Towards effectiveness of cell free DNA based liquid biopsy in head and neck squamous cell carcinoma.

Liquid biopsy is a minimally invasive procedure, that uses body fluids sampling to detect and characterize cancer fingerprints. It is of great potential in oncology, however there are challenges associated with the proper handling of liquid biopsy samples that need to be addressed to implement such analysis in patients' care. Therefore, in this study we performed optimization of pre-analytical conditions and detailed characterization of cfDNA fraction (concentration, length, integrity score) in surgically treated HNSCC patients (n = 152) and healthy volunteers (n = 56). We observed significantly higher cfDNA concentration in patients compared to healthy controls (p < 0.0001) and a time dependent decrease of cfDNA concentration after tumor resection. Our results also revealed a significant increase of cfDNA concentration with age in both, healthy volunteers (p = 0.04) and HNSCC patients (p = 0.000002). Moreover, considering the multitude of HNSCC locations, we showed the lack of difference in cfDNA concentration depending on the anatomical location. Furthermore, we demonstrated a trend toward higher cfDNA length (range 35-10380 and 500-10380 bp) in the group of patients with recurrence during follow-up. In conclusion, our study provide a broad characterization of cfDNA fractions in HNSCC patients and healthy controls. These findings point to several aspects necessary to consider when implementing liquid biopsy in clinical practice including: (I) time required for epithelial regeneration to avoid falsely elevated levels of cfDNA not resulting from active cancer, (II) age-related accumulation of nucleic acids accompanied by less efficient elimination of cfDNA and (III) higher cfDNA length in patients with recurrence during follow-up, reflecting predominance of tumor necrosis.

The presence of additional cytogenetic aberrations in chronic myeloid leukemia cells at the time of diagnosis or their appearance on tyrosine kinase inhibitor therapy predicts the imatinib treatment failure.

Currently used treatment of CML dramatically improved the prognosis of disease. However, additional chromosome aberrations (ACA/Ph+) are still one of the adverse prognostic factors. OBJECTIVES: evaluation of the impact of ACA/Ph+ appearance during disease outcome on the response to treatment. THE STUDY GROUP: consisted of 203 patients. The median time of follow-up was 72 months. ACA/Ph+ was found in 53 patients. RESULTS: patients were divided into four groups: standard risk, intermediate, high and very high risk. When ACA/Ph+ presence was documented at diagnosis time the optimal response was observed in 41.2%, 25%, and 0% of pts with intermediate, high and very high risk, respectively. If ACA/Ph+ were detected during imatinib treatment the optimal response was in 4.8% of patients. The risk of blastic transformation for patients with standard risk, intermediate, high and very high risk was 2.7%, 18.4%, 20% and 50%, respectively. CONCLUSIONS: the presence of ACA/Ph+ at diagnosis time or their appearance on therapy seems to be clinically relevant not only in terms of the risk of blastic transformation but also in terms of the treatment failure. Gathering patients with various karyotypes and their responses to treatment would allow to set better guidelines and predictions.

Identification of two unannotated miRNAs in classic Hodgkin lymphoma cell lines.

MicroRNAs (miRNAs) are small non coding RNAs responsible for posttranscriptional regulation of gene expression. Even though almost 2000 precursors have been described so far, additional miRNAs are still being discovered in normal as well as malignant cells. Alike protein coding genes, miRNAs may acquire oncogenic properties in consequence of altered expression or presence of gain or loss of function mutations. In this study we mined datasets from miRNA expression profiling (miRNA-seq) of 7 classic Hodgkin Lymphoma (cHL) cell lines, 10 non-Hodgkin lymphoma (NHL) cell lines and 56 samples of germinal center derived B-cell lymphomas. Our aim was to discover potential novel cHL oncomiRs not reported in miRBase (release 22.1) and expressed in cHL cell lines but no other B-cell lymphomas. We identified six such miRNA candidates in cHL cell lines and verified the expression of two of them encoded at chr2:212678788-212678849 and chr5:168090507-168090561 (GRCh38). Interestingly, we showed that one of the validated miRNAs (located in an intron of the TENM2 gene) is expressed together with its host gene. TENM2 is characterized by hypomethylation and open chromatin around its TSS in cHL cell lines in contrast to NHL cell lines and germinal centre B-cells respectively. It indicates an epigenetic mechanism responsible for aberrant expression of both, the TENM2 gene and the novel miRNA in cHL cell lines. Despite the GO analysis performed with the input of the in silico predicted novel miRNA target genes did not reveal ontologies typically associated with cHL pathogenesis, it pointed to several interesting candidates involved in i.e. lymphopoiesis. These include the lymphoma related BCL11A gene, the IKZF2 gene involved in lymphocyte development or the transcription initiator GTF2H1.

