Magdalena Kowalewska

Systemic inflammation as a confounding factor in cancer biomarker discovery and validation

A crucial aspect of tumour biology, inflammation, is often overlooked in biomarker studies and needs to be urgently addressed.

Sdf-1 (CXCL12) improves skeletal muscle regeneration via the mobilisation of Cxcr4 and CD34 expressing cells

The regeneration of skeletal muscles involves satellite cells, which are muscle‐specific precursor cells. In muscles, injured either mechanically or as a consequence of a disease, such as muscular dystrophy, local release of the growth factors and cytokines leads to satellite cells activation, proliferation and differentiation of the resulting myoblasts, followed by the formation of new myofibres. Various cell types, such as stem and progenitor cells, originating from other tissues different than the muscle, are also able to follow a myogenic program. Participation of these cells in the repair process depends on their precise mobilisation to the site of the injury.

The accuracy of the sentinel lymph node concept in early stage squamous cell vulvar carcinoma

OBJECTIVE The purpose of the study was to determine the feasibility and accuracy of the sentinel lymph node (SLN) identification in vulvar carcinoma patients. METHODS Sixty-two patients with clinical early stage vulvar cancer underwent SLN detection procedure, followed by a complete inguinofemoral lymphadenectomy. The SLN was identified intraoperatively using lymphoscintigraphy with technetium-99m as well as patent blue V staining. The resected lymph nodes (LN) were submitted for histological examination by hematoxylin-eosin staining (H-E) and cytokeratin immunohistochemistry (IHC) and examined by the reverse transcriptase-polymerase chain reaction (RT-PCR) assay. RESULTS A total of 109 inguinal LN were dissected in 56 patients. SLNs were identified in 76% groins with patent blue V and in 99% with the use of Tc-99m. The accuracy differed significantly (p<0.0001). An H-E examination combined with IHC revealed 7 false-negative SLNs. The sensitivity of this method was 73% (95% CI, 64% to 81%) and the negative predictive value for a negative SLN finding was 92% (95% CI, 87% to 97%). The RT-PCR assay showed 8 false-negative SLNs. The sensitivity of the RT-PCR-based assay was 83% (95% CI, 75% to 90%) and the negative predictive value for a negative SLN was 88% (95% CI, 82% to 94%). The two diagnostic methods were found not to differ significantly. CONCLUSIONS In SLN mapping, the Tc-99m colloid lymphoscintigraphy is superior to the blue dye staining. Our data do not support the concept of the SLN identification as a highly accurate procedure in predicting the inguinofemoral LN status in patients with early stage vulvar cancer.

Detection of carbonic anhydrase 9–expressing tumor cells in the lymph nodes of vulvar carcinoma patients by RT‐PCR

Regional lymph node status is an important prognostic factor for vulvar cancer. The goal of our study was to elaborate a reliable test for detecting micrometastases, undetectable by traditional methods, in the lymph nodes of vulvar squamous carcinoma patients. For this purpose, carbonic anhydrase‐9 (CA9) was investigated as a cancer‐related marker by RT‐PCR. Firstly, primary carcinoma specimens were examined for CA9 expression by immunohistochemistry with M75 monoclonal antibody. All 19 tissues exhibited a variable degree of staining, which was mostly confined to the plasma membranes of tumor cells. Correspondingly, all primary tumor specimens and the control A‐431 vulvar cancer cell line gave a positive signal in the nested RT‐PCR assay designed to detect CA9‐expressing cells with a high sensitivity. Analysis of 77 lymph node specimens from 20 patients revealed a full correlation between RT‐PCR results and standard hematoxylin–eosin staining in 75% of samples, whereas 25% of specimens were negative by the standard method and positive for CA9 mRNA, accounting for 28% of all histologically negative lymph nodes. There were no false‐negatives with RT‐PCR. A positive inguinal lymph node with a negative sentinel node was observed in the same groin only once in 38 specimens. Our findings clearly indicate potential value of CA9 as a molecular marker for the assessment of regional lymph node status in vulvar cancer patients and support a possible utility of our RT‐PCR assay in the detection of micrometastases.

