Irene Wessel

Human small cell lung cancer NYH cells resistant to the bisdioxopiperazine ICRF-187 exhibit a functional dominant Tyr165Ser mutation in the Walker A ATP binding site of topoisomerase IIα

Bisdioxopiperazine anti‐cancer agents are catalytic inhibitors of topoisomerase II which by unknown means lock the enzyme in a closed clamp form and inhibit its ATPase activity. In order to demarcate a putative pharmacophore, we here describe a novel Tyr165Ser mutation in the enzymes Walker A ATP binding site leading to specific bisdioxopiperazine resistance when transformed into a temperature‐conditional yeast system. The Tyr165Ser mutation differed from a previously described Arg162Gln by being heterozygous and by purified Tyr165Ser enzyme being drug‐resistant in a kinetoplast DNA decatenation enzymatic assay. This suggested dominant nature of Tyr165Ser was supported by co‐transformation studies in yeast of plasmids carrying wild type and mutant genes. These results enable a model of the bisdioxopiperazine pharmacophore using the proposed asymmetric ATP hydrolysis of the enzyme.

N‐terminal and core‐domain random mutations in human topoisomerase II α conferring bisdioxopiperazine resistance

Random mutagenesis of human topoisomerase II α cDNA followed by functional expression in yeast cells lacking endogenous topoisomerase II activity in the presence of ICRF‐187, identified five functional mutations conferring cellular bisdioxopiperazine resistance. The mutations L169F, G551S, P592L, D645N, and T996L confer >37, 37, 18, 14, and 19 fold resistance towards ICRF‐187 in a 24 h clonogenic assay, respectively. Purified recombinant L169F protein is highly resistant towards catalytic inhibition by ICRF‐187 in vitro while G551S, D645N, and T996L proteins are not. This demonstrates that cellular bisdioxopiperazine resistance can result from at least two classes of mutations in topoisomerase II; one class renders the protein non‐responsive to bisdioxopiperazine compounds, while an other class does not appear to affect the catalytic sensitivity towards these drugs. In addition, our results indicate that different protein domains are involved in mediating the effect of bisdioxopiperazine compounds.

Activation of the interleukin-6/Janus kinase/STAT3 pathway in pleomorphic adenoma of the parotid gland

The interleukin‐6 (IL‐6)/Janus kinase (JAK)/signal transducer and activator of transcription 3 (STAT3) pathway is of crucial importance in promoting tumorigenesis in several malignant tumors but may also be active in benign tumors, e.g., of pleomorphic adenoma (PA). In this study we characterize the expression of the pathway components with immunohistochemistry and selected mRNAs and microRNAs (miRNAs) regulated by this pathway in isolated duct‐ and myoepithelial cells in PA. 46 PAs were immunostained and 10 of these were used for in situ hybridization (ISH). Six frozen specimens were analyzed using reverse transcription‐polymerase chain reaction (RT‐PCR). Using immunohistochemistry, IL‐6, JAK1, JAK2 and STAT3 were detected significantly more frequently in PA cells than in cells from normal salivary gland tissue. Using RT‐PCR cyclin D1, fibroblast growth factor 2, and p21 were found to be overexpressed while matrix metallopeptidase 9 was detected at low levels in PA compared to normal salivary gland. ISH showed significant overexpression of miR‐181b in PA, while miR‐21 was undetectable in PA and normal tissue. Overexpression of the pathway components and its mRNA and miRNA products provide important clues regarding the growth of PAs. Our findings brings us one step closer to targeted treatment of this tumor entity, although in vitro studies are warranted to confirm this.

Increasing incidence and survival in oral cancer: a nationwide Danish study from 1980 to 2014

Abstract Background: Oral carcinomas (OCs) make up a significant proportion of head and neck carcinomas (HNCs) and are an important cause of morbidity and mortality globally. The purpose of this population-based study was to determine trends in incidence and survival in OC in the Danish population from 1980 to 2014. Material and methods: This study covered all patients registered in the nationwide Danish cancer registry (DCR) in the period 1980–2014. Age-adjusted incidence rate (AAIR) per 100,000 and annual percentage change (APC) were evaluated. Also, 5-year overall survival (OS) was calculated with Cox regression analysis in relation to location, gender, age, and calendar year at diagnosis. Results: Altogether, 8299 patients with oral cancer were identified, 5062 (61%) of whom were males and 3237 (39%) were females. The median age at diagnosis was 63 years. The AAIR of patients with OC increased from 1.9 per 100,000 in 1980 to 3.5 per 100,000 in 2014, and we observed a significant increase in 5-year OS of 12% points (a relative increase of 38%) from the period 1980–1984 to 2005–2009. Women were found to have a better prognosis than men. Conclusions: We found an unexpected increase in the age-standardized incidence of OC during the last 30 years in Denmark, and also an improvement in survival. The 5-year OS was significantly better in recent years even when we adjusted the analysis for relevant covariates.

