The Day-To-Day Practice of MMR and MSI Assessment in Colorectal Adenocarcinoma: What We Know and What We Still Need to Explore.

BACKGROUND: The DNA mismatch repair (MMR) system is a highly preserved protein complex recognizing short insertions, short deletions, and single base mismatches during DNA replication and recombination. MMR protein status is identified using immunohistochemistry. Deficit in one or more MMR proteins, configuring deficient MMR status (dMMR), leads to frameshift mutations particularly clustered in microsatellite repeats. Thus, microsatellite instability (MSI) is the epiphenomenon of dMMR. In colorectal cancer (CRC), MMR/MSI status is a biomarker with prognostic and predictive value of resistance to 5-fluorouracil and response to immune checkpoint inhibitor therapy. SUMMARY: In this Review, we describe the challenges the practicing pathologist may face in relation to the assessment of MMR/MSI status and any open issues which still need to be addressed, focusing on pre-analytic issues, pitfalls in the interpretation, and technical aspects of the different assays. KEY MESSAGES: The current methods of detecting dMMR/MSI status have been optimized for CRCs, and whether these techniques can be applied to all tumor and specimen types is still not fully understood. Following the Food and Drug Administration (FDA), tissue/site agnostic drug approval of pembrolizumab for advanced/metastatic MSI tumors, MMR/MSI status in gastrointestinal tract is a common request from the oncologist. In this setting, several issues still need to be addressed, including criteria for sample adequacy.

Robotic resection of a single adenoid cystic tumor liver metastasis using ICG fluorescence. A case report and literature review.

Adenoid cystic carcinoma (AdCC) is a rare tumor that typically develops in the salivary glands and less frequently in other sites of the head and neck region. Only a few cases of resected metachronous liver metastases have been reported. Minimally invasive surgery is currently the gold standard of care for liver resections; furthermore, the use of Indocyanine Green (ICG) is continuously increasing in surgical practice, especially in cases of primary liver tumors and colorectal liver metastases, due to its capacity to enhance liver nodules. We report the case of a 54-year-old male with a single liver metastasis of AdCC, located in SIII, who presented in our center 9 months after resection of a primary tumor of the laryngotracheal junction and adjuvant proton therapy. A 25-mg injection of ICG (0.3 mg/kg) was administered 48 h before surgery in order to highlight the tumor and perform an ICG-guided resection. The lesion was clearly visible during surgery, and, given its position and the proximity to the main lobar vessels of the left lobe, we opted for a left lateral sectionectomy. The outcome was unremarkable, with no major postoperative complications. The administration of ICG 48 h before surgery seems to be a valid tool even in cases of AdCC liver metastases, providing surgeons with better visualization of the lesion and improving the precision of the resection.

HER2-low in gastro-oesophageal adenocarcinoma: a real-world pathological perspective.

AIMS: In the DESTINY-Gastric01 trial, a novel HER2-targeted antibody-drug conjugate trastuzumab deruxtecan proved to be effective in HER2-low gastro-oesophageal adenocarcinomas. The aim of our study is to investigate the clinicopathological and molecular features of HER2-low gastric/gastro-oesophageal junction cancers in the real-world setting of a large multi-Institutional series. METHODS: We retrospectively evaluated 1210 formalin-fixed paraffin-embedded samples of gastro-oesophageal adenocarcinomas which were analysed by immunohistochemistry for HER2 protein expression in 8 Italian surgical pathology units from January 2018 to June 2022. We assessed the prevalence of HER2-low (ie, HER2 1+ and HER2 2+ without amplification) and its correlation with clinical and histopathological features, other biomarkers' status, including mismatch repair/microsatellite instability status, Epstein-Barr encoding region (EBER) and PD-L1 Combined Positive Score. RESULTS: HER2 status could be assessed in 1189/1210 cases, including 710 HER2 0 cases, 217 HER2 1+, 120 not amplified HER2 2+, 41 amplified HER2 2+ and 101 HER2 3+. The estimated prevalence of HER2-low was 28.3% (95% CI 25.8% to 31.0%) overall, and was higher in biopsy specimens (34.9%, 95% CI 31.2% to 38.8%) compared with surgical resection specimens (21.0%, 95% CI 17.7% to 24.6%) (p<0.0001). Moreover, HER2-low prevalence ranged from 19.1% to 40.6% among centres (p=0.0005). CONCLUSIONS: This work shows how the expansion of the HER2 spectrum might raise problems in reproducibility, especially in biopsy specimens, decreasing interlaboratory and interobserver concordance. If controlled trials confirm the promising activity of novel anti-HER2 agents in HER2-low gastro-oesophageal cancers, a shift in the interpretation of HER2 status may need to be pursued.

