by AKT, which leads to a decreased ATR protein level, resulting in p53 delayed accumulation in the cell and persistence of thymine dimmers, and consequently the cell ability to defend against UVB declines –58. It hOn the other hand, the interference of E6 with the Notch signaling pathway was highlighted . Beta HPE7 of beta HPV types binds retinoblastoma protein (pRb) and modulates the cell cycle by inducing a quiescent state during G0 and G1 phases . The stuIt has been observed that E7 is able to induce the proliferation of stem cells-like keratinocytes . In addiα3 chain expression, which may contribute to malignant transformation [In an animal model, it has been found that HPV 8-E7 induces a decreased E cadherin expression and an increased N cadherin expression, which may promote epithelial-mesenchymal transition. In addition, E7-positive keratinocytes showed increased fibronectin and integrin ormation .The aforementioned studies have revealed a high incidence of beta HPV types in healthy skin. In this context, it has been speculated that beta HPV types need cofactors to induce a carcinogenic process . The rolIt is well known that SCC occurs in areas characterized by chronic inflammation such as scarring or burns . InflammThe innate immune response of the host plays an important role in the evolution of HPV infection, influencing the duration and regression of the lesions. During HPV infection, keratinocytes, fibroblasts, and inflammatory cells  release an important amount of proinflammatory mediators. In some cases, HPV escape the host's defence mechanisms resulting in persistent infections. It should be noted that persistent infections are closely related to chronic inflammation, an important cofactor in carcinogenesis . Tumour β pathways results in the release of chemokines and the attraction of immune cells. HPV induces CCL2 synthesis in monocytes, with a chemotactic effect on myelomonocytes. Myelomonocytes will release MMP-9, which will promote the inflammatory response [In the early stages of HPV infection, antiviral immunity attempts to eliminate the virus, but HPV has developed mechanisms that allow its persistence in the cells, by inhibiting acute inflammation and preventing immune recognition. In the late stages, the infected cells modify the tumour microenvironment by promoting a chronic inflammatory process. IL-6 represents an important player in this stage . The actresponse .κB in keratinocytes, which allows their survival in a cytokine-dominated microenvironment [It has been established that alpha HPV types are associated with chronic inflammation in cervical cancer . Recent ironment .It has been shown that SCC may present a population of cells called myeloid-derived suppressor cells that are involved in the suppression of the immune response, their presence being associated with an unfavorable prognosis. Studies have revealed that HPV can stimulate these cells. Moreover, HPV seems to induce alterations in NK cells that participate in chronic latent viral infections. Certain subpopulations of NK cells exhibit immunological memory and contribute to the antitumour defence .The prevalence of HPV infection is higher in immunosuppressed individuals than in the general population, and they have an increased risk of developing HPV-associated tumours, characterized by an aggressive behavior and a rapid growth . IncreasκB pathway is one of the mechanisms by which HPV participates in carcinogenesis in immunosuppressed patients [Clinical and histopathological analysis showed that in the case of transplantation patients most of the cSCCs arise on common warts and are located especially on the sun-exposed areas . The immpatients . In the patients .The study by Weissenborn et al.  analysedκB. and inducible nitric oxide synthase (iNOS) were identified [Exposure to UV produces changes in the function of keratinocytes and immune cells that will release proinflammatory cytokines and promote processes such as cell proliferation and angiogenesis . It seementified .However, Akgul et al.  demonstrTomlins and Storey  have shoHowever, Guerrini et al.  using HaIt has been postulated that certain genetic variations may increase the susceptibility to develop HPV-mediated skin cancers. It seems that genetic variations of p53 may act as a risk factor .The association between beta HPV infection and cSCC is proven in the case of EV, these patients having an increased susceptibility to the infection with HPV . EV is aThe mechanisms that are involved in persistent HPV 8 infections and participate in the development of neoplasms are not completely elucidated. Recent research has focused on the role of chronic inflammation. Starting from the idea that S100A8 and S100A9 proteins promote chronic inflammation and carcinogenesis in skin neoplasms, Podgorska et al.  have stuThe presence of beta HPV DNA in cSCC samples and the detection of antibodies against HPV in patients with cSCC are arguments in favor of the participation of beta HPV types in cSCC pathogenesis. Beta HPV infection seems to play an important role in initiating carcinogenesis, but not in tumour progression. This hypothesis is supported by the identification of an increased viral load in premalignant lesions such as actinic keratoses compared to cSCC. However, the high incidence of beta HPV types in healthy skin denotes that some cofactors contribute to its pathogenesis. We consider that chronic inflammation is a key player in beta HPV-related carcinogenesis; therefore, a deeper research is needed to understand how the inflammatory pathways are involved in the malignant process in order to counteract their activation. The data available in the literature on this topic suggest that beta HPV types should not be neglected in the onset of cSCC.


The causative agent of cervical cancer referred to as Human papillomavirus (HPV) remains a real public health problem. Many countries in West Africa, such as Togo have no data on the high-risk HPV (HR-HPV) infection and genotypes distribution. In order to fill the knowledge gap in the field in Togo, the main objective of this study was to determine the prevalence of pre-cancerous lesions of the cervix and HR-HPV genotypes among Togolese women.Samples were collected from 240 women by introducing a swab in the cervix. Then, the screening of precancerous cervical lesions using the visual inspection with acetic acid and lugol (VIA / VIL) was conducted. The HR-HPV genotypes were characterised by real-time multiplex PCR.Out of 240 women recruited, 128 (53.3%) were infected by HR-HPV. The most common genotypes were HPV 56 (22.7%), followed by HPV 51 (20.3%), HPV 31 (19.5%), HPV 52 (18.8%) and HPV 35 (17.2%). The least common genotypes were HPV 33 (2.3%) and HPV 16 (2.3%). Among the women, 1.3% (3/240) were positive to VIA/VIL.This study allowed HR-HPV genotypes to be characterised for the first time in Lomé, Togo. This will help in mapping the HR-HPV genotypes in West Africa.The online version contains supplementary material available at 10.1186/s12879-021-05956-5. Infection by papillomavirus is considered as a sexually transmitted disease (STD) affecting the general population . Accordi2.This transversal study is both descriptive and analytical and was conducted between December 2016 and March 2017 in the maritime region of Lomé, the economic capital city of Togo. This city is located in the south of the country, covering an area of 6100 kmClinique Victoire, Centre Médical de Santé Source de Vie, Clinique Autel d’Elie and Centre de Santé Saint Camille. From this population, a random sample of 240 sexually active women was taken to collect endocervical swab samples. Each woman responded to a questionnaire  and Burkina Faso (CERS). All patients were asked to give their written consent before sampling.The gynaecologist placed the patients in the lithotomy position and examined their external genitalia. Then, he inserted a sterile speculum into the vagina and introduced a cotton swab into the endocervical canal. With three counter clockwise movements, he collected cells from the squamocolumnar junction (SCJ). The swab sample