The PRKD1 E710D hotspot mutation is highly specific in separating polymorphous adenocarcinoma of the palate from adenoid cystic carcinoma and pleomorphic adenoma on FNA: PRKD1 Hotspot Mutation in PAC

Polymorphous adenocarcinoma (PAC) of the palatal minor salivary glands, previously known as polymorphous low‐grade adenocarcinoma, is the second most common intraoral malignant salivary gland carcinoma after adenoid cystic carcinoma (ACC) and carries an excellent prognosis. Unfortunately, PAC demonstrates cytological overlap with 2 other salivary gland tumors frequently encountered in the same location, namely ACC and pleomorphic adenoma (PA). Recently, the protein kinase D1 (PRKD1) hotspot mutation E710D was demonstrated to be specific for PAC and to be present in the majority of cases. The objective of the current study was to investigate the value of PRKD1 hotspot sequencing in identifying PAC in paired fine‐needle aspiration (FNA) and surgical specimens from cases of PAC, ACC, and PA.

Swallowing therapy and progressive resistance training in head and neck cancer patients undergoing radiotherapy treatment: randomized control trial protocol and preliminary data

Abstract Background: Head and neck cancer (HNC) patients are often challenged by treatment induced dysphagia and trismus. Traditionally, rehabilitation is initiated when loss of function has already occurred. There is increasing evidence that it is of benefit to patients to initiate an early rehabilitation process before and during treatment. HNC patients have a unique set of functional challenges such as pre- and post-treatment dysphagia, pain and weight loss. The aim of the trial is to investigate the effects of swallowing and mouth-opening exercises combined with progressive resistance training (PRT) during radiotherapy. This report presents the protocol, interim inclusion and feasibility data. Material and methods: The trial (clinicaltrials.gov NCT02385929) is a multicenter randomized controlled trial (RCT) with a parallel-group randomization (1:1). The planned sample size of 240 HNC patients is randomly assigned to either (1) twice weekly PRT and three times weekly swallowing exercises by physio- and occupational therapists, respectively, as well as daily home exercises throughout radiotherapy or (2) standard care. Inclusion criteria are patients with cancer in the larynx, pharynx, oral cavity, or unknown primary tumor who are referred to radiotherapy with curative intent. Outcomes are measured at end-of-treatment and two, five, and 12 months post-treatment. Interim results: In 16 months, 321 HNC patients were screened for eligibility. Of these, 131 (41%) were eligible according to inclusion criteria. One-hundred-and-fifteen patients were invited to participate of which 69 (60%) were enrolled in the trial and randomized for either intervention or control group with 10 drop-outs (14%). The six pilot patients adhered more than 90% to the program. Conclusion: Preliminary results show that exercise according to protocol is tolerable and feasible.

Adenoid cystic carcinomas of the salivary gland, lacrimal gland, and breast are morphologically and genetically similar but have distinct microRNA expression profiles

Adenoid cystic carcinoma is among the most frequent malignancies in the salivary and lacrimal glands and has a grave prognosis characterized by frequent local recurrences, distant metastases, and tumor-related mortality. Conversely, adenoid cystic carcinoma of the breast is a rare type of triple-negative (estrogen and progesterone receptor, HER2) and basal-like carcinoma, which in contrast to other triple-negative and basal-like breast carcinomas has a very favorable prognosis. Irrespective of site, adenoid cystic carcinoma is characterized by gene fusions involving MYB, MYBL1, and NFIB, and the reason for the different clinical outcomes is unknown. In order to identify the molecular mechanisms underlying the discrepancy in clinical outcome, we characterized the phenotypic profiles, pattern of gene rearrangements, and global microRNA expression profiles of 64 salivary gland, 9 lacrimal gland, and 11 breast adenoid cystic carcinomas. All breast and lacrimal gland adenoid cystic carcinomas had triple-negative and basal-like phenotypes, while salivary gland tumors were indeterminate in 13% of cases. Aberrations in MYB and/or NFIB were found in the majority of cases in all three locations, whereas MYBL1 involvement was restricted to tumors in the salivary gland. Global microRNA expression profiling separated salivary and lacrimal gland adenoid cystic carcinoma from their respective normal glands but could not distinguish normal breast adenoid cystic carcinoma from normal breast tissue. Hierarchical clustering separated adenoid cystic carcinomas of salivary gland origin from those of the breast and placed lacrimal gland carcinomas in between these. Functional annotation of the microRNAs differentially expressed between salivary gland and breast adenoid cystic carcinoma showed these as regulating genes involved in metabolism, signal transduction, and genes involved in other cancers. In conclusion, microRNA dysregulation is the first class of molecules separating adenoid cystic carcinoma according to the site of origin. This highlights a novel venue for exploring the biology of adenoid cystic carcinoma.

