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Immunohistochemistry (IHC) and polymerase chain reaction (PCR) and fragment separation by capillary electrophoresis represent the current clinical laboratory standard for the evaluation of microsatellite instability (MSI) status. The importance of reporting MSI status in colorectal cancer is based on its potential for guiding treatment and as a prognostic indicator. It is also used to identify patients for Lynch syndrome testing. Our aim was to evaluate pre-analytical factors, such as age of formalin-fixed and paraffin-embedded (FFPE) block, neoplastic cell percentage, mucinous component, and DNA integrity, that may influence the accuracy of MSI testing and assess the concordance between three different MSI evaluation approaches. We selected the mucinous colorectal cancer (CRC) histotype for this study as it may possibly represent an intrinsic diagnostic issue due to its low tumor cellularity. Seventy-five cases of mucinous CRC and corresponding normal colon tissue samples were retrospectively selected. MMR proteins were evaluated by IHC. After DNA quality and quantity evaluation, the Idylla™ and TapeStation 4200 platforms were adopted for the evaluation of MSI status. Seventy-three (97.3%) cases were successfully analyzed by the three methodologies. Overall, the Idylla™ platform showed a concordance rate with IHC of 98.0% for microsatellite stable (MSS)/proficient MMR (pMMR) cases and 81.8% for MSI/deficient MMR (dMMR) cases. The TapeStation 4200 system showed a concordance rate with IHC of 96.0% for MSS/pMMR cases and 45.4% for MSI/dMMR cases. The concordance rates of the TapeStation 4200 system with respect to the Idylla™ platform were 98.1% for MSS profile and 57.8% for MSI profile. Discordant cases were analyzed using the Titano MSI kit. Considering pre-analytical factors, no significant variation in concordance rate among IHC analyses and molecular systems was observed by considering the presence of an acellular mucus cut-off >50% of the tumor area, FFPE year preparation, and DNA concentration. Conversely, the Idylla™ platform showed a significant variation in concordance rate with the IHC approach by considering a neoplastic cell percentage >50% (p-value = 0.002), and the TapeStation 4200 system showed a significant variation in concordance rate with the IHC approach by considering a DNA integrity number (DIN) ≥4 as cut-off (p-value = 0.009). Our data pinpoint a central role of the pre-analytical phase in the diagnostic outcome of MSI testing in CRC.

Background: Since liquid-based cytology (LBC) has replaced the conventional Papanicolaou test in cervical cancer screening programs, pre-analytical procedures—particularly the choice of LBC collection media—have become crucial to ensure the accuracy of high-risk (HR) HPV DNA testing. This study aims to evaluate whether the newly developed CytoPath® LBC medium can serve as a reliable alternative to standard solutions. Methods: This study exploited cell lines to evaluate the stability, integrity, and recovery rate of genomic DNA at different fixation time points (1, 7, 14 and 40 days) and serial dilutions (1:5, 1:10 and 1:20) extracted from cell lines. These samples have also undergone quantitative Real-Time PCR (qPCR) based HR-HPV test to assess the relative performance of the new preservative solution in detecting viral DNA with respect to the standard reference. Results: Cervical cell lines preserved in both media demonstrated consistent DNA stability over time. DNA yields were comparable between the two media. Notably, the DNA Integrity Number (DIN) was higher in samples fixed with the CytoPath® solution. HR-HPV detection by qPCR showed equivalent performance, regardless of the fixative used. Conclusions: The CytoPath® fixative solution represents a valid alternative to standard preservation media, offering improved DNA integrity while maintaining equivalent performance in HR-HPV qPCR testing.

Background/Objectives: The non-small-cell lung cancer (NSCLC) therapeutic landscape has undergone a profound transformation with the introduction of multiple personalized treatment options. Mutations in ERBB2 (HER2) have recently emerged as promising novel targets for the treatment of non-squamous NSCLC (nsNSCLC). Accurate, rapid, and efficient molecular profiling is crucial for identifying patients who may benefit from targeted therapies, including HER2-directed agents. Materials and Methods: Here, we aimed to retrospectively assess the performance of the Oncomine™ Precision Assay* (OPA) in combination with the Ion Torrent Genexus™ Integrated Sequencer* (Thermo Fisher Scientific. Waltham, MA, USA) for detecting ERBB2 mutations in nsNSCLC. A total of 108 archived nsNSCLC samples, consisting of biopsies, resections, and cytological specimens, were used to assess concordance with in-house-validated orthogonal tests. Results: The OPA showed high sensitivity and specificity with an overall accuracy of 100% for single-nucleotide variants (SNVs) and insertions and deletions (Indels). SNVs and Indels with allele frequencies as low as 5% were correctly identified across samples with a tumor cell content ranging from 5% to 95%. Additionally, the assay demonstrated high reproducibility across the six participating laboratories. The turnaround time of the OPA was notably shorter compared to traditional orthogonal methods, facilitating rapid molecular report generation. Conclusions: The OPA in combination with the Ion Torrent Genexus™ System allows for highly sensitive and specific detection of relevant ERBB2 mutations. The assay’s streamlined workflow, coupled with its automated data analysis pipeline, enables a fast turnaround time for testing across a range of sample types. This includes samples with reduced tumor cell content and limited available input. This study demonstrates the future potential of using this assay in a clinical setting.