Counting mitoses in gastrointestinal stromal tumours (GISTs): variable practices in the real-world setting and their clinical implications.

Mitotic count (MC) is an important prognostic indicator in gastrointestinal stromal tumours (GISTs). Though MC evaluation was initially proposed in 50 HPFs, recent international guidelines recommend that MC be performed on 5 mm2 because HPFs may have different areas depending on the ocular field number (FN) of the utilized light microscope. Performing MC on different areas leads to a non-standardized evaluation and erroneous risk stratification. The aim of the study was to audit real-life MC practices with special emphasis on possible risk stratification errors. A survey was administered to Italian pathologists to evaluate the following: method used for MC (5 mm2 versus 50 HPF); FN of the light microscope; prognostic scheme for risk stratification. Based on the results of the survey, 100 GISTs (25/risk class using Miettinen prognostic scheme) were retrieved and MC performed using 5 mm2 versus the corresponding mm2 area sizes of 50 HPFs with variable FNs (18, 20, 22). The survey demonstrated that the majority of pathologists (64.5%) use 50 HPFs with various FNs leading to excessive area size. The most frequently used prognostic scheme is that by Miettinen. Using this prognostic scheme and counting mitoses in 5 mm2 versus 50 HPFs with FNs 18, 20 and 22, a change in risk class was identified ranging from 10 to 41%, depending on FN. In conclusion, this study demonstrates that MC is still frequently performed on 50 HPF, with area sizes exceeding the specified 5 mm2 by far.

A practical approach for PD-L1 evaluation in gastroesophageal cancer.

PD-L1 is an established predictive immunohistochemical biomarker of response to immune checkpoint inhibitors. At present, PD-L1 is routinely assessed on biopsy samples of advanced gastroesophageal cancer patients before initiating first-line treatment. However, PD-L1 is still a suboptimal biomarker, due to changing cut-off values and scoring systems, interobserver and interlaboratory variability.This practical illustrated review discusses the range of staining patterns of PD-L1 and the potential pitfalls and challenges that can be encountered when evaluating PD-L1, focusing on gastric and gastroesophageal adenocarcinoma (G/GEA) and esophageal squamous cell carcinoma (ESCC).

Dasatinib-induced Crohn's-like colitis.

Dasatinib is a second-generation multityrosine kinase inhibitor used in the first-line and second-line treatment of Philadelphia chromosome-positive leukaemia. The most frequent type of Dasatinib-induced intestinal injury is haemorrhagic colitis; other morphologic patterns include apoptotic colopathy, CD8+ T-cell-mediated colitis and non-specific colitis. Aim of this study is to describe a novel Crohn's-like histopathologic pattern of Dasatinib-induced colitis. Four patients developed diarrhoea during Dasatinib treatment; colonoscopy was performed and biopsy sets were taken for histological analysis. All patients showed patchy, chronic active inflammation with cryptitis and microgranulomas (two patients). Ileal and rectal biopsies showed either no or mild, focal inflammation. An increase in lamina propria eosinophils was seen (two patients) and apoptoses were seen (three patients). Complete remission was observed after interruption of treatment. Dasatinib-induced colitis and Crohn's disease may share histologic features including microgranulomas, which can potentially lead to misdiagnosis if no information on treatment is provided.

Assessment and Response to Neoadjuvant Treatments in Breast Cancer: Current Practice, Response Monitoring, Future Approaches and Perspectives.

Neoadjuvant treatments (NAT) for breast cancer (BC) consist in the administration of chemotherapy-more rarely endocrine therapy-before surgery. Firstly, it was introduced 50 years ago to downsize locally advanced (inoperable) BCs. NAT are now widespread and so effective to be used also at the early stage of the disease. NAT are heterogeneous in terms of therapeutic patterns, class of used drugs, dosage, and duration. The poly-chemotherapy regimen and administration schedule are established by a multi-disciplinary team, according to the stage of disease, the tumor subtype and the age, the physical status, and the drug sensitivity of BC patients. Consequently, an accurate monitoring of treatment response can provide significant clinical advantages, such as the treatment de-escalation in case of early recognition of complete response or, on the contrary, the switch to an alternative treatment path in case of early detection of resistance to the ongoing therapy. Future is going toward increasingly personalized therapies and the prediction of individual response to treatment is the key to practice customized care pathways, preserving oncological safety and effectiveness. To gain such goal, the development of an accurate monitoring system, reproducible and reliable alone or as part of more complex diagnostic algorithms, will be promising.