was introduced in a sterile Eppendorf tube with a transport medium provided with the DNA-Sorb-A kit . All swabs samples were then collected and preserved at − 20 °C in one of the 4 health centres before being transported to Ouagadougou (Burkina Faso) at the Pietro Annigoni Biomolecular Research Centre (CERBA) for molecular analysis.Following cervical swabs samples collection for HR-HPV detection, the screening of cervical precancerous lesions or dysplasia was performed by the gynaecologist using visual inspection of the cervix with acetic acid (VIA) and then with lugol (VIL). VIA was conducted by applying a 4% acetic acid solution to the cervix. The test was considered positive if distinct, well-defined, and dense acetowhite areas were seen or as negative if there were no acetowhite lesions. To conduct VIL, the gynaecologist delicately applied 10% lugol solution to the cervix. Clinically, the test was considered as “negative” for dysplasia when no unstained lesion area was observed, or as “positive” when an unstained lesion area is observed..Genomic DNA was isolated using the DNA-Sorb-A kit  in compliance with the instruction manualDNA was amplified by multiplex RT-PCR using the « HPV Genotypes 14 Real-TM Quant » V67–100 FRT kit of SACACE biotechnologies®, Italia following the protocol provided by the manufacturer. The kit allowed us to detect 14 genotypes of HR-HPV: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68.Pre-PCR steps consisted in preparing the Mix containing the DNA polymerase, primers, and deoxy ribonucleotide triphosphates. We introduced into each tube, 15 μL of the Mix solution and 10 μL of the DNA extract to obtain a volume of 25 μL. This mixture is placed on a SaCycler-96 Real Time PCR v.7.3  platform for amplification.The amplification schedule was as follows: one (1) cycle at 95 °C for 15 min; five (5) cycles at 95 °C for 5 s, followed by incubation at 60 °C for 20 s and at 72 °C for 15 s; and finally 40 cycles at 95 °C for 5 s followed by 60 °C for 30 s and 72 °C for 15 s.Based on the answers given by women to the questionnaire, we analysed the relation between some factors and the carrying of HPV infection. These were: age, age at first intercourse, number of sexual partners and the result of VIA/VIL.p < 0.05.The data were processed and analysed using SPSS software version 21.0, the Microsoft EXCEL 2010 software and Epi Info software version 6. Chi-square was used to compare proportions and averages. The difference was statistically significant for This study was conducted on 240 endocervical samples from sexually active women. We used the results of the questionnaire to analyse the socio-demographic, sexual, and behavioural characterisation of the women. The age of the women varied from 17 to 67 years with an average of 37.2 ± 12.7 years. In 39.6 and 35% of the cases, the women had secondary and higher education level respectively. The others (6.3%) were illiterate or had a primary education level (19.2%). The unemployed women and housewives represented respectively 49.6, and 10.8%. Only 21.2% women were salaried, 2.1% were retired and 16.3% were students. Married women represented 48.8%. The age at which occurred the first sexual relation varied between 8 and 30 years. More than half of women in this study did not use condoms during sexual intercourse (59.2%). Those having at least one sexual intercourse a week represented 80.76%. Table Screening by VIA/VIL conducted on all the 240 women allowed the detection of 1.3% (3/240) dysplastic lesions  of women were infected by high-risk HPV. The kit used in the study allowed the characterisation of 14 genotypes of HR-HPV: HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68. The most common genotypes found in women were HPV 56 (22.7%) and HPV 51 (20.3%), followed by HPV 31 (19.5%), HPV 52 (18.8%) and HPV 35 (17.2%). Genotypes 33 and 16 with the same prevalence at 2.3% were the less common. HPV 16 and 18, the target of the vaccines currently available represented together 16.4% of cases. Figure 28/240 ofThe number of HR-HPV genotypes per women ranged from 1 to 5. Considering multiple infections, we observed a total of 204 genotypes in 128 women infected by high-risk HPV. Among the 128 women infected with HR-HPV, 56.0% (72/128) were infected by only one type of HPV. There were 38 cases (30%) of multiple infections representing 2 genotypes, 12 cases (9%) for 3 genotypes, 5 cases (4.0%) for 4 genotypes and 1 case (1%) for 5 genotypes.The analysis of HPV infection frequency according to women’s sexual behaviour revealed that women with two or more sexual partners were all HPV-positive Table . Those wThe current study on the distribution of high-risk HPV genotypes in the general population of women was conducted in Lomé (Togo). This kind of study on HPV in this city is, to the best of our knowledge, the first one. The objective of this study was to have a precise estimate of the prevalence of high-risk HPV among women in Lomé and identify the most common genotypes in the city in order to create a representative regional map.In this study, the reported age was between 17 and 61 years with an average of 34.67 ± 1.2 years. This population is similar to the study population of Traore et al.  in Bobo-We found a percentage of 52.2% of women which do not use condoms. This can be explained by the fact that most of the women in this study had a married life and therefore used other methods of contraception. The level of knowledge about cervical cancer was low. This could be explained by the relatively low level of education amongst these women and by the lack of information concerning cervical cancer and its high-risk factors.The prevalence of cervical dysplasia screened by VIA/VIL was 1.3% (3/240). This prevalence is lower than that obtained in Bobo-Dioulasso in Burkina Faso (3.9%) .We found a percentage of 53.3% of women infected with at least one high-risk HPV genotype. This result is greater than that obtained by Zohoncon et al. in 2013  who founThe high prevalence of HPV in the current study can be explained by three main reasons: First, the type of population recruited . Secondly, the relatively low level of education of these women and the lack of information concerning cervical cancer and its high-risk factors. Thirdly, the low rate of screening of cervical cancer in Togo since it has been demonstrated that the participation in screening for cervical cancer is associated with a low infection rate of HPV .Furthermore, Singh et al. in 2012 , demonstSome authors reported a link between the number of sexual partners and HPV infection . In the The most frequently encountered genotypes were HPV 56, followed by HPV 51 and 31. HPV 16 and 18, targets of the currently available vaccines, were found respectively at 2.3 and 14.01% of cases.. In Thailand, the genotypes found were HPV 72 and 52 [However, our results are similar to those found in several other studies, particularly in West Africa. Indeed, in the study of Traore et al. in Bobo-Dioulasso, Burkina Faso , the mos2 and 52  while in2 and 52 . All theHowever, our results are different from the literature that suggests that HPV genotypes 16 and 18 are the most frequent in the world. Indeed, several authors noticed that, in women with a normal cytology and women with an abnormal cytology  or womenThis work allowed us to collect data on the epidemiology and distribution of high-risk HPV genotypes in Lomé. The most frequent genotypes were HPV 56 (22.7%) and HPV 51 (20.3%), followed by HPV 31 (19.5%), HPV 52 (18.8%), and HPV 35 (172%). The five first most common genotypes belong to HPV 30′S and HPV 50′S families. The prognosis of cervical cancer in developing countries is often bad due to its late diagnosis and limited therapy options.It would be necessary to focus on preventive actions such as information, education and sensitization targeting the population of these countries on HPV infection. Screening of pre-cancerous cervical lesions must be performed on all women while research for new polyvalent vaccines targeting the most frequent genotypes in Africa must continue.Additional file 1.