Global DNA Methylation Profiling Reveals Differentially Methylated CpGs between Salivary Gland Pleomorphic Adenomas with Distinct Clinical Course.

Pleomorphic adenomas (PAs) are the most frequently diagnosed benign salivary gland tumors. Although the majority of PAs are characterized by slow growth, some develop very fast and are more prone to recur. The reason for such differences remains unidentified. In this study, we performed global DNA methylation profiling using the Infinium Human Methylation EPIC 850k BeadChip Array (Illumina) to search for epigenetic biomarkers that could distinguish both groups of tumors. The analysis was performed in four fast-growing tumors (FGTs) and four slow-growing tumors (SGTs). In all, 85 CpG dinucleotides differentiating both groups were identified. Six CpG tags (cg06748470, cg18413218, cg10121788, cg08249296, cg18455472, and cg19930657) were selected for bisulfite pyrosequencing in the extended group of samples. We confirmed differences in DNA methylation between both groups of samples. To evaluate the potential diagnostic accuracy of the selected markers, ROC curves were constructed. We indicated that CpGs included in two assays showed an area under the curve with an acceptable prognostic value (AUC > 0.7). However, logistic regression analysis allowed us to indicate a more optimal model consisting of five CpGs ((1) cg06748470, (2) cg00600454, (3) CpG located in chr14: 77,371,501−77,371,502 (not annotated in GRCh37/hg19), (4) CpG2 located in chr16: 77,469,589−77,469,590 (not annotated GRCh37/hg19), and (5) cg19930657) with AUC > 0.8. This set of epigenetic biomarkers may be considered as differentiating factors between FGT and SGT during salivary gland tumor diagnosis. However, this data should be confirmed in a larger cohort of samples.

Loss of function mutations of BCOR in classical Hodgkin lymphoma.

BCOR is a component of a variant Polycomb repressive complex 1 (PRC1.1). PRC1 and PRC2 complexes together constitute a major gene regulatory system critical for appropriate cellular differentiation. The gene is upregulated in germinal center (GC) B cells and mutated in a number of hematologic malignancies. We report BCOR inactivating alterations in 4/7 classic Hodgkin lymphoma (cHL) cell lines, subclonal somatic mutations in Hodgkin and Reed-Sternberg (HRS) cells of 4/10 cHL cases, and deletions in HRS cells of 7/17 primary cHL cases. In mice, conditional loss of Bcor driven by AID-Cre in GC B cells resulted in gene expression changes of 46 genes (>2-fold) including upregulated Lef1 that encodes a transcription factor responsible for establishing T-cell identity and Il9r (interleukin-9 receptor), an important member of the cytokine network in cHL. Our findings suggest a role for BCOR loss in cHL pathogenesis and GC-B cell homeostasis.

Prognostic Roles of BRAF, KIT, NRAS, IGF2R and SF3B1 Mutations in Mucosal Melanomas.

