Journal of Molecular Pathology

Adenoid cystic carcinoma (AdCC) is known to behave differently based on its location, histologic features, and molecular profile. Despite this understanding, efforts to use these molecular findings to develop personalized treatments have not yet been successful. The purpose of this retrospective study is to examine the molecular characteristics of AdCC with various histologic features in three different locations. A reference group of 20 classic cribriform AdCC cases from the parotid gland was included, along with 10 salivary AdCCs (Group 1), 10 sinonasal AdCCs with hyalinization (Group 2), and 10 solid mammary AdCCs with basaloid features (Group 3). Tissue samples were processed and tested using various molecular techniques, and the Wilcoxon signed-rank test was used to compare the different groups. Molecular data were obtained for both common and rare cases of sinonasal, salivary, and mammary AdCCs, revealing differences in molecular features depending on the tumor’s location. The molecular profile of the AdCCs in the experimental group varied depending on the site, with MYB gene rearrangements being common in all cases. We report the first MYB::KMT2C/D fusions in a subset of salivary AdCCs and sinonasal AdCCs but not in mammary adenoid cystic carcinoma with basaloid features. We conclude that co-occurring genetic alterations may vary among different sites and may have implications for the prognosis and treatment plan of AdCC. More research is needed to fully understand the mechanisms of KMT2C and KMT2D mutations in the development and progression of head and neck cancer, including their interactions with the NOTCH pathway. Full article

According to the ESMO and ASCO clinical guidelines, the main role of liquid biopsy in EGFR+ advanced NSCLC patients is represented by T790M detection after erlotinib/gefitinib/afatinib progression. However, the general international expert consensus regards osimertinib as the preferred upfront treatment in this setting; therefore, this role has been scaled back in recent years. As of today, liquid biopsy has no ASCO or ESMO recommendation following first-line osimertinib; in the same vein, no targeted therapy has received ASCO or ESMO recommendation following post upfront Osimertinib progression. However, this standard could change in the near future. Therefore, adopting a clinical point of view, this paper aims to provide a comprehensive review on the previous, the current and the possible future role of liquid biopsy in the framework of the diagnostic–therapeutic algorithm of EGFR+ advanced NSCLC. Full article

Background: A plethora of data supports HPV-based screening to be the preferred strategy for cervical cancer prevention. The shift to a more sensitive first-line test brings the need of effective triage up for discussion. Currently, most algorithms apply cytology as a triage of HPV-DNA positive women. This study compared the performance of a 7-type HPV-mRNA test to cytology. Methods: From 1 January 2019 until 31 December 2021, cervical samples from 58,029 women were examined at the University Hospital of North Norway. A total of 30.5% (17,684/58,029) fulfilled the criteria for HPV-DNA primary screening. All positive samples were triaged by cytology and followed-up according to national guidelines through 2022. Additionally, a 7-type HPV-mRNA test was applied. The study endpoint was a histologically confirmed high-grade lesion (CIN2+). Results: A total of 5.6% (990/17,684) had positive HPV-DNA test, 97.2% (962/990) with valid HPV-mRNA results. A total of 55.5% (534/962) had abnormal cytology (ASC-US+), and 35.1% (338/962) had a positive HPV-mRNA test. A total of 13.9% (134/962) had CIN2+. The sensitivity (CIN2+) of cytology versus the HPV-mRNA test was 76.1% (102/134) versus 73.1% (98/134), p = 0.67. The specificity was 47.8% (396/828) versus 71.0% (588/624), p < 0.001. PPV was 19.1% (102/534) and 29.0% (98/338), p < 0.001, respectively. The number of colposcopies per CIN2+ detected by cytology and HPV-mRNA test was 5.2 and 3.1. Conclusion: The 7-type HPV mRNA test was significantly more specific than cervical cytology in a triage of HPV-DNA positive women. Using this biomarker as the threshold for colposcopy may better balance the benefits and harms of screening. Full article