Cancer burden in sub-Saharan Africa is on the rise with one-third of cancers estimated to be caused by infectious agents. Head and neck squamous cell cancer (HNSCC) is the sixth most common malignancy in sub-Saharan Africa and includes tumors in the Upper Aero-digestive Tract (UADT). The established risk factors are tobacco and alcohol exposure with a recent recognition of the role of Human Papilloma Virus (HPV). The HPV related HNC is seen predominantly in the oropharynx, presents at a younger age and has a better prognosis. With a rapidly increasing incidence of these cancers in the developed world, it was important to study HPV in HNC in Uganda. The HPV can be detected using P16 immunohistochemistry as a surrogate marker thus making it suitable for screening. The study aimed at establishing the presence of HPV and the commonly affected sites in UADT squamous cell carcinoma (SCC) at Uganda Cancer Institute (UCI) using P16 immunohistochemistry.This was a cross sectional study in which 59 patients with histologically proven SCC from the oral cavity, oropharynx, larynx and hypopharynx were recruited. These patients’ demographics and clinical data were collected. Tissue sections from retrieved histology samples were stained by Haematoxylin and Eosin to reconfirm SCC. Subsequently, P16 expression was determined using P16 immunohistochemistry.Seventy-one patients were enrolled and 59 patients with confirmed SCC of the sites of interest were analyzed. The majority (79.7%) of the participants were male and over 50 years. 59.3% were tobacco smokers, 66.1% used alcohol, 52.2% used both. Only 27.1% used none of the substances. Only 27.1% of the participants were HIV positive. Most of the tumors were in the larynx (37.3%) and 64.4% were overall TNM stage 4. The overall prevalence of HPV in UADT SCC at UCI was 20.3, 95%CI 10.9–32.8. The oropharynx had the highest prevalence (30.8%).The prevalence of HPV in UADT SCC at UCI is significant at 20.3%. The most affected site, is the oropharynx. Vigilant HPV screening of these sites with confirmation where possible is recommended. Head and neck carcinoma (HNC) ranks sixth among the most common cancers seen worldwide . The comThe etiology of head and neck squamous cell carcinomas (HNSCC) has mainly been attributed to tobacco and alcohol consumption . HoweverHigh risk HPV causes dysregulation of the cell cycle at the molecular level. The HPV produces onco-proteins that affect three tumor suppressor genes  in the host cell. When the HPV onco-protein E7 binds to the retinoblastoma gene in the host cell, it consequently releases the inhibition of P16 gene. This in turn causes an increased expression of p16 protein which can be detected by immunohistochemistry. P16 is currently used as a surrogate marker for HPV , 13. PriThis study was approved by Makerere University’s School of Medicine Research and Ethics Committee and that of Uganda Cancer Institute. This was a cross sectional study conducted from October 2018 to May 2019.The study was conducted at Uganda Cancer Institute (UCI), a public, specialised tertiary care center for cancer treatment, research and training. It is located in the urban centre of Uganda’s Capital City Kampala. It serves the 42 million Ugandan population in addition to referrals from neighbouring countries like South Sudan and DRC. Approximately 200 cancer patients are seen daily with an average of 8 new HN cases. 400 patients with HNC per year are seen on referral basis with their histologically confirmed biopsies  for staging and treatment planning in a tumor board setting.For this study, upper aero-digestive tract (UADT) SCC comprised of the oral cavity, oropharynx, hypopharynx and larynx only. We recruited all patients that consented to participating in the study. On the other hand, patients with history of prior radiotherapy, those whose tissue blocks could not be accessed from the pathology labs and those whose retrieved blocks showed no malignancy or had insufficient tissue for histological analysis were excluded.We captured age, gender, education level, occupation, socioeconomic status, history of smoking and alcohol ingestion, HIV status (documented evidence), sexual history and the Tumor, Node, Metastasis (TNM) stage of the patient (American Joint Committee on Cancer AJCC 7th edition). TNM staging was captured after it was agreed upon in the HN tumor board. Our dependant variable was the HPV status. For purposes of this study, it was assumed that a p16 positive stain meant HPV positive and negative staining for p16 expression meant HPV negative..We used the Kish Leslie (1965) formula for sample size estimation [using the study in Sudan, , adjusteDuring the study period, patients with result slips showing histologically confirmed UADT SCC were interviewed. Their tissue blocks were retrieved from accessible pathology labs. The tissue blocks were then collected at the UCI pathology laboratory for histological re-confirmation followed by p16 IHC.Retrieved Formalin Fixed Paraffin Embedded tissues were trimmed into 3–5 μm’ thickness and prepared serially for routine H&E staining method by use of standard operating procedure (SOP)  Table . The medMost of the participants used tobacco 59.3%), 66.1% used alcohol and 52.5% used both tobacco and alcohol. The HIV positive participants constituted 27.1%  followed by the oral cavity as shown in Fig. The overall prevalence of HPV in UADT SCC at UCI using P16 IHC was 20.3, 95% Confidence interval (CI) of 10.9–32.8.The site specific prevalence of HPV in UADT at UCI; the oropharynx had the highest prevalence (30.8%), followed by the oral cavity 29.4%) as summarized in the Table 9.4% as sThe overall prevalence of HPV in UADT SCC using p16 IHC in this study was significant at 20.3%, lower than the global incidence of 25% but similar to some studies in Sub Saharan Africa (SSA) , 15, 16.The commonest detection methods for HPV in HNC worldwide are PCR and ISH  However,The sites of interest in our study were the oral cavity, oropharynx, larynx and hypopharynx. Several studies from SSA often combine other sites from the head and neck like the esophagus, paranasal sinuses, nasopharynx and even tumours of unknown origin. In addition, the distinction between the oral cavity and oropharynx is seldom made and the pharyngeal subsites are not grouped into naso/oro or hypopharyngeal sites –16. ThisThe oropharynx(OP) had the highest HPV prevalence (30.8%) compared to the non- oropharyngeal (NOP) sites in agreement with most studies , 7, 9. IIn SSA, Ahmed et al. made no distinction of oropharynx or hypopharynx . FaggonsIn the developed world, higher proportions of 39–56% are seen compared to our study , 22–24. The prevalence of HPV in the oral cavity (29.4%) in our study was slightly higher than that of Ahmed et al. 22%) . This si% . This Our study shows a lower proportion of HPV in laryngeal carcinoma (9.1%) compared to other studies that used P16 IHC. Hernandez et al. in their laryngeal cancer-focused study found a 12.5% HPV positivity . Ahmed eThe prevalence of HPV in hypopharyngeal carcinoma (14.3%) in our study was lower than that found by Yang et al. (26.1%) in China that similarly used P16 IHC . This hiWe secondarily compared HPV status in the UADT SCC with gender, age, substance use and sexual habits and did not find any statistical significance.All our participants were black with a male predominance  Several studies similarly found the majority of patients with HPV related cancer to be male , 22. TheOf the HPV positive group, 41% used both alcohol and tobacco and the same percentage did not use any of the mentioned substances. In contrast, in the HPV negative group, 23% neither smoked nor took alcohol while 55% used both alcohol and tobacco. These findings suggest a higher chance of HPV positivity in non- substance users as shown in Herero’s study .In our study, participants under 40 years were all HPV negative, with majority being HIV negative non- substance users. This would suggest another pathway that may not be viral, probably genetic or dietary and poses an opportunity for further research in our setting.Concerning sexual behavior, the trends for lifetime sexual partners and oral sexual habits in both groups were similar. Oral sexual practices were low and 18% declined to answer questions on oral sexual habits. This was not surprising as it is generally considered a taboo to talk about or disclose sexual practices in most of our cultural settings.This study was done at Uganda Cancer Institute which receives patient referrals from across the country so the findings of this study may be generalizable to Uganda. We were able to get histology samples within a year of diagnosis increasing our chances of viability in the tissues and the slides were read by two consultant pathologists further strengthening the reliability of the results.P16 is a surrogate marker and this limits the findings of our study since we were unable to confirm them by PCR or ISH. Furthermore, it was beyond the scope of this study to infer correlation between P16 status (denoting HPV) and age, gender, substance use and sexual habits.The prevalence of HPV using P16 IHC in UADT SCC at UCI was significant at 20.3%. The commonest site affected by HPV was the oropharynx closely followed by the oral cavity.Particular attention with routine screening of patients with SCC involving the oropharynx/ oral cavity should be taken. We recommend studies using ISH and PCR to confirm our findings with P16 IHC.Additional file 1. Data Collection Form.Additional file 2. Standard Harris’ Haematoxylin and Eosin stain for Paraffin sections .Additional file 3. P16 Staining.Additional file 4. Scoring of P16 gene expression immuno-staining.Additional file 5. AMERICAN JOINT COMMITTEE ON CANCER (AJCC).