Mitochondrial genotype in vulvar carcinoma - cuckoo in the nest

Vulvar squamous cell carcinoma (VSCC) is a rare female genital neoplasm. Although numerous molecular changes have been reported in VSCC, biomarkers of clinical relevance are still lacking. On the other hand, there is emerging evidence on the use of mtDNA as a diagnostic tool in oncology. In order to investigate mtDNA status in VSCC patients, haplogroup distribution analysis and D-loop sequencing were performed. The results were compared with available data for the general Polish population, cancer free-centenarians as well as patients with endometrial and head and neck cancer. The obtained data were also compared with the current status of mitochondrial databases. Significant differences in haplogroup distribution between VSCC cohort, general Polish population and cancer-free centenarians cohort were found. Moreover, a correlation between the VSCC patients haplogroup and HPV status was observed. Finally, a specific pattern of mtDNA polymorphisms was found in VSCC. Our results suggest that the mitochondrial genetic background may influence the risk of VSCC occurrence as well as susceptibility to HPV infection.

The frequency of human papillomavirus infection in polish patients with vulvar squamous cell carcinoma.

Introduction: Vulvar cancer is a rare condition representing about 4% of all female genital tract tumors. In contrast to the established relationship of virtually all cervical cancer cases with the human papillomavirus (HPV) infection, the reported HPV positivity in vulvar carcinoma ranges widely. Methods: Using the Linear Array HPV Genotyping Test, we investigated the HPV incidence in a group of 46 Polish patients with vulvar squamous cell carcinoma (age range, 37-93 years; median age, 70.2 years) in clinical stages T1-2, N0-2, and M0. Results: The presence of HPV DNA was confirmed in 7 of 46 (15%) primary tumor samples. HPV 16 was found in 5 tumors (71%). HPVs 6 and 58 were detected in the remaining 2 cases of virus-associated tumors. Conclusions: We conclude that a fraction of cancers of vulva associated with HPV is insignificant, given the HPV prevalence of 8.6% in the Polish population aged 55 to 59 years (the oldest cohort of Polish women studied to date).

Squamous cell carcinoma antigen 1 and 2 expression in cultured normal peripheral blood mononuclear cells and in vulvar squamous cell carcinoma

Squamous cell carcinoma antigen (SCCA) is expressed in normal squamous cell epithelia and in squamous cell carcinomas (SCC). Two nearly identical genes encode the inhibitory serpins SCCA1 (SERPINB3) and SCCA2 (SERPINB4). Serum levels of SCCA are elevated in patients with benign skin diseases and in patients with SCC. SCCA, used for the monitoring of SCC patients, presents no satisfactory diagnostic specificity. As we have shown previously, the reverse transcription polymerase chain reaction (RT-PCR)-based SCCA messenger RNA (mRNA) testing aimed at detecting disseminated cancer cells may be hampered by the false-positive results due to SCCA expression in activated peripheral blood mononuclear cells (PBMC). The aim of this study was to assess the expression of SCCA at mRNA and protein levels in cultured normal PBMC, compared to that in vulvar SCC (VSCC) samples. High SCCA concentrations were found in vulvar tumours and in metastatic lymph nodes, while negative inguinal lymph nodes from the same patients often presented significantly less SCCA. In normal activated PBMC, the level of SCCA protein was the lowest. At the mRNA level SCCA was detectable in normal PBMC even in cultures with no mitogen stimulation, but only by the nested RT-PCR, contrary to VSCC samples found to be SCCA positive already in one-step PCR. Both SCCA1 and SCCA2 transcripts were present in cultured PBMC; SCCA1 was expressed at a higher level than SCCA2. In conclusion, both SCCA forms are detectable in normal PBMC cultured in vitro. SCCA expression level in normal PBMC is much lower than in the squamous epithelium-derived cells. In VSCC, in addition to tumour itself, metastatic lymph nodes seem also to be a potential source of serum SCCA.