PD-L1 evaluation in the gastrointestinal tract: from biological rationale to its clinical application.

Immune-checkpoint inhibitors targeting the PD-1/PD-L1 axis have brought significant clinical benefit in many solid cancer types, including gastrointestinal malignancies. However, it has been estimated that only 20-40% of patients respond to treatment. The pattern of expression and potential predictive value of PD-L1 as an immunohistochemical biomarker has been extensively studied in gastrointestinal neoplasms. Until now, its predictive value has been demonstrated, and is currently in use only in upper gastrointestinal malignancies (gastroesophageal adenocarcinoma and esophageal squamous cell carcinoma).In this Review, we describe the technical aspects and challenges related to PD-L1 immunohistochemical assays, the current role of PD-L1 as a biomarker in clinical practice and we outline the main studies and clinical trials analyzing the prognostic and predictive value of PD-L1 in gastrointestinal cancers.

Lymphoplasmacyte-rich meningioma with hematologic signs and PD-L1 over-expression.

Lymphoplasmacyte-rich meningioma (LPRM) is one of the rarest variants of grade I meningiomas. It can be clinically associated with prominent peripheral blood abnormalities, anemia, and/or various gammopathy, which usually disappear after surgical removal of the tumor. We document a case of right frontal LPRM in a 72-year-old male who presented general cognitive decadence. The patient suffered from mild anemia. The LPRM is a rare variant of meningioma, with only a few cases globally reported in the literature. It has been categorized as a grade I tumor in the 2021 World Health Organization (WHO) classification central nervous system. Due to the rarity, this meningioma variant origin and biological behavior are still not clear. Immunohistochemistry profile showed prominent PD-L1 expression, leading to additional interrogation on LPRM immunomorphological characteristics, the significance of the inflammatory tumoral microenvironment and its correlation with the immune-checkpoints.

HLA-G as a prognostic marker in stage II/III colorectal cancer: not quite there yet.

Identifying innovative molecules involved in the tumor immune escape process could help refine the survival stratification of colorectal cancer (CRC) patients. HLA-G, a non-classical HLA molecule, physiologically involved in tolerogenic mechanisms, has recently emerged as a relevant prognostic marker in other tumor types, but ambiguous data are reported in the CRC setting. This study aims to evaluate the HLA-G expression and prognostic potential in a series of stage II/III CRCs. HLA-G expression was evaluated in 100 pT3 CRC cases by means of immunohistochemistry using the 4H84 and MEM-G/2 monoclonal antibodies. We observed heterogeneous expression of HLA-G showing different ranges: 4H84 expression ranged from > 1 to 40%-median 7%; MEM-G/2 expression ranged from 20 to 90%-median 50%. HLA-G positivity (any intensity > 1%) varied according to the antibody employed, identifying: 8 4H84 positive, 34 MEM-G/2 positive, 6 double-positive and 52 negative cases. Correlation with clinico-pathologic data showed a significant association with a poor tumor differentiation in stage III right-sided CRC subgroup (p = 0.043), while no other pathologic variable was significantly associated. Survival analysis revealed a reduced disease-free survival rate (HR 4.304613; p = 0.031) in the subgroup of CRC-related death cases, while no correlations were observed considering the whole series and the overall survival. In conclusion, HLA-G is a promising CRC prognostic marker however much work is still required regarding technical aspects and evaluation of expression.

Pathologist's approach to paediatric and neonatal eosinophilic gastrointestinal disorders.

Children are not simply miniature adults. The evaluation of their gastrointestinal disorders is therefore different from that in full-grown adults and requires a particular clinical/pathologic approach.Different studies have tried to assess the normal eosinophil distribution in the gastrointestinal tract in adults while very few studies have investigated the paediatric population, consequently complicating the pathologist's ability in identifying an abnormal number of eosinophils in this setting of patients.When evaluating gastrointestinal tract biopsies with eosinophilia, eosinophilic count must be considered along with other histological features like eosinophil distribution in the gastrointestinal wall, their degranulation, cryptitis and crypt abscesses, other accompanying inflammatory cells, apoptotic bodies, foreign material or microorganisms; these findings, although rarely specific, may be a useful aid for diagnosis.Reports should not include a diagnosis of primary eosinophilic gastrointestinal disorders (EoGID) if clinical data and test results do not rule out other forms of gastrointestinal eosinophilia. A more descriptive definition like "with eosinophilic pattern" should be favoured over a specific diagnosis of "eosinophilic disorder" in order to avoid potential confusion between different entities.