Over the past decade, molecular cytopathology has emerged as a relevant area of modern pathology. Notably, in patients with advanced-stage cancer, cytological samples could be the only material available for diagnosis and molecular biomarker testing to identify patients suitable for targeted therapies. As a result, the contemporary cytopathologist’s role extends beyond morphological assessments to include critical skills such as evaluating the adequacy of the cytological samples and managing these specimens for molecular testing. This case collection can be a valuable source of insight, especially for young pathologists, who should learn to combine the opportunities offered by molecular biology with the basis of morphological evaluation.

Microplastics (MPs) are defined as plastic particles smaller than 5 mm. They have been found almost everywhere they have been searched for and recent discoveries have also demonstrated their presence in human placenta, blood, meconium, and breastmilk, but their location and toxicity to humans have not been reported to date. The aim of this study was twofold: 1. To locate MPs within the intra/extracellular compartment in human placenta. 2. To understand whether their presence and location are associated with possible structural changes of cell organelles. Using variable pressure scanning electron microscopy and transmission electron microscopy, MPs have been localized in ten human placentas. In this study, we demonstrated for the first time the presence and localization in the cellular compartment of fragments compatible with MPs in the human placenta and we hypothesized a possible correlation between their presence and important ultrastructural alterations of some intracytoplasmic organelles (mitochondria and endoplasmic reticulum). These alterations have never been reported in normal healthy term pregnancies until today. They could be the result of a prolonged attempt to remove and destroy the plastic particles inside the placental tissue. The presence of virtually indestructible particles in term human placenta could contribute to the activation of pathological traits, such as oxidative stress, apoptosis, and inflammation, characteristic of metabolic disorders underlying obesity, diabetes, and metabolic syndrome and partially accounting for the recent epidemic of non-communicable diseases.

Transition to parenthood represents an important life event increasing vulnerability to psychological disorders. Postpartum depression and parenting distress are the most common psychological disturbances and a growing scientific evidence suggests that both mothers and fathers are involved in this developmental crisis. This paper aims to explore maternal and paternal experience of transition to parenthood in terms of parenting distress and risk of postpartum depression. Seventy-five couples of first-time parents were invited to compile the Edinburgh Postnatal Depression Scale and the Parenting Stress Index-Short Form in the first month of children life. Study sample reported very high levels of parenting distress and a risk of postpartum depression in 20.8% of mothers and 5.7% of fathers. No significant correlation between parenting distress and the risk of postpartum depression emerged, both in mothers than in fathers group while maternal distress levels are related to paternal one. The first month after partum represents a critical phase of parents life and it could be considered a developmental crisis characterized by anxiety, stress and mood alterations that could have important repercussions on the child psycho-physical development.

AimsAny reference laboratory testing non-small cell lung cancer samples for predictive biomarkers needs to develop and validate a wide range of different molecular techniques, each with a specific time requirement and application. Updated international guidelines suggest that next generation sequencing (NGS) to be the initial procedure. However, in a non-negligible subset of cases, library generation may fail or amplicon coverage may be insufficient. In these NGS ‘invalid’ cases, the Idylla system may represent a viable option for rapid epidermal growth factor receptor (EGFR) genotyping.MethodsThis retrospective study included 68 archival DNA samples previously processed by Ion Torrent NGS assay. Out of these, 43 cases, including 24 EGFR mutant samples, had a valid NGS result, whereas 25/68 (37%) were invalid. All samples were retested by directly pipetting the DNA inside the EGFR Idylla assay cartridge.ResultsIn all 43 cases with a valid NGS result, Idylla confirmed the EGFR mutational status. In particular, 24/24 (100%) of EGFR mutant samples as detected by NGS were confirmed by Idylla. Moreover, a large portion of cases (20/25; 80%) whose assessment by NGS was invalid were adequately processed by Idylla. Noteworthy, in 4/25 (16%) of cases, Idylla detected actionable EGFR mutations.ConclusionsIdylla assay could be very useful to quickly process cases for which NGS does not allow genotyping.

