Fast and reliable Sanger POLE sequencing protocol in FFPE tissues of endometrial cancer.

BACKGROUND: The new classification of endometrial carcinoma (EC) requires molecular interpretation of somatic polymerase epsilon (POLE) exonuclease domain mutations. The identification of pathogenic mutations within the POLE gene defines the important subtype of ultramutated tumours ("POLE-ultramutated") with specified prognostic and predictive utility. POLE somatic mutations are present in 7-12% of ECs, usually high-grade tumours with aggressive appearance. Molecular analysis of the POLE gene can be performed using a qPCR test, the Sanger sequencing method, a next generation sequencing (NGS) panel test and also in situ hybridisation (IHC) assay. We describe our current approach of identification of POLE mutations using Sanger sequencing technology, which is still the most robust, accurate and fast technique to sequence DNA. MATERIALS AND METHODS: We present a reliable protocol for Sanger sequencing of the entire sequence coding exonuclease domain of POLE - exons 9, 10, 11, 12, 13 and 14 (codons 268-491) with 5-10 nucleotides in exon/intron boundaries (reference sequences: NM_006231.4, NP_006222.2). RESULT: The protocol has been optimized for formalin-fixed, paraffin-embedded (FFPE) EC tissues. CONCLUSION: The method developed in our laboratory allows better diagnosis of patients with EC according to current standards.

Detection of BRCA1/2 pathogenic variants in patients with breast and/or ovarian cancer and their families. Analysis of 3,458 cases from Lower Silesia (Poland) according to the diagnostic algorithm of the National Cancer Control Programme.

Breast and ovarian cancers are among the most common malignancies in the female population, with approximately 5-10% of cases being hereditary. BRCA1 and BRCA2 with other homologous recombination genes are the most tested genes in hereditary breast and ovarian cancer (HBOC) patients. As next-generation sequencing (NGS) has become a standard and popular technique, such as for HBOC, it has greatly simplified and accelerated molecular diagnosis of cancer. The study group included 3,458 HBOC patients or their relatives from Lower Silesia (Poland) (a voivodeship located in south-west Poland inhabited by 2.9 million people). All patients were tested according to the recommendations from the National Cancer Control Programme of the Ministry of Health for the years 2018-21. We tested 3,400 patients for recurrent pathogenic variants for the Polish population: five BRCA1 founder variants (c.5266dup, c.181T>G, c.4035del, c.3700_3704del, and c.68_69del), two PALB2 variants (c.509_510del, c.172_175del) and three CHEK2 variants [c.1100del, c.444+1G>A, g.27417113-27422508del (del5395)]. Next 260 patients from the study group were chosen for the BRCA1/2 NGS panel, and additionally selected marker pathogenic variants were tested using Sanger sequencing and MLPA methods in 45 and 13 individuals, respectively. The analysis of BRCA1/2 in the 3,458 patients with HBOC or their relatives revealed 144 carriers of 37 different pathogenic variants (22 in BRCA1 and 15 in BRCA2). Among all detected variants, 71.53% constituted founder pathogenic BRCA1 variants. Our study has revealed that for the Lower Silesian population, the first-line BRCA1/2 molecular test may be limited to only three variants in BRCA1-c.5266dup, c.181T>G, and c.4035del-but the aim should be to provide a full screening test of HBOC critical genes. The key and still growing role of molecular diagnostics of neoplasms, which includes HBOC, is undeniable. Therefore, it is necessary to provide complete and optimal therapeutic and prophylactic algorithms in line with current medical knowledge.

Analysis of lung cancer measures of the National Cancer Network pilot study in Poland for potential improvement in the quality of advanced-stage lung cancer therapy.