"Stranger things" in the gut: uncommon items in gastrointestinal specimens.

Organic (such as parasites or vegetable remnants) and inorganic substances may be encountered during routine pathology diagnostic work up of endoscopic gastrointestinal biopsy samples and major resections, causing possible diagnostic conundrums for the young and not so young pathologists. The main aim of this review is the description of the most frequent oddities one can encounter as foreign bodies, in gastrointestinal pathology, on the basis of the current literature and personal experience. The types of encountered substances are divided into four principal categories: parasites (helminths such as Enterobius vermicularis, Strongyloides, Schistosoma, and Anisakis, and protozoa such as Entamoeba, Giardia and some intestinal coccidia); drugs and pharmaceutical fillers (found as deposits and as bystanders, innocent or not); seeds (possibly confused with worms) and plant remnants; pollutants (secondary to post-resection or post-biopsy contamination of the sample). An ample library of images is provided in order to consent easy referencing for diagnostic routine.

Lymphoplasmacyte-rich meningioma with hematologic signs and PD-L1 over-expression

ABSTRACT Lymphoplasmacyte-rich meningioma (LPRM) is one of the rarest variants of grade I meningiomas. It can be clinically associated with prominent peripheral blood abnormalities, anemia, and/or various gammopathy, which usually disappear after surgical removal of the tumor. We document a case of right frontal LPRM in a 72-year-old male who presented general cognitive decadence. The patient suffered from mild anemia. The LPRM is a rare variant of meningioma, with only a few cases globally reported in the literature. It has been categorized as a grade I tumor in the 2021 World Health Organization (WHO) classification central nervous system. Due to the rarity, this meningioma variant origin and biological behavior are still not clear. Immunohistochemistry profile showed prominent PD-L1 expression, leading to additional interrogation on LPRM immunomorphological characteristics, the significance of the inflammatory tumoral microenvironment and its correlation with the immune-checkpoints.

Extension of Collagen Deposition in COVID-19 Post Mortem Lung Samples and Computed Tomography Analysis Findings.

Lung fibrosis has specific computed tomography (CT) findings and represents a common finding in advanced COVID-19 pneumonia whose reversibility has been poorly investigated. The aim of this study was to quantify the extension of collagen deposition and aeration in postmortem cryobiopsies of critically ill COVID-19 patients and to describe the correlations with qualitative and quantitative analyses of lung CT. Postmortem transbronchial cryobiopsy samples were obtained, formalin fixed, paraffin embedded and stained with Sirius red to quantify collagen deposition, defining fibrotic samples as those with collagen deposition above 10%. Lung CT images were analyzed qualitatively with a radiographic score and quantitatively with computer-based analysis at the lobe level. Thirty samples from 10 patients with COVID-19 pneumonia deceased during invasive mechanical ventilation were included in this study. The median [interquartile range] percent collagen extension was 6.8% (4.6-16.2%). In fibrotic compared to nonfibrotic samples, the qualitative score was higher (260 (250-290) vs. 190 (120-270), p = 0.036) while the gas fraction was lower (0.46 (0.32-0.47) vs. 0.59 (0.37-0.68), p = 0.047). A radiographic score above 230 had 100% sensitivity (95% confidence interval, CI: 66.4% to 100%) and 66.7% specificity (95% CI: 41.0% to 92.3%) to detect fibrotic samples, while a gas fraction below 0.57 had 100% sensitivity (95% CI: 66.4% to 100%) and 57.1% specificity (95% CI: 26.3% to 88.0%). In COVID-19 pneumonia, qualitative and quantitative analyses of lung CT images have high sensitivity but moderate to low specificity to detect histopathological fibrosis. Pseudofibrotic CT findings do not always correspond to increased collagen deposition.

The Seeds of Doubt: Finding Seeds in Intriguing Places.

Introduction: Seeds may be found in gastrointestinal tissue samples, and their multifaceted appearance may be challenging. The aim is to report a rough incidence of pathology samples which show seeds, specify the most frequent sample types and show an iconography of the most commonly identified seeds. Materials and Methods: Between 2017 and 2020, all gastrointestinal pathology cases in which seeds/seed parts were found, were collected and seed type described by referencing a seed image library. Results: Fifty cases with complete seeds/seed parts were collected: 16 colonic resections for colorectal cancer and diverticulosis, 13 appendiceal resections for appendicitis, 1 gastric resection. Fifteen cases were found in polypectomy specimens and 5 cases in colorectal endoscopic biopsies. Most frequent seed types were tomato, kiwi, blueberry, and blackberry seeds. Conclusion: Seeds may be found in up to 4% of specimens; their recognition may be useful to exclude parasitic infections as well as in forensic sciences.

