Cost-effectiveness of primary human papillomavirus triage approaches among vaccinated women in Norway: A model-based analysis.

As Norway considers revising triage approaches following their first adolescent cohort with human papillomavirus (HPV) vaccination entering the cervical cancer screening program, we analyzed the health impact and cost-effectiveness of alternative primary HPV triage approaches for women initiating cervical cancer screening in 2023. We used a multimodeling approach that captured HPV transmission and cervical carcinogenesis to evaluate the health benefits, harms and cost-effectiveness of alternative extended genotyping and age-based triage strategies under five-yearly primary HPV testing (including the status-quo screening strategy in Norway) for women born in 1998 (ie, age 25 in 2023). We examined 35 strategies that varied alternative groupings of high-risk HPV genotypes ("high-risk" genotypes; "medium-risk" genotypes or "intermediate-risk" genotypes), number and types of HPV included in each group, management of HPV-positive women to direct colposcopy or active surveillance, wait time for re-testing and age at which the HPV triage algorithm switched from less to more intensive strategies. Given the range of benchmarks for severity-specific cost-effectiveness thresholds in Norway, we found that the preferred strategy for vaccinated women aged 25 years in 2023 involved an age-based switch from a less to more intensive follow-up algorithm at age 30 or 35 years with HPV-16/18 genotypes in the "high-risk" group. The two potentially cost-effective strategies could reduce the number of colposcopies compared to current guidelines and simultaneously improve health benefits. Using age to guide primary HPV triage, paired with selective HPV genotype and follow-up time for re-testing, could improve both the cervical cancer program effectiveness and efficiency.

Randomized Implementation of a Primary Human Papillomavirus Testing-based Cervical Cancer Screening Protocol for Women 34 to 69 Years in Norway.

BACKGROUND: Cervical cancer screening programs are facing a programmatic shift where screening protocol based on human papillomavirus testing (HPV-Screening protocol) is replacing the liquid-based cytology (LBC-Screening protocol). For safe technology transfer within the nationwide screening programme in Norway, HPV-Screening protocol was implemented randomized to compare the real-world effectiveness of HPV-Screening protocol and LBC-Screening protocol at the first screening round. METHODS: Among 302,295 women ages 34 to 69 years scheduled to attend screening from February 2015 to June 2017, 157,447 attended. A total of 77,207 were randomly allocated to the HPV-Screening protocol and 80,240 were allocated to the LBC-Screening protocol. All women were followed up for 18 months. RESULTS: The HPV-Screening protocol resulted in a relative increase of 60% in the detection of cervical intraepithelial neoplasia (CIN) grade 2 or worse [risk ratio (RR) = 1.6, 95% confidence interval (CI) = 1.5-1.7], 40% in CIN grade 3 or worse (RR = 1.4, 95% CI = 1.3-1.6), 40% in cancer (RR = 1.4, 95% CI = 1.0-2.1), and 60% in colposcopy referrals (RR = 1.6, 95% CI = 1.5-1.6) compared with LBC-Screening. The performance of both protocols was age dependent, being more effective in women ages under 50 years. CONCLUSIONS: The HPV-Screening protocol was well accepted by women in Norway and detected more CIN2, CIN3, and cancers compared with the LBC-Screening protocol. IMPACT: A randomized implementation of the HPV-Screening protocol with real-world assessment enabled a gradual, quality assured, and safe technology transition. HPV-based screening protocol may further be improved by using HPV genotyping and age-specific referral algorithms.

A Gene Signature Identifying CIN3 Regression and Cervical Cancer Survival.

