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Hydraulic fracturing in shale formations induces microseismic events in a region we refer to as the microseismic volume. Many of these microseismic events are signatures of failure in the formation that are believed to be a result of induced unpropped (IU) fractures beyond the primary propped fracture. Areally extensive microseismicity may be evidence that these IU fractures occur and extend spatially beyond the propped fracture during pumping in many unconventional reservoirs. We present evidence that these fractures close over time after pumping is stopped and that this closure of IU fractures can have a significant impact on stress interference between fractures. To illustrate these effects, microseismic and radioactive-tracer data are presented for four laterals drilled and fractured from a single pad. Two wells on this pad were fractured with the consecutive-fracturing sequence, and the other two wells were fractured with the zipper-fracturing sequence. Geomechanical simulations were performed to model the pad scenario and explain the micro-seismic and tracer observations, with emphasis on the two different fracturing sequences. Our simulations show that the opening of the IU fractures results in significant temporary changes to the stress field in the rock. One consequence of this is that later fracture stages tend to propagate into the open-fracture networks of IU fractures created earlier because of stress reorientation. This can lead to inefficient usage of fluid, proppant, and capital because the region that is being stimulated has already been stimulated by the previous stage. By analyzing the net pressure, radioactive-tracer data, and microseismic data from the four-well pad, we show that these IU fractures close over time because the fracture fluid leaks off. This reduces the stress shadow, and subsequent induced fractures are no longer subjected to the significantly altered stresses, allowing for more-efficient fracture-network coverage by subsequent fractures in a horizontal well. On the basis of the data presented and computer simulations, we propose the idea of maximizing the time between fracturing in the microseismic volume of a recently fractured region (within operational constraints). The time required for the IU fractures to close can be estimated from our models and varies on the basis of the reservoir and fluid properties from several hours to days. One example of how this is accomplished in practice is zipper fractures. However, our work suggests that there also may be other fracture-sequencing strategies for accomplishing this. [ABSTRACT FROM AUTHOR]

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

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., (Copyright © 2024 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Objective: Patients with human papillomavirus DNA positive (HPVDNA + ) and p16 ink4a 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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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.

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 3‑kinase (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 Administration‑approved PI3K, cyclin‑dependent kinase‑4/6 (CDK4/6), poly‑ADP‑ribose‑polymerase (PARP) and WEE1 G2 checkpoint kinase (WEE1) inhibitors (BYL719, PD‑0332991, BMN673 and MK‑1775, respectively), were used to treat NB cell lines SK‑N‑AS, SK‑N‑BE(2)‑C, SK‑N‑DZ, SK‑N‑FI and SK‑N‑SH and viability (assessed by WST‑1 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 PD‑0332991 or BMN673 with MK‑1775 resulted in additive or synergistic effects in most cell lines., except for SK‑N‑SH for the former and for SK‑N‑AS for the latter. Moreover, combining MK‑1775 and BMN673 decreased the numbers of cells in S phase to a greater extent than either drug alone, while when combining PD‑0332991 and BYL719 the observed effect was close to that of PD‑0332991 alone. To summarize, PI3K and CDK4/6 or PARP and WEE1 exhibited synergistic anti‑NB effects and lower doses of the inhibitors could be utilized, thereby potentially reducing adverse side effects.

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., (Copyright © 2023 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Medulloblastoma (MB), grouped as either WNT‑activated, 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 side‑effects. The present study therefore examined the effects of the poly‑ADP‑ribose polymerase (PARP) and WEE1‑like protein kinase (WEE1) inhibitors, BMN673 and MK‑1775, respectively, alone or in combination on four MB cell lines. More specifically, the MB cell lines, DAOY, UW228‑3, MED8A and D425, were tested for their sensitivity to BMN673 and MK‑1775 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 MK‑1775 exerted dose‑dependent inhibitory effects on the viability of almost all MB cell lines. Notably, when BMN673 and MK‑1775 were used in combination, synergistic effects were noted in the SHH group cell lines (DAOY and UW228‑3), but not in the already WEE1‑sensitive 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 UW228‑3 cells in the S and G2/M phases, with the UW228‑3 cells exhibiting a greater delay. To conclude, MK‑1775 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 MK‑1775 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.