Genetic and drug sensitivity assays on primary cultures are not only of basic but also of translational interest and could eventually aid oncologists in the selection of treatments. However, cancer cells need to be identified and differentiated from the non-tumor cells always present in primary cultures. Also, successive passages can change the proportions of these two subpopulations. In this study, we propose fluorescence in situ hybridization (FISH) analysis on cell smears to determine the presence of tumor cells in primary cultures obtained from patients carrying translocations or copy number gains. FISH proved to be an easy, fast, economic, and reliable method of characterizing cell populations, which could be used repeatedly at different passages to monitor variations and to confirm the maintenance of translocations and copy number gains throughout the culture process. Full article

Somatic MET exon 14 skipping mutations (MET ex14) are targetable driver mutations for non-small cell lung cancer (NSCLC), responsive to MET inhibitors. Objective: This study seeks to further characterize the clinicopathologic features and mutational profile of MET ex14 variant NSCLC. Design: Retrospective review of all MET ex14 tested NSCLC. Testing for selected BRAF, EGFR, HER2, KRAS, and MET mutations was performed using a clinically validated NGS assay, followed by MiSeq sequencing. Variants were classified as significant (Tier1/2) or variants of uncertain significance (VUS) per 2017 AMP/ASCO/CAP Joint Consensus Guidelines. PD-L1 expression was assessed by immunohistochemistry. Results: Of 2296 NSCLCs tested between 2017-7/2019, MET ex14 variants were present in 44 (1.9%). A total of 32 of 44 variants were MET exon 14 skipping, while the other 12 mutations were significant missense (3) or VUS (9). Of nine VUS, five were adjacent to the canonical splice site and likely to impact splicing. Four cases had concomitant mutations. Of 35 cases with known clinical staging, stage 1–2 = 20 (57%), stage 3 = 3 (9%), and stage 4 = 12 (34%). Of 19 resected NSCLSs, histological types and growth pattern included 7 lepidic pattern-predominant. A high percentage of tumors with MET ex14 mutations are positive for PD-L1, and the percentage of cases with PD-L1 expression >50% trends higher in more advanced disease. Conclusions: Most MET variants identified in our cohort (73%) are MET ex14 skipping. The prevalence of MET ex14 variants is 1.9%, and a large percentage of tumors has lower clinical stage and less aggressive pathologic features. Full article

Lung cancer is one of the most common cancers and a leading cause of cancer-related mortality in Malaysia. This analysis aimed to evaluate the prevalence of actionable and common mutations, as well as co-mutations frequently occurring with EGFR variants in lung cancer. Mutational profiling of lung tumour samples was performed using next generation sequencing (NGS) panels at the Subang Jaya Medical Centre laboratory. A total of 469 lung tumour samples referred from several medical facilities in Malaysia were analysed and 84% were of the adenocarcinoma subtype. The three most frequent mutations found were EGFR (46.5%), TP53 (37.5%) and KRAS (14.3%). Actionable mutations with approved drug targets for lung cancer were detected in 63.5% of patient samples. Among patients with EGFR mutations, deletions in exon 19 were detected in 44.5% and p.L858R in 38.5% of samples. The most common co-mutations for samples with EGFR mutations were found in the TP53 gene (38.1%). A median turnaround time (TAT) of 3 working days was achievable with an automated NGS platform. NGS testing can provide valuable information on the mutational landscape and the prevalence of common or actionable mutations present in lung cancer patients. This real-world experience demonstrates the high percentage of actionable mutations detected and highlights the value of NGS testing in a clinical diagnostic setting. Full article