In the human placenta, we have detected the MPs by Raman microspectroscopy analysis and, for the first time, with transmission electron microscopy. MPs fragments have been localized in different compartments of placental tissue, free in the cytoplasm and within organelles like lysosomes. Moreover, their presence has been correlated with ultrastructural alterations of some cell organelles, typical of metabolic stress, mainly dilated rough endoplasmic reticulum and numerous swollen electrodense mitochondria, as well as signs derived from involuting organelles. As a result, we have speculated that microplastics in the placenta could be responsible for pathological traits activation such as oxidative stress, apoptosis, and inflammation causing long-term effects on the health of the mother and child. To demonstrate the cytotoxicity of PS-NPs on the placenta and confirm the results, we performed experiments on a trophoblast human cell line, the HTR8/SVneo cells. HTR8/SVneo cells were treated, for 24 h and 48h, with increasing concentrations (10, 25, 50, 75, and 100 μg/mL) of 0.05 µm polystyrene (PS) and cellular viability was evaluated by Counting Kit-8. Fluorescent PS-NPs examined under fluorescence/confocal microscopy were used to investigate the internalization of plastics in the placenta cells. Transmission electron microscopy was used to evaluate possible PS-NPs-dependent ultrastructural alterations of cells and organelles. Our study shows that starting from 24 h exposure, PS-NPs treatment, at 50 μg/mL dose, has a cytotoxic effect on placental cells, causing the death of 40% of cells and affecting the morphology of the surviving cells. In addition, PS-NPs alter the ultrastructure of some organelles in the surviving cells, like those we have already described . We found that NPs enter the cells, affecting the endoplasmic reticulum and mitochondria morphology, accumulating as aggregates within lysosome-like organelles. Interestingly these aggregates become larger as the concentration of NPs increases. We speculated that the accumulation of NPs inside lysosome-like organelles could result from a prolonged and impossible attempt by the cell to remove and destroy PS. This would lead to ER and mitochondrial stress, impairing mitochondria/ER functions and oxidative stress, thus activating the apoptotic pathway and suggesting that PS-NPs could act as a cell stressor, leading to the death of cells. In support of our hypothesis, we also found NPs associated with morphological signs of cellular regression and degeneration, such as the presence of a highly vacuolized cytoplasm, dilatation, and vesiculation of ER, associated with the uncoupling/loss of associated mitochondria, cytoplasmic fragments, and free organelles deriving from cellular lysis. Based on electron microscopy and immunofluorescence analysis and study, we demonstrate the cytotoxicity of PS-NPs in trophoblast cells together with ultrastructural alterations associated with cellular regression and degeneration typical of metabolic stress. An abnormal amount of NPs in the cells might determine a persistent cellular alarm CDR (cell danger response), the evolutionarily conserved metabolic response that protects the cells and hosts from harm triggered by chemical (as in the case of NPs/MPs), physical, or biological agents that exceed the cellular capacity for homeostasis. This study could further help to demonstrate that the inevitable exposure of MPs/NPs in the environment, which characterizes the modern world, might be partially responsible for the epidemic of non-transmissible disease.

Immune-checkpoint inhibitors (ICIs) play a key role in the treatment of advanced stage colorectal cancer (CRC) patients featuring a deficient DNA mismatch repair (dMMR) system or a high microsatellite instability (MSI-H) profile. However, beyond the established role in CRC patients, ICIs have highly proven efficacy in other solid tumors featuring MSI-H/dMMR status represented by endometrial, gastric, ovarian, prostatic, and pancreatic carcinomas (EC, GC, OC, PrC, and PaC). Our aim was to compare the concordance rates among the Idylla™ MSI test, TapeStation 4200, and immunohistochemical (IHC) analysis in assessing MSI-H/dMMR status in EC, GC, OC, PrC, and PaC patients. The Sanger sequencing-based Titano MSI test was used in discordant cases. One hundred and eighty-five cases (n = 40 PrC, n = 39 GC, n = 38 OC, n = 35 PaC, and n = 33 EC) were retrospectively selected. MMR protein expression was evaluated by IHC. After DNA quality and quantity evaluations, the IdyllaTM and TapeStation 4200 platforms were adopted for the evaluation of MSI status. Remarkably, compared to IHC, the Idylla™ platform achieved a global concordance rate of 94.5% (154/163) for the microsatellite stable (MSS)/proficient MMR (pMMR) cases and 77.3% (17/22) for the MSI-H/dMMR cases. Similarly, a global concordance rate of 91.4% (149/163) and 68.2% (15/22) for MSS/pMMR and MSI-H/dMMR cases was also identified between IHC and the TapeStation 4200 microfluidic system. In addition, a global concordance of 93.1% (148/159) and 69.2% (18/26) for MSS/pMMR and MSI-H/dMMR cases was observed between the Idylla™ and TapeStation 4200 platforms. Discordant cases were analyzed using the Titano MSI kit. Overall, our data pinpointed a central role for molecular techniques in the diagnostic evaluation of dMMR/MSI-H status not only in CRC patients but also in other types of solid tumors.