Prognostic implications of p16 and HPV discordance in oropharyngeal cancer (HNCIG-EPIC-OPC): a multicentre, multinational, individual patient data analysis.

BACKGROUND: p16INK4a (p16) immunohistochemistry is the most widely used biomarker assay for inferring HPV causation in oropharyngeal cancer in clinical and trial settings. However, discordance exists between p16 and HPV DNA or RNA status in some patients with oropharyngeal cancer. We aimed to clearly quantify the extent of discordance, and its prognostic implications. METHODS: In this multicentre, multinational individual patient data analysis, we did a literature search in PubMed and Cochrane database for systematic reviews and original studies published in English between Jan 1, 1970, and Sept 30, 2022. We included retrospective series and prospective cohorts of consecutively recruited patients previously analysed in individual studies with minimum cohort size of 100 patients with primary squamous cell carcinoma of the oropharynx. Patient inclusion criteria were diagnosis with a primary squamous cell carcinoma of oropharyngeal cancer; data on p16 immunohistochemistry and on HPV testing; information on age, sex, tobacco, and alcohol use; staging by TNM 7th edition; information on treatments received; and data on clinical outcomes and follow-up (date of last follow-up if alive, date of recurrence or metastasis, and date and cause of death). There were no limits on age or performance status. The primary outcomes were the proportion of patients of the overall cohort who showed the different p16 and HPV result combinations, as well as 5-year overall survival and 5-year disease-free survival. Patients with recurrent or metastatic disease or who were treated palliatively were excluded from overall survival and disease-free survival analyses. Multivariable analysis models were used to calculate adjusted hazard ratios (aHR) for different p16 and HPV testing methods for overall survival, adjusted for prespecified confounding factors. FINDINGS: Our search returned 13 eligible studies that provided individual data for 13 cohorts of patients with oropharyngeal cancer from the UK, Canada, Denmark, Sweden, France, Germany, the Netherlands, Switzerland, and Spain. 7895 patients with oropharyngeal cancer were assessed for eligibility. 241 were excluded before analysis, and 7654 were eligible for p16 and HPV analysis. 5714 (74·7%) of 7654 patients were male and 1940 (25·3%) were female. Ethnicity data were not reported. 3805 patients were p16-positive, 415 (10·9%) of whom were HPV-negative. This proportion differed significantly by geographical region and was highest in the areas with lowest HPV-attributable fractions (r=-0·744, p=0·0035). The proportion of patients with p16+/HPV- oropharyngeal cancer was highest in subsites outside the tonsil and base of tongue (29·7% vs 9·0%, p<0·0001). 5-year overall survival was 81·1% (95% CI 79·5-82·7) for p16+/HPV+, 40·4% (38·6-42·4) for p16-/HPV-, 53·2% (46·6-60·8) for p16-/HPV+, and 54·7% (49·2-60·9) for p16+/HPV-. 5-year disease-free survival was 84·3% (95% CI 82·9-85·7) for p16+/HPV+, 60·8% (58·8-62·9) for p16-/HPV-; 71·1% (64·7-78·2) for p16-/HPV+, and 67·9% (62·5-73·7) for p16+/HPV-. Results were similar across all European sub-regions, but there were insufficient numbers of discordant patients from North America to draw conclusions in this cohort. INTERPRETATION: Patients with discordant oropharyngeal cancer (p16-/HPV+ or p16+/HPV-) had a significantly worse prognosis than patients with p16+/HPV+ oropharyngeal cancer, and a significantly better prognosis than patients with p16-/HPV- oropharyngeal cancer. Along with routine p16 immunohistochemistry, HPV testing should be mandated for clinical trials for all patients (or at least following a positive p16 test), and is recommended where HPV status might influence patient care, especially in areas with low HPV-attributable fractions. FUNDING: European Regional Development Fund, Generalitat de Catalunya, National Institute for Health Research (NIHR) UK, Cancer Research UK, Medical Research Council UK, and The Swedish Cancer Foundation and the Stockholm Cancer Society.

Prevalence of human papillomavirus (HPV) types 16 and 18 in cervical cancer in Stockholm, Sweden during 2019-2023 compared to 2003-2008.