BACKGROUND: This study aimed to present the performance of the National Cancer Network's (NCN) pilot program in the Lower Silesian Voivodeship (southwestern province of Poland with a population of 2,9 million in 2019), to analyse measures describing lung cancer patients and to determine whether those measures can be used to improve the treatment outcomes of stage III and IV patients with lung cancer in Poland. METHODS: Three measures of the NCN pilot programme were analysed: "Percentage of patients with genetic and molecular testing for predictive factors", "Assessment of the completeness of a pathological examination", and "Percentage of stage III and IV cancer patients". As many as 2,218 patients with ICD-10-CM Diagnosis Code C34 were included in the NCN pilot program from 1 to 2019 to 31 December 2020, in the Lower Silesian Voivodeship. The scores of each measure were calculated quarterly by the Regional Coordinating Centre, Wroclaw Comprehensive Cancer Center, Wroclaw, Poland. RESULTS: Genetic and molecular testing among stage III and IV patients was performed in only 40% and 60% of patients, respectively. The incompleteness of histopathological examinations did not exceed 0.5%. Stage III and stage IV patients accounted for 37% and 35% of the analysed patients, respectively. CONCLUSIONS: The NCN pilot program measures presented in this study appear to be highly sensitive, simple, and transparent tools to monitor the quality of lung cancer diagnosis and assess clinical staging in patients within a specific region. An increase in the proportion of stage III and IV patients who will undergo genetic and molecular testing in the era of modern drug therapies should result in improved treatment outcomes in this patient group. In the present analysis, the values of the main analysed measure, which evaluates the number of genetic and molecular tests for predictive factors for lung cancer, were subject to significant fluctuations during the pilot project. Both upwards and downwards trends were observed. Further analysis in the future is warranted to eliminate the unfavourable factors influencing the obtained values of the measure.

Next generation sequencing of glioblastoma circulating tumor cells: non-invasive solution for disease monitoring.

Treatment of aggressive glioblastoma multiforme (GBM) must be based on very precise histological and molecular diagnostic of GBM type. According to the WHO guidelines, only tissue biopsy is a relevant source of cellular material evaluated in the diagnostic process to specify the tumor features. Nevertheless, obtaining a GBM biopsy is complicated and relies mostly on resection surgery. Evaluating circulating free DNA and/or circulating tumor cells (CTCs) in the clinic, using a liquid biopsy could represent a non-invasive cancer care optimization. In the present study, the peripheral blood of patients undergoing GBM resection (n = 18) was collected and examined for CTCs. The feasibility of GBM molecular diagnostics from a simple non-invasive peripheral blood withdrawal was evaluated. The size-based enriched CTCs were analyzed using cytomorphology and their origin confirmed based on mutational analysis. In addition, shared DNA mutations in CTCs and in primary tumor tissue were searched. For the identification of CTCs, next generation sequencing (NGS) was used. The GeneReader™ sequencing platform enables targeted sequencing of a 12-gene panel and direct evaluation of detected gene variations using QIAGEN Clinical Insight Analyze (QCI-A) software with a special algorithm for liquid biopsy sequencing analysis. Herein, we present a standard operating procedure for CTC enrichment in GBM patients, CTC in vitro culture, CTC cytomorphological evaluation, and NGS analysis of CTCs using the QIAGEN Actionable Insights Tumor (ATP) Panel. CTCs were present in all tested patients (18/18). The NGS data generated for formalin-fixed paraffin-embedded (FFPE) primary tumor tissues and CTCs reached significantly high-quality parameters. The comparisons between different sample types (CTCs vs. primary tumors) and sampling area (different primary tumor regions) showed a significant level of concordance, indicating CTC testing could be used for patient monitoring and recurrence awareness. Notably, more mutations were detected when analyzing CTC samples compared with the paired primary tumors (n = 3). The results confirm the feasibility of using CTCs as a source of tumor DNA in a diagnostic process, especially when evaluating the molecular characteristics of GBMs. A major advantage of the presented NGS approach for detecting CTCs is the simultaneous identification of several markers relevant for GBM diagnostics, allowing molecular diagnostics on cytological specimens and potential administration of innovative targeted therapies.

Detection of Circulating Tumor Cells in Renal Cell Carcinoma: Disease Stage Correlation and Molecular Characterization.

The presence of circulating tumor cells (CTCs) in patients with solid tumors is associated with poor prognosis. However, there are limited data concerning the detection of CTCs in renal cell cancer (RCC). The aim of this study is to evaluate the presence of CTCs in peripheral blood of patients with RCC undergoing surgery (n = 186). CTCs were tested before and after surgery as well as during the follow-up period afterwards. In total 495 CTC testing in duplicates were provided. To enrich CTCs, a size-based separation protocol and tube MetaCell® was used. CTCs presence was evaluated by single cell cytomorphology based on vital fluorescence microscopy. Additionally, to standardly applied fluorescence stains, CTCs viability was controlled by mitochondrial activity. CTCs were detected independently on the sampling order in up to 86.7% of the tested blood samples in patients undergoing RCC surgery. There is higher probability of CTC detection with growing tumor size, especially in clear cell renal cell cancer (ccRCC) cases. Similarly, the tumor size corresponds with metastasis presence and lymph node positivity and CTC detection. This paper describes for the first-time successful analysis of viable CTCs and their mitochondria as a part of the functional characterization of CTCs in RCC.