The purpose of this study was to establish a gene signature that may predict CIN3 regression and that may aid in selecting patients who may safely refrain from conization. Oncomine mRNA data including 398 immune-related genes from 21 lesions with confirmed regression and 28 with persistent CIN3 were compared. L1000 mRNA data from a cervical cancer cohort was available for validation (n = 239). Transcriptomic analyses identified TDO2 (p = 0.004), CCL5 (p < 0.001), CCL3 (p = 0.04), CD38 (p = 0.02), and PRF1 (p = 0.005) as upregulated, and LCK downregulated (p = 0.01) in CIN3 regression as compared to persistent CIN3 lesions. From these, a gene signature predicting CIN3 regression with a sensitivity of 91% (AUC = 0.85) was established. Transcriptomic analyses revealed proliferation as significantly linked to persistent CIN3. Within the cancer cohort, high regression signature score associated with immune activation by Gene Set enrichment Analyses (GSEA) and immune cell infiltration by histopathological evaluation (p < 0.001). Low signature score was associated with poor survival (p = 0.007) and large tumors (p = 0.01). In conclusion, the proposed six-gene signature predicts CIN regression and favorable cervical cancer prognosis and points to common drivers in precursors and cervical cancer lesions.

High-Grade Cervical Intraepithelial Neoplasia (CIN) Associates with Increased Proliferation and Attenuated Immune Signaling.

Implementation of high-risk human papilloma virus (HPV) screening and the increasing proportion of HPV vaccinated women in the screening program will reduce the percentage of HPV positive women with oncogenic potential. In search of more specific markers to identify women with high risk of cancer development, we used RNA sequencing to compare the transcriptomic immune-profile of 13 lesions with cervical intraepithelial neoplasia grade 3 (CIN3) or adenocarcinoma in situ (AIS) and 14 normal biopsies from women with detected HPV infections. In CIN3/AIS lesions as compared to normal tissue, 27 differential expressed genes were identified. Transcriptomic analysis revealed significantly higher expression of a number of genes related to proliferation, (CDKN2A, MELK, CDK1, MKI67, CCNB2, BUB1, FOXM1, CDKN3), but significantly lower expression of genes related to a favorable immune response (NCAM1, ARG1, CD160, IL18, CX3CL1). Compared to the RNA sequencing results, good correlation was achieved with relative quantitative PCR analysis for NCAM1 and CDKN2A. Quantification of NCAM1 positive cells with immunohistochemistry showed epithelial reduction of NCAM1 in CIN3/AIS lesions. In conclusion, NCAM1 and CDKN2A are two promising candidates to distinguish whether women are at high risk of developing cervical cancer and in need of frequent follow-up.

Real-world data on cervical cancer risk stratification by cytology and HPV genotype to inform the management of HPV-positive women in routine cervical screening.

BACKGROUND: HPV16/18 detection may improve cervical cancer risk stratification and better guide which HPV-positive women warrant immediate colposcopy/biopsy. We estimated risks of cervical precancer and cancer by HPV genotype and cytology during the implementation phase of primary HPV testing in Norway. METHODS: A total of 3111 women, aged 34-69 years, testing HPV-positive at baseline and undergoing cytology testing from February 2015 to April 2018 had data available for analysis. Risk estimates with 95% confidence intervals (95%CIs) of cervical intraepithelial neoplasia grade 3 or more severe (CIN3+) were estimated for cytology results and HPV genotypes (HPV16, HPV18, and other high-risk HPV). RESULTS: CIN3+ risks were higher for HPV16/18 than other high-risk HPV genotypes. Among women with any cytologic abnormality [atypical squamous cells of undetermined significance or worse], immediate risks were 57.8% (95%CI = 53.0-62.6%) for HPV16, 40.2% (95%CI = 32.3-49.2%) for HPV18, and 31.4% (95%CI = 28.7-34.3%) for other high-risk HPV. Among those with normal cytology, CIN3+ risks were 19.9% (95%CI = 15.0-26.1%) for HPV16 positives, 10.8% (95%CI = 5.6-20.5%) for HPV18 positives, and 5.5% (95%CI = 4.2-7.1%) for other high-risk HPV. CONCLUSIONS: The benefits and harms of managing women based on HPV positivity and cytology results can be better balanced by inclusion of HPV genotyping in screening and choosing more conservative management for other high-risk HPV compared to HPV16/18.

The legal framework for European cervical cancer screening programmes.