BACKGROUND: The prevalence of different HPV types, especially HPV16 and 18 in cervical cancer in patients diagnosed 2019-2023 in Stockholm was compared to corresponding data from 2003-2008 before the introduction of HPV vaccination in Sweden. MATERIAL AND METHODS: Cervical cancer samples from 125 patients diagnosed 2019-2023 in Stockholm were analysed for 27 HPV types by multiplex assay and the HPV type prevalence data was compared to data obtained in 154 cervical samples from 2003-2008. RESULTS: Patient median age was higher 2019-2023 compared to 2003-2008 (55-years vs. 42-years, p = 0.046). Overall HPV prevalence was 93.6%, HPV16 and 18 accounted for 62.2% of all squamous cell carcinoma cases (SCC) and 63.6% of all adenocarcinoma cases (ADC) vs. 92.9%, 69.7% and 88.6% respectively 2003-2008. CONCLUSION: The joint prevalence of HPV16 and 18 in SCC and ADC tended to be slightly lower in 2019-2023 as compared to 2003-2008, but the difference was not statistically significant.

Human papilloma virus (HPV) prevalence upon HPV vaccination in Swedish youth: a review based on our findings 2008-2018, and perspectives on cancer prevention.

PURPOSE: Three human papillomavirus (HPV) vaccines are available against up to nine HPV types. In Sweden, from 2012, Gardasil was offered to 10-12 year old girls through the school-based vaccination program, and as catchup vaccination for women up to 26 years. To obtain a baseline, and follow HPV vaccination effects, during 2008-2018, cervical and oral HPV prevalence were followed at a youth clinic in Stockholm, and in 2013 for comparison oral HPV prevalence was examined in high-school youth in a middle-sized county in Sweden. METHODS: In this review, we discuss all our data with cervical and oral mouthwash samples that were collected and tested for 24-27 HPV types by a bead-based multiplex assay from 2008. RESULTS: Compared with 2008-2011, with ~ 35% HPV16 and > 60% high risk (HR) HPV cervical prevalence at the youth clinic, a decrease of vaccine HPV types was observed between 2013 and 2018, with e.g., HPV16 falling to 5% in catchup vaccinated women and 15-18% in nonvaccinated women. Most common cervical HR-HPV types were HPV39, 51, 52, 56, and 59 together accounting for ~ 10% of cervical cancer, and where only HPV52 is included in Gardasil-9. At baseline 2009-2011, oral HPV prevalence was ~ 10% in unvaccinated youth at the youth clinic, but after 2013 it dropped to < 2% at the youth clinic and high schools. CONCLUSION: To conclude, Gardasil HPV types have decreased, but it is still important to follow remaining HR-HPV types and cancer development, since there is an ongoing increase in the incidence of HPV-associated tonsillar and base of tongue cancer, and cervical cancer in Sweden.

Development and external validation of nomograms in oropharyngeal cancer patients with known HPV-DNA status: a European Multicentre Study (OroGrams).

BACKGROUND: The proxy marker for human papillomavirus (HPV), p16, is included in the new AJCC 8th/UICC 8th staging system, but due to incongruence between p16 status and HPV infection, single biomarker evaluation could lead to misallocation of patients. We established nomograms for overall survival (OS) and progression-free survival (PFS) in patients with oropharyngeal squamous cell carcinoma (OPSCC) and known HPV-DNA and p16 status, and validated the models in cohorts from high- and low-prevalent HPV countries. METHODS: Consecutive OPSCC patients treated in Denmark, 2000-2014 formed the development cohort. The validation cohorts were from Sweden, Germany, and the United Kingdom. We developed nomograms by applying a backward-selection procedure for selection of variables, and assessed model performance. RESULTS: In the development cohort, 1313 patients, and in the validation cohorts, 344 German, 503 Swedish and 463 British patients were included. For the OS nomogram, age, gender, combined HPV-DNA and p16 status, smoking, T-, N-, and M-status and UICC-8 staging were selected, and for the PFS nomogram the same variables except UICC-8 staging. The nomograms performed well in discrimination and calibration. CONCLUSIONS: Our nomograms are reliable prognostic methods in patients with OPSCC. Combining HPV DNA and p16 is essential for correct prognostication. The nomograms are available at www.orograms.org .

