From sampling to cellblock: The fully automated journey of cytological specimens.

In recent years, technological innovation have emerged to standardize pathology laboratory processes and reduce the handling of diagnostic samples. Among them is an automatic tissue embedding system that eliminates the need for manual activity in tissue paraffin embedding, thereby improving sample preservation. Unfortunately, this system cannot be used for cytological specimens due to the lack of an effective holder to support the procedure steps. In this study, we evaluated the performance of a commercial polymer matrix to enable and standardize the automatic paraffin embedding of cytological material from different organs and sources. Cytological samples from 40 patients were collected on the matrices and submitted for fully automatic workflow preparation, from formalin fixation until paraffin block, using the Sakura embedding system. Our results demonstrated the feasibility of the automated procedure, from loading cytological sample onto the matrix to obtaining the paraffin cellblock, thereby avoiding manual manipulation of cellular material. All samples resulted adequately processed and paraffin-embedded showing satisfactory tissue permeation by processing reagents, optimal preservation of cytoplasmic and nuclear details, and good quality of staining results on paraffin sections. Automated embedding of cytological samples eliminates the risk of lost specimens, reduces laboratory burden, standardizes procedures, increases diagnostic yield, and ultimately improves patients' management.

Molecular profiling of a bladder cancer with very high tumour mutational burden.

The increasing incidence of urothelial bladder cancer is a notable global concern, as evidenced by the epidemiological data in terms of frequency, distribution, as well as mortality rates. Although numerous molecular alterations have been linked to the occurrence and progression of bladder cancer, currently there is a limited knowledge on the molecular signature able of accurately predicting clinical outcomes. In this report, we present a case of a pT3b high-grade infiltrating urothelial carcinoma with areas of squamous differentiation characterized by very high tumor mutational burden (TMB), with up-regulations of immune checkpoints. The high TMB, along with elevated expressions of PD-L1, PD-L2, and PD1, underscores the rationale for developing a personalized immunotherapy focused on the use of immune-checkpoint inhibitors. Additionally, molecular analysis revealed somatic mutations in several other cancer-related genes, including TP53, TP63 and NOTCH3. Mutations of TP53 and TP63 genes provide mechanistic insights on the molecular mechanisms underlying disease development and progression. Notably, the above-mentioned mutations and the elevated hypoxia score make the targeting of p53 and/or hypoxia related pathways a plausible personalized medicine option for this bladder cancer, particularly in combination with immunotherapy. Our data suggest a requirement for molecular profiling in bladder cancer to possibly select appropriate immune-checkpoint therapy.

Programmed Cell Death Pathways in Cholangiocarcinoma: Opportunities for Targeted Therapy.

Cholangiocarcinoma is a highly aggressive cancer arising from the bile ducts. The limited effectiveness of conventional therapies has prompted the search for new approaches to target this disease. Recent evidence suggests that distinct programmed cell death mechanisms, namely, apoptosis, ferroptosis, pyroptosis and necroptosis, play a critical role in the development and progression of cholangiocarcinoma. This review aims to summarize the current knowledge on the role of programmed cell death in cholangiocarcinoma and its potential implications for the development of novel therapies. Several studies have shown that the dysregulation of apoptotic signaling pathways contributes to cholangiocarcinoma tumorigenesis and resistance to treatment. Similarly, ferroptosis, pyroptosis and necroptosis, which are pro-inflammatory forms of cell death, have been implicated in promoting immune cell recruitment and activation, thus enhancing the antitumor immune response. Moreover, recent studies have suggested that targeting cell death pathways could sensitize cholangiocarcinoma cells to chemotherapy and immunotherapy. In conclusion, programmed cell death represents a relevant molecular mechanism of pathogenesis in cholangiocarcinoma, and further research is needed to fully elucidate the underlying details and possibly identify therapeutic strategies.

Radioimmune Imaging of α4ß7 Integrin and TNFα for Diagnostic and Therapeutic Applications in Inflammatory Bowel Disease.