Human papillomavirus (HPV)-positive oropharyngeal squamous cell carcinoma (OPSCC) is associated with a favourable prognosis. It has therefore been suggested that treatment should be individualized and separated by HPV status. However, additional prognostic markers are still needed before treatment can be individualized for this patient group. For this purpose, all patients diagnosed with HPV and p16-positive OPSCC in Stockholm 2000-2009, identified as having a partial/nonresponse to treatment and having viable tumour cells in their neck specimen with material available were categorized as cases. These were matched to controls (complete responders), and the differences in the gene expression were analysed. Two separate verification cohorts were identified including patients with HPV- and p16-positive OPSCC, and the data from the case-control study were verified by qPCR and immunohistochemistry (IHC) in the respective cohorts. A separation of gene expression in correlation with survival was observed in the case-control study, and FGF11 expression was identified as significantly differently expressed between the two groups. The prognostic role of FGF11 was validated in the two cohorts on the RNA and protein levels, respectively. Taken together, our findings suggest that FGF11 may indicate a poor prognosis in HPV-positive OPSCC and may serve as a prognostic biomarker.

Human papillomavirus positive (HPV + ) tonsillar and base of tongue cancer (TSCC/BOTSCC) is rising in incidence, but chemoradiotherapy is not curative for all. Therefore, targeted therapy with PI3K (BYL719), PARP (BMN-673), and WEE1 (MK-1775) inhibitors alone or combined was pursued with or without 10 Gy and their effects were analyzed by viability, proliferation, and cytotoxicity assays on the TSCC/BOTSCC cell lines HPV + UPCI-SCC-154 and HPV - UT-SCC-60A. Effective single drug/10 Gy combinations were validated on additional TSCC lines. Finally, APR-246 was assessed on several TSCC/BOTSCC cell lines. BYL719, BMN-673, and MK-1775 treatments induced dose dependent responses in HPV + UPCI-SCC-154 and HPV - UT-SCC-60A and when combined with 10 Gy, synergistic effects were disclosed, as was also the case upon validation. Using BYL719/BMN-673, BYL719/MK-1775, or BMN-673/MK-1775 combinations on HPV + UPCI-SCC-154 and HPV - UT-SCC-60A also induced synergy compared to single drug administrations, but adding 10 Gy to these synergistic drug combinations had no further major effects. Low APR-246 concentrations had limited usefulness. To conclude, synergistic effects were disclosed when complementing single BYL719 BMN-673 and MK-1775 administrations with 10 Gy or when combining the inhibitors, while adding 10 Gy to the latter did not further enhance their already additive/synergistic effects. APR-246 was suboptimal in the present context.

Objectives: Tumor cells release fragments of their DNA into the circulation, so called cell-free tumor DNA (ctDNA) or liquid biopsy. Here, we analyze if cell-free human papillomavirus DNA (ctHPV DNA) is detectable before, during and after treatment, in patients with cervical cancer or pre-malignant lesions that may develop into cervical cancer, and whether ctHPV DNA levels were correlated to patient or tumor characteristics and outcome. Furthermore, total cell-free DNA load is studied using cfAlbumin DNA as a surrogate marker., Methods: 18 patients with locally advanced CC (LACC), 15 patients with early stage CC (ESCC) and 21 patients with pre-malignant lesions, all with verified HPV16, 18 or 45-positive lesions, were included. Pre- during- and post-treatment plasma were tested for HPV16, 18 & 45 and total cfDNA load using droplet digital PCR., Results: ctHPV DNA was found in 94.4% and 26.7% of pre-treatment plasma of patients with LACC and ESCC respectively, while all samples from patients with pre-malignant lesions were negative. Higher levels of ctHPV DNA were correlated to higher FIGO2018 stage. Patients with LACC and persistent ctHPV DNA at end-of-treatment had significantly worse progression-free survival (PFS) than patients who had cleared the ctHPV DNA (p = 0.007). Patients with total ctDNA-levels above median in pre-treatment plasma had a worse PFS (p = 0.026), compared to patients with total ctDNA-levels below median., Conclusion: ctHPV DNA is a promising prognostic biomarker in locally advanced cervical cancer that should be studied further for clinical use., Competing Interests: Declaration of Competing Interest The authors have no relevant conflicts of interest to disclose., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Human papillomavirus positive (HPV + ) tonsillar and base of tongue squamous cell carcinoma (TSCC/BOTSCC) have a favorable outcome, but upon relapse, survival is poor and new therapeutical options are needed. Recently, we found synergistic effects by combining the food and drug administration approved (FDA) phosphoinositide 3-kinase (PI3K) and fibroblast-growth-factor-receptor (FGFR) inhibitors BYL719 and JNJ-42756493 on TSCC cell lines. Here this approach was extended and Cyclin-Dependent-Kinase-4/6 (CDK4/6) and Poly-ADP-ribose-polymerase (PARP) and WEE1 inhibitors PD-0332991, and MK-1775 respectively were also examined. HPV + CU-OP-2, -3, -20, and HPV - CU-OP-17 TSCC cell lines were treated with either BYL719 and JNJ-42756493, PD-0332991 BMN-673 and MK-1775 alone or in different combinations. Viability, proliferation, and cytotoxicity were followed by WST-1 assays and the IncuCyte S3 Live ® Cell Analysis System. All inhibitors presented dose-dependent inhibitory effects on tested TSCC lines. Synergy was frequently obtained when combining CDK4/6 with PI3K inhibitors, but only sometimes or rarely when combining CDK4/6 with FGFR inhibitors or PARP with WEE1 inhibitors. To conclude, using CDK4/6 with PI3K or FGFR inhibitors, especially PD-0332991 with BYL719 presented synergy and enhanced the decrease of viability considerably, while although dose dependent responses were obtained with PARP and WEE1 inhibitors (BMN-673 and MK-1775 resp.), synergy was rarely disclosed.