Since the first days of organ transplantation, it has been accepted that solid transplant recipients have a high risk of developing cancer. Chronic immunosuppression and environmental factors play a role in cancer development in recipients. In the present study, we tried to evaluate the cumulative incidence of cervical dysplasia after renal transplantation, risk factors for disease development, and the time until high-grade dysplasia occurred.A total of 50 patients with renal transplantation who presented for gynecologic follow-up was included in the study. The medical records of the patients were reviewed until the last clinical visit, their demographic characteristics, transplant history, gynecologic history, and gynecologic examination results  were reviewed.Of the 50 women in the study population, 29  developed cervical dysplasia after the first transplant at a median follow-up of 7.8 (range: 4.6-12.9) years. Twenty-one women with benign cervical cytology before transplantation had evidence of low-grade intraepithelial lesions + after transplant (47% of these were within 2 years after transplant). During the follow-up, 8 women (18.2%) were diagnosed as having high-grade intraepithelial lesions + (within 5 years after transplantation).Renal transplant patients were found to have higher abnormal cervical cytology and histology rates than the normal population. PRECIS: Incidence of cervical dysplasia increased in patients with renal transplant.de novo) malignancy that develops after renal transplant is seen as an unfortunate complication of a successful surgery. The development of such malignancies may be caused by various factors such as individual and regional susceptibility, pre-transplant disease states, viral status of the recipient, and high doses of various immunosuppressive drugs to protect the graft. Lymphoproliferative disease, skin cancer, Kaposi’s sarcoma, and cervical dysplasia have been reported with high incidence in these patient groups after transplantation. The persistence of human papillomavirus (HPV) is the most important factor in the development of dysplasia. Therefore, if immunologic control of the virus is interrupted, this adverse event poses a higher risk to patients. Immunocompromised women are at high risk for a variety of premalignant and malignant conditions, including cervical dysplasia. There are many factors in the development of cervical malignant lesions but immunosuppression on high-risk HPV clearance is the most important,3. The long-term consequences of the suppressed immune system are becoming increasingly important for improving life expectancy and quality. Cervical cancer screening guidelines are constantly updated due to the increase in knowledge about the etiopathogenesis of the disease; however, the ideal method and frequency for cervical cancer screening after organ transplantation is still uncertain. Therefore, in this study, we aimed to evaluate the abnormal findings resulting from cervical cancer screening and histology in women with renal transplantation. We tried to evaluate the cumulative incidence of cervical dysplasia after renal transplantation, to determine risk factors for disease development, and to evaluate the time to high-grade dysplasia. In addition, we aimed to determine whether the risk of abnormal cervical testing approached the general population risk. The point we wanted to draw attention to in our study was to emphasize the factors that might be cofactors of cervical dysplasia and to stress the importance of multidisciplinary evaluation of patients with organ transplants who are difficult to follow-up.Primary . Patients with renal transplant who presented for gynecologic follow-up were determined from our hospital’s database. Written informed consent was obtained from participants to use their medical records for research. As the inclusion criteria, it was decided to have documented gynecologic follow-up for at least 1 year after renal transplant and to have at least one cervical pathology sample before or after transplant. Patients who had a hysterectomy prior to transplantation or within 1 year were excluded. After applying the inclusion and exclusion criteria, a total of 50 patients were included in the study. The medical records of the patients were reviewed until the last clinical visit, and demographic characteristics, transplant history, gynecologic history and results of gynecologic exams  were reviewed.We created three categories according to the cytologic and histologic features:1. Cervicitis, inflammation, atypical squamous cells of undetermined significance (similar lesions were classified as benign)2. Low-grade intraepithelial lesions (LSIL), cervical intraepithelial neoplasia (CIN) I were classified as LSIL+3. High-grade intraepithelial lesions (HSIL), CIN II, and CIN III were classified as HSIL+Descriptive statistics were reported as means and standard deviations (SD), and interquartile medians, frequencies, and percentages as indicated. The cumulative incidence of cervical dysplasia in the general population was calculated based on the gold standard tissue diagnosis. Findings of abnormal screening tests  have also been reported. Patient demographics such as age, transplant age, body mass index (BMI) (weight in kilograms divided by height in square meters), parity, immunosuppression agent, and dialysis type and time status are tabulated.Data are presented as the number of observations , mean ± SD, range. The results of homogeneity (Levene’s test) and normality (Shapiro-Wilk test) were used to decide the statistical methods for comparing the study groups. Among normally distributed groups with homogeneous variances, dependent groups were compared using Student’s t-test. According to the test results, parametric test assumptions were not available for some variables; therefore, the independent groups were compared using the Mann-Whitney U test. Categorical data were analyzed using Fisher’s Exact test and the chi-square test. In cases in which the expected counts for inclusion were not met in less than 20% of the cells, Monte Carlo simulation was used and the values were determined. Logistic regression analysis was performed to determine whether cervical dysplasia was positive or negative. Statistical analyses were performed using the IBM SPSS Statistics for Windows Version 25.0 software package . P-values <0.05 were considered statistically significant. Nonparametric cumulative incidence analyses were performed using the Stata 11.0/MP for Linux package . Non-parametric cumulative incidence estimates were produced using the stcompet command, and multivariate comparisons of cumulative incidence functions were completed using stcrreg.A total of 50 women with renal transplantation were evaluated for gynecologic follow-up and cervical screening. Transplantation was performed when the patients were aged 41.6-14.2 years on average, and gynecologic follow-up was started at least 1 year before transplantation. The mean follow-up period was 5.07 (4.98) years in patients with cervical dysplasia and 6.76 (4.72) years in patients without dysplasia. The most common renal diseases were diabetes mellitus (66%), hypertension (14%), and lupus nephritis (10%). For 11 (22%) patients, the diagnosis was other renal diseases. Twenty-one women with benign cervical cytology before transplantation had evidence of LSIL+ after transplant (47% of these were within 2 years after transplant). During the follow-up, eight women (18.2%) were diagnosed as having HSIL+ (within 5 years after transplantation). Factors associated with an increased risk of developing cervical dysplasia in univariate and multivariate analysis were: age , time after transplant , the use of cyclosporine-A (OR=1.381 95% CI: 0.554-2.336), the use of tacrolimus (OR=1.731 95% CI: 0.224-2.382), the use of azathioprine , the use of mycophenolate mofetil , hemodialysis , peritoneal dialysis , and hemodialysis + peritoneal dialysis . Among the factors that reduce the risk of the development of cervical dysplasia were the following parameters: age at transplantation , years of follow-up , current smoker or quit within the past 1 year , and BMI . . The risk of malignancy is estimated as 20% after 10 years of chronic immunosuppression. Possible mechanisms for malignancy development include replication of oncogenic viruses , immunity disorders , and direct carcinogenic effects of immunosuppressives. The most important risk for cervical cancer is infection with high-risk HPV types. The most common sexually transmitted disease seen in the general population and patients with renal transplantation is HPV infection. Patients with renal transplants have a higher rate of permanent disease and disease burden compared with the general population. It was reported that the risk of developing cervical neoplasia  was 14 times higher in female patients who had renal and liver transplantation compared with controls. Chapman and Webster reported that 46 of 13,077 patients (6.6%) who had a renal transplant were diagnosed as having cervical cancer. It has been reported that the incidence of primary (de novo) malignancy after organ transplantation is between 2.92% and 3.36% in renal transplant recipients in Turkey. Haberal et al. reported that the incidence of malignancy among renal transplant recipients was 3.7%, a 47-year-old female patient was diagnosed as having cervical cancer 4 months after renal transplant, and the incidence of gynecologic malignancy was 2% among renal transplant recipients. Akgun et al. reported that of 347 renal and 24 liver transplants performed in Organ Transplantation Centers, malignancy developed in 15 renal transplant patients (3.36%) and one liver transplant patient (3.84%) during 13 years of follow-up, and one in situ cervical carcinoma developed 4 months after renal transplantation. It has been reported that the HPV prevalence can be as low as 5% and as high as 63% in studies