Imaging using radiolabelled monoclonal antibodies can provide, non-invasively, molecular information which allows for the planning of the best treatment and for monitoring the therapeutic response in cancer, as well as in chronic inflammatory diseases. In the present study, our main goal was to evaluate if a pre-therapy scan with radiolabelled anti-α4ß7 integrin or radiolabelled anti-TNFα mAb could predict therapeutic outcome with unlabelled anti-α4ß7 integrin or anti-TNFα mAb. To this aim, we developed two radiopharmaceuticals to study the expression of therapeutic targets for inflammatory bowel diseases (IBD), to be used for therapy decision making. Both anti-α4ß7 integrin and anti-TNFα mAbs were successfully radiolabelled with technetium-99m with high labelling efficiency and stability. Dextran sulfate sodium (DSS)-induced colitis was used as a model for murine IBD and the bowel uptake of radiolabelled mAbs was evaluated ex vivo and in vivo by planar and SPECT/CT images. These studies allowed us to define best imaging strategy and to validate the specificity of mAb binding in vivo to their targets. Bowel uptake in four different regions was compared to immunohistochemistry (IHC) score (partial and global). Then, to evaluate the biomarker expression prior to therapy administration, in initial IBD, another group of DSS-treated mice was injected with radiolabelled mAb on day 2 of DSS administration (to quantify the presence of the target in the bowel) and then injected with a single therapeutic dose of unlabelled anti-α4ß7 integrin or anti-TNFα mAb. Good correlation was demonstrated between bowel uptake of radiolabelled mAb and immunohistochemistry (IHC) score, both in vivo and ex vivo. Mice treated with unlabelled α4ß7 integrin and anti-TNFα showed an inverse correlation between the bowel uptake of radiolabelled mAb and the histological score after therapy, proving that only mice with high α4ß7 integrin or TNFα expression will benefit of therapy with unlabelled mAb.

Antigenicity Preservation Is Related to Tissue Characteristics and the Post-Mortem Interval: Immunohistochemical Study and Literature Review.

The main aim of this study was to investigate the post-mortem proteolytic degradation process of selected tissue antigens and correlate it to the post-mortem interval. During the autopsy of 12 cadavers (time interval ranging 1 day-2 years after death) samples of skin, liver, kidney, and spleen were collected. All samples were formalin-fixed and paraffin-embedded. Four µm paraffin sections were used for hematoxylin-eosin staining and immunohistochemical analysis (Ki67, Vimentin, Pan cytokeratin, and CD20). Data reported here show that immunohistochemical reactivity preservation was related to the characteristics of the tissues. In particular, the most resistant tissue was the skin, where the autolysis phenomena were not appreciable before 5 days. On the contrary, the liver and the spleen underwent early autolysis, while the kidney displayed an early autolysis of the tubules and a late one of the glomeruli. As concerns specific antigens, immunoreactivity was lost earliest for nuclear antigens as compared to cytoplasmic ones. In conclusion, our results demonstrate that immunohistochemical detection of specific antigens may be useful in estimating the post-mortem interval, especially when we need to know whether the post-mortem interval is a few days or more than 7-10 days.

[99Tc]Sestamibi bioaccumulation induces apoptosis in prostate cancer cells: an in vitro study.

The main aim of this preliminary in vitro study was to evaluate both the uptake of [99Tc]Sestamibi into prostate cancer cells and the relationship among [99Tc]Sestamibi bioaccumulation, cancer cells proliferation and apoptosis. An in vitro study in which PC3 prostate cancer cell line was cultured with increasing doses of decayed sestamibi has been developed. Specifically, PC3 cells were incubated with three different concentrations of [99Tc]Sestamibi: 10 µg/mL, 1 µg/mL, and 0.1 µg/mL Expression of apoptotic caspase-3 and AIF, as well as the ultrastructure of PC3 cells, were evaluated at T0 and after 24, 48, 72, and 120 h following [99Tc]Sestamibi incubation. Data here reported showed the bioaccumulation of sestamibi in prostate cancer cells. As concern the cancer cell homeostasis, the treatment of PC3 cells with [99Tc]Sestamibi strongly influenced the cells proliferation. Indeed, a significant reduction in the number of mitosis was observed. Noteworthy, the accumulation of sestamibi in prostate cancer cells was associated with the appearance of morphological signs of apoptosis. The increase in AIF and caspase 3 expression in prostate cancer cells treated with 10 µg/mL of [99Tc]Sestamibi confirmed that this radiopharmaceutical can trigger the apoptosis. To the best of our knowledge, this preliminary study reported for the first time in vitro data about the uptake of sestamibi in prostate cancer cells. The evidence about the accumulation of sestamibi in prostate cancer cells and its role in the apoptosis process could open new clinical perspectives on the use of this radiopharmaceutical in both the diagnosis and treatment of prostate cancers.