BACKGROUND: The prognostic value of commonly recurrent mutations remains unclear in mucosal melanomas. METHODS: Clinicopathologic parameters of 214 cases of mucosal melanomas diagnosed in 1989-2020 in several clinical institutions were analyzed. NRAS, KIT, BRAF, IGF2R and SF3B1 mutational analyses by Sanger sequencing and next generation sequencing-based assay were performed in a subset of cases. RESULTS: Of the triple (BRAF, NRAS, NF1)-negative cases, APC, KIT and KRAS are detected mainly in sinonasal, vulvovaginal and anorectal melanomas, respectively. NRAS, KIT, BRAF, IGF2R and SF3B1 mutations are detected in 19% (37/198), 22% (44/197), 12% (25/201), 16% (22/138) and 15% (20/133) of cases, respectively. In univariate analyses, advanced stage (p = 0.016), 65 years or older (p = 0.048) and presence of ulceration (p = 0.027) are significantly correlated with worse overall survival (OS), respectively. NRAS mutation significantly correlates with worse OS (p = 0.028) and worse melanoma-specific survival (MSS) (p = 0.03) for all cases of mucosal melanomas. In multivariate analyses, NRAS mutation remains as an independent predictor of worse OS (p = 0.036) and worse MSS (p = 0.024). CONCLUSION: NRAS mutation is a predictor of worse survival, independent of stage in mucosal melanomas. The significance of frequently mutated IGF2R in mucosal melanomas remains unclear.

Genetic syndromes with vascular malformations - update on molecular background and diagnostics.

Vascular malformations are present in a great variety of congenital syndromes, either as the predominant or additional feature. They pose a major challenge to the clinician: due to significant phenotype overlap, a precise diagnosis is often difficult to obtain, some of the malformations carry a risk of life threatening complications and, for many entities, treatment is not well established. To facilitate their recognition and aid in differentiation, we present a selection of notable congenital disorders of vascular system development, distinguishing between the heritable germinal and sporadic somatic mutations as their causes. Clinical features, genetic background and comprehensible description of molecular mechanisms is provided for each entity.

Loss of the MAF Transcription Factor in Laryngeal Squamous Cell Carcinoma.

MAF is a transcription factor that may act either as a tumor suppressor or as an oncogene, depending on cell type. We have shown previously that the overexpressed miR-1290 influences MAF protein levels in LSCC (laryngeal squamous cell carcinoma) cell lines. In this study, we shed further light on the interaction between miR-1290 and MAF, as well as on cellular MAF protein localization in LSCC. We confirmed the direct interaction between miR-1290 and MAF 3'UTR by a dual-luciferase reporter assay. In addition, we used immunohistochemistry staining to analyze MAF protein distribution and observed loss of MAF nuclear expression in 58% LSCC samples, of which 10% showed complete absence of MAF, compared to nuclear and cytoplasmatic expression in 100% normal mucosa. Using TCGA data, bisulfite pyrosequencing and CNV analysis, we excluded the possibility that loss-of-function mutations, promoter region DNA methylation or CNV are responsible for MAF loss in LSCC. Finally, we identified genes involved in the regulation of apoptosis harboring the MAF binding motif in their promoter region by applied FIMO and DAVID GO analysis. Our results highlight the role of miR-1290 in suppressing MAF expression in LSCC. Furthermore, MAF loss or mislocalization in FFPE LSCC tumor samples might suggest that MAF acts as a LSCC tumor suppressor by regulating apoptosis.

Deregulated miRNAs Contribute to Silencing of B-Cell Specific Transcription Factors and Activation of NF-κB in Classical Hodgkin Lymphoma.

A hallmark of classical Hodgkin lymphoma (cHL) is the attenuation of B-cell transcription factors leading to global transcriptional reprogramming. The role of miRNAs (microRNAs) involved in this process is poorly studied. Therefore, we performed global miRNA expression profiling using RNA-seq on commonly used cHL cell lines, non-Hodgkin lymphoma cell lines and sorted normal CD77+ germinal centre B-cells as controls and characterized the cHL miRNome (microRNome). Among the 298 miRNAs expressed in cHL, 56 were significantly overexpressed and 23 downregulated (p < 0.05) compared to the controls. Moreover, we identified five miRNAs (hsa-miR-9-5p, hsa-miR-24-3p, hsa-miR-196a-5p, hsa-miR-21-5p, hsa-miR-155-5p) as especially important in the pathogenesis of this lymphoma. Target genes of the overexpressed miRNAs in cHL were significantly enriched (p < 0.05) in gene ontologies related to transcription factor activity. Therefore, we further focused on selected interactions with the SPI1 and ELF1 transcription factors attenuated in cHL and the NF-ĸB inhibitor TNFAIP3. We confirmed the interactions between hsa-miR-27a-5p:SPI1, hsa-miR-330-3p:ELF-1, hsa-miR-450b-5p:ELF-1 and hsa-miR-23a-3p:TNFAIP3, which suggest that overexpression of these miRNAs contributes to silencing of the respective genes. Moreover, by analyzing microdissected HRS cells, we demonstrated that these miRNAs are also overexpressed in primary tumor cells. Therefore, these miRNAs play a role in silencing the B-cell phenotype in cHL.