of female kidney transplant patients. The risk of persistent infection with HPV type 16-18 genotypes is higher in immunocompromised patients than in the general population. Similarly, in another study conducted in patients with kidney transplantation, although the incidence of HPV-related malignancy was found to be increased after transplantation, the same increase was not observed in patients who developed end-stage renal failure but who are not currently transplanted. These findings support the role of immunosuppressive agents in increasing the risk of HPV-related diseases in patients with renal transplantation.After the spread of organ transplantation worldwide, lymphoid and non-lymphoid tissue malignancies, especially skin cancers, have started to be seen in organ transplant recipients with a high incidence. Organ transplant recipients are at a 3- to 4-fold risk of malignancy due to chronic immunosuppression. However, compared with the general population, the relative risk for certain cancers increases 100-foldIn our clinic, a total of 50 female patients underwent renal transplantation between 2016-2017 and received immunosuppressive therapy. Of the 50 women in the study population, 29  developed cervical dysplasia after the first transplant at a median follow-up of 7.8 years. Twenty-one women with benign cervical cytology before transplantation had evidence of LSIL + after transplant (47% of these were within 2 years of transplantation). During the follow-up, eight women (18.2%) were diagnosed as HSIL + (within 5 years after transplantation). Cervical cancer was not detected in any patients in our study. reported that gynecologic malignancies  in women who underwent organ transplantation were 1.9 times less frequent than in the normal population and concluded that this was due to the active mammographic and gynecologic examination policy before and after liver transplantation. In a study conducted among 1,778 patients who underwent organ transplantation in the United Kingdom, it was reported that cervical cancer was detected in one of 78 women who developed primary (de novo) non-lymphoid tissue malignancy, the expected incidence of cancer in terms of cervical cancer was 0.79%, and no increased risk of cervical and breast cancer was observed. However, the role and extent of immune dysfunction in the development of cervical dysplasia are not clear in this patient population. In a study evaluating the incidence of cervical cancer after transplantation, a similar incidence was found in groups with and without systemic lupus erythematosus (SLE); however, specific immunosuppressive drugs or the severity of SLE have not been evaluated.There are also studies reporting that there is no increase in the risk of developing gynecologic malignancy after organ transplantation, on the contrary, the relative frequency of gynecologic tumors decreased compared with the general population. Fung et al.. In our study, we saw that a significant number of LSIL+ or worse lesions occurred within 5 years after transplantation; this increases the importance of annual screening especially in this patient group in the first 5 years. The 5-year cumulative HSIL+ incidence in our study was 0.33%, similar to the 0.3% reference cohort rate. Our cohort consisted of women with lower levels of abnormal cytology, LSIL+ and HSIL+ histology, compared with a cohort of about one million women.In our study, 0.71% of patients developed LSIL+, and 0.33% developed HSIL or worse lesions within the first 5 years after transplantation. In a study examining the relationship between organ transplantation and invasive cervical cancer, an average interval of 3.8 years was found between transplant and cancerde novo) malignancies in renal transplant patients is the same as in normal non-transplant patients. In patients with immunosuppressive therapy, especially with solid organ transplantation, Papanicolaou (PAP) tests can be obtained at the first examination, if there is a positive PAP test, examination with colposcopy and biopsy should be taken from these lesions in the presence of suspicious lesions,23. However, in these patients, the dose of immunosuppression is reduced to the lowest possible level immediately after tumor diagnosis. It is very important to diagnose and stage malignancies in organ transplant recipients as soon as possible. When these lesions are detected as either in situ or low-grade malignancies, oncologic results are undoubtedly better.The treatment of primary  should be followed up closely, including colposcopy.


Besides these traditional HPV detection tools, novel diagnostic approaches are being evaluated for HPV-induced OPSCC “ultrastaging.” E6 humoral response and ddPCR-detecting HPVct DNA are two techniques performed on blood and are therefore non-invasive. Baseline E6 humoral levels could have a prognostic value, and HPVct DNA could be helpful for HPV OPSCC recurrence monitoring. At last, next-generation sequencing (NGS)-based “capture HPV” is a technique feasible on biopsies and circulating DNA material. It helps characterize HPV integration status and sites, and it could define prognostic subgroups in HPV-induced OPSCC. These novel precision detection tools could be further integrated in the care of patients with HPV-induced OPSCC.Besides classic tobacco and alcohol risk factors, human papillomavirus (HPV) plays a role in the development of a subset of head and neck squamous cell carcinomas (HNSCCs), and notably oropharynx squamous cell carcinomas (OPSCCs). HPV-induced OPSCCs have a different biological behavior and a better prognosis compared to non-HPV-induced OPSCCs and the eighth-edition TNM classification now separates these two entities. Therefore, determining the HPV status of patients with OPSCC is now essential for treatment, prognosis, and development of clinical trials. In this review, after reminding essential steps of HPV implication in the cell cycle, we describe the existing tools that are currently feasible in routine practice according to facilities available in health structures, with their benefits and drawbacks: HPV PCR, E6/E7 mRNA RT-PCR, E6/E7 mRNA  Head and neck squamous cell carcinomas (HNSCCs) constitute a group of malignant tumors located in the oropharynx, larynx, hypopharynx, nasopharynx, and oral cavity. All together, they represent approximately 800,000 new cases and 400,000 deaths per year . ClassicHPV involved in mucosal cancer can be divided into two main groups, depending on their oncogenic associated risk. Low-risk HPV are very rarely associated with the development of cancers, and HPV-induced OPSCCs are usually developed after a high-risk HPV infection. Conversely, high-risk HPV genotypes encompass HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, and HPV68. The high-risk genotypes produce E6 and E7 oncoproteins. E6 protein binds to tumor suppressor p53 by the formation of a trimeric complex E6/E6AP/p53 , leadingin situ hybridization targeting DNA (DNA ISH), and in situ hybridization targeting RNA (RNA ISH) , RT-PCR (E6 and E7 mRNA detection), p16 immunohistochemistry, RNA ISH) . All theRNA ISH) . AccordiRNA ISH) . SeveralRNA ISH) –35 and (RNA ISH) , 36. TheV status .TM Oral Test (Arbor Vita Corp©) to reference tests performed on FFPE material: p16 and high-risk HPV mRNA. Agreement between fine-needle aspirates OncoE6TM and FFPE p16 was good (kappa = 0.53). Agreement between oral samples and FFPE p16 and high-risk HPV mRNA was poor (kappa = 0.02 for both), probably due to lower concentrations of E6 protein in these analytes . Depending on the DNA targets, DNA ISH can focus only on high-risk 16 and 18 genotypes, or on broader high-risk HPV-like genotypes 16, 18, 31, 33, 51 . Ventana© Inform HPV III Family 16 Probe cocktail is also able to detect 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 66 types. Finally, some screening probe cocktails can detect most frequent high-risk HPV , as well as some low-risk HPV (6 and 11 types for Enzo©’s screening probe for example).Many studies have evaluated the use of DNA ISH to diagnose HPV infection in oropharynx carcinomas , 62–66. Data about consistency of DNA ISH results in OPSCCs are quite controversial. For Schlecht et al., comparison of high-risk HPVs probe cocktail  and HPV16/18 DNA probe cocktail  showed better performances by the first manufacturer . ConversStudies about RNA ISH have been rising in the last 10 years and showing excellent diagnostic performances. Sensitivities vary from 87 to 100%, and specificities vary from 88 to 100%, being more frequently around 95% , 69–72. The main inconvenient of RNA ISH is its cost, rendering this diagnostic option poorly available for numerous pathology laboratories. A secondary limit of this assay is its incapacity to assess which one of the high-risk HPV types is present in the tumor tissue, whereas this information could be useful to precise prognosis of HPV-positive OPSCCs .in situ hybridization is equivalent for appreciation of prognosis compared to RT-PCR. They showed better survival for patients with HPV-driven OPSCC sought by E6/E7 in situ hybridization   . NowadayImmunostaining against p16 protein is a cost-effective method to diagnose a high-risk HPV infection within tissues. Overexpression of p16 protein may be an indirect sign of expression of E6 and E7 proteins with cell-cycle upregulation , 30. NevSensitivity of p16 immunostaining in the oropharynx is around 80–90% , 92–95. Several studies and meta-analyses have shown that p16-positive OPSCCs have a better overall survival and a better disease-free survival compared to p16-negative OPSCCs , 99–101,n = 62) and with p16−negative non−OP HNSCC (n = 559) were not significantly different (p = 0.26) (p = 0.34) between the p16-positive and p-16 negative patients  . More sppatients .p = 0.0115). This difference was not shown for p16-positive patients (p = 0.998)  and RT-alone treatment arms . Regardi= 0.998) .Finally, concerning the