Integration of Next-generation sequencing technologies in pathological diagnostics

Today, with the better understanding of the molecular events involved in malignancy and the mechanisms of pharmacotherapy, larger gene panels are more helpful than single biomarker detection. After the completion of the first human genome sequence in 2004, the growing need to sequence a large number of individual genomes in a fast, low-cost and accurate way has directed a shift from traditional Sanger sequencing methods towards new high-throughput genomic technologies. In 2005, the development of next generation sequencing (NGS) methods has represented one of the more significant technical advances in molecular biology. NGS, also known as massive parallel sequencing because of the ability to allow the parallel analysis of a very large number of DNA molecules, is beginning to show its full potential for diagnostic and therapeutic applications. Until recently, NGS platforms were envisioned for large-scale applications,focused on whole genome sequencing, with protocols, consumable costs and a turnaround time (TAT) unsuitable for the needs of small diagnostic laboratories. The development of miniaturised technology by benchtop NGS sequencers decreased sequencing costs, moving NGS from a few large sequencing core centers to a much larger number of individual laboratories. Currently, most pathology departments acquired and NGS benchtop sequencer, thus NGS is adopted for routine molecular diagnostics,including cytological samples. To understand the current and future application of NGS in the field of pathology,modern pathologists need to understand its basic principles. This thesis describes my research on the integration of NGS technologies in pathological diagnostics, both concerning histological and cytological specimens. Moreover, a research application of NGS on mouse xenograft cytological samples is described

2-deoxy-2[F-18] fluoro-D-glucose positron emission tomography Deauville scale and core-needle biopsy to determine successful management after six doxorubicin, bleomycin, vinblastine and dacarbazine cycles in advanced-stage Hodgkin lymphoma.

Abstract Background The clinical impact of the positivity of the Deauville scale (DS) of positron emission tomography (PET) performed at the end of doxorubicin, bleomycin, vinblastine and dacarbazine (ABVD) in patients with advanced Hodgkin lymphoma (HL), in terms of providing rationale to shift poor responders onto a more intensive regimen, remain to be validated by histopathology. Patients and methods This prospective trial involved patients with stage IIB/IV HL who after six ABVD cycles underwent PET (PET6) and core-needle cutting biopsy (CNCB) of 2-deoxy-2[F-18] fluoro- d -glucose (FDG)-avid lymph nodes. Patients received high-dose chemotherapy/autologous haematopoietic stem cell rescue (HDCT/AHSCR) if CNCB was positive for HL, alternatively, if CNCB or PET was negative, received observation or consolidation radiotherapy (cRT) on residual nodal masses, as initially planned. The end-point was 5-year progression-free survival (PFS). Results In all, 43 of the 169 (25%) evaluable patients were PET6 positive (DS 4, 32; DS 5, 11). Among them, histology showed malignancy (HL) in 100% of DS 5 scores and in 12.5% of DS 4 scores. Fifteen patients with positive biopsy received HDCT/AHSCR, whereas 28 patients with negative biopsy, as well as 126 patients with negative PET6, continued the original plan (cRT, 78 patients; observation, 76 patients). The 5-year PFS in the negative PET6 group, negative biopsy group and positive biopsy group was 95.4%, 100% and 52.5%, respectively. Conclusion DS positivity of end-of-ABVD PET in advanced HL carried a certain number of CNCB-proven non-malignant FDG-uptakes. The DS 4 scores which were found to have negative histology appeared to benefit from continuing the original non-intensive therapeutic plane as indicated by the successful outcome in more than 95% of them by obtaining similar 5-year PFS to the PET6-negative group. By contrast, the DS 5 score had consistently positive histology and was associated with unsuccessful conventional therapy, promptly requiring treatment intensification or innovative therapeutic approaches

Liposomal doxorubicin supercharge-containing front-line treatment in patients with advanced-stage diffuse large B-cell lymphoma or classical Hodgkin lymphoma: Preliminary results of a single-centre phase II study