An aetiological role of human papillomavirus (HPV) and/or human polyomaviruses (HPyVs) has been proposed in adenoid cystic carcinoma (AdCC). Moreover, HPV-related multiphenotypic carcinoma (HMSC) was recently introduced as an emerging entity of the sinonasal region. Here, we primarily want to study the role of HPV/HPyV in a large AdCC cohort and, secondly, possibly identify and characterize HMSC. Tumour DNA from 68 patients initially diagnosed with AdCC between 2000 and 2012 was, therefore, tested for 27 HPV types and 10 HPyVs. HPV DNA-positive samples were micromorphologically re-evaluated, further stained for p16 INK4a , S100, p63 and CD117 and tested for the presence of the MYB-NFIB fusion transcript. Notably, no samples were HPyV-positive, while one sinonasal and two tonsillar carcinomas were HPV- and p16-positive. After re-evaluating the micromorphology, immunohistochemistry and presence of fusion transcripts, all tumours had the same appearance and fitted within the diagnosis of HMSC, but in all these three cases, the morphology of the HMSC and basaloid squamous cell carcinoma was overlapping. We conclude that HPV and HPyV have no major role in AdCC. However, based on our data, we also suggest that HMSC should be considered as a basaloid variant of squamous cell carcinoma, and not its own entity, until better characterized.

To identify predictive/targetable markers in human papillomavirus positive (HPV + ) tonsillar and base of tongue cancer (TSCC/BOTSCC), whole-exome sequencing (WES) of tumours of patients with/without recurrence was performed. Forty primary tumours and adjacent normal tissue were separated by micro-dissection from formalin-fixed paraffin-embedded tissue from patients treated with curative intent 2000-2014 at Karolinska University Hospital. Successful sequencing was obtained in primary tumours of 18 patients without and primaries of 17 with local or distant recurrence, as well as in 10 corresponding recurrences (i.e., five local relapses and five distant metastases) from these 17 patients. One variant-a high-impact deletion in the CDC27 gene-was observed only in primaries of 5/17 patients that had a recurrence after full treatment but in none of those without recurrence. In addition, 3 variants and 26 mutated genes, including CDC27 , BCLAF1 and AQP7 , were present in at least 30% of all primary tumours independent of prognosis. To conclude, a CDC27 deletion was specific and found in ~30% of samples from patients with a local relapse/distant metastasis and could, therefore, potentially be a prospective marker to predict prognosis. Commonly mutated genes, such as BCLAF1 , should be further studied in the context of targeted therapy.

Objectives: Medulloblastoma (MB) is treated with surgery and chemotherapy, with or without irradiation, but unfortunately >20% of the patients are not cured, and treatment comes with serious long-term side effects, so novel treatments are urgently needed. Phosphoinositide 3-kinases (PI3K), fibroblast growth factor receptors (FGFR), and cyclin-D kinases (CDK) play critical roles in cancer, and especially PI3K is crucial in MB, so here targeted therapies against them were explored., Methods: MB cell lines DAOY and UW228-3 were exposed to PI3K (BYL719), FGFR (JNJ-42756493), and CDK4/6 (PD-0332991) inhibitors, as single or combined treatments, and their viability, cell confluence, apoptosis, and cytotoxicity were examined. Moreover, the inhibitors were combined with cisplatin, vincristine, or irradiation., Results: Single treatments with FGFR, PI3K, or CDK4/6 inhibitors decreased viability and proliferation slightly; however, when combining two inhibitors, or the inhibitors with irradiation, sensitivity was enhanced and lower doses could be used. A more complex pattern was obtained when combining the inhibitors with cisplatin and vincristine., Conclusions: The data suggest that combination treatments with PI3K, FGFR, and CDK4/6 inhibitors for MB could be beneficial and their use should be pursued further. Likewise, their combination with irradiation gave positive effects, while the addition of cisplatin and vincristine resulted in more complex patterns, which need to be investigated further., Competing Interests: The 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., (Copyright © 2021 Lukoseviciute, Maier, Poulou-Sidiropoulou, Rosendahl, Holzhauser, Dalianis and Kostopoulou.)