response to immunotherapies there are again few data available, but p16 status is quite consistently used. In KEYNOTE-012, for the head-and-neck cohorts, the percentage of p16 + patients was relatively small with 45 (23%) being p16 + and 147 (77%) being p16- . When stin vitro data suggest that E6 and E7 oncoproteins and spliced isoforms of E6 oncoprotein would be associated with higher levels of IL6, responsible of an immunosuppressive environment within cancer  based on detection of plasmatic HPV DNA (E6 or E7 genes) with new ultrasensitive methods appears to have a clinical interest in HPV OPSCC. Indeed, the liquid-biopsy approach using the detection of ctDNA released from tumor cells and detectable in blood has garnered growing interest  particulediction . HPV-relediction , 117 andediction . This quediction . Our teaediction . Indeed,ediction  and for Finally, the performance of HPVctDNA has also been evaluated to monitor treatment response early, showing that HPVctDNA kinetics are clearly correlated with treatment failure or success and this feature would be more precocious than classical Response Evaluation Criteria in Solid Sumours (RECIST) criteria , 133. InIn cervical carcinomas, integration of HPV DNA into the host genome seems to be the main critical etiological event in the progression from normal cervix to intraepithelial neoplasm, and finally to invasive cervical cancer. This HPV oncogenic process is considered to be identical in OPSCC, but with no scientific certainties as pretumoral lesions are not yet characterized in head and neck cancer. However, for cervical cancers, different studies have already shown that a part of HPV-driven tumor does not present any integration and is associated only with episomal HPV –136. TheRecently, using a next-generation sequencing (NGS) technology called “Capture HPV”  on biops“HPV capture” technology has already been performed on HPV cervical and anal cancers , 139 to Depending on the material obtained from patients, different HPV assays are feasible. Some samples require more invasive procedures than others. For this reason, except for the specific context of a clinical trial, performing a second “fresh” biopsy for RT-PCR is not standard because it requires invasive procedures. The new generation of HPV assays is highly sensitive and can be performed on non-invasive or minimally invasive samples, such as blood puncture and oral rinse. These approaches will undoubtedly be complementary to current classical routine practice HPV assays and will help to stratify and monitor HPV-positive HNSCCs. Considering the availability of human samples and technical aspects of assays cited above, we have briefly, through this review, given an overview of the techniques feasible on each kind of sample.in situ hybridization assays, the popularity of RNA ISH stems from its excellent diagnostic performances and the biological value of the assay, because positive cases show evidence of transcriptionally active HPV infection. Nevertheless, the price of this assay hampers its use in routine practice. DNA ISH is more difficult to read, and the technique process is highly dependent on the level of expertise of pathology laboratories. This variability leads to moderate diagnostic performances, and this assay is becoming unpopular. RT-PCR and PCR are non-spatial assays but are powerful tools to detect HPV infection. RT-PCR is more performant on fresh and frozen tissues which are often not available in routine practice. For PCR, several commercial assays have been developed for cervical cancers and could be used for HNSCCs, but an important work of comparative evaluation of these tools is needed in HNSCCs and some pre-PCR steps might be optimized to enhance the yield of the technique. Pragmatically, the high sensitivity of p16 immunostaining and the value of PCR to specify HPV type make these tools really interesting in routine practice. Indeed, using p16 immunostaining as a screening tool than PCR constitutes a performant way to diagnose and specify the HPV type since this information is important because of its prognostic value even among high-risk HPV types. In case RNA ISH is feasible, using it as a standalone test might be a seductive solution but it does not provide any precision on the HPV type. We think that further studies evaluating the impact of high-risk HPV type in the prognosis of patients should be conducted to be sure that this information requires a second PCR assay. Among new HPV biomarkers, HPVctDNA detection could be a useful monitoring tool to detect early disease recurrence. This latter tool also seems to have prognostic value, since quantification of HPVctDNA is correlated with T and N stages in OPSCCs. Finally, HPV capture, based on next-generation sequencing, gives insights into the integration process of various genotypes of HPV. In the near future, this assay could be a stratification and prognostic tool for patients with HPV-induced OPSCC.In this review, we have explored main HPV detection tools available in routine practice on fresh, frozen, and formalin-fixed tissues in the HNSCC context. If p16 immunostaining is the most affordable technique, it seems that the threshold of 70% of positive tumor cells recommended by the College of American Pathologists might be a little too high because a fraction of cases with a nuclear and cytoplasmic staining in 50–70% of tumor cells are clearly associated with high-risk HPV infection. Of the two JA, CL, and CBa designed the manuscript. JA, CL, and HP wrote the first draft. AM and AB illustrated the manuscript. JA, CL, CBa, AM, AB, DV, CBr, HP, and HM edited and revised the manuscript. All authors contributed to the article and approved the submitted version.CBa is consultant for Roche, MSD and HP is consultant for MSD Vaccin. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Optimality principles have been used to explain the structure and behavior of living matter at different levels of organization, from basic phenomena at the molecular level, up to complex dynamics in whole populations. Most of these studies have assumed a single-criteria approach. Such optimality principles have been justified from an evolutionary perspective. In the context of the cell, previous studies have shown how dynamics of gene expression in small metabolic models can be explained assuming that cells have developed optimal adaptation strategies. Most of these works have considered rather simplified representations, such as small linear pathways, or reduced networks with a single branching point, and a single objective for the optimality criteria.S. cerevisiae and B. subtilis. In particular, we consider metabolic dynamics during nutrient shift experiments.Here we consider the extension of this approach to more realistic scenarios, i.e. biochemical pathways of arbitrary size and structure. We first show that exploiting optimality principles for these networks poses great challenges due to the complexity of the associated optimal control problems. Second, in order to surmount such challenges, we present a computational framework which has been designed with scalability and efficiency in mind, including mechanisms to avoid the most common pitfalls. Third, we illustrate its performance with several case studies considering the central carbon metabolism of We show how multi-objective optimal control can be used to predict temporal profiles of enzyme activation and metabolite concentrations in complex metabolic pathways. Further, we also show how to consider general cost/benefit trade-offs. In this study we have considered metabolic pathways, but this computational framework can also be applied to analyze the dynamics of other complex pathways, such as signal transduction or gene regulatory networks. This paper is based on two main concepts: (i) genetic and biochemical dynamics are key to understand biological function, and (ii) optimality hypotheses enable predictions in biology. We start by briefly reviewing the relevant previous literature, with emphasis on early works. Then, we discuss the integration of these two main ideas in an optimal control framework which can then be used to analyze and understand complex biochemical pathways. We finally illustrate this approach with case studies related with metabolic networks.Mathematical modelling allows us to understand complex biological systems –3. WolkeThe dynamic behaviour of biological systems is often explained in terms of feedback regulation mechanisms , 18. FeeCan biology be predictive? It has been argued that the systems approach to biology will enable us to predict biological outcomes despite the complexity of the organism under study –32. Acco(i)analytic (in the sense of explanatory), i.e. helping us to understand, or even predict, the way in which a biological system occurs. Many examples can be found in ecology  and evol(ii)synthetic (in the sense of design and/or decision making), where it helps us to decide how to optimally manipulate, change or even build a bioprocess or biosystem. Examples can be found in biomedicine , metabolThe optimality hypothesis as an underlying principle in living matter has a long history. It was already used in the early 1900s to explain structural and functional organization in physiology , 35. A fIn the remainder of this paper, we will focus on the explanatory role of optimality in the context of cellular systems. Savageu  and HeinChapters specifically devoted to the interrelation between evolution and optimality in the context of biochemical pathways can be found in , 66. FroHeinrich et al  noted thIn this context, it is worth remembering that any optimization problem involves at least two elements: the objective (or cost) function, i.e. the criterion being maximized or minimized, and the decision variables, i.e. the degrees of freedom of the system which can vary to seek the optimal cost. Additionally, in most realistic situations the optimization