The ETS Homologous Factor (EHF) Represents a Useful Immunohistochemical Marker for Predicting Prostate Cancer Metastasis.

The main aim of this study was to investigate the risk of prostate cancer metastasis formation associated with the expression of ETS homologous factor (EHF) in a cohort of bioptic samples. To this end, the expression of EHF was evaluated in a cohort of 152 prostate biopsies including primary prostate cancers that developed metastatic lesions, primary prostate cancers that did not develop metastasis, and benign lesions. Data here reported EHF as a candidate immunohistochemical prognostic biomarker for prostate cancer metastasis formation regardless of the Gleason scoring system. Indeed, our data clearly show that primary lesions with EHF positive cells ≥40% had a great risk of developing metastasis within five years from the first diagnosis. Patients with these lesions had about a 40-fold increased risk of developing metastasis as compared with patients with prostate lesions characterized by a percentage of EHF positive cells ≤30%. In conclusion, the immunohistochemical evaluation of EHF could significantly improve the management of prostate cancer patients by optimizing the diagnostic and therapeutic health procedures and, more important, ameliorating the patient's quality of life.

High Sensitivity C-Reactive Protein Increases the Risk of Carotid Plaque Instability in Male Dyslipidemic Patients.

BACKGROUND: The aim of this study was to evaluate how the high sensitivity C-reactive protein (hs-CRP) values influence the risk of carotid plaque instability in association with other cardiovascular risk factors. METHODS: One hundred and fifty-six carotid plaques from both symptomatic and asymptomatic patients requiring surgical carotid endarterectomy were retrospectively collected. According to the modified American Heart Association, atherosclerosis plaques have been histologically distinguished into unstable and stable. The following anamnestic and hematochemical data were also considered: age, gender, hypertension, diabetes mellitus, smoking habit, therapy, low-density lipoprotein (LDL)-C, kidney failure and hs-CRP. RESULTS: The results of our study clearly show that high levels of hs-CRP significantly increase the carotid plaque instability in dyslipidemic patients. Specifically, a 67% increase of the risk of carotid plaque instability was observed in patients with high LDL-C. Therefore, the highest risk was observed in male dyslipidemic patients 2333 (95% CI 0.73-7.48) and in aged female patients 2713 (95% CI 0.14-53.27). DISCUSSION: These data strongly suggest a biological relationship between the hs-CRP values and the alteration of lipidic metabolism mostly in male patients affected by carotid atherosclerosis. The measurement of hs-CRP might be useful as a potential screening tool in the prevention of atheroscletotic disease.

The risk of carotid plaque instability in patients with metabolic syndrome is higher in women with hypertriglyceridemia.

BACKGROUND: Metabolic syndrome certainly favors growth of carotid plaque; however, it is uncertain if it determines plaque destabilization. Furthermore, it is likely that only some components of metabolic syndrome are associated with increased risk of plaque destabilization. Therefore, we evaluated the effect of different elements of metabolic syndrome, individually and in association, on carotid plaques destabilization. METHODS: A total of 186 carotid endarterectomies from symptomatic and asymptomatic patients were histologically analysed and correlated with major cardiovascular risk factors. RESULTS: Metabolic syndrome, regardless of the cluster of its components, is not associated with a significant increase in risk of plaque destabilization, rather with the presence of stable plaques. The incidence of unstable plaques in patients with metabolic syndrome is quite low (43.9 %), when compared with that seen in the presence of some risk factors, but significantly increases in the subgroup of female patients with hypertriglyceridemia, showing an odds ratio of 3.01 (95% CI, 0.25-36.30). CONCLUSIONS: Our data may help to identify patients with real increased risk of acute cerebrovascular diseases thus supporting the hypothesis that the control of hypertriglyceridemia should be a key point on prevention of carotid atherosclerotic plaque destabilization, especially in post-menopausal female patients.