The incidence of Human-papillomavirus-positive (HPV + ) tonsillar and base-of-tongue squamous cell carcinoma (TSCC and BOTSCC, respectively) is increasing epidemically, but they have better prognosis than equivalent HPV-negative (HPV - ) cancers, with roughly 80% vs. 50% 3-year disease-free survival, respectively. The majority of HPV + TSCC and BOTSCC patients therefore most likely do not require the intensified chemoradiotherapy given today to head and neck cancer patients and would with de-escalated therapy avoid several severe side effects. Moreover, for those with poor prognosis, survival has not improved, so better-tailored alternatives are urgently needed. In line with refined personalized medicine, recent studies have focused on identifying predictive markers and driver cancer genes useful for better stratifying patient treatment as well as for targeted therapy. This review presents some of these endeavors and briefly describes some recent experimental progress and some clinical trials with targeted therapy.

Objectives: Human papillomavirus positive (HPV + ) tonsillar and base of tongue squamous cell carcinoma (TSCC/BOTSCC), the major subsites of oropharyngeal squamous cell carcinoma (OPSCC) have favorable outcome, but upon relapse, outcome is poor and new therapies needed. Since, phosphatidyl-inositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA) and fibroblast-growth-factor-receptor-3 (FGFR3) mutations often occur in such tumors, here, we tested targeted therapy directed to such genes in TSCC/BOTSCC cell lines. We also combined the two types of inhibitors with each other, and cisplatin or docetaxel that are used clinically., Methods: The HPV + CU-OP-2, -3, -20, UPCI-SCC-154, and HPV - CU-OP-17 and UT-SCC-60A cell lines were first tested for common PIK3CA/FGFR3 mutations by competitive-allele-specific TaqMan-PCR. They were then treated with the food and drug administration (FDA) approved drugs, alpelisib (BYL719) and erdafitinib (JNJ-42756493) alone and in combination with cisplatin or docetaxel. Viability, proliferation, apoptosis and cytotoxicity responses were thereafter followed by WST-1 assays and the IncuCyte S3 Live ® Cell Analysis System., Results: HPV + CU-OP-2 had a pS249C-FGFR3, and like CU-OP-20, a pE545K-PIK3CA mutation, while no other lines had such mutations. Irrespectively, dose dependent responses to all PI3K/FGFR inhibitors were obtained, and upon combining the inhibitors, positive effects were observed. Cisplatin and docetaxel also induced dose dependent responses, and upon combination with the inhibitors, both positive and neutral effects were found., Conclusions: The data suggest that FDA approved drugs alpelisib and erdafitinib efficiently inhibit TSCC/BOTSCC cell line growth, especially when combined irrespective of presence of corresponding mutations and should be further explored, for use upon recurrent disease., Competing Interests: The 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., (Copyright © 2021 Holzhauser, Wild, Zupancic, Ursu, Bersani, Näsman, Kostopoulou and Dalianis.)

Neuroblastoma (NB) is a heterogenous disease with treatment varying from observation for low‑risk tumors, to extensive therapy with chemotherapy, surgery, radiotherapy, and autologous bone‑marrow‑transplantation and immunotherapy. However, a high frequency of primary‑chemo‑refractory disease and recurrences urgently require novel treatment strategies. The present study therefore investigated the anti‑NB efficacy of the recently FDA‑approved phosphoinositide 3‑kinase (PI3K) and fibroblast growth factor receptor (FGFR) inhibitors, alpelisib (BYL719) and erdafitinib (JNJ‑42756493), alone and in combination with or without cisplatin, vincristine, or doxorubicin on 5 NB cell lines. For this purpose, the NB cell lines, SK‑N‑AS, SK‑N‑BE(2)‑C, SK‑N‑DZ, SK‑N‑FI and SK‑N‑SH (where SK‑N‑DZ had a deletion of PIK3C2G and none had FGFR mutations according to the Cancer Program's Dependency Map, although some were chemoresistant), were tested for their sensitivity to FDA‑approved inhibitors alone or in combination, or together with cytostatic drugs by viability, cytotoxicity, apoptosis and proliferation assays. The results revealed that monotherapy with alpelisib or erdafitinib resulted in a dose‑dependent inhibition of cell viability and proliferation. Notably, the combined use of PI3K and FGFR inhibitors resulted in an enhanced efficacy, while their combined use with the canonical cytotoxic agents, cisplatin, vincristine and doxorubicin, resulted in variable synergistic, additive and antagonistic effects. Collectively, the present study provides pre‑clinical evidence that PI3K and FGFR inhibitors exhibit promising anti‑NB activity. The data presented herein also indicate that the incorporation of these inhibitors into chemotherapeutic regimens requires careful consideration and further research in order to obtain a beneficial efficacy. Nevertheless, the addition of PI3K and FGFR inhibitors to the treatment arsenal might reduce the occurrence of refractory and relapsing disease in NB without FGFR and PI3K mutations.