problem also needs to incorporate constraints, i.e. relationships describing what is feasible or acceptable . Both in biology and physics, the set of constraints include physicochemical laws . But a main difference between physics and biology is the presence of additional functional constraints in the latter . That isMost of the above mentioned early studies of optimality in biology considered stationary (i.e. steady state) systems. However, as remarked at the beginning of this paper, biological function is closely linked to dynamics. Thus we now focus on the question of explaining and/or predicting dynamics (i.e. function) exploiting optimality principles.The optimization of dynamical systems is studied by optimal control theory , 68. Opts, with notable impetus in the case of optimal decision making in biomedical engineering . Aroundheduling ).In recent years, optimal control has been increasingly used to explain cellular phenomena e.g. –73). In . In 73])There are several important messages in the pioneering work of Klipp et al : (i) it The work of Klipp et al  spurred Trade-offs are ubiquitous in evolutionary biology: very often one trait can not improve without a decrease in another, as already noted by Darwin . Numerous [s for metabolic steady-state conditions [s, the approach gained traction: El-Samad et al [A mathematical framework that combines in a natural way the concepts of optimality and trade-offs is multicriteria optimization, where one seeks the best  trade-offs  that correspond to the simultaneous optimization of several objectives. The concept of Pareto optimality was originally developed in the field of economics, but it was already suggested for applications to ecology in the early 1970s . In the nditions , 65, 112ad et al  studied ad et al , 115 andad et al –118. Morad et al  and biolad et al . Higueraad et al  analyzedad et al –125. Manad et al –128.Considering the prediction of dynamics in biochemical pathways, to the best of our knowledge the study of de Hijas-Liste et al  was the Both in single and multicriteria formulations there is an underlying key question that needs to be addressed: the selection of the objective functions. In other words, we need to formulate objective functions which encapsulate the fitness and the trade-offs of the biological system under study. Heinrich and Schuster  suggesteAs discussed above, optimal control can provide important insights in the domain of systems biology. So far, existing studies have made use of rather simple dynamic models . In manHowever, for many applications, more complex models need to be used. Can optimal control be applied at these larger scales? This is one of the key questions considered here. The answer ultimately relies on two key issues: (i) the availability of the detailed (time-series) measurements need to build these more complex dynamic models, and (ii) the existence of numerical optimal control methods that can solve these problems in a reliable and efficient way. Regarding (i), Ewald et al. mention In contrast, regarding (ii), the situation currently remains an open question, serving as the main motivation for this work. Despite the many significant advances during the last decades, reliably solving nonlinear optimal control problems can be very challenging, even for small problems, due to the existence of local solutions and high sensitivity to initial guesses . This isIn the case of biochemical pathways, the application of optimal control faces important challenges and pitfalls, in line with those identified for chemical reaction systems , 134, inHere, our goal is to provide a multi-objective optimal control approach that can be applied to dynamic models of complex biochemical pathways, surmounting the above mentioned challenges. In particular, we present a computational workflow that is reliable (robust), avoiding convergence to local solutions, but efficient enough (in terms of reasonable computation times) to handle realistic network topologies and arbitrary nonlinear kinetics. We will show how this workflow is capable of scaling up well with network size, and how it can handle multi-criteria formulations.S. cerevisiae and B. subtilis. In particular, we use our workflow to explain metabolic dynamics during nutrient shift experiments.We illustrate the performance of this workflow considering three different case studies, based on dynamic models of the central carbon metabolism of optimal control (OCP) consists of computing the optimal decision variables  and time-invariant parameters that minimize (or maximize) a given cost functional (performance index), subject to the set of ODEs and possibly algebraic inequality constraints. Mathematically, the OCP is usually stated as follows:We consider dynamic systems described by nonlinear ordinary differential equations (ODEs). The problem of entclass1pt{minimaN different criteria considered:In the case of a multicriteria formulation, the cost functional In multicriteria optimization, the objectives are usually in conflict (improving one damages the others), so the optimal solution is not unique, but a set of optimal trade-offs . Many methods have been developed for multicriteria optimal control, and they are usually classified in three categories : scalariThe curse of dimensionality, so the latter two are the most promising strategies for realistic problems. Indirect approaches were historically the first developed and are based on the transformation of the original optimal control problem into a multi-point boundary value problem using Pontryagin’s necessary conditions [Methods for the numerical solution of nonlinear optimal control problems can be classified under three categories: dynamic programming, indirect and direct approaches Fig. . Dynamicnditions , 68. Indnditions ). DirectIn the sequential strategy –143, alsi:The control vector parameterization (CVP) approach proceeds by dividing the time horizon into a number of elements  with stepsize The direct collocation approach is probably the most well known complete discretization approach. In this method the solution of the infinite dimensional problem is transcribed into a non-linear programming problem discretizing both the states and the controls  by meansualities . Readersualities , 146 andConstraints play a key role in mathematical modeling in biology. From the computational optimization point of view, constraints limit the solution space, adding further complexity to already complex mathematical formulations. However, from the biological point of view, constraints add information to the model, making it more realistic.In general, constraints can have physical and biological meaning . For exaApart from physical constraints, biological limitations can be of even higher importance and complexity . These lIn this context, it would be interesting to analyze the interplay between constraints in biological models with the optimal trade-offs (Pareto set). In some cases, constraints can act as the operating principles we are trying to identify and understand . It woulcostate variables) can be estimated and provided along with the results as part of the solution of the optimal control problem. However, an interpretation of their significance is not necessarily intuitive. As mentioned previously, in nonlinear optimal control theory and while using direct methods, the problem is formulated as a constrained NLP optimization problem where both algebraic and differential equations are constraining the search space. In such a problem, the Lagrange multipliers represent what the cost would be if those constraints were violated. Additionally the Lagrange multipliers with respect to the state variables are discrete approximation of the adjoint variables, the approximation be depending on the transcription method of choice. Consequently, in nonlinear optimal control the adjoint variables can be used to explain the possible variation on the cost function associated with an incremental change in the states [In mathematical optimization, once an optimal solution is found, it is always interesting to analyze the role of the different constraints on such solution. In constrained optimization in economics, especially in linear programming, the concept of shadow price is used to quantify how much the optimal value of the objective function changes by relaxing a constraint , 151. Ine states .n variables Using direct optimization methods, the general optimal control problem is transcribed into a NLP problem. Let us consider a general NLP problem with cost function fined as :15\documLet us now consider an optimal control problem where we seek to find the controls Consider now that this infinite-dimensional optimal control problem is transcribed into a finite-dimensional NLP problem using a complete parameterization strategy. The relationship between the Lagrange multipliers  run without good initial guesses, (ii) avoid convergence to local solutions, (iii) approximate complicated control profiles with good accuracy and reasonable computation cost, (iv) scale up well in terms of control and state variables. Further, the approach is able to handle multiobjective optimal control problems by transforming them into a set of nonlinear optimal control problems, as depicted in Fig. first phase using AMIGO2_DO, a hybrid stochastic-deterministic method based on control vector parameterizationsecond phase using ICLOCS, a simultaneous (complete discretization) fast local methodThe main justification for the first phase is the handling of the multimodality issues using a robust approach that has the additional benefit of not requiring good initial guesses. We have used the latest version of the AMIGO2_DO solver, a sequential optimal control solver included in the AMIGO2 toolbox [In order to meet these requirements, we tested a number of different options and finally arrived to the following two-phase strategy named AMIGO2_DO+ICLOCS: toolbox . Althoug