ECIL guidelines for the prevention, diagnosis and treatment of BK polyomavirus-associated haemorrhagic cystitis in haematopoietic stem cell transplant recipients.

OBJECTIVES: To define guidelines for BK polyomavirus (BKPyV)-associated haemorrhagic cystitis (BKPyV-HC) after paediatric and adult HSCT. METHODS: Review of English literature and evidence-based recommendations by expert consensus. RESULTS: BKPyV-HC occurs in 8%-25% of paediatric and 7%-54% of adult recipients undergoing allogeneic HSCT. Diagnosis requires the triad of cystitis, macro-haematuria and high urine BKPyV loads >7 log10 copies/mL, and exclusion of other relevant aetiologies. BKPyV viraemia is frequent and may serve as a more specific semiquantitative follow-up marker. No randomized controlled trials are available to inform antiviral prophylaxis or treatment. However, hyper-hydration and/or bladder irrigation showed limited prophylactic value. Fluoroquinolones are not effective for prophylaxis or treatment, but rather increase antibiotic resistance. Hyperbaric oxygen or fibrin glue is marginally effective based on small case series from correspondingly equipped centres. Although cidofovir has been reported to improve and/or reduce BKPyV viraemia or viruria, the current data do not support its regular use. CONCLUSIONS: BKPyV-HC remains a disabling unmet clinical need in HSCT that requires novel approaches supported by proper clinical trials.

Protein Expression in Tonsillar and Base of Tongue Cancer and in Relation to Human Papillomavirus (HPV) and Clinical Outcome.

Human papillomavirus (HPV) is a major etiological factor for tonsillar and the base of tongue cancer (TSCC/BOTSCC). HPV-positive and HPV-negative TSCC/BOTSCC present major differences in mutations, mRNA expression and clinical outcome. Earlier protein studies on TSCC/BOTSCC have mainly analyzed individual proteins. Here, the aim was to compare a larger set of cancer and immune related proteins in HPV-positive and HPV-negative TSCC/BOTSCC in relation to normal tissue, presence of HPV, and clinical outcome. Fresh frozen tissue from 42 HPV-positive and 17 HPV-negative TSCC/BOTSCC, and corresponding normal samples, were analyzed for expression of 167 proteins using two Olink multiplex immunoassays. Major differences in protein expression between TSCC/BOTSCC and normal tissue were identified, especially in chemo- and cytokines. Moreover, 34 proteins, mainly immunoregulatory proteins and chemokines, were differently expressed in HPV-positive vs HPV-negative TSCC/BOTSCC. Several proteins were potentially related to clinical outcome for HPV-positive or HPV-negative tumors. For HPV-positive tumors, these were mostly related to angiogenesis and hypoxia. Correlation with clinical outcome of one of these, VEGFA, was validated by immunohistochemistry. Differences in immune related proteins between HPV-positive and HPV-negative TSCC/BOTSCC reflect the stronger activity of the immune defense in the former. Angiogenesis related proteins might serve as potential targets for therapy in HPV-positive TSCC/BOTSCC.

A novel approach for HLA-A typing in formalin-fixed paraffin-embedded-derived DNA.

The aim of this study was to establish a novel approach for human leukocyte antigen (HLA)-typing from formalin-fixed paraffin-embedded-derived DNA. HLAs can be a prognostic factor in cancer and have an extensive polymorphism. This polymorphism is predominantly restricted to exons, which encode the peptide-binding domain of the protein. Formalin-fixed paraffin-embedded material is routinely collected in the clinic and therefore a great source of DNA for genetic analyses. However, its low quality due to fragmentation and nucleotide changes has often created obstacles in designing genetic assays. In this study, we amplified the most polymorphic exons of the HLA-A gene, exons 2, 3, and 4, in 16 formalin-fixed paraffin-embedded samples >10 years old. These tissue samples belonged to patients already HLA-typed by peripheral blood samples at the routine laboratory. Acquired amplification products were used for sequencing, which provided enough information to establish an HLA allele. The same method was applied to DNA extracted from peripheral blood from a healthy volunteer with known HLA type. Of the samples, 14/16 (88%) were successfully typed, in one sample only one of the alleles could be determined, and in one sample no allele could be determined. The amplification of the most polymorphic exons of HLA-A was a successful alternative when DNA quality prevented positive results with previously described methods. The method is usable when an HLA type is needed but the patients are deceased and/or no whole blood samples can be collected. It has thus potential to be used in several fields such as the clinic, research, and forensic science.