Laryngeal squamous cell carcinoma cell lines show high tolerance for siRNA-mediated CDK1 knockdown.

Alterations of the cell cycle checkpoints lead to uncontrolled cell growth and result in tumorigenesis. One of the genes essential for cell proliferation and cell cycle regulation is CDK1. This makes it a potential target in cancer therapy. In our previous study we have shown upregulation of this gene in laryngeal squamous cell carcinoma (LSCC). Here we analyze the impact of siRNA-mediated CDK1 knockdown on cell proliferation and viability, measured with cell growth monitoring and colorimetric test (CCK8 assay), respectively. We proved that a reduction of CDK1 expression by more than 50% has no effect on these cellular processes in LSCC cell lines (n=2). Moreover, using microarrays, we analyzed global gene expression deregulation in these cell lines after CDK1 knockdown. We searched for enriched ontologies in the group of identified 137 differentially expressed genes (>2-fold change). Within this group we found 3 enriched pathways: protein binding (GO:0005515), mitotic nuclear division (GO:0007067) and transmembrane receptor protein tyrosine kinase signaling pathway (GO:0007169) and a group of 11 genes encoding proteins for which interaction with CDK1 was indicated with the use of bioinformatic tools. Among these genes we propose three: CDK6, CALD1 and FYN as potentially dependent on CDK1.

Assessing Various Control Samples for Microarray Gene Expression Profiling of Laryngeal Squamous Cell Carcinoma.

Selection of optimal control samples is crucial in expression profiling tumor samples. To address this issue, we performed microarray expression profiling of control samples routinely used in head and neck squamous cell carcinoma studies: human bronchial and tracheal epithelial cells, squamous cells obtained by laser uvulopalatoplasty and tumor surgical margins. We compared the results using multidimensional scaling and hierarchical clustering versus tumor samples and laryngeal squamous cell carcinoma cell lines. A general observation from our study is that the analyzed cohorts separated according to two dominant factors: "malignancy", which separated controls from malignant samples and "cell culture-microenvironment" which reflected the differences between cultured and non-cultured samples. In conclusion, we advocate the use of cultured epithelial cells as controls for gene expression profiling of cancer cell lines. In contrast, comparisons of gene expression profiles of cancer cell lines versus surgical margin controls should be treated with caution, whereas fresh frozen surgical margins seem to be appropriate for gene expression profiling of tumor samples.

European context of the diversity and phylogenetic position of SARS-CoV-2 sequences from Polish COVID-19 patients.

