There is still a role for cytology in the 'liquid biopsy' era. A lesson from a TKI-treated patient showing adenocarcinoma to squamous cell carcinoma transition during disease progression

Non-small cell lung carcinoma harbouring epidermal growth factor receptor (EGFR) mutation, usually progress after an initial response to tyrosine-kinase inhibitors (TKI). Liquid biopsy enables with a simple blood draw the accurate detection of EGFR p.T790M mutation, the most common resistance mechanism, avoiding the more invasive tissue re-biopsy. However, in a subset of cases, resistance mechanisms are more complex featuring both genetic and morphological changes. Here we report the case of a 67 years-old woman, affected by an EGFR mutated lung adenocarcinoma and treated by TKI. At disease progression, the patient developed a morphological transition to squamous cell carcinoma in association to the arising of a PIK3CA p.E542K mutant subclone. This case illustrates that, even in the "liquid biopsy" era, cytology can have still a role by providing an overall assessment of both morphology and genetic TKI resistance mechanisms

A Critical Issue in Lung Cancer Cytology and Small Biopsies: DNA and RNA Extraction from Archival Stained Slides for Biomarker Detection through Real Time PCR and NGS—The Experience in Pathological Anatomy Unit

The handling of biomaterials is crucial for precision medicine in advanced-stage lung patients with only cytology or small biopsies available. The main purpose of the study was to evaluate the quantity and quality of nucleic acids extracted from mixed stained slides (MSSs), including H&E, IHC and FISH, compared to the extraction from unstained slides (USs). A series of 35 lung adenocarcinoma surgical samples was selected to set up the method and the technical approach was validated in a series of 15 small biopsies and 38 cytological samples. DNA extracted from MSSs was adequate in all samples and the Real Time PCR was successful in 30/35 surgical samples (86%), 14/15 small biopsies (93%), and 33/38 cytological samples (87%). NGS using DNA extracted from MSSs was successful in 18/35 surgical samples (51%), 11/15 small biopsies (73%), and 26/38 cytological samples (68%). RNA extracted from MSSs was unsatisfactory in all cases showing an inadequate degree of fragmentation. Our technical approach based on the recovery of stained slides could represent a strategic way forward for DNA-based biomarker testing in lung cancer cases without biomaterials. The RNA extracted from MSSs did not represent a successful approach

Application of CytoMatrix for the diagnosis of melanoma metastases on FNA cytology samples: Performance of a novel cell block method

BackgroundThe management of cytological samples can significantly impact diagnostic interpretation. Cell blocks (CB) are a popular method due to their ability to provide additional morphological information and be used for immunocytochemistry and molecular tests. Recently, a new CB technique called the synthetic matrix CytoMatrix (CM) has been introduced, which can gather and hold cytological material within its three-dimensional structure. MethodsIn this study, 40 cytological samples from patients with melanoma metastases were analyzed to evaluate the diagnostic performance of CM compared to another CB method used in the laboratory. The researchers assessed the morphological adequacy of the two techniques, as well as their performance in immunocytochemical analysis and molecular. ResultsThis study showed that CM was quicker and equally effective compared to the other method, with the laboratory technician having less of an impact on the CM approach across all passages. Additionally, all CMs were adequate, whereas the other method was adequate in 90% of cases. Immunocytochemistry confirmed the diagnosis of melanoma metastases in all cases, and all 40 CMs and 36 of the other method were adequate for fluorescence in situ hybridization analysis. ConclusionCM is a low-time-consuming technology that is unaffected by technicians during all setup phases, making it simpler to standardize the procedure. Moreover, a low loss of diagnostic cells ensures greater benefits for morphological analysis, immunocytochemistry, and molecular testing. Overall, the study highlights the potential of CM as a valuable technique for managing cytological samples

FOXE1 gene dosage affects thyroid cancer histology and differentiation in vivo

The transcription factor Forkhead box E1 (FOXE1) is a key player in thyroid development and function and has been identified by genome-wide association studies as a susceptibility gene for papillary thyroid cancer. Several cancer-associated polymorphisms fall into gene regulatory regions and are likely to affect FOXE1 expression levels. However, the possibility that changes in FOXE1 expression modulate thyroid cancer development has not been investigated. Here, we describe the effects of FOXE1 gene dosage reduction on cancer phenotype in vivo. Mice heterozygous for FOXE1 null allele (FOXE1+/−) were crossed with a BRAFV600E-inducible cancer model to develop thyroid cancer in either a FOXE1+/+ or FOXE1+/− genetic background. In FOXE1+/+ mice, cancer histological features are quite similar to that of human high-grade papillary thyroid carcinomas, while cancers developed with reduced FOXE1 gene dosage maintain morphological features resembling less malignant thyroid cancers, showing reduced proliferation index and increased apoptosis as well. Such cancers, however, appear severely undifferentiated, indicating that FOXE1 levels affect thyroid differentiation during neoplastic transformation. These results show that FOXE1 dosage exerts pleiotropic effects on thyroid cancer phenotype by affecting histology and regulating key markers of tumor differentiation and progression, thus suggesting the possibility that FOXE1 could behave as lineage-specific oncogene in follicular cell-derived thyroid cancer

Current Water Conditions in Massachusetts (2011-01-13)