We evaluated the impact of liposomal doxorubicin (NPLD) supercharge-containing therapy on interim fluorodeoxyglucose positron emission tomography (interim-FDG-PET) responses in high-risk diffuse large B-cell lymphoma (DLBCL) or classical Hodgkin lymphoma (c-HL). In this phase II study (2016-2021), 81 adult patients with advanced-stage DLBCL (n = 53) and c-HL (n = 28) received front-line treatment with R-COMP-dose-intensified (DI) and MBVD-DI. R-COMP-DI consisted of 70 mg/m2 of NPLD plus standard rituximab, cyclophosphamide, vincristine and prednisone for three cycles (followed by three cycles with NPLD de-escalated at 50 mg/m2 ); MBVD-DI consisted of 35 mg/m2 of NPLD plus standard bleomycin, vinblastine and dacarbazine for two cycles (followed by four cycles with NPLD de-escalated at 25 mg/m2 ). Patients underwent R-COMP-DI and MBVD-DI with a median dose intensity of 91% and 94% respectively. At interim-FDG-PET, 72/81 patients (one failed to undergo interim-FDG-PET due to early death) had a Deauville score of ≤3. At end of treatment, 90% of patients reached complete responses. In all, 20 patients had Grade ≥3 adverse events, and four of them required hospitalisation. At a median 21-months of follow-up, the progression-free survival of the entire population was 77.3% (95% confidence interval 68%-88%). Our data suggest that the NPLD supercharge-driven strategy in high-risk DLBCL/c-HL may be a promising option to test in phase III trials, for improving negative interim-FDG-PET cases incidence

Generation and Characterization of a Tumor Stromal Microenvironment and Analysis of Its Interplay with Breast Cancer Cells: An In Vitro Model to Study Breast Cancer-Associated Fibroblast Inactivation

Breast cancer-associated fibroblasts (BCAFs), the most abundant non-cancer stromal cells of the breast tumor microenvironment (TME), dramatically sustain breast cancer (BC) progression by interacting with BC cells. BCAFs, as well as myofibroblasts, display an up regulation of activation and inflammation markers represented by α-smooth muscle actin (α-SMA) and cyclooxygenase 2 (COX-2). BCAF aggregates have been identified in the peripheral blood of metastatic BC patients. We generated an in vitro stromal model consisting of human primary BCAFs grown as monolayers or 3D cell aggregates, namely spheroids and reverted BCAFs, obtained from BCAF spheroids reverted to 2D cell adhesion growth after 216 h of 3D culture. We firstly evaluated the state of activation and inflammation and the mesenchymal status of the BCAF monolayers, BCAF spheroids and reverted BCAFs. Then, we analyzed the MCF-7 cell viability and migration following treatment with conditioned media from the different BCAF cultures. After 216 h of 3D culture, the BCAFs acquired an inactivated phenotype, associated with a significant reduction in α-SMA and COX-2 protein expression. The deactivation of the BCAF spheroids at 216 h was further confirmed by the cytostatic effect exerted by their conditioned medium on MCF-7 cells. Interestingly, the reverted BCAFs also retained a less activated phenotype as indicated by α-SMA protein expression reduction. Furthermore, the reverted BCAFs exhibited a reduced pro-tumor phenotype as indicated by the anti-migratory effect exerted by their conditioned medium on MCF-7 cells. The deactivation of BCAFs without drug treatment is possible and leads to a reduced capability of BCAFs to sustain BC progression in vitro. Consequently, this study could be a starting point to develop new therapeutic strategies targeting BCAFs and their interactions with cancer cells

RNA-Based Assay for Next-Generation Sequencing of Clinically Relevant Gene Fusions in Non-Small Cell Lung Cancer