To provide a comprehensive analysis of the SARS-CoV-2 sequence diversity in Poland in the European context. All publicly available (n = 115; GISAID database) whole-genome SARS-Cov-2 sequences from Polish samples, including those obtained during coronavirus testing performed in our COVID-19 Lab, were examined. Multiple sequence alignment of Polish isolates, phylogenetic analysis (ML tree), and multidimensional scaling (based on the pairwise DNA distances) were complemented by the comparison of the coronavirus clades frequency and diversity in the subset of over 5000 European GISAID sequences. Approximately seventy-seven percent of isolates in the European dataset carried frequent and ubiquitously found haplotypes; the remaining haplotype diversity was population-specific and resulted from population-specific mutations, homoplasies, and recombinations. Coronavirus strains circulating in Poland represented the variability found in other European countries. The prevalence of clades circulating in Poland was shifted in favor of GR, both in terms of the diversity (number of distinct haplotypes) and the frequency (number of isolates) of the clade. Polish-specific haplotypes were rare and could be explained by changes affecting common European strains. The analysis of the whole viral genomes allowed detection of several tight clusters of isolates, presumably reflecting local outbreaks. New mutations, homoplasies, and, to a smaller extent, recombinations increase SARS-CoV-2 haplotype diversity, but the majority of these variants do not increase in frequency and remains rare and population-specific. The spectrum of SARS-CoV-2 haplotypes in the Polish dataset reflects many independent transfers from a variety of sources, followed by many local outbreaks. The prevalence of the sequences belonging to the GR clade among Polish isolates is consistent with the European trend of the GR clade frequency increase.

New genetic variants of TET2 and ASXL1 identified by next generation sequencing and pyrosequencing in a patient with MDS-RS-MLD and secondary acute myeloid leukemia.

Myelodysplastic syndromes (MDS) are a heterogeneous group of myeloid neoplasms characterized by the presence of cytopenias, ineffective hematopoiesis and frequent transformation into secondary acute myeloid leukemia (secAML). Recent genomic studies provide unprecedented insight into the molecular landscape of clonal proliferation in MDS. Genetic diversity of both MDS and secAML subclones cannot be defined by a single somatic mutation. Mutations of the founding clone may survive over implemented chemotherapy and allogenic hematopoietic cell transplantation (alloHCT), but new subclonal mutations may also appear. Next generation sequencing (NGS) makes it possible to define the mutational profile of disease subclones during the treatment course and has a potential in pre- and post-alloHCT monitoring. Understanding the molecular pathophysiology of MDS may soon allow for monitoring the course of disease and personalized treatment depending on the mutational landscape. In the present paper we report, for the first time in MDS, ASXL1 c.1945G>T, TET2 c.4044+2dupT and c.4076G>T sequence variants. Moreover, we detected RUNX1 c.509-2A>C and SF3B1 c.1874G>T sequence variants. Furthermore, we verify the clinical utility of NGS and pyrosequencing in MDS and secAML.

The Tumor Suppressive mir-148a Is Epigenetically Inactivated in Classical Hodgkin Lymphoma.

DNA methylation was shown previously to be a crucial mechanism responsible for transcriptional deregulation in the pathogenesis of classical Hodgkin lymphoma (cHL). To identify epigenetically inactivated miRNAs in cHL, we have analyzed the set of miRNAs downregulated in cHL cell lines using bisulfite pyrosequencing. We focused on miRNAs with promoter regions located within or <1000 bp from a CpG island. Most promising candidate miRNAs were further studied in primary Hodgkin and Reed-Sternberg (HRS) cells obtained by laser capture microdissection. Last, to evaluate the function of identified miRNAs, we performed a luciferase reporter assay to confirm miRNA: mRNA interactions and therefore established cHL cell lines with stable overexpression of selected miRNAs for proliferation tests. We found a significant reverse correlation between DNA methylation and expression levels of mir-339-3p, mir-148a-3p, mir-148a-5p and mir-193a-5 demonstrating epigenetic regulation of these miRNAs in cHL cell lines. Moreover, we demonstrated direct interaction between miR-148a-3p and IL15 and HOMER1 transcripts as well as between mir-148a-5p and SUB1 and SERPINH1 transcripts. Furthermore, mir-148a overexpression resulted in reduced cell proliferation in the KM-H2 cell line. In summary, we report that mir-148a is a novel tumor suppressor inactivated in cHL and that epigenetic silencing of miRNAs is a common phenomenon in cHL.

The Potential Role of Selected miRNA in Uveal Melanoma Primary Tumors as Early Biomarkers of Disease Progression.