Background: Oropharyngeal squamous cell carcinoma (OPSCC) is dominated by tonsillar and tongue base carcinomas (TSCC/BOTSCC), but there are carcinomas at other sites, such as uvula/soft palate/pharyngeal wall here defined as other OPSCC. Human papillomavirus (HPV) positive TSCC/BOTSCC have favorable outcome, and the TNM-classification separates OPSCC into HPV mediated (p16 INK4a overexpressing, p16+) and HPV unrelated OPSCC (p16 INK4a non-overexpressing, p16-) cancer, but the prognostic role of p16+ in other OPSCC is unclear., Aims/objectives: This study therefore aimed to further investigate the prognostic role of p16+, presence of HPV DNA, or both combined in other OPSCC., Material and Methods: 195 other OPSCC, from patients diagnosed 2000-2018 were tested for p16, and/or presence of HPV DNA and the data correlated to outcome., Results: Neither overall survival, nor disease free survival correlated to presence of p16+ or HPV DNA in other OPSCC. p16+ and HPV DNA presence were correlated ( p  < .0001), but the sensitivity of p16 as a surrogate marker for presence of HPV DNA was low (49%)., Conclusions and Significance: The data suggest that p16+ (and p16+/HPV DNA) positive other OPSCC should be analyzed cautiously and possibly separately from the HPV mediated OPSCC staging group.

Background: TNM-8 staging separates oropharyngeal squamous cell carcinomas (OPSCC) into human papillomavirus (HPV)-mediated and -unrelated OPSCC based on p16INK4a overexpression (p16+), as surrogate marker for HPV. However, OPSCC is histologically and clinically heterogenous including tonsillar and base of tongue squamous cell carcinomas (TSCC and BOTSCC respectively), and carcinomas of soft palate and walls (otherOPSCC). The significance of HPV is established in TSCC/BOTSCC, while its role in otherOPSCC is unclear, which is not considered in TNM-8. Here, p16+ was therefore evaluated in relation to overall survival (OS) and tumor stage per OPSCC subsite., Patients and Methods: All 932 patients, treated with curative intent in Stockholm 2000-2016 with OPSCC, previously analyzed for p16 expression, were included. Clinical data, including stage and OS, was collected retrospectively., Results: Patients with p16+ otherOPSCC had significantly poorer OS compared to patients with p16+ TSCC/BOTSCC (p = 0.005) and their survival was similar to that of patients with p16-otherOPSCC/TSCC/BOTSCC. Moreover, patients with TNM-8 stage I-II and p16+ otherOPSCC had a significant poorer OS compared to patients with p16+ TSCC/BOTSCC and similar stage (p = 0.02). Lastly, patients with otherOPSCC and low TNM-7 stage had a significant better OS, as compared to those with a high stage (p = 0.019) while no hazard discrimination was observed with TNM-7 in TSCC/BOTSCC., Conclusion: Our results indicate a risk of misclassification of patients with otherOPSCC and low TNM-8 stage. We suggest that p16 should only be evaluated in TSCC/BOTSCC and that patients with otherOPSCC should all be staged as patients with HPV-unrelated (p16-) OPSCC., Competing Interests: Conflict of interest statement The authors have declared no conflicts of interest., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Human papillomavirus‑positive (HPV+) oropharyngeal squamous cell carcinoma (OPSCC) has increased in incidence and has a much better prognosis than HPV‑negative (HPV‑) OPSCC with radiotherapy alone, but exactly why is unknown. The present study therefore aimed to further examine the sensitivity and possible changes in gene expression of several HPV+ and HPV‑ OPSCC, including various novel cell lines, upon ionizing irradiation (IR). Previously established HPV+ UM‑SCC‑47, UPCI‑SCC‑90, CU‑OP‑2, CU‑OP‑3 and HPV‑ UM‑SCC‑4, UM‑SCC‑6, UM‑SCC‑74a, UM‑SCC‑19 and newly established CU‑OP‑17 and CU‑OP‑20, characterised here, were subjected to 0‑6 Gy. Surviving fractions of each cell line were tested by clonogenic assays, and irregularities in cell cycle responses were examined by flow cytometry, while changes in gene expression were followed by mRNA sequencing. HPV+ OPSCC cell lines showed greater variation in sensitivity to ionizing irradiation (IR) and tended to be more sensitive than HPV‑ OPSCC cell lines. However, their IR sensitivity was not correlated to the proportion of cells in G2 arrest, and HPV‑ cell lines generally showed lower increases in G2 after IR. Upon IR with 2 Gy, mRNA sequencing revealed an increase in minor HPV integration sites in HPV+ cell lines, and some changes in gene expression in OPSCC cell lines, but not primarily those associated with DNA repair. To conclude, HPV+ OPSCC cell lines showed greater variation in their sensitivity to IR, with some that were radioresistant, but overall the HPV+ OPSCC group still tended to be more sensitive to IR than the HPV‑ OPSCC group. In addition, HPV+ OPSCC lines were more frequently in G2 as compared to HPV‑ cell lines, but the increase in G2 arrest upon IR in HPV+ OPSCC was not correlated to sensitivity to IR. Increases in minor HPV integration sites and changes in gene expression were also demonstrated after irradiation with 2 Gy.