Extensive Histopathological Characterization of Inflamed Bowel in the Dextran Sulfate Sodium Mouse Model with Emphasis on Clinically Relevant Biomarkers and Targets for Drug Development.

This study aims to develop a reliable and reproducible inflammatory bowel disease (IBD) murine model based on a careful spatial-temporal histological characterization. Secondary aims included extensive preclinical studies focused on the in situ expression of clinically relevant biomarkers and targets involved in IBD. C57BL/6 female mice were used to establish the IBD model. Colitis was induced by the oral administration of 2% Dextran Sulfate Sodium (DSS) for 5 days, followed by 2, 4 or 9 days of water. Histological analysis was performed by sectioning the whole colon into rings of 5 mm each. Immunohistochemical analyses were performed for molecular targets of interest for monitoring disease activity, treatment response and predicting outcome. Data reported here allowed us to develop an original scoring method useful as a tool for the histological assessment of preclinical models of DSS-induced IBD. Immunohistochemical data showed a significant increase in TNF-α, α4ß7, VEGFRII, GR-1, CD25, CD3 and IL-12p40 expression in DSS mice if compared to controls. No difference was observed for IL-17, IL-23R, IL-36R or F480. Knowledge of the spatial-temporal pattern distribution of the pathological lesions of a well-characterized disease model lays the foundation for the study of the tissue expression of meaningful predictive biomarkers, thereby improving translational success rates of preclinical studies for a personalized management of IBD patients.

The Paradox Effect of Calcification in Carotid Atherosclerosis: Microcalcification is Correlated with Plaque Instability.

BACKGROUND: this study aims to investigate the possible association among the histopathologic features of carotid plaque instability, the presence of micro- or macrocalcifications, the expression of in situ inflammatory biomarkers, and the occurrence of the major risk factors in this process in a large series of carotid plaques. METHODS: a total of 687 carotid plaques from symptomatic and asymptomatic patients were collected. Histological evaluation was performed to classify the calcium deposits in micro or macrocalcifications according to their morphological features (location and size). Immunohistochemistry was performed to study the expression of the main inflammatory biomarkers. RESULTS: results here reported demonstrated that calcifications are very frequent in carotid plaques, with a significant difference between the presence of micro- and macrocalcifications. Specifically, microcalcifications were significantly associated to high inflamed unstable plaques. Paradoxically, macrocalcifications seem to stabilize the plaque and are associated to a M2 macrophage polarization instead. DISCUSSION: the characterization of mechanisms involved in the formation of carotid calcifications can lay the foundation for developing new strategies for the management of patients affected by carotid atherosclerosis. Data of this study could provide key elements for an exhaustive evaluation of carotid plaque calcifications allowing to establish the risk of associated clinical events.

Breast cancer metastasis to bone: From epithelial to mesenchymal transition to breast osteoblast-like cells.

In this review we highlighted the newest aspects concerning the physiopathology of breast cancer metastatization into the bone including: a) in situ biomarkers of breast cancer metastatic diseases, b) biological processes related to the origin of metastatic cells (epithelial to mesenchymal transition), c) the nature and the possible role of Breast Osteoblast-Like Cells in the formation of bone lesions and d) the prognostic value of breast microcalcifications for the bone metastatic disease. In addition, the more recent data about the biology of breast cancer metastatic process and the origin and function of Breast Osteoblast-Like Cells have been analyzed to propose the use of molecular imaging investigations able to identify early neoplastic lesions with high propensity to form bone metastasis in vivo.

Molecular Aspects and Prognostic Significance of Microcalcifications in Human Pathology: A Narrative Review.

The presence of calcium deposits in human lesions is largely used as imaging biomarkers of human diseases such as breast cancer. Indeed, the presence of micro- or macrocalcifications is frequently associated with the development of both benign and malignant lesions. Nevertheless, the molecular mechanisms involved in the formation of these calcium deposits, as well as the prognostic significance of their presence in human tissues, have not been completely elucidated. Therefore, a better characterization of the biological process related to the formation of calcifications in different tissues and organs, as well as the understanding of the prognostic significance of the presence of these calcium deposits into human tissues could significantly improve the management of patients characterized by microcalcifications associated lesions. Starting from these considerations, this narrative review highlights the most recent histopathological and molecular data concerning the formation of calcifications in breast, thyroid, lung, and ovarian diseases. Evidence reported here could deeply change the current point of view concerning the role of ectopic calcifications in the progression of human diseases and also in the patients' management. In fact, the presence of calcifications can suggest an unfavorable prognosis due to dysregulation of normal tissues homeostasis.