Uveal melanoma (UM) is the most common primary tumor of the eye diagnosed in adults, associated with a high risk of metastasis and thereby, poor prognosis. Among known risk factors for the development of metastatic disease is the loss of BAP1 expression and chromosome 3 monosomy in the primary tumor. However, the expression levels of specific micro RNAs (miRNA) in tumor tissue may also serve as a valuable marker for determining the risk of metastatic disease in patients with primary uveal melanoma. In our study, we analyzed the miRNA expression data of cases selected from The Cancer Genome Atlas study on uveal melanoma, and determined a panel of 15 miRNAs differentially expressed between patients with primary and metastatic disease. Next, 6 miRNAs were validated on a group of 46 tumor samples from primary and metastatic patients. We have shown, that expression of hsa-miR-592, hsa-miR-346, and hsa-miR-1247 was significantly increased, while hsa-miR-506 and hsa-miR-513c were decreased in the tumors of patients with metastatic disease. Hsa-miR-196b expression did not differ between the two subgroups, however, we showed significant correlation with BAP1 expression. Moreover, hsa-miR-592 also showed correlation with monosomy 3 tumors. Gene ontology analysis revealed involvement of those miRNAs with cellular processes mediating the metastatic process. Our results showed that miRNAs play an important role in the deregulation of several oncogenic pathways in UM and can, thereby, promote metastatic spread to distant organs. Moreover, differentially expressed miRNAs may be used as an interesting biomarker for the assessment of metastatic risk in uveal melanoma patients.

Fibroblasts in Nodular Sclerosing Classical Hodgkin Lymphoma Are Defined by a Specific Phenotype and Protect Tumor Cells from Brentuximab-Vedotin Induced Injury.

Classical Hodgkin lymphoma (cHL) is one of the most common malignant lymphomas in Western Europe. The nodular sclerosing subtype of cHL (NS cHL) is characterized by a proliferation of fibroblasts in the tumor microenvironment, leading to fibrotic bands surrounding the lymphoma infiltrate. Several studies have described a crosstalk between the tumour cells of cHL, the Hodgkin- and Reed-Sternberg (HRS) cells, and cancer-associated fibroblasts. However, to date a deep molecular characterization of these fibroblasts is lacking. Thus, the aim of the present study is a comprehensive characterization of these fibroblasts. Gene expression profiling and methylation profiles of fibroblasts isolated from primary lymph node suspensions revealed persistent differences between fibroblasts obtained from NS cHL and lymphadenitis. NS cHL derived fibroblasts exhibit a myofibroblastic phenotype characterized by myocardin (MYOCD) expression. Moreover, TIMP3, an inhibitor of matrix metalloproteinases, was strongly upregulated in NS cHL fibroblasts, likely contributing to the accumulation of collagen in sclerotic bands of NS cHL. As previously shown for other types of cancer-associated fibroblasts, treatment by luteolin could reverse this fibroblast phenotype and decrease TIMP3 secretion. NS cHL fibroblasts showed enhanced proliferation when they were exposed to soluble factors released from HRS cells. For HRS cells, soluble factors from fibroblasts were not sufficient to protect them from Brentuximab-Vedotin induced cell death. However, HRS cells adherent to fibroblasts were protected from Brentuximab-Vedotin induced injury. In summary, we confirm the importance of fibroblasts for HRS cell survival and identify TIMP3 which probably contributes as a major factor to the typical fibrosis observed in NS cHL.

Downregulation of CEACAM6 gene expression in laryngeal squamous cell carcinoma is an effect of DNA hypermethylation and correlates with disease progression.