Background/aim: Medulloblastoma (MB) accounts for ~20% of pediatric malignant central nervous system tumors. Treatment strategies, including surgery, radiation therapy and/or chemotherapy, are effective, but recurrence and metastasis frequently occur. Therefore, novel therapies are required. Herein, the effects of fibroblast growth factor receptor (FGFR) and phosphoinositide 3-kinase (PI3K) inhibitors on MB cells lines were evaluated., Materials and Methods: MB cell lines (UW228-3, DAOY, Med8a, D425, D283) were tested for sensitivity to FGFR (AZD4547) and PI3K (BEZ235 and BYL719) inhibitors by viability, cytotoxicity, apoptosis, and proliferation assays., Results: Single treatments with FGFR and PI3K inhibitors decreased viability and proliferation in a dose-dependent pattern in most cell lines. Combinination of the two type of drugs, increased sensitivity, especially of the most resistant cell line UW228-3., Conclusion: Combination treatments with FGFR and PI3K inhibitors were superior to single treatments with FGFR and PI3K inhibitors, especially with BEZ235, for MB cell lines., (Copyright© 2020, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Fibroblast growth factor receptor (FGFR)3 and phosphatidylinositol‑4,5‑bisphosphate 3‑kinase, catalytic subunit alpha (PIK3CA) mutations are found in various types of cancer, such as urinary bladder cancer, human papillomavirus‑positive tonsillar and base of the tongue squamous cell carcinoma, breast cancer and some childhood sarcomas. Several drugs can target these genes, some of which have been used for the treatment of urinary bladder cancer. Much less is known about childhood cancer. For this reason, the present study investigated the presence of such mutations in neuroblastomas (NBs) and tested NB cell lines for sensitivity to FGFR and phosphoinositide 3‑kinase (PI3K) inhibitors. In total, 29 NBs were examined for the presence of the three most common FGFR3 and PIK3CA mutations using a competitive allele‑specific TaqMan PCR (CAST‑PCR). Furthermore, the SK‑N‑AS, SK‑N‑BE(2)‑C, SK‑N‑DZ, SK‑N‑FI and SK‑N‑SH NB cell lines (where SK‑N‑DZ had a deletion of PIK3C2G, none had FGFR mutations according to the Cancer Program's Dependency Map, but some were chemoresistant), were tested for sensitivity to FGFR (AZD4547) and PI3K (BEZ235 and BKM120) inhibitors by viability, cytotoxicity, apoptosis and proliferation assays. CAST‑PCR detected one FGFR3 mutation in 1/29 NBs. Following treatment with FGFR and PI3K inhibitors, a decrease in viability and proliferation was observed in the majority, but not all, the cell lines. Following combination treatment with both drugs, the sensitivity of all cell lines was increased. On the whole, the findings of this study demonstrate that FGFR3 and PIK3CA mutations are uncommon in patients with NB. However, certain NB cell lines are rather sensitive to both FGFR and PI3K inhibitors alone, and even more so when the different drugs are used in combination.