Sexual experiences in relation to HPV vaccination status in female high school students in Sweden.

OBJECTIVES: To investigate sexual experiences, sexually transmitted infections (STIs) and use of condoms in relation to human papillomavirus (HPV)-vaccination status in female high school students. METHODS: In 2013, 355 female students with a median age of 18 years from randomly selected high schools in Sweden answered a classroom questionnaire on sexual experiences and HPV-vaccination status. RESULTS: In total 227/348 (65%) of the women reported having received at least one HPV vaccine dose. Median age at first intercourse was 15 and 16 years respectively, in the 141/227 (62%) vaccinated after, and the 86/227 (38%) vaccinated before their sexual debut. There were no differences between the HPV-vaccinated and non-vaccinated groups regarding condom use, STIs, and experiences of oral and anal sex, or friends- with-benefit relationships. However, having had sexual intercourse and 'one-night stands' were more common in the vaccinated group (both p < 0.05). CONCLUSION: Many students (62%) were vaccinated against HPV, with two-thirds after their sexual debut. There were no differences in condom use and STIs, and only a few differences in sexual experiences between the HPV-vaccinated and non-vaccinated groups. Initiating HPV vaccination before sexual debut is important, as is information about the link between HPV, sexual behaviour and cancer.

Adenoid Cystic Carcinoma, Clinical Presentation, Current Treatment and Approaches Towards Novel Therapies.

Adenoid cystic carcinoma (AdCC) is a rare cancer originating from secretory glands with unknown aetiology. It is one of the most dominant malignant salivary tumours (MST). However, it can arise in other areas of the head and neck region and in secretory glands outside this area. It occurs at all ages, but is more frequent between 50-70 years of age and more common in females than in males. The symptoms of AdCC are generally unspecific and the clinical diagnosis of AdCC maybe challenging, partially due to its heterogenous histopathology and indolent growth. Moreover, there is a lack of good prognostic markers, and due to its rarity, it is difficult to predict which therapeutic methods are the most optimal for each patient, especially since very late recurrences occur. This review presents some major characteristics of AdCC and some current treatments for this disease.

Combined targeted therapy with PI3K and CDK4/6, or FGFR inhibitors show synergistic effects in a neuroblastoma spheroid culture model.

AIM: Neuroblastoma (NB) is, in spite of current intensive therapy with severe side effects, still not cured so new therapies are needed. Recently, we showed combining phosphoinositide 3-kinase (PI3K) (BYL719), fibroblast growth factor receptor (FGFR) (JNJ-42756493) and cyclin-dependent kinase 4/6 (CDK4/6) (PD-0332991) inhibitors, in vitro in NB cell lines grown as monolayers had synergistic effects. However, there were variations depending on the combinations used and the targeted NB cell lines. To obtain further information and to mimic more natural circumstances, we investigated the effects of single and combined administrations of the above inhibitors in spheroid NB-cultures. MATERIAL AND METHODS: Spheroid cultures of NB cell lines SK-N-AS, SK-N-BE(2)-C, SK-N-FI and SK-N-SH were established and treated with single and combined administrations of BYL719, JNJ-42756493, and PD-0332991 and followed for growth, viability, proliferation, cytotoxicity and migration. KEY FINDINGS: Single inhibitor administrations gave dose dependent responses with regard to growth and viability and their combinations were efficient and resulted in a range of additive and synergistic effects. The responses to individual drugs and their various combinations were predominantly alike regardless of whether the cells were cultivated in monolayer or D spheroid NB models. However, in general, slightly higher drug concentrations were necessary in spheroidcultures. SIGNIFICANCE: This study provides pre-clinical evidence that single PI3K, FGFR, and CDK4/6, inhibitors exhibit promising anti-NB activity and when combined lower doses of the drugs could be also used in spheroid NB-cultures, supporting the pursuit of further in vitro and in vivo studies in preparation for future potential clinical use.