BACKGROUND: A comprehensive legal framework needs to be developed to run the health services and to regulate the information systems required to manage and to ensure the quality of cancer screening programmes. The aim of our study was to document and to compare the status of legal basis for cervical screening registration in European countries. METHODS: An electronic questionnaire including questions on governance, decision-making structures and legal framework was developed. The primary responses were collected by September 2016. RESULTS: We sent the questionnaire to representatives of 35 European countries (28 countries of the EU, with the United Kingdom included as 4 countries; 4 EFTA member countries: Iceland, Liechtenstein, Norway, and Switzerland); responses were collected from 33 countries. The legal framework makes it possible to personally invite individuals in 29 countries (88%). Systematic screening registration in an electronic registry is legally enshrined in 23 countries (70%). Individual linkage of records between screening and cancer registries is allowed in 19 of those countries. Linkage studies involving cancer and screening registries have been conducted in 15 countries. CONCLUSION: Although the majority of EU/EFTA countries have implemented population-based screening, only half of them have successfully performed record linkage studies, which are nevertheless a key recommendation for quality assurance of the entire screening process. The European legislation is open to the possibility of using health data for these purposes; however, member states themselves must recognize the public interest to create a legal basis, which would enable all the necessary functions for high-quality cancer screening programmes.

Psychological effect of cervical cancer screening when changing primary screening method from cytology to high-risk human papilloma virus testing.

From 2015, Norway has implemented high-risk human papilloma virus (hrHPV) testing in primary screening for cervical cancer. Women aged 34-69 years, living in four counties, have been pseudo-randomly assigned (1:1 randomization) to either hrHPV testing every 5 years (followed by cytology if hrHPV is positive), or cytology testing every 3 years (followed by hrHPV testing if low-grade cytology is detected). We compared anxiety and depression scores among participants by screening arm and results. In total, 1,008 women answered a structured questionnaire that included the validated Patient Health Questionnaire-4 (PHQ-4). The Relative Risk Ratio (RRR) of mild vs. normal anxiety and depression scores, and moderate/severe vs. normal anxiety and depression scores, were estimated by multinomial logistic regression with 95% confidence intervals (95% CIs). Compared to women who were screened with cytology, women randomized to hrHPV testing were not more likely to have mild anxiety and depression scores (RRR 0.96, CI 0.70-1.31) nor more likely to have moderate/severe anxiety and depression scores (RRR 1.14, CI 0.65-2.02). Women with five different combinations of abnormal screening test results were not more likely to have mild or moderate/severe vs. normal anxiety and depression scores than women with normal screening results. The likelihood of having abnormal long-term (4-24 months after the screening) anxiety or depression scores among women 34 years and older was not affected by screening method or screening results. The results of our study suggest that a change to hrHPV testing in primary screening would not increase psychological distress among participants.

hvTRA, a novel TRAIL receptor agonist, induces apoptosis and sustained growth retardation in melanoma.

In recent years, new treatment options for malignant melanoma patients have enhanced the overall survival for selected patients. Despite new hope, most melanoma patients still relapse with drug-resistant tumors or experience intrinsic resistance to the therapy. Therefore, novel treatment modalities beneficial for subgroups of patients are needed. TRAIL receptor agonists have been suggested as promising candidates for use in cancer treatment as they preferentially induce apoptosis in cancer cells. Unfortunately, the first generation of TRAIL receptor agonists showed poor clinical efficacy. hvTRA is a second-generation TRAIL receptor agonist with improved composition giving increased potency, and in the present study, we showed hvTRA-induced activation of apoptosis leading to an efficient and sustained reduction in melanoma cell growth in cell lines and xenograft models. Furthermore, the potential of hvTRA in a clinical setting was demonstrated by showing efficacy on tumor cells harvested from melanoma patients with lymph node metastasis in an ex vivo drug sensitivity assay. Inhibition of mutated BRAF has been shown to regulate proteins in the intrinsic apoptotic pathway, making the cells more susceptible for apoptosis induction. In an attempt to increase the efficacy of hvTRA, combination treatment with the mutated BRAF inhibitor vemurafenib was investigated. A synergistic effect by the combination was observed for several cell lines in vitro, and an initial cytotoxic effect was observed in vivo. Unfortunately, the initial increased reduction in tumor growth compared with hvTRA mono treatment was not sustained, and this was related to downregulation of the DR5 level by vemurafenib. Altogether, the presented data imply that hvTRA efficiently induce apoptosis and growth delay in melanoma models and patient material, and the potential of this TRAIL receptor agonist should be further evaluated for treatment of subgroups of melanoma patients.