We have turned our attention to CEACAM6 gene, already described as deregulated in various types of cancer. By using the expression microarrays performed on the set of 16 laryngeal squamous cell carcinoma (LSCC) samples: 11 cell lines and 5 primary tumors we have shown downregulation of CEACAM6 gene as compared to non cancer controls from head and neck region. CEACAM6 gene downregulation, further confirmed by quantitative PCR on 25 LSCC cell lines, was observed in cell lines derived from recurrent tumors in comparison to controls. A significant gene downregulation was observed in cell lines derived from advanced, high grade tumors in comparison to controls. Intrigued by the recurrent transcriptional loss of CEACAM6 we searched for the mechanism potentially responsible for its downregulation and hence we analyzed DNA copy number changes (a-CGH), promoter DNA methylation status and occurrence of gene mutations (in silico). Neither the analysis of gene copy number, nor the mutation screen has shown recurrent deletions or mutations, that could contribute to the observed downregulation of the gene. However, by using bisulfite pyrosequencing, we have shown DNA hypermethylation (mean DNA methylation > 78%) of CEACAM6 promoter region in 9/25 (36%) LSCC cell lines. Importantly, the 5-aza-2-deoxycytidine-induced inhibition of DNA methylation resulted in restoration of CEACAM6 expression in the two LSCC cell lines on mRNA level. In summary, we have shown that recurrent downregulation of CEACAM6 in LSCC is dependent on the gene's promoter DNA methylation and is observed predominantly in large, poorly differentiated tumors and recurrences.

Single nucleotide polymorphism rs11614913 associated with CC genotype in miR-196a2 is overrepresented in laryngeal squamous cell carcinoma, but not salivary gland tumors in Polish population.

The miRNA-196a2 has shown significance in the development of various neoplasms, including head and neck squamous cell carcinoma (HNSCC). The oncogenic functionality of this miRNA is mediated via its potential to target annexin A1 mRNA, a tumor suppressor gene involved in inhibition of the NF-κB pathway. Interestingly, recent data indicate a susceptibility for aforementioned neoplasms in patients with the CC genotype vs the CT and TT genotypes of the rs11614913 SNP located within the DNA sequence of the miR-196a2 that results in elevated expression of the gene. To further investigate this phenomenon, we genotyped this SNP in 40 patients with laryngeal squamous cell carcinoma (LSCC), the most common tumor of the head and neck region and 60 patients with salivary gland tumors (SGT) that show a yet unexplained incidence increase in the last two decades. In agreement with previous reports, we have identified a statistically significant (p < 0.05) overrepresentation of the CC genotype in LSCC patients and demonstrated in LSCC cell lines that it results in elevated expression of miR-196a2 as compared to cell lines with the TT genotype of the respective SNP. Importantly, none of these correlations was found in patients with SGT. These findings underline the importance of the SNP rs11614913 for LSCC development in the Polish population and moreover highlight the different genetic background of the two studied neoplasms of the head and neck region.

Recurrent epigenetic silencing of the PTPRD tumor suppressor in laryngeal squamous cell carcinoma.

Cellular processes like differentiation, mitotic cycle, and cell growth are regulated by tyrosine kinases with known oncogenic potential and tyrosine phosphatases that downmodulate the first. Therefore, tyrosine phosphatases are recurrent targets of gene alterations in human carcinomas. We and others suggested recently a tumor suppressor function of the PTPRD tyrosine phosphatase and reported homozygous deletions of the PTPRD locus in laryngeal squamous cell carcinoma. In this study, we investigated other gene-inactivating mechanisms potentially targeting PTPRD, including loss-of-function mutations and also epigenetic alterations like promoter DNA hypermethylation. We sequenced the PTPRD gene in eight laryngeal squamous cell carcinoma cell lines but did not identify any inactivating mutations. In contrast, by bisulfite pyrosequencing of the gene promoter region, we identified significantly higher levels of methylation (p = 0.001 and p = 0.0002, respectively) in 9/14 (64%) laryngeal squamous cell carcinoma cell lines and 37/79 (47%) of primary laryngeal squamous cell carcinoma tumors as compared to normal epithelium of the upper aerodigestive tract. There was also a strong correlation (p = 0.0001) between methylation and transcriptional silencing for the PTPRD gene observed in a cohort of 497 head and neck tumors from The Cancer Genome Atlas dataset suggesting that DNA methylation is the main mechanism of PTPRD silencing in these tumors. In summary, our data provide further evidence of the high incidence of PTPRD inactivation in laryngeal squamous cell carcinoma. We suggest that deletions and loss-of-function mutations are responsible for PTPRD loss only in a fraction of cases, whereas DNA methylation is the dominating mechanism of PTPRD inactivation.