Diagnostic concordance of phyllodes tumour of the breast.

AIMS: Phyllodes tumours (PT) are rare and distinct breast tumours, which span a morphological continuum. Classification into benign, borderline and malignant categories reflects their biology and clinical behaviour and is essential to guide management. This study aims to assess the diagnostic agreement of PT using the UK National Health Service Breast Screening Programme (NHSBSP) breast pathology external quality assurance (EQA) scheme data. METHODS AND RESULTS: Twenty-six PTs were identified in the EQA scheme, which were diagnosed by an average of 607 participants/circulation. Data on diagnostic categories were collected and representative slides were reviewed. The level of concordance between reporting pathologists was assessed. There were 14 benign, six borderline and six malignant PT. The overall rate of diagnosis agreement was 86% when analysed as benign lesions, borderline PT and malignant lesions, which decreased to 79% when diagnosed as PT (irrespective of grade) and to 63% when the diagnosis was further refined to PT categories (benign, borderline and malignant PTs). The highest agreement rate was observed in malignant PT (86%) and the lowest in borderline PT (42%). Malignant heterologous elements, stromal overgrowth and leaf-like architecture are features associated with higher concordance rates. Lower-priority features were stromal expansion, clefting and multinodularity. CONCLUSION: The concordance of PT diagnosis, as an entity, is high, but its classification into benign, borderline and malignant has variable agreement levels, with borderline tumours having the lowest concordance rate. More research to refine the diagnostic criteria for categorisation of PT is warranted to improve concordance between pathologists.

Methods for restoration of ki67 antigenicity in aged paraffin tissue blocks.

Pathology archives are a treasure trove of paraffin embedded tissue spanning many years and covering a wide variety of tissues and diseases. The possibility of using old archival formalin fixed paraffin embedded (FFPE) tissues for diagnostic updates and research projects is a widespread need and it requires archives of stable, well-preserved samples. Immunohistochemistry performed on old archival paraffin blocks may give unreliable results, in particular for some antigens, such as Ki67. In consideration of this phenomenon, our aim is to comprehensively test and identify methods which may be used to obtain Ki67 immunohistochemical reactions of good quality from old archival FFPE blocks. Various methods were tested in order to evaluate their possible efficacy in increasing Ki67 immunointensity in a collection of 40-year-old, archival blocks including re-embedding, with deeper sectioning of tissue from the block and increasing heat-based pretreatment times (20 cases) and re-processing (20 cases). All reactions were performed using an automated immunostainer and Ki67 stained immunosections compared using a visual colour-based scale (the first immunostained section was considered as baseline). The combination of deep sectioning (1000 µM) and prolonged heat-based pretreatment (64 min) markedly increased immunoreactivity for Ki67. Re-embedding and reprocessing did not have a significant effect. Large tissue samples showed heterogeneity of Ki67 immunoexpression between the periphery of the sample and the central area. In conclusion, the study defines a useful protocol to increase antigen retrieval applicable to dated archival tissues.

Neuroendocrine neoplasms of the esophagus and stomach.

Esophageal neuroendocrine neoplasms (E-NENs) are much rarer than other gastro-entero-pancreatic neuroendocrine neoplasms, the majority showing aggressive behavior with early dissemination and poor prognosis. Among E-NENs, exceptionally rare well differentiated neuroendocrine tumors (E-NET) and more frequent esophageal poorly differentiated neuroendocrine carcinomas (E-NEC) and mixed neuroendocrine-non neuroendocrine neoplasms (MiNEN) can be recognized. E-NECs usually exhibit a small cell morphology or mixed small and large cells. Esophageal MiNEN are composed of NEC component admixed with adenocarcinoma or squamous cell carcinoma. Gastric (G) NENs encompass a wide spectrum of entities ranging from indolent G-NETs to highly aggressive G-NECs and MiNENs. Among G-NETs, ECL-cell NETs are the most common and, although composed of histamine-producing cells, are a heterogeneous group of neoplastic proliferations showing different clinical and prognostic features depending on the patient's clinico-pathological background including the morphology of the peri-tumoral mucosa, gastrin serum levels, presence or absence of antral G-cell hyperplasia, and presence or absence of MEN1 syndrome. In general, NET associated with hypergastrinemia show a better outcome than NET not associated with hypergastrinemia. G-NECs and MiNENs are aggressive neoplasms more frequently observed in males and associated with a dismal prognosis.