Quality assurance of human papillomavirus (HPV) testing in the implementation of HPV primary screening in Norway: an inter-laboratory reproducibility study.

BACKGROUND: Human papillomavirus (HPV) testing as primary screening for cervical cancer is currently being implemented in Norway in a randomized controlled fashion, involving three laboratories. As part of the quality assurance programme of the implementation, an evaluation of the inter-laboratory reproducibility of the HPV test was initiated, to ensure satisfactory HPV test reliability in all three laboratories. METHODS: The HPV test used is the cobas 4800 HPV Test, detecting 14 high-risk types with individual HPV genotype results for HPV16 and HPV18. In addition to the three laboratories involved in the implementation, the Norwegian HPV reference laboratory was included as a fourth comparative laboratory. A stratified sample of 500 cervical liquid based cytology (LBC) samples was used in the evaluation, with an aim towards a high-risk HPV positivity of ~25%. Samples were collected at one laboratory, anonymized, aliquoted, and distributed to the other laboratories. RESULTS: Comparison of the test results of all four laboratories revealed a 95.6% agreement, an 86.3% positive agreement and a kappa value of 0.94 (95% CI 0.92-0.97). For negative cytology specimens, there was a 95.8% overall agreement, a 67.4% positive agreement, and a kappa value of 0.88 (95% CI 0.80-0.93). For abnormal cytology specimens, there was a 95.8% overall agreement, a 95.5% positive agreement, and a kappa value of 0.86 (95% CI 0.71-0.97). CONCLUSIONS: The study showed a high inter-laboratory reproducibility of HPV testing, implying satisfactory user performance and reliability in the laboratories involved in the implementation project. This is important knowledge and we recommend similar studies always to be performed prior to the introduction of new screening routines.

Fibroblast-induced switching to the mesenchymal-like phenotype and PI3K/mTOR signaling protects melanoma cells from BRAF inhibitors.

The knowledge on how tumor-associated stroma influences efficacy of anti-cancer therapy just started to emerge. Here we show that lung fibroblasts reduce melanoma sensitivity to the BRAF inhibitor (BRAFi) vemurafenib only if the two cell types are in close proximity. In the presence of fibroblasts, the adjacent melanoma cells acquire de-differentiated mesenchymal-like phenotype. Upon treatment with BRAFi, such melanoma cells maintain high levels of phospho ribosomal protein S6 (pS6), i.e. active mTOR signaling, which is suppressed in the BRAFi sensitive cells without stromal contacts. Inhibitors of PI3K/mTOR in combination with BRAFi eradicate pS6high cell subpopulations and potentiate anti-cancer effects in melanoma protected by the fibroblasts. mTOR and BRAF co-inhibition also delayed the development of early-stage lung metastases in vivo. In conclusion, we demonstrate that upon influence from fibroblasts, melanoma cells undergo a phenotype switch to the mesenchymal state, which can support PI3K/mTOR signaling. The lost sensitivity to BRAFi in such cells can be overcome by co-targeting PI3K/mTOR. This knowledge could be explored for designing BRAFi combination therapies aiming to eliminate both stroma-protected and non-protected counterparts of metastases.

Impact of scheduled appointments on cervical screening participation in Norway: a randomised intervention.