Circulating tumor cells in gynaecological malignancies.

New non-invasive approaches have developed for diagnosis and treatment of malignant diseases. Cells shed from the primary tumor circulating in the bloodstream with metastasis potential are called Circulating Tumor Cells (CTCs). These cells are easily acquired from the peripheral blood of patients, while several enrichment and isolation methods are available nowadays with different benefits and positive detection rates. A brief characterization of three major categories of detection is described (nucleic acid-based, physical properties-based, antibody-based). In this review we concentrate on gynecological malignancies and how CTCs could be used in the diagnosis of cancer, treatment management and its effective prognosis and early recurrence detection. Presence of CTCs in endometrial cancer patients show worse overall survival, while gene analysis could identify patients in need of systemic therapy after surgical treatment to prevent metastasis and recurrence. Based on the influence of human papillomavirus (HPV) in the etiology of cervical cancer, viral oncogene transcripts could be used as an ideal marker for cervical cancer cells detection. In ovarian cancer, CTCs could help in the differentiation from benign adnexal masses and show a high independence from other biomarkers such as CA125 and HE4. While detection of CTC after complete cytoreductive surgery could indicate invisible lesions, combination of tumor associated genes rises the specificity of CTC detection.

Circulating Endometrial Cells in Women With Spontaneous Pneumothorax.

BACKGROUND: The occurrence of catamenial pneumothorax (CP) is rare, and the awareness of this diagnosis among physicians is insufficient. CP is highly correlated with pelvic endometriosis and remains the most common form of thoracic endometriosis syndrome. Circulating endometrial cells (CECs) have been previously detected in patients with pelvic endometriosis. Could CECs bring new insights into pneumothorax management? METHODS: This study aims to describe the occurrence and molecular characteristics of CECs in women with spontaneous pneumothorax (SP) (N = 20) with high suspicion of its catamenial character. CECs were enriched from peripheral blood by size-based separation (MetaCell). In addition to cytomorphology, gene expression profiling of captured cells was performed for 24 endometriosis-associated genes. RESULTS: CECs were present in all 20 patients with SP. Enriched CECs exhibited four character features: epithelial, stem cell-like, stroma-like, and glandular. However, not all of them were present in every sampling. Gene expression profiling revealed two distinct phenotypes of CECs in SP and/or CP: one of them refers to the diaphragm openings syndrome and the other to endometrial tissue pleural implantations. Comparisons of the gene expression profiles of CECs in pneumothorax (CECs-SP group) with CECs in pelvic endometriosis (CECs-non-SP group) have revealed significantly higher expression of HER2 in the CECs-SP group compared with the CECs-non-SP group. CONCLUSIONS: This proof-of-concept study demonstrates successful isolation and characterization of CECs in patients with SP. Identification of CECs in SP could alert endometriosis involvement and help early referral to gynecologic consultation for further examination and treatment.

Del Nido cardioplegia - what is the current evidence?

Del Nido cardioplegia is believed to be both clinically and economically efficient. The interest in this myocardial protection method has been continuously growing. However, the evidence is not clear. The article summarizes recent reports regarding del Nido cardioplegia.

Immunomagnetic detection of cancer cells in pleural effusion of generalized cancer.

Malignant pleural effusions (MPE) are a common clinical problem in patients with neoplastic disease. Pleural fluid cytology is the simplest definitive method for obtaining a diagnosis of MPE. We describe a method that may increase the cancer cell detection rate using immunomagnetic separation in MPE. In comparison to standard MPE cytodiagnostic methods, we report a more streamlined method of isolation living cells that are able to proliferate. These captured cells can then be used for additional downstream analysis e.g. chemosensitivity testing. Several case studies of MPE diagnostics using immunomagnetic separation are presented in the following report. The immunomagnetic separation of cancer cells from MPE could be used for more accurate staging of patients with routine effusions.