Human papillomavirus positive (HPV + ) tonsillar and base of tongue squamous cell carcinoma (TSCC/BOTSCC) have better outcomes than corresponding HPV - negative (HPV - ) cancer cases. Our previous study demonstrated that fibroblast growth factor receptor 3 (FGFR3) and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit a (PIK3CA) are often mutated in HPV + cancer. To investigate whether targeted therapy is an option for TSCC/BOTSCC, two HPV + and one HPV - TSCC/BOTSCC cell lines were tested for their sensitivity towards FGFR and PI3K inhibitors. The HPV + cell lines UM-SCC-47 and UPCI-SCC-154, and the HPV - cell line UT-SSC-60A were tested by competitive allele-specific TaqMan-PCR for presence/absence of frequently occurring FGFR3 and PIK3CA mutations. All cells were then treated with FGFR inhibitor AZD4547 and PI3K inhibitors BEZ235 and BKM120 alone, or with AZD4547 and BEZ235 in combination. Viability was analyzed using a WST-1 assay, cytotoxicity tested by a CellTox Green cytotoxicity assay, apoptosis analyzed by a Caspase Glo 3/7 assay and proliferation examined with the xCELLigence system. HPV + UM-SCC-47 and UPCI-SCC-154 cells, and HPV - UT-SSC-60A cells, did not exhibit any common FGFR3 or PIK3CA mutations, but were all sensitive to FGFR inhibitor AZD4547 and PI3K inhibitors BEZ235 and BKM120. Notably, HPV + UPCI-SCC-154 cells were more sensitive than the other two cell lines. Furthermore, when AZD4547 and BEZ235 treatment was combined in HPV + UPCI-SCC-154 and HPV - UT-SSC-60A cells, potentiated combination effects were observed. HPV + UM-SCC-47 and UPCI-SCC-154 cells, and HPV - UT-SSC-60A cells had no common FGFR3 or PIK3CA mutations, but were sensitive to FGFR inhibitor AZD4547, and PI3K inhibitors BEZ235 and BKM120. Furthermore, the latter two cell lines were particularly sensitive to combinations of AZD4547 and BEZ235., (Copyright © 2019, Spandidos Publications.)

Background: Tonsillar and base of tongue squamous cell carcinoma (TSCC/BOTSCC) has increased. In Stockholm, the proportion of human papillomavirus (HPV)-positive cases and the incidence of TSCC rose between 1970 and 2006 then stabilized. Here, HPV-prevalence, and TSCC/BOTSCC incidence 2000-2016, in Stockholm and Sweden were followed., Methods: Incidence data for 2000-2016 were obtained from the Swedish Cancer Registry. TSCC/BOTSCC biopsies, 2013-2016 from Stockholm, were examined for HPV DNA and p16 INK4a , or data obtained from medical reports. For cases 2000-2012, data were available from previous studies., Results: The incidence of TSCC/BOTSCC has continued to rise in Stockholm and Sweden 2000-2016, especially after 2008. HPV DNA and p16 INK4a analysis was determined for 795 Stockholm cases from 2000 to 2016, with 72% being HPV DNA and p16 INK4a positive 2013-2016, and 70% positive 2000-2016., Conclusion: During 2000-2016, especially after 2008, the incidence of TSCC/BOTSCC has continued to increase in Stockholm and Sweden, with an HPV-prevalence of approximately 70% in Stockholm., (© 2018 Wiley Periodicals, Inc.)

The incidence of Human Papillomavirus (HPV)-associated oropharyngeal squamous cell carcinoma (OPSCC) is increasing rapidly in the UK. Patients with HPV-positive OPSCC generally show superior clinical responses relative to HPV-negative patients. We hypothesised that these superior responses could be associated with defective repair of DNA double strand breaks (DSB). The study aimed to determine whether defective DNA repair could be associated with sensitivity to inhibition of DNA repair using the PARP inhibitor Olaparib. Sensitivity to Olaparib, and induction and repair of DNA damage, were assessed in a panel of 8 OPSCC cell-lines, including 2 novel HPV-positive lines. Effects on cell cycle distribution and levels of PARP1 and p53 were quantified. RNA-sequencing was used to assess differences in activity of DNA repair pathways. Two HPV-positive OPSCC lines were sensitive to Olaparib at potentially therapeutic doses (0.1-0.5 μM). Two HPV-negative lines were sensitive at an intermediate dose. Four other lines, derived from HPV-positive and HPV-negative tumours, were resistant to PARP inhibition. Only one cell-line, UPCISCC90, showed results consistent with the original hypothesis i.e. that in HPV-positive cells, treatment with Olaparib would cause accumulation of DSB, resulting in cell cycle arrest. There was no evidence that HPV-positive tumours exhibit defective repair of DSB. However, the data suggest that a subset of OPSCC may be susceptible to PARP-inhibitor based therapy., Competing Interests: The authors have declared that no competing interests exist.