Curcumin Alone and Combined With PI3K Inhibitors Elicits Positive Effects on Oropharyngeal Cancer Cell Lines Regardless of HPV Status.

BACKGROUND/AIM: Human papillomavirus positive (HPV+) oropharyngeal squamous cell carcinoma (OPSCC) is rising in incidence. Compared to HPV-negative (HPV-) OPSCC, HPV+ cases have a better 5-year survival. With its severe side-effects, today's chemoradiotherapy has not improved outcome compared to radiotherapy alone, so new therapies are needed. Mutations in phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA), fibroblast growth factor receptor 3 (FGFR3) and cell division cycle 27 (CDC27) are found in HPV+ OPSCC, and in vitro targeted therapy combining PI3K and FGFR inhibitors showed synergistic effects. Here the effects of targeting CDC27 with curcumin with/without various inhibitors or cisplatin on OPSCC cell lines were examined. MATERIALS AND METHODS: Curcumin was administered to HPV+ OPSCC cell lines CU-OP-2, CU-OP-3 and CU-OP-20, and HPV- CU-OP-17 with/without PI3K, cyclin-dependent kinase 4/6, FGFR, poly (ADP-ribose) polymerase or WEE1 inhibitors (BYL719, PD-0332991, JNJ-42756493, BMN-673 and MK-1775, respectively), or cisplatin. The cell lines were then assessed for 72 h after treatment for viability, proliferation and cytotoxicity. RESULTS: Curcumin led to dose-dependent responses with reduced viability and proliferation; upon combining it with BYL719, additional positive effects were found for most OPSCC lines grown as monolayers, and these effects were validated in CU-OP-2 cells grown as spheroids. Curcumin with MK-1775 or PD-0332991 also elicited some positive effects on CU-OP-2 and CU-OP-17 cells. CONCLUSION: Curcumin alone led to dose-dependent responses and when combined with BYL719, positive effects were revealed, as they were when it was combined with MK-1775 or PD-0332991, suggesting a potential use of some of these combinations for HPV+ OPSCC.

Human papillomavirus (HPV) load is higher in HPVDNA/p16 positive than in HPVDNA positive/p16 negative oropharyngeal squamous cell carcinoma but does not differ significantly between various subsites or correlate to survival.

OBJECTIVE: Patients with human papillomavirus DNA positive (HPVDNA+) and p16ink4a overexpressing (p16+) oropharyngeal squamous cell carcinoma (OPSCC), especially those with cancer in the tonsillar and base of tongue subsites as compared to other OPSCC subsites have a better outcome than those with only HPVDNA+ or only p16+ cancer. Likewise having a high viral load has been suggested to be a positive prognostic factor. We therefore hypothesized, that HPV viral load could vary depending on OPSCC subsite, as well as with regard to whether the cancer was HPVDNA+ and p16+, or only HPVDNA+, or only p16+ and that this affected outcome. MATERIAL AND METHODS: To address these issues HPV viral load was determined by HPV digital droplet (dd) PCR in tumor biopsies with previously known HPVDNA/p16 status from 270 OPSCC patients diagnosed 2000-2016 in Stockholm, Sweden. More specifically, of these patients 235 had HPVDNA+/p16+, 10 had HPVDNA+/p16-, 13 had HPVDNA-/p16+ and 12 had HPVDNA-/p16- cancer. RESULTS: We found that HPVDNA+/p16+ OPSCC had a significantly higher viral load than HPVDNA+/p16- OPSCC. Moreover, there was a tendency for a higher viral load in the tonsillar and base of tongue OPSCC subsites compared to the other subsites and for a low viral load to correlate to a better clinical outcome but none of these tendencies reached statistical significance. CONCLUSION: To conclude, the mean viral load in HPVDNA+/p16+ OPSCC was higher than in HPVDNA+/p16- OPSCC, but there was no statistically significant difference in viral load depending on OPSCC subsite or on clinical outcome.

