Clinical application of the HM-1000 image processing for HER2 fluorescence in situ hybridization signal quantification in breast cancer.

BACKGROUND: Accurate quantification of human epidermal growth factor receptor 2 (HER2) gene amplification is important for predicting treatment response and prognosis in patients with breast cancer. Fluorescence in situ hybridization (FISH) is the gold standard for the diagnosis of HER2 status, particularly in cases with equivocal status on immunohistochemistry (IHC) staining, but has some limitations of non-classical amplifications and such cases are diagnosed basing on additional IHC and FISH. This study investigated the clinical utility of a novel super-resolution fluorescence microscopy technique for the better FISH signal visualization and HER2 FISH classification. METHODS: Fourteen breast cancer tissue samples were retrospectively collected between September 2018 and February 2022, and FISH HER2 signal quantification was evaluated by determining the HER2/chromosome 17 centromere (CEP17) ratio and the number of HER2 signals per nucleus in super- versus conventional-resolution images. RESULTS: Super-resolution images maintained the same overall HER2 diagnosis from routine, but HER2 FISH amplification changed negative to monosomy in two cases. Two Letrozole non-response relapses coincided to monosomy samples. The median number of HER2 signals per nucleus was 7.5 in super-resolution images and 4.0 in conventional-resolution images in HER2-positive samples and 2.8 and 2.1 signals per nucleus, respectively, in HER2-negative samples. CONCLUSIONS: Super-resolution images improved signal visualization, including a significant difference in the number of countable HER2 and CEP17 signals in a single nucleus compared with conventional-resolution images. Increased accuracy of signal quantification by super-resolution microscopy may provide clinicians with more detailed information regarding HER2 FISH status that allows to better FISH classification such as HER2-low samples.

Glyoxal acid-free (GAF) histological fixative is a suitable alternative to formalin: results from an open-label comparative non-inferiority study.

Formalin, an aqueous solution of formaldehyde, has been the gold standard for fixation of histological samples for over a century. Despite its considerable advantages, growing evidence points to objective toxicity, particularly highlighting its carcinogenicity and mutagenic effects. In 2016, the European Union proposed a ban, but a temporary permission was granted in consideration of its fundamental role in the medical-diagnostic field. In the present study, we tested an innovative fixative, glyoxal acid-free (GAF) (a glyoxal solution deprived of acids), which allows optimal tissue fixation at structural and molecular level combined with the absence of toxicity and carcinogenic activity. An open-label, non-inferiority, multicentric trial was performed comparing fixation of histological specimens with GAF fixative vs standard phosphate-buffered formalin (PBF), evaluating the morphological preservation and the diagnostic value with four binary score questions answered by both the central pathology reviewer and local center reviewers. The mean of total score in the GAF vs PBF fixative groups was 3.7 ± 0.5 vs 3.9 ± 0.3 for the central reviewer and 3.8 ± 0.5 vs 4.0 ± 0.1 for the local pathologist reviewers, respectively. In terms of median value, similar results were observed between the two fixative groups, with a median value of 4.0. Data collected indicate the non-inferiority of GAF as compared to PBF for all organs tested. The present clinical performance study, performed following the international standard for performance evaluation of in vitro diagnostic medical devices, highlights the capability of GAF to ensure both structural preservation and diagnostic value of the preparations.

Dissecting Breast Cancer Circulating Tumor Cells Competence via Modelling Metastasis in Zebrafish.

BACKGROUND: Cancer metastasis is a deathly process, and a better understanding of the different steps is needed. The shedding of circulating tumor cells (CTCs) and CTC-cluster from the primary tumor, its survival in circulation, and homing are key events of the metastasis cascade. In vitro models of CTCs and in vivo models of metastasis represent an excellent opportunity to delve into the behavior of metastatic cells, to gain understanding on how secondary tumors appear. METHODS: Using the zebrafish embryo, in combination with the mouse and in vitro assays, as an in vivo model of the spatiotemporal development of metastases, we study the metastatic competency of breast cancer CTCs and CTC-clusters and the molecular mechanisms. RESULTS: CTC-clusters disseminated at a lower frequency than single CTCs in the zebrafish and showed a reduced capacity to invade. A temporal follow-up of the behavior of disseminated CTCs showed a higher survival and proliferation capacity of CTC-clusters, supported by their increased resistance to fluid shear stress. These data were corroborated in mouse studies. In addition, a differential gene signature was observed, with CTC-clusters upregulating cell cycle and stemness related genes. CONCLUSIONS: The zebrafish embryo is a valuable model system to understand the biology of breast cancer CTCs and CTC-clusters.