Aim: To investigate whether the rise during the past decades in the incidence of tonsillar and base of tongue squamous cell carcinoma (TSCC and BOTSCC) and the proportion of human papillomavirus (HPV) positive cancer has continued in Stockholm., Patients and Methods: Pre-treatment biopsies (n=252) available from 280 patients diagnosed with TSCC and BOTSCC during 2008-2012 in the County of Stockholm were tested for HPV DNA by a multiplex bead-based assay. Incidence records were acquired from the Swedish Cancer Registry. The data obtained were evaluated together with previous figures from 1970 to 2007., Results: HPV DNA was present in 186/252 (74%) of TSCC and BOTSCC biopsies obtained during 2008-2012 in Stockholm. In this region the age-standardised incidence, including the prevalence of HPV-positive and HPV-negative TSCC stabilised 2007-2012 compared to 2000-2006, while for BOTSCC throughout 1998-2012 the same parameters increased moderately (p<0.05, for all). In parallel, from 2000 to 2006 through 2007-2012 in Sweden, the age-standardised incidence of both TSCC and BOTSCC continued to rise (p=0.012 and p=0.001 respectively)., Conclusion: During 2000-2012 the age-standardised incidence and the proportion of HPV-positive TSCC have stabilised at a high level, while the proportion of HPV-negative cancer has remained at a low level in Stockholm, whereas for BOTSCC all parameters are increasing moderately. In contrast, in Sweden the incidence of both TSCC and BOTSCC is still increasing. We hypothesise that the HPV epidemic could be stabilising, first for TSCC, but so far not for BOTSCC, in e.g. some urban areas, while previous trends for both tumours persist at other geographic locations., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

APC(min/+) mice, carrying a nonsense mutation in the adenomatous polyposis coli (APC) gene, appear as a perfect model to study development or therapy of intestinal neoplasia. We tested whether the flavonoid flavone is able to affect adenoma development in APC(min/+) mice. Tumor sizes were significantly increased by flavone selectively in small intestine. This was associated with reduced cell numbers displaying cleaved caspase-3 and enhanced expression of phosphoglycoprotein (P-gp). However, according to great variability in P-gp expression in all parts of mice intestines, an association between expression of P-gp and inhibition of apoptosis was demonstrated in human Caco-2 colorectal cancer cells.

ATP-driven efflux pumps such as phosphoglycoprotein-170 (P-gp), multidrug-resistance-associated protein-2 (MRP-2), or breast cancer resistance protein (BCRP) play a crucial role in limiting the efficacy of tumor pharmacotherapy. Selected flavonoids have been suggested to inhibit individual efflux-transporters and to act therefore as multidrug-resistance reversing agents. In the present study it is shown that the flavonoid chrysin acts as a potent inhibitor of P-gp, MRP-2, and BCRP in Caco-2 colon carcinoma cells. As a consequence, cells accumulated higher rates of the apoptosis-inducing chemotherapeutic topotecan in the presence of chrysin, even though under these conditions the expression of the transporters was markedly increased. Interestingly, in spite of the enhanced cellular drug accumulation the topotecan-induced apoptosis, assessed according to DNA-fragmentation, chromatin condensation, and by determination of sub-G1 peaks using fluorescence-assisted-cell sorting (FACS), was potently inhibited by chrysin. Suggested transport-independent apoptosis inhibiting activities of ATP-binding cassette (ABC)-transporters, such as the inhibition of caspases, were shown to be necessary for the inhibition of topotecan-induced apoptosis and were found to be associated with stabilization of beta-catenin especially in the cytosol. Inhibition of topotecan-induced intracellular acidification, however, was proven not to prevent caspase-activation and apoptosis. In conclusion, our studies show that chrysin in spite of raising the cellular concentrations of topotecan potently inhibits the apoptosis-inducing activities of the anti-tumor drug. Inhibition of caspase-activation was identified as the underlying mechanism and is suggested to be caused by transport-independent functions of ABC-transporters., (2010 Elsevier Inc. All rights reserved.)