MHC class I expression in HPV positive and negative tonsillar squamous cell carcinoma in correlation to clinical outcome.

Human papillomavirus (HPV) is an important factor for the development of tonsillar squamous cell carcinoma (TSCC). In addition, patients with HPV-positive TSCC have a better clinical outcome than patients with HPV-negative TSCC. Although, HPV is an important prognostic marker, additional biomarkers are needed to better predict clinical outcome to individualize treatment. Hence, we examined if classical HLA HLA-A,B,C and nonclassical HLA-E,G could serve as such marker. Formalin-fixed paraffin-embedded TSCC from 150 patients diagnosed 2000-2006, earlier analyzed for HPV DNA and p16(INK4a), and treated with intention to cure were evaluated for the expression of HLA-A,B,C and HLA-E,G by immunohistochemistry. For HPV-positive TSCC a low expression of HLA-A,B,C, whereas for HPV-negative TSCC, a normal expression of HLA-A,B,C was significantly correlated to a favorable clinical outcome. These correlations were more pronounced for membrane staining of HLA-A,B,C when compared with cytoplasmatic staining. No significant correlation was found between HLA-E,G and HPV status or clinical outcome. The unexpected contrasting correlation between HLA-A,B,C expression, and clinical outcome depending on HPV, indicates essential differences between HPV-positive and HPV-negative TSCC. Furthermore, our data demonstrate that for both HPV-positive and HPV-negative TSCC, the expression of HLA-A,B,C together with HPV may serve as a useful biomarker for predicting clinical outcome.

Oral human papillomavirus prevalence in high school students of one municipality in Sweden.

The rise in human papillomavirus (HPV) infection has been suggested to be responsible for the increased incidence of oropharyngeal cancer in the Western world. This has boosted interest in oral HPV prevalence and whether HPV vaccines can prevent oral HPV infection. In a previous study we showed oral HPV prevalence to be almost 10% in youth aged 15-23 y attending a youth clinic in Stockholm, Sweden. However, this may not be a generalizable sample within the Swedish population. Therefore, mouthwashes were used to investigate oral HPV prevalence in 335 Swedish high school students aged 17-21 y (median age 18 y), from 1 municipality with 140,000 inhabitants. The presence of HPV DNA in the oral samples, as examined by a Luminex-based assay, was significantly lower in this cohort, only 1.8% (3.1% in females and 0.6% in males), as compared to our previous study.

New Approaches Towards Targeted Therapy for Childhood Neuroblastoma.

Neuroblastoma (NB), comprising around 10% of all childhood neoplasms and 15% of pediatric cancer deaths is a heterogenous disease and can be divided into very low-, low-, intermediate- and high-risk NB. Treatment of very low/low-risk NB is usually based on observation, or surgery alone, whereas intermediate-risk NB is in addition to surgery treated with mild chemotherapy and roughly 80-95% of the patients are cured. In contrast, high-risk NB patients receive multimodal therapy with e.g. induction, consolidation, and maintenance therapy, which can include induction chemotherapy and surgery and in severe cases adding intensive chemotherapy and even autologous stem cell transplantation and radiotherapy. Unfortunately, however this treatment does not cure all patients and around 50% succumb to disease. For this purpose, new treatment options are urgently needed. In this review, we describe the complex molecular heterogeneity of NB, and potential new options for targeted therapy.

Special Issue "HPV in the Head and Neck Region 2.0".

Members of the human papillomavirus (HPV) family have been known for causing cancers and condylomas in the anogenital tract for some time, as reflected by the Nobel Prize in Medicine given to Professor Harald zur Hausen 2008 [...].

Adenoid Cystic Carcinoma (AdCC): A Clinical Survey of a Large Patient Cohort.