Publisher Correction: ITGB3-mediated uptake of small extracellular vesicles facilitates intercellular communication in breast cancer cells.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Characterization of Kelch domain-containing protein 7B in breast tumours and breast cancer cell lines.

Adenocarcinomas exhibit great heterogeneity, with many genetic and epigenetic alterations. The Kelch domain-containing protein 7B (KLHDC7B) has recently been identified as epigenetically modified and upregulated in breast cancer. The potential reversibility of epigenetic states offers exciting possibilities for novel cancer diagnostics and drugs. However, to properly evaluate specific inhibitors, the role of KLHDC7B in the development and progression of breast cancer should be established. With that objective in mind, the present study investigated a series of human breast tumours and correlated their clinicopathology, according to the Elston-Ellis modification of the Scarff-Bloom-Richardson (SBR) grading system, with KLHDC7B mRNA expression, analysed using quantitative PCR (qPCR). The results revealed that KLHDC7B was significantly upregulated in grade 3 tumours, and that KLHDC7B expression varied according to the tumour grade and the individual, being downregulated in well-differentiated and moderately-differentiated tumours (grade 1-2) and upregulated in poorly-differentiated tumours (grade 3). Immunohistochemical staining revealed that ductal tumours and tumours with a higher percentage of Ki67 positive cells showed the highest levels of KLHDC7B. Receptor expression, HER, p53 status, presence of metastasis, and vascular invasion showed no association with KLHDC7B expression. Previous studies have proposed KLHDC7B as an epigenetic marker of breast cancer. We propose that KLHDC7B should be used as a marker for poorly-differentiated tumours only; use of KLHDC7B without considering tumour grade could lead to an inaccurate diagnosis. Finally, we suggest the appropriate breast cancer cell lines to use to determine the functions of KLHDC7B. KLHDC7B expression was tested in the non-tumour cell line MCF-10A and in the breast cancer cell lines MCF-7, MDA-MB-231 and MDA-MB-468, using qPCR and western blotting. The results revealed that all tested cancer cell lines overexpressed KLHDC7B mRNA, but MDA-MB-468 exhibited a much lower level of protein expression relative to mRNA. Although the breast cancer cell lines used may be appropriate for studying KLHDC7B epigenetic status, MDA-MB-468 should be excluded from functional experiments.

The impossible interview with the man of the neuron doctrine. Interview by Edward G Jones.

This paper follows the form of that by Mazzarello that precedes it (Mazzarello, 2006) and presents an imaginary interview with Santiago Ramón y Cajal in December 1906. A few days earlier Cajal had been awarded the Nobel Prize for Physiology or Medicine, an award that he shared equally with Professor Camillo Golgi. Golgi had been recognized for his work as a pioneer into investigations of the nervous system, primarily on account of his discovery of the "black reaction" of silver chromate impregnation of whole nerve cells and their processes. Cajal had been recognized for his implementation of that method and for laying with it the foundations of what was to become modern neuroanatomical science. Paradoxically, the two awardees had been led by their researches to diametrically opposed views of the organization of the nervous system. Golgi believed in a continuous network of axons that formed the basis of all the integrative properties of the nervous system, while Cajal had provided the information that led to the formulation of the neuron doctrine that saw the nervous system as being made up of chains of discontinuous cells joined by polarized functional contacts that we now call synapses. The paper takes the form of an interview with Professor Cajal in the Grand Hotel Stockholm. His responses to questions posed by the imaginary interviewer are all taken from Cajal's own writings.