Gene fusions represent novel predictive biomarkers for advanced non-small cell lung cancer (NSCLC). In this study, we validated a narrow NGS gene panel able to cover therapeutically-relevant gene fusions and splicing events in advanced-stage NSCLC patients. To this aim, we first assessed minimal complementary DNA (cDNA) input and the limit of detection (LoD) in different cell lines. Then, to evaluate the feasibility of applying our panel to routine clinical samples, we retrospectively selected archived lung adenocarcinoma histological and cytological (cell blocks) samples. Overall, our SiRe RNA fusion panel was able to detect all fusions and a splicing event harbored in a RNA pool diluted up to 2 ng/µL. It also successfully analyzed 46 (95.8%) out of 48 samples. Among these, 43 (93.5%) out of 46 samples reproduced the same results as those obtained with conventional techniques. Intriguingly, the three discordant results were confirmed by a CE-IVD automated real-time polymerase chain reaction (RT-PCR) analysis (Easy PGX platform, Diatech Pharmacogenetics, Jesi, Italy). Based on these findings, we conclude that our new SiRe RNA fusion panel is a valid and robust tool for the detection of clinically relevant gene fusions and splicing events in advanced NSCLC

Cytology of Eyelid Squamous cell carcinoma

Macroscopy: Plaque, nodular and/or ulcerative lesion. Microscopy: Scraping samples of squamous cell carcinoma are better observed with Papanicolaou-stained in which squamous cytoplasmic differentiation is more evident. The cytological pattern may vary according to cellular differentiation, which ranges from extremely well-differentiated to anaplastic. When almost all cells show indirect signs of paracheratosis, such as wide and extremely keratinizing cytoplasm with retention of small pyknotic nuclei, but the smear lacks cells from a possible lesion beneath, cytological diagnosis should be delayed. A variable number of granulocytes may also be present in the smears, depending on the presence of necrosis. Inflammatory infiltrate may also characterize non-tumuoral ulcerated lesions in which scraping may be requested, in this case the scraping should be effected at the edge of the lesion and not at the centre. It may also be useful to remember that benign ulcerated lesions are generally more painful when scraped than tumours, produce fewer cellular smears and obviously yield cells with a lesser degree of atypia. Immunophenotype Not applicable Molecular features Not applicable. Differential diagnosis Bowen disease, BC

Cytology of the orbit and ocular adnexa

The orbit and ocular adnexa, which cytopathologists are rarely requested to investigate, is one of the most complex and difficult anatomical regions to investigate cytologically. Furthermore, the scarcity of routine orbit and ocular adnexa cytology increases the difficulties of cytopathologists who often have to deal with scanty cellular samples from different and sometimes complex or rare pathologies; moreover, the specific clinical and anatomical features require close cooperation between ophthalmologists and cytopathologists. In fact, whereas cytopathologists routinely perform fine needle cytology (FNC) on much of the palpable and impalpable lesions by themselves, in this specific field the insertion of the needle is an almost exclusive task of ophthalmologists. The cytopathologist’s role is that of making smears, evaluating adequacy, managing diagnostic material, selecting ancillary techniques and making the final diagnosis. Finally, orbital and ocular adnexa cytology, as in other regions, has been improved by immunocytochemistry (ICC), flow cytometry (FC) fluorescence in-situ hybridization (FISH) and molecular techniques, however, the usage of these procedures is often impeded by the scanty material obtainable, the management of which further increases the complexity of the cytological approach

Eyelids cytology: Squamous papilloma

Squamous papilloma is the most frequent benign tumour of the eyelid. It may be sessile or pedunculated and composed of projections of acanthotic and parakeratosic epithelium arising from a fibro-vascular stalk. Cells may show atypical nuclei with coilocytotic modifications, as observed in HPV infections

Cytology of Conjunctival inflammatory lesions

Allergic and vernal conjunctivitis are characterized by a prevalence of eosinophils in the inflammatory infiltrate. Bacterial and viral infections are generally diagnosed on the basis of clinical presentation or sometimes by microbiology. Nonetheless, cytological features may be helpful for a presumptive diagnosis, mainly in clinically unexpected cases, and in the general management of the patients. Infections by verruca vulgaris, measles, herpes may be suggested by specific cytological features. Trachoma is a “historical” bacterial conjunctival infection caused by Chlamydia trachomatis. The cytological features are almost the same as those described in the female genital tract and are characterized by cytoplasmic basophilic inclusions with halos. Cytological features of Candida, Aspergillus, mucormycosis conjunctivitis have also been described; diagnosis and treatment must be timely and effective because of the risk of corneal damage