Adenoid cystic carcinoma (AdCC), a rare heterogenous disease, presents diagnostic, prognostic, and therapeutic challenges. To obtain more knowledge, we conducted a retrospective study on a cohort of 155 patients diagnosed in 2000-2022 with AdCC of the head and neck in Stockholm and investigated several clinical parameters in correlation to treatment and prognosis in the 142/155 patients treated with curative intent. The strongest favourable prognostic factors were early disease stage (stage I and II) as compared to late disease (stage III and IV) and major salivary gland subsite as compared to other subsites, with the best prognosis in the parotid gland, irrespective of the stage of the disease. Notably, in contrast to some studies, a significant correlation to survival was not found for perineural invasion or radical surgery. However, similar to others, we confirmed that other common prognostic factors, e.g., smoking, age, and gender, did not correlate to survival and should not be used for prognostication of AdCC of the head and neck. To conclude, in AdCC early disease stage, major salivary gland subsite and multimodal treatment were the strongest favourable prognostic factors, while this was not the case for age, gender and smoking nor perineural invasion and radical surgery.

Combination of PARP and WEE1 inhibitors in vitro: Potential for use in the treatment of SHH medulloblastoma.

Medulloblastoma (MB), grouped as either WNT­activated, Sonic hedgehog (SHH)­activated, or non-WNT/non-SHH group 3, accounts for almost 20% of all childhood brain cancers. In spite of current intensive treatments, not all patients are cured and survivors suffer from severe side­effects. The present study therefore examined the effects of the poly­ADP­ribose polymerase (PARP) and WEE1­like protein kinase (WEE1) inhibitors, BMN673 and MK­1775, respectively, alone or in combination on four MB cell lines. More specifically, the MB cell lines, DAOY, UW228­3, MED8A and D425, were tested for their sensitivity to BMN673 and MK­1775 alone or in combination, using cell viability, cell confluency and cytotoxicity assays. The effects on the cell cycle phases were also examined using FACS analysis. Monotherapy with BMN673 and MK­1775 exerted dose­dependent inhibitory effects on the viability of almost all MB cell lines. Notably, when BMN673 and MK­1775 were used in combination, synergistic effects were noted in the SHH group cell lines (DAOY and UW228­3), but not in the already WEE1­sensitive group 3 (MED8A and D425) lines. Moreover, the combination treatment decreased the percentage of cells in the G1 phase and induced the novel distribution of both DAOY and UW228­3 cells in the S and G2/M phases, with the UW228­3 cells exhibiting a greater delay. To conclude, MK­1775 was efficient in all and BMN673 in most cell lines, and their combined use exerted synergistic effects on the SHH, but not the group 3 cell lines. These data suggest that MK­1775 alone may be of interest for all MB cell lines, and that the combination of PARP/WEE1 inhibitors may provide possible therapeutic opportunities for the therapy of SHH MBs. Their use warrants further investigations in the future.

Efficacy of combined targeted therapy with PI3K and CDK4/6 or PARP and WEE1 inhibitors in neuroblastoma cell lines.

Neuroblastoma (NB), the most frequent solid extracranial tumor in children, is not always cured by current aggressive therapies that have notable adverse effects; therefore, novel treatments are necessary. Phosphoinositide 3­kinase (PI3K) and fibroblast growth factor receptor inhibitors exhibit synergistic effect in NB cell lines. In the present study, mono­ and combination therapy of the United States Food and Drug Administration­approved PI3K, cyclin­dependent kinase­4/6 (CDK4/6), poly­ADP­ribose­polymerase (PARP) and WEE1 G2 checkpoint kinase (WEE1) inhibitors (BYL719, PD­0332991, BMN673 and MK­1775, respectively), were used to treat NB cell lines SK­N­AS, SK­N­BE(2)­C, SK­N­DZ, SK­N­FI and SK­N­SH and viability (assessed by WST­1 assay), proliferation (incucyte analysis) and cell cycle (FACS) changes were assessed. Treatments with all single drugs presented dose­-dependent responses with decreased viability and proliferation and combining BYL719 with PD­0332991 or BMN673 with MK­1775 resulted in additive or synergistic effects in most cell lines., except for SK­N­SH for the former and for SK­N­AS for the latter. Moreover, combining MK­1775 and BMN673 decreased the numbers of cells in S phase to a greater extent than either drug alone, while when combining PD­0332991 and BYL719 the observed effect was close to that of PD­0332991 alone. To summarize, PI3K and CDK4/6 or PARP and WEE1 exhibited synergistic anti­NB effects and lower doses of the inhibitors could be utilized, thereby potentially reducing adverse side effects.