Lack of microsatellite instability in squamous cell vulvar carcinoma

Mutator phenotypes with microsatellite instability (MSI) are observed in a subset of solid tumors. The objective of our study was to investigate the occurrence of MSI in vulvar squamous cell carcinoma (VSCC) and a possible relation between MSI and the presence of human papilloma virus (HPV). DNA samples from 44 tissue specimens of the primary VSCC as well as from six metastatic lymph node samples were analysed and compared with matched reference DNA from blood samples. The MSI status was examined by polymerase chain reaction (PCR) using the Bethesda panel of five microsatellite markers. PCR products were analysed by fluorescent capillary electrophoresis. No microsatellite instability was detected in tumor samples or in metastatic lymph nodes from any of the VSCC patients examined. Microsatellite instability seems not to play a major role in the carcinogenesis of VSCC and is probably not associated with the HPV‐related genetic background of this neoplasm.

BCOR Internal Tandem Duplication in High-grade Uterine Sarcomas

Endometrial stromal sarcomas (ESSs) are mesenchymal uterine tumors characterized by recurrent genetic events, most commonly chromosomal rearrangements, that create oncogenic gene fusions. High-grade endometrial stromal sarcomas (HG-ESSs), as defined in the 2014 World Health Organization Classification, typically contain oncogenic YWHAE-NUTM2 fusions; however, although not well characterized, there are tumors morphologically overlapping with HG-ESS that do not contain the YWHAE-NUTM2 fusions. These fusions are also found in certain pediatric primitive sarcomas, including clear cell sarcoma of the kidney and soft tissue undifferentiated round cell sarcoma of infancy. A subset of these same pediatric sarcomas lack YWHAE-NUTM2 fusions and instead have internal tandem duplications (ITDs) involving exon 15 of BCOR (BCOR ITD). We investigated the presence of BCOR ITD by targeted sequencing in a series of 31 uterine sarcomas, comprising 5 low-grade ESS, 13 uterine sarcomas diagnosed as HG-ESS, and 13 undifferentiated uterine sarcomas. BCOR ITD were present in 1 uterine sarcoma diagnosed as HG-ESS and 2 undifferentiated sarcomas with uniform nuclear features, all of which lacked any of the recurrent chromosome translocations known to occur in ESS. These 3 high-grade sarcomas with BCOR ITD affected young patients (average age, 24) and morphologically were composed of nonpleomorphic spindle cells admixed with epithelioid and round cell areas. Focal myxoid stroma was present in 2 cases. Mitotic activity was brisk, necrosis was present, and there was lymphovascular involvement in all cases. The 3 uterine sarcomas with BCOR ITD exhibited diffuse cyclin D1 immunohistochemical expression and there was diffuse BCOR expression in the 2 cases tested. Long-term follow-up in 2 patients revealed 1 to be tumor-free after 22 years and the other to die of disease after 8 years. In conclusion, BCOR ITD is an oncogenic alternative to YWHAE-NUTM2 fusion in high-grade uterine sarcomas with uniform nuclear features. We propose that neoplasms with the morphology described and BCOR ITD be regarded as a unique subtype of high-grade uterine sarcoma, possibly within the family of endometrial stromal neoplasia.

Molecular Mechanism for Cellular Response to β-Escin and Its Therapeutic Implications.

β-escin is a mixture of triterpene saponins isolated from the horse chestnut seeds (Aesculus hippocastanum L.). The anti-edematous, anti-inflammatory and venotonic properties of β-escin have been the most extensively clinically investigated effects of this plant-based drug and randomized controlled trials have proved the efficacy of β-escin for the treatment of chronic venous insufficiency. However, despite the clinical recognition of the drug its pharmacological mechanism of action still remains largely elusive. To determine the cellular and molecular basis for the therapeutic effectiveness of β-escin we performed discovery and targeted proteomic analyses and in vitro evaluation of cellular and molecular responses in human endothelial cells under inflammatory conditions. Our results demonstrate that in endothelial cells β-escin potently induces cholesterol synthesis which is rapidly followed with marked fall in actin cytoskeleton integrity. The concomitant changes in cell functioning result in a significantly diminished responses to TNF-α stimulation. These include reduced migration, alleviated endothelial monolayer permeability, and inhibition of NFκB signal transduction leading to down-expression of TNF-α—induced effector proteins. Moreover, the study provides evidence for novel therapeutic potential of β-escin beyond the current vascular indications.