Erratum: Mapelli, S.N., et al. A Novel Prostate Cell Type-Specific Gene Signature to Interrogate Prostate Tumor Differentiation Status and Monitor Therapeutic Response (Running Title: Phenotypic Classification of Prostate Tumors). Cancers 2020, 12, 176.

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Imaging Diagnostics and Pathology in SARS-CoV-2-Related Diseases.

In December 2019, physicians reported numerous patients showing pneumonia of unknown origin in the Chinese region of Wuhan. Following the spreading of the infection over the world, The World Health Organization (WHO) on 11 March 2020 declared the novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) outbreak a global pandemic. The scientific community is exerting an extraordinary effort to elucidate all aspects related to SARS-CoV-2, such as the structure, ultrastructure, invasion mechanisms, replication mechanisms, or drugs for treatment, mainly through in vitro studies. Thus, the clinical in vivo data can provide a test bench for new discoveries in the field of SARS-CoV-2, finding new solutions to fight the current pandemic. During this dramatic situation, the normal scientific protocols for the development of new diagnostic procedures or drugs are frequently not completely applied in order to speed up these processes. In this context, interdisciplinarity is fundamental. Specifically, a great contribution can be provided by the association and interpretation of data derived from medical disciplines based on the study of images, such as radiology, nuclear medicine, and pathology. Therefore, here, we highlighted the most recent histopathological and imaging data concerning the SARS-CoV-2 infection in lung and other human organs such as the kidney, heart, and vascular system. In addition, we evaluated the possible matches among data of radiology, nuclear medicine, and pathology departments in order to support the intense scientific work to address the SARS-CoV-2 pandemic. In this regard, the development of artificial intelligence algorithms that are capable of correlating these clinical data with the new scientific discoveries concerning SARS-CoV-2 might be the keystone to get out of the pandemic.

BMP-2 Variants in Breast Epithelial to Mesenchymal Transition and Microcalcifications Origin.

This study aims to investigate the possible different roles of the BMP-2 variants, cytoplasmic and nuclear variant, in both epithelial to mesenchymal transition and in microcalcifications origin in human breast cancers. To this end, the in situ expression of cytoplasmic and nuclear BMP-2 was associated with the expression of the main epithelial to mesenchymal transition biomarkers (e-cadherin and vimentin) and molecules involved in bone metabolisms (RUNX2, RANKL, SDF-1) by immunohistochemistry. In addition, the expression of cytoplasmic and nuclear BMP-2 was associated with the presence of microcalcifications. Our data showed a significant association among the number of cytoplasmic BMP-2-positive cells and the number of both vimentin (positive association) and e-cadherin (negative association) positive breast cells. Conversely, no associations were found concerning the nuclear BMP-2-positive breast cells. Surprisingly, the opposite result was obtained by analyzing the variants of BMP-2 and both the expression of RANKL and SDF-1 and the presence of microcalcifications. Specifically, the presence of microcalcifications was related to the expression of nuclear BMP-2 variant rather than the cytoplasmic one, as well as a strong association between the number of nuclear BMP-2 and the expression of the main breast osteoblast-like cells (BOLCs) biomarkers. To further corroborate these data, an in vitro experiment for demonstrating the co-expression of nBMP-2 and RANKL or vimentin or SDF-1 in breast cancer cells that acquire the capability to produce microcalcifications was developed. These investigations confirmed the association between the nBMP-2 expression and both RANKL and SDF-1. The data supports the idea that whilst cytoplasmic BMP-2 can be involved in epithelial to mesenchymal transition phenomenon, the nuclear variant is related to the essential mechanisms for the formation of breast microcalcifications. In conclusion, from these experimental and translational perspectives, the complexity of BMP-2 signaling will require a detailed understanding of the involvement of specific BMP-2 variants in breast cancers.