Electrochemotherapy of mucosal head and neck tumors: a systematic review

Abstract Background: Electrochemotherapy, the combination of electroporation and chemotherapy, is mainly used in the palliative setting for treatment of cutaneous and subcutaneous metastases; however, new applications are continuously being explored. Patients with head and neck cancer are primarily treated with surgery and/or radio-chemotherapy. In the setting of local recurrence with no further curative treatment options available, electrochemotherapy could be of value. We therefore performed a systematic search of the present literature. Materials and methods: Eligible studies presented data from patients with head and neck cancer treated across the mucosal surface with electrochemotherapy. The search resulted in 11 studies with a total of 72 patients. Results: Overall complete response was reported as good, especially in primary small tumors. Side effects were minor in primary tumors whereas large, recurrent tumors displayed more frequent side effects and some serious adverse events. Design and structure of the studies differed considerably, making general comparisons difficult. Conclusion: Few studies concerning electrochemotherapy on mucosal head and neck tumors are available and they are not easily comparable. Overall response to treatment is good; nonetheless, further systematic studies are warranted.

MicroRNA dysregulation in adenoid cystic carcinoma of the salivary gland in relation to prognosis and gene fusion status: a cohort study

Adenoid cystic carcinoma (ACC) is among the most frequent malignancies of the salivary gland, and is notorious for its prolonged clinical course characterized by frequent recurrences often years after initial treatment. No molecular marker has been shown to have independent prognostic value in ACC, including characteristic gene fusions involving MYB, MYBL1, and NFIB. MicroRNA has been shown to be associated with clinical outcome in numerous malignancies, including one study of ACC, warranting further validation of this class of markers in this disease. Here, we investigate the prognostic value of microRNA in two ACC cohorts: a training cohort (n = 64) and a validation cohort (n = 120) with microarray and qPCR. In the training cohort, multivariate analysis of microarray data found high expression of hsa-miR-6835-3p to be associated with reduced recurrence-free survival (RFS) (p = 0.016). Measuring the highest ranking microRNAs identified in survival analysis in the same cohort, qPCR identified high expression of hsa-miR-4676 to be associated with reduced overall survival (OS) and high expression of hsa-mir-1180 to be associated with improved RFS. This was not confirmed in the validation cohort, in which qPCR identified high expression of hsa-mir-21, hsa-mir-181a-2, and hsa-mir-152 to be associated with reduced OS and high expression of hsa-miR-374c to be associated with improved RFS. Interestingly, two distinct subsets of ACC separated in microRNA expression irrespective of gene fusion status, but without significant difference in outcome. Collectively, qPCR identified several microRNAs associated with OS and RFS, and different subsets of ACC separated according to microRNA expression, suggestive of ACC being a heterogeneous group of malignancies in its microRNA profile.

Genetic rearrangements, hotspot mutations, and microRNA expression in the progression of metastatic adenoid cystic carcinoma of the salivary gland

Adenoid cystic carcinoma (ACC) is among the most common salivary gland malignancies, and is notorious for its unpredictable clinical course with frequent local recurrences and metastatic spread. However, the molecular mechanisms for metastatic spread are poorly understood. This malignancy is known to frequently harbor gene fusions involving MYB, MYBL1, and NFIB, and to have a low mutational burden. Most studies have focused on primary tumors to understand the biology of ACC, but this has not revealed a genetic cause for metastatic dissemination in the majority of cases. Hence, other molecular mechanisms are likely to be involved. Here, we characterize the genetic and microRNA expressional landscape of primary ACC and corresponding metastatic lesions from 11 patients. FISH demonstrated preservation of MYB aberrations between primary tumors and metastases, and targeted next-generation sequencing identified mutations exclusive for the metastatic lesions in 3/11 cases (27.3%). Global microRNA profiling identified several differentially expressed miRNAs between primary ACC and metastases as compared to normal salivary gland tissue. Interestingly, individual tumor pairs differed in miRNA profile, but there was no general difference between primary ACCs and metastases. Collectively, we show that MYB and NFIB aberrations are consistently preserved in ACC metastatic lesions, and that additional mutations included in the 50-gene hotspot panel used are infrequently acquired by the metastatic lesions. In contrast, tumor pairs differ in microRNA expression and our data suggest that they are heterogeneous according to their microRNA profile. This adds an additional layer to the complex process of ACC metastatic spread.