BACKGROUND: The main barrier to optimal effect in many established population-based screening programmes against cervical cancer is low participation. In Norway, a routine health service integrated population-based screening programme has been running since 1995, using open invitations and reminders. The aim of this randomised health service study was to pilot scheduled appointments and assess their potential for increased participation. METHODS: Within the national screening programme, we randomised 1087 women overdue for screening to receive invitations with scheduled appointments (intervention) or the standard open reminders (control). Letters were sent 2-4 weeks before the scheduled appointments at three centres: a midwife clinic, a public healthcare centre and a general practitioner centre. The primary outcome was participation at 6 months of follow-up. Secondary outcomes were participation at 1 and 3 months. Risk ratios (RRs) overall, and stratified by screening centre, age group and previous participation, were calculated using log-binomial regression. RESULTS: At 6 months, 20% of the 510 women in the control group and 37% of the 526 women in the intervention group had participated in screening, excluding 51 women in total from analysis due to participation just before invitation and therefore not yet visible in the central records. The RR for participation at 6 months was 1.9 (95% CI 1.5 to 2.3). There was no significant heterogeneity between centres or age groups. Participation increased among women both with (RR 1.7; 95% CI 1.4 to 2.1) and without (RR 3.5; 95% CI 1.3 to 9.2) previous participation. The RRs for participation at 1 and 3 months were 4.0 (95% CI 2.6 to 6.2) and 2.7 (95% CI 2.1 to 3.5), respectively. CONCLUSIONS: Scheduled appointments increased screening participation consistently across all target ages and screening centres among women overdue for screening. Participation increased also among women with no previous records of cervical screening.

Combined inhibition of the cell cycle related proteins Wee1 and Chk1/2 induces synergistic anti-cancer effect in melanoma.

BACKGROUND: Malignant melanoma has an increasing incidence rate and the metastatic disease is notoriously resistant to standard chemotherapy. Loss of cell cycle checkpoints is frequently found in many cancer types and makes the cells reliant on compensatory mechanisms to control progression. This feature may be exploited in therapy, and kinases involved in checkpoint regulation, such as Wee1 and Chk1/2, have thus become attractive therapeutic targets. METHODS: In the present study we combined a Wee1 inhibitor (MK1775) with Chk1/2 inhibitor (AZD7762) in malignant melanoma cell lines grown in vitro (2D and 3D cultures) and in xenografts models. RESULTS: Our in vitro studies showed that combined inhibition of Wee1 and Chk1/2 synergistically decreased viability and increased apoptosis (cleavage of caspase 3 and PARP), which may be explained by accumulation of DNA-damage (increased expression of γ-H2A.X)--and premature mitosis of S-phase cells. Compared to either inhibitor used as single agents, combined treatment reduced spheroid growth and led to greater tumour growth inhibition in melanoma xenografts. CONCLUSIONS: These data provide a rationale for further evaluation of the combination of Wee1 and Chk1/2 inhibitors in malignant melanoma.

Low-dose anisomycin sensitizes melanoma cells to TRAIL induced apoptosis.

Tumor necrosis factor related apoptosis-inducing ligand (TRAIL) has been shown to induce apoptosis in malignant cells while leaving normal cells unharmed, making it a desirable anticancer target. In the present study, metastatic melanoma cell lines were treated with lexatumumab (Human Genome Sciences, Inc.) a high-affinity monoclonal antibody agonistic to TRAIL receptor 2 (DR5). Binding of the antibody to the receptor led to activation of the extrinsic apoptosis pathway in approximately 20% of the treated cells. However, by combining subtoxic concentrations of the protein translation inhibitor anisomycin with lexatumumab, we obtained synergistic effects on cell viability compared with single agent treatment. Even the low doses of anisomycin could inhibit protein synthesis in melanoma cells with up to 30%, which might result in the shift in the levels of the proteins involved in apoptosis. Co-treatment with anisomycin increased activation of caspases and cleavage of the anti-apoptotic protein Livin, leading to formation of truncated p30-Livin α and p28-Livin ß proteins with potential pro-apoptotic functions. Furthermore, ansiomcycin treatment decreased levels of antiapototic XIAP. In summary our results suggest that combinational treatment with anicomycin and lexatumumab represents a novel therapeutic strategy in the treatment of melanoma.

Dacarbazine and the agonistic TRAIL receptor-2 antibody lexatumumab induce synergistic anticancer effects in melanoma.