BACKGROUND: Neutrophil functions have long been regarded as limited to acute inflammation and the defense against microbes. The role(s) of neutrophils in cancer remain poorly understood. Neutrophils infiltrate tumors and are key effector cells in the orchestration of inflammatory responses. Thyroid cancer (TC) is the most recurrent endocrine malignant tumor and is responsible for 70% of deaths due to endocrine cancers. No studies are so far available on the role of neutrophils in TC. OBJECTIVE: Our purpose was to study the involvement of tumor-associated neutrophils in TC. METHODS: Highly purified human neutrophils (>99%) from healthy donors were stimulated in vitro with conditioned media derived from TC cell lines TPC1 and 8505c (TC-CMs). Neutrophil functions (e.g., chemotaxis, activation, plasticity, survival, gene expression, and protein release) were evaluated. RESULTS: TC-derived soluble factors promoted neutrophil chemotaxis and survival. Neutrophil chemotaxis toward a TC-CM was mediated, at least in part, by CXCL8/IL-8, and survival was mediated by granulocyte-macrophage colony-stimulating factor (GM-CSF). In addition, each TC-CM induced morphological changes and activation of neutrophils (e.g., CD11b and CD66b upregulation and CD62L shedding) and modified neutrophils' kinetic properties. Furthermore, each TC-CM induced production of reactive oxygen species, expression of proinflammatory and angiogenic mediators (CXCL8/IL-8, VEGF-A, and TNF-α), and a release of matrix metalloproteinase 9 (MMP-9). Moreover, in TC patients, tumor-associated neutrophils correlated with larger tumor size. CONCLUSIONS: TC cell lines produce soluble factors able to "educate" neutrophils toward an activated functional state. These data will advance the understanding of the molecular and cellular mechanisms of innate immunity in TC

TC-derived soluble factors promoted neutrophil survival.

<p><b>A.</b> Neutrophils were cultured in a TC-CM or the control medium. At the indicated time points, live cells were evaluated by flow cytometry with FITC-conjugated annexin V and PI. Results were expressed as percentages of live cells (mean ± SEM of five independent experiments); ***p < 0.005; **p < 0.01; *p < 0.05. <b>B.</b> Representative flow cytometric panels of dot plots of PMNs cultured in a TC-CM or control medium and stained with FITC-conjugated annexin V and propidium iodide (PI) at 24 (upper panels) and 48 (lower panels) hours. <b>C</b>. The GM-CSF release by TPC1 and 8505c cells was evaluated by an ELISA in a TC-CM or in the control medium. Results were expressed as mean ± SEM of seven independent experiments; ****p < 0.001; ***p < 0.005. <b>D-F.</b> Neutrophil survival in a TPC1-derived <b>(D-E)</b> or 8505c-derived <b>(F-G)</b> conditioned medium was evaluated in the presence of an anti-GM-CSF blocking antibody or the relative isotype control (10 μg/ml). At 24 hours, live cells were stained with FITC-conjugated annexin V and PI and analyzed by flow cytometry. <b>Figs E and G</b> illustrate representative flow cytometric panels of one out of five independent experiments. The results were expressed as mean ± SEM of five independent experiments; **p < 0.01; *p < 0.05.</p

TC-derived soluble factors modified neutrophils’ kinetic properties.

<p>Neutrophils were stimulated with a TC-CM or the control medium for 18 hours. Within this time window, digital phase contrast images of 15 fields/well were captured every 15 minutes via a 20× objective in the Operetta high-content imaging system. PhenoLOGIC (PerkinElmer) was employed for image segmentation and to calculate the single-cell kinetic properties <b>(A)</b>, accumulated distance, <b>(B)</b> speed and straightness <b>(C)</b> in dedicated analysis sequence. The results were expressed as mean ± SEM of six independent experiments; ***p < 0.005; **p < 0.01; *p < 0.05. <b>D-E.</b> Timepoint analyses of time-dependent properties such as current step size (D) and current speed (E) illustrating dynamic changes of the behavior of neutrophils (in presence or absence of TC-CMs) in function of the elapsed time from treatment. The results were expressed as mean ± SEM of six independent experiments; ****p < 0.001.</p

The number of tumor-infiltrating neutrophils positively correlated with tumor size in human TC specimens.

<p><b>A and B.</b> Histological analysis of TC specimens stained with a monoclonal anti-CD66b antibody. Whole-tumor section density of CD66b⁺ neutrophils was scored at 200× magnification. Representative cases of papillary thyroid carcinomas with a high <b>(A)</b> and low <b>(B)</b> CD66b⁺ neutrophil count (arrows; hematoxylin counterstaining, 200×). <b>C.</b> CD66b⁺ neutrophil counts in tumor specimens were distributed according to the tumor size. The median value of tumor size served as a cutoff level. Results are shown as the median, the 25th and 75th percentiles (boxes), and 5th and 95th percentiles (whiskers); *p < 0.05, according to the two-tailed Mann–Whitney <i>U</i> test. <b>D.</b> Neutrophil density positively correlated with larger tumor size in TC patients (r = 0.43; p = 0.01; Pearson’s correlation test).</p

TC-CMs induced morphological changes in neutrophils.

<p>Neutrophils were stimulated with a TC-CM or the control medium for 16 hours and then were imaged by means of an Operetta high-content imaging system at 20× magnification. The images were analyzed in the Harmony software with PhenoLOGIC (PerkinElmer) and a dedicated analysis sequence (morphological properties, method STAR) to evaluate cell area <b>(A)</b>, roundness <b>(B)</b>, radial mean <b>(C)</b>, symmetry <b>(D)</b>, width-to-length ratio <b>(E)</b>, and axial length <b>(F)</b>. The results were expressed as an increase or decrease compared to the control (mean ± SEM of five independent experiments); ***p < 0.005.</p