Ratios between the blood cells are indirect measures of the imbalance in the pro-inflammatory status observed in carcinogenesis and have been proposed as accessible and feasible biomarkers to predict cancer prognosis. We aim to evaluate the prognostic significance of neutrophil/lymphocyte (NLR), monocyte/lymphocyte (MLR), and platelet/lymphocyte (PLR) ratios in Brazilian patients with luminal breast cancer (LBC) treated with tamoxifen. A retrospective cohort of 72 operable LBC patients. Preoperative leukocyte and platelet absolute values permitted to calculate NLR, MLR, and PLR. Area under curve (ROC) determined the cutoff value associated with relapse and death. Univariate and multivariate analyses were used to assess the relationship of the platelet and PLR to disease-free survival (DFS) and overall survival (OS). Lower DFS was associated with >297 × 10³/mm³ (54 vs. 60.9 months in <297, p = 0.04). Platelet > 279 × 10³/mm³ are related to higher OS (p = 0.03). Univariate analysis revealed that platelet concentration was associated with DFS (p = 0.04) and OS (p = 0.04), but not as an independent factor (HR = 1.31, 95%CI: 0.42–4.07, p = 0.65) and OS (HR = 1.64, 95%CI: 0.28–9.52, p = 0.58). Both univariate (p = 0.01) and multivariate analysis revealed that PLR < 191.5 was a significant independent predictor of higher OS/better prognosis (HR = 16.16, 95%CI: 2.83–109.25, p = 0.00). Pretreatment platelet indices (absolute count and PLR) are prognosis predictors in LBC patients. Platelet > 279 × 10³/mm³ and PRL < 191.5 was associated with a higher OS, with the PRL being an independent predictor of higher OS. Full article

Breast cancer is among the most frequent malignancies in women worldwide. While early detection and effective treatment provide many women with a cure and prevent their cancer from spreading, metastases to distant sites still occur in around 20% of women suffering from breast cancer. These relapses occur in many forms and locations and are as varied as the primary breast tumors. Metastatic spread makes a cancer incurable and potentially lethal, but new, targeted treatments can offer control of the cancer cells if the features of new targets are unlocked by advanced diagnostic testing. The article offers an overview of the pathomechanisms of metastatic progression and describes the types of metastases, such as hormone-receptor-positive and -negative breast cancers, and HER2-overexpressing or triple-negative types. Once distant metastatic spread occurs, cytology allows a precise diagnosis to confirm the breast origin. Other molecular targets include ESR1 and PIK3CA mutations, MSI, NTRK fusion, PD-L1 expression and others, which can be obtained also from cytology material and used to determine eligibility for emerging targeted therapeutic options. We outline the diagnostic features of metastatic breast cancer in cytology samples, together with validated and emergent biomarkers that may provide new, targeted treatment options. Full article

Huntington’s disease (HD) is a progressive neurodegenerative disorder characterized by abnormal progressive involuntary movements, cognitive deficits, sleep disturbances, and psychiatric symptoms. The onset and progression of the clinical symptoms have been linked to impaired adult neurogenesis in the brains of subjects with HD, due to the reduced neurogenic potential of neural stem cells (NSCs). Among various pathogenic determinants, an altered clock pathway appears to induce the dysregulation of neurogenesis in neurodegenerative disorders. Notably, gamma-aminobutyric acid (GABA)-ergic neurons that express the vasoactive intestinal peptide (VIP) in the brain play a key role in the regulation of circadian rhythm and neuroplasticity. While an abnormal clock gene pathway has been associated with the inactivation of GABAergic VIP neurons, recent studies suggest the activation of this neuronal population in the brain positively contributes to neuroplasticity. Thus, the activation of GABAergic VIP neurons in the brain might help rectify the irregular circadian rhythm in HD. Chemogenetics refers to the incorporation of genetically engineered receptors or ion channels into a specific cell population followed by its activation using desired chemical ligands. The recent advancement of chemogenetic-based approaches represents a potential scientific tool to rectify the aberrant circadian clock pathways. Considering the facts, the defects in the circadian rhythm can be rectified by the activation of VIP-expressing GABAergic neurons using chemogenetics approaches. Thus, the chemogenetic-based rectification of an abnormal circadian rhythm may facilitate the neurogenic potentials of NSCs to restore the neuroregenerative plasticity in HD. Eventually, the increased neurogenesis in the brain can be expected to mitigate neuronal loss and functional deficits. Full article