Mapatumumab and lexatumumab (targeting death receptor 4 (DR4) and 5 (DR5), respectively) are agonistic TRAIL receptor antibodies that induce apoptosis in a wide range of cancer cells. The potency of mapatumumab and lexatumumab was assessed in mono therapy protocols, and the ability to sensitize for dacarbazine (DTIC) treatment was explored in ten different melanoma cell lines. Our data indicated that melanoma cell lines tend to be resistant to mapatumumab, most likely due to low expression of DR4, while a dose dependent response to lexatumumab was observed. Combining DTIC and lexatumumab induced an additive or synergistic effect on cell death in the various melanoma cell lines. The synergistic effect observed in the FEMX-1 cell line was related to enhanced cleavage of Bid in parallel with elevated expression of the pro-apoptotic proteins Bim, Bax and Bak. Furthermore, the anti-apoptotic proteins Bcl-XL, cIAP-1, XIAP and livin were down regulated. Cleavage of Bid and down regulation of cIAP-2 and livin were observed in vivo. Altogether, these data suggest a change in the balance between pro- and anti-apoptotic proteins favoring induction of apoptosis. In the more therapy resistant cell line, HHMS, no changes in the pro- and anti-apoptotic proteins were observed. FEMX-1 xenografts treated with DTIC and lexatumumab showed reduced growth and increased level of apoptosis compared to the control groups, providing arguments for further evaluation of this combination in melanoma patients.

Synthetic retinoid CD437 induces apoptosis and acts synergistically with TRAIL receptor-2 agonist in malignant melanoma.

The novel synthetic retinoid, CD437, shows potent anti-tumor activity in a range of different cancer cell lines and now serves as a prototype for development of new retinoid related molecules (RRMs). The purpose of this study was to examine the effect and cellular targets of CD437 in the human metastatic melanoma cell lines FEMX-1 and WM239. We showed that treatment with CD437 led to cell cycle arrest and induced apoptosis through both the extrinsic- and intrinsic pathways (caspase 8, -9 and PARP cleavage) in both cell lines. Interestingly, apoptosis was induced independently of DNA-fragmentation in FEMX-1 cells, and appeared partially caspase-independent in the WM239 cells. Additionally, up-regulation of CHOP mRNA and cathepsin D protein expression, following retinoid treatment, suggests involvement of the endoplasmatic reticulum (ER) and lysosomes, respectively. Combination of suboptimal concentrations of CD437 and lexatumumab, a TRAIL death receptor-2 agonist, resulted in synergistic reduction of viable cells, along with increased PARP cleavage. These results indicate that CD437 has a strong anti-neoplastic effect alone and in combination with lexatumumab in melanoma cell lines.

Light-directed delivery of nucleic acids.

A major barrier within the field of non-viral gene therapy toward therapeutic strategies, e.g., tumor therapy, has been lack of appropriate specific delivery strategies to the intended target tissues or cells. In this chapter, we describe a protocol for light-directed delivery of nucleic acids through the use of photochemical internalization (PCI) technology. PCI is based on a photosensitizing compound that localizes to endocytic membranes. Upon illumination, the photosensitizing compound induces damage to the endocytic membranes, resulting in release of endocytosed material, i.e., nucleic acids into cytosol. The main benefit of the strategy described is the possibility for site-specific delivery of nucleic acids to a place of interest.

Targeting inhibitor of apoptosis proteins in combination with dacarbazine or TRAIL in melanoma cells.

Melanoma is a highly aggressive malignant tumor with an exceptional ability to develop resistance and no curative therapy is available for patients with distant metastatic disease. The inhibitor of apoptosis protein (IAP) family has been related to therapy resistance in cancer. We examined the importance of the IAPs in the resistance to the commonly used chemotherapeutic agent dacarbazine (DTIC) and the apoptosis inducer TRAIL (TNF-related apoptosis inducing ligand) in malignant melanoma. The data presented show that the expression of IAPs is universal, concomitant and generally high in melanoma cell lines and in patient samples. Depleting IAP expression by siRNA tended to reduce cell viability, with XIAP reduction being the most efficient in all four cell lines examined (FEMX-1, LOX, SKMEL-28 and WM115). The combined treatment of XIAP siRNA and DTIC showed a weak improvement in two of four cell lines, while all four cell lines showed enhanced sensitivity towards TRAIL (AdhCMV-TRAIL) after XIAP depletion. In addition, cIAP-1, cIAP-2 and survivin down-regulation sensitized to TRAIL treatment in several of the cell lines. Cells exposed to TRAIL and XIAP siRNA showed increased DNA-fragmentation and cleavage of Bid, procaspase-8, -9, -7 and -3 and PARP, and change in the balance between pro- and anti-apoptotic proteins, indicating an enhanced level of apoptosis. Furthermore, the combined treatment reduced the ability of melanoma cells to engraft and form tumors in mice, actualizing the combination for future therapy of malignant melanoma.