toolbox  providedFor the second phase we used ICLOCS , a very Choosing the right settings for these solvers is of key importance, but this task can be a real challenge for non-expert users. Here we provide the best settings that we found for the class of problems considered.control discretization options: an initial piecewise-linear discretization of the control using 5–10 elements proved successful. When used alone, the AMIGO2_DO mesh-refinement options can be used to approximate better difficult controls. When used as part of AMIGO2_DO+ICLOCS, we found more efficient to refine the controls during the ICLOCS phaseoptimization solver: best results were obtained using the eSS (enhanced scatter search) global solver  with FSQinitial value problem solver: CVODES , with reRecommended settings for the ICLOCS phase:initial guesses: a good initial guesses for the unknown parameters and state trajectories is key to improve convergence. When used in the hybrid approach, the solution obtained by AMIGO2_DO was used as the initial guess for ICLOCS.transcription method: trapezoidalderivative generation: analytic. Providing analytic expressions for the gradient of the cost function, Jacobian of the constraints and Hessian of the Lagrangian was found extremely important. Generating this information can be very cumbersome for non-trivial problems, so we have coded an auxiliary function that automates it using symbolic manipulationNLP solver: IPOPT Recommended settings for the AMIGO2_DO phase:Some optimal control problems can have non-unique solutions, i.e. different control trajectories corresponding to the same cost function value. This is an often overlooked issue that, although mathematically correct, might have unforseen consequences when trying to explain or predict biological behaviour.In a strict mathematical sense, such an issue is related with the system’s structure. For example, a dynamic system with highly correlated controls that can compensate their impact on the cost function might result in multiple solutions with different controls, yet identical cost function. In particular, in optimal control theory, a very well known issue is the existence of a singular arc. In a singular arc, the controls appear linearly in the system’s Hamiltonian and therefore can not be uniquely identified.However, from a computational point of view, practical multiplicity of solutions can also be defined. When solving a non-linear optimal control problem one can obtain solutions of different control trajectories that correspond to very similar or, even, tolerance-wise same cost function values. Such a finding implies that the cost function is practically insensitive to at least one of the controls in at least a certain interval of the time horizon.In this work we investigate the non-uniqueness of solutions from a practical and numerical point of view considering the ensemble of solutions found in the close vicinity of the global solution . Software source code with the implementations of the above case studies for these solvers is available at 10.5281/zenodo.3793620.Here we illustrate and evaluate our new two-phase approach, AMIGO2_DO+ICLOCS, by solving three challenging case studies:This case study is a relatively simple yet non-trivial problem presented in , extendiN is:The mathematical formulation of the multiobjective optimal control problem is:Here we computed the Pareto front of this multi-objective optimal control with the three different numerical strategies described above. The results are summarized in the Pareto front presented in Fig. These results are in close agreement with the best Pareto set obtained in . HoweverWe further support this claim by providing additional detailed comparisons of the optimal controls and optimal state trajectories computed by the three approaches for several points in the Pareto front 27\documeWe solved the above multicriteria optimal control problem with the three approaches, obtaining essentially the same Pareto front with all of them, as depicted in Fig. In contrast with the previous case study, we found that AMIGO2_DO+ICLOCS was the best numerical strategy for this problem, arriving to the best solution in only 5 min of computation using a standard PC, and avoiding convergence to local solutions. Interestingly, msICLOCS converged to local solutions or crashed rather frequently, even with the use of fine-tuning. In Fig. We also performed an analysis of solution multiplicity and sensitivity of the cost function with respect to the controls by analyzing 100 runs of the hybrid approach. Considering the solutions within entclass1pt{minimaThis case study includes two path constraints representing the critical values of ATPc and NADHc which are needed to ensure the survival of the cell. We performed a numerical analysis to evaluate the impact of these path constraints on the optimal solution. In Fig. Next we performed an analysis of the adjoint variables  to get further insights regarding the local sensitivity of the performance index with respect to the constraints. That is, the adjoint variables for specific constraints or bounds indicate the local sensitivity of the solution with respect to their incremental change. Detailed results for the Lagrange multipliers are presented in the Additional file B.subtilis taken from [This case study is based on a simplified kinetic model of the central carbon metabolism of ken from . The modHere, the objectives considered are the maximization of the overall ATP production and the minimization of the protein investment (enzyme synthesis). We aim to find the cost/benefit trade-offs, where the benefit is computed as the integral of ATP levels along the time horizon, and the cost is given by the integral of the total enzyme concentrations for the whole time horizon.The corresponding multicriteria optimal control formulation is as follows:In this case study we are considering two dynamic scenarios. In the first scenario (named G-M) we consider a culture growing in an environment where glucose is the only substrate. At a certain time-point we add malate and we observe the dynamic behavior. The second scenario (M-G) is vice-versa. That is, the culture is initially growing on malate, before instantly adding glucose as a substrate and then observe the dynamics. The first phase of both scenarios is computed starting from the same initial values and reaching a steady state for the consumption of the substrate available.The initial conditions for scenario G-M are:Even though the three methods arrived to essentially the same multicriteria optimal control Pareto sets, msICLOCS was the fastest, converging to very good results in very modest computation times . As in the previous case studies, fine-tuning ICLOCS was found to be critical to improve its convergence speed and to avoid convergence to local solutions. In Additional file Finally, we performed an analysis of possible solution multiplicity and sensitivity of the cost function with respect to the controls. We were not able to identify multiplicity of solutions as shown in the respective figures in Additional file The above results show that the novel combined strategy AMIGO2_DO+ICLOCS was the best in terms of robustness, solving these three challenging case studies without any issues, while requiring a very reasonable computational cost for all of them. Although the multi-start strategy msICLOCS was faster in two of the problems, it often failed when attempting the solve the SC case study.The performance of the AMIGO2_DO+ICLOCS strategy also compares very favorably with the previous results of the different optimal control methods considered by de Hijas-Liste et al  for the dered in . FinallyHere we have considered the exploitation of optimality principles to explain dynamics in biochemical networks in the absence of complete prior knowledge about the regulatory mechanisms. After carefully reviewing the previous literature, we have presented a multicriteria optimal control framework to generate biologically meaningful predictions for rather complex metabolic pathways. This strategy requires, as a starting point, a calibrated dynamic model of the pathway and the set of cost functions to be considered in the multicriteria formulation. Next, the approach uses a Pareto optimality hypothesis to compute the dynamic effects of regulation in a metabolic pathway. Such computation is performed without the need of making any assumption about the pathway regulation.S. cerevisiae and B. subtilis. Our results indicate that the two-phase strategy AMIGO2_DO+ICLOCS presented excellent scalability and a good compromise between efficiency and robustness. We also show how this framework allows us to explore the interplay and trade-offs between different biological cost functions and constraints in biological systems.We have also discussed the many possible challenges and pitfalls involved in the solution of these nonlinear multicriteria optimal control problems. In order to surmount these difficulties, here we have considered several numerical strategies with the aim of finding a method suitable to handle realistic networks of arbitrary topology. We have carefully evaluated their performance with several challenging case studies considering the central carbon metabolism of Our vision is that multicriteria optimal control can play a major role in fully exploiting the explanatory and predictive power of kinetic models in computational systems biology. In future work, we will illustrate its usefulness for the analysis of cell signaling pathways, and for the solution of metabolic engineering problems considering kinetic models. Another interesting area for future research is that of optimal control using Boolean networks, a topic attracting increasing attention –177. ThiAdditional file 1. Solution details for case study LPN3B.Additional file 2. Solution details for case study SC.Additional file 3. Solution details for case study BSUB.