Aldehyde dehydrogenase (ALDH) activity does not select for cells with enhanced aggressive properties in malignant melanoma.

BACKGROUND: Malignant melanoma is an exceptionally aggressive, drug-resistant and heterogeneous cancer. Recently it has been shown that melanoma cells with high clonogenic and tumourigenic abilities are common, but markers distinguishing such cells from cells lacking these abilities have not been identified. There is therefore no definite evidence that an exclusive cell subpopulation, i.e. cancer stem cells (CSC), exists in malignant melanoma. Rather, it is suggested that multiple cell populations are implicated in initiation and progression of the disease, making it of importance to identify subpopulations with elevated aggressive properties. METHODS AND FINDINGS: In several other cancer forms, Aldehyde Dehydrogenase (ALDH), which plays a role in stem cell biology and resistance, is a valuable functional marker for identification of cells that show enhanced aggressiveness and drug-resistance. Furthermore, the presence of ALDH(+) cells is linked to poor clinical prognosis in these cancers. By analyzing cell cultures, xenografts and patient biopsies, we showed that aggressive melanoma harboured a large, distinguishable ALDH(+) subpopulation. In vivo, ALDH(+) cells gave rise to ALDH(-) cells, while the opposite conversion was rare, indicating a higher abilities of ALDH(+) cells to reestablish tumour heterogeneity with respect to the ALDH phenotype. However, both ALDH(+) and ALDH(-) cells demonstrated similarly high abilities for clone formation in vitro and tumour initiation in vivo. Furthermore, both subpopulations showed similar sensitivity to the anti-melanoma drugs, dacarbazine and lexatumumab. CONCLUSIONS: These findings suggest that ALDH does not distinguish tumour-initiating and/or therapy-resistant cells, implying that the ALDH phenotype is not associated with more-aggressive subpopulations in malignant melanoma, and arguing against ALDH as a "universal" marker. Besides, it was shown that the ability to reestablish tumour heterogeneity is not necessarily linked to the more aggressive phenotype.

Combined treatment with Ad-hTRAIL and DTIC or SAHA is associated with increased mitochondrial-mediated apoptosis in human melanoma cell lines.

BACKGROUND: Currently, dacarbazine (DTIC) is the only approved systemic treatment for metastatic malignant melanoma. However, the modest treatment effect encourages studies on novel therapeutic molecules, delivery systems and combination therapies. Full-length TRAIL, delivered from an adenoviral vector (Ad-hTRAIL), was studied in combination with DTIC or the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) in human melanoma cell lines. METHODS: The cytotoxic potential of the combination treatments was assessed by cell viability measurements and CalcuSyn analysis. Involvement of apoptosis was analyzed by TUNEL staining, mitochondrial membrane potential measurements, and activation and expression levels of caspases and other mediators of apoptosis. RESULTS: Ad-hTRAIL in combination with DTIC or SAHA resulted in additive or synergistic growth inhibition compared to each treatment used as single agent. Both combinations augmented apoptosis, which was mediated through the death receptor (DR) pathway by enhanced activation of caspase-8, and through increased loss of mitochondrial integrity. Provoked cleavage of Bid, which bridges the extrinsic and intrinsic apoptosis pathways, and downregulation of the anti-apoptotic mediators Bcl-X(L), Mcl-1 and XIAP (but not Bcl-2) were critical contributing factors. Increased levels of DR4 and DR5 were not a common underlying mechanism as DTIC did not affect the levels of either of the receptors. However, SAHA-induced expression of DR4 may have reduced the TRAIL resistance in the SKMEL-28 cell line. CONCLUSION: Administration of Ad-hTRAIL in combination with DTIC or SAHA enhances apoptosis in human melanoma cell lines, and suggests that the therapeutic potential of such treatment strategies should be further evaluated for possible clinical use.