Background and Aims: Multiple laboratory methods are used to screen patients with colorectal cancer (CRC) for mismatch repair (MMR) protein deficiency to identify possible Lynch syndrome patients. The goal of this study was to compare the agreement between ready-to-use immunohistochemistry (IHC) assays for MLH-1, PMS-2, MSH-2, MSH-6, and mutated BRAF at V600E and molecular methods in CRC cases. The inclusion of the BRAF V600E mutation testing is important for the identification of patients with sporadic CRC, as the BRAF V600E mutation is very rarely observed in patients with Lynch syndrome tumors. Methods: CRC cases were analyzed by ColoSeq^TM tumor sequencing assay and VENTANA MMR IHC Panel that included anti-MLH1, anti-PMS2, anti-MSH2, anti-MSH6, and anti-BRAF V600E antibodies. Additionally, CRC cases with MLH1 IHC loss were evaluated for MLH1 promoter hypermethylation. Results: One hundred and eighteen cases were analyzed. The overall percent agreement (OPA) for each evaluated marker status compared to next-generation sequencing (NGS) exceeded 96%. Twenty-three cases were positive for the BRAF V600E mutation by IHC and NGS, and twenty cases showed loss of MLH1 protein and were positive for MLH1 hypermethylation. Samples with loss of MMR protein expression by IHC demonstrated genetic and/or epigenetic alterations that were consistent with the observed protein expression patterns. Conclusions: The results of this study indicate that ready-to-use IHC assays can correctly identify the loss of MMR proteins and the presence of mutated BRAF V600E protein, supporting the utility of the VENTANA MMR IHC Panel as an aid to stratify patients with sporadic CRC vs. potential Lynch syndrome. Full article

Metastatic breast cancer (MBC) remains in most cases an incurable disease with genetic complexity and heterogeneity. Improvements in classification and management have been introduced, in addition to the development of endocrine and anti-HER2 targeted therapies. Currently, efforts are being made to delineate the best approach for the genomic landscape of MBC and, as result, molecular therapeutic targets. Here, we highlight the recent developments in the cytopathology of MBC, discussing cytological diagnostic approaches in the characterization of hallmarks, such as immunocytochemistry and genomic biomarkers. Cytological material can be processed for ancillary testing for diagnostic and therapeutic purposes. Reassessment of receptor status is indicated due to changes in tumor biology and metastatic presentation. PD-L1 expression is the only approved biomarker for predicting immune checkpoint inhibitor response in metastatic TNBC, evaluated by immunostaining. The feasibility of applying PD-L1 assays in MBC cytological samples can be recommended, with the adoption of a combined positive score. Non-formalin cytological samples provide higher purity, cellular yield, and better tumor fraction for single-multi gene assays. In MBC, molecular tests enable personalized therapy such as PIK3CA, NTRK fusion genes, and MSI. Cytopathology combined with molecular analysis must be performed effectively in routine clinical practice, through procedure standardization and experience dissemination. Full article

Gankyrin has a household function in essentially all cells by acting as a chaperone in the assembly of the 26S proteasome, but also functions as a tumor-promoting protein by antagonizing the tumor suppressors retinoblastoma protein, p16, and p53. While gankyrin is overexpressed in many neoplasms outside the skin, its expression in normal skin and cutaneous neoplasms has not been reported previously. We studied the expression of gankyrin in archival human formalin-fixed tissues of cutaneous neoplasms by immunohistochemistry with a monoclonal antibody, and found gankyrin to be overexpressed in 3 of 20 squamous cell carcinomas, none of 10 basal cell carcinomas, 13 of 18 melanocytic nevi, and 7 of 10 melanomas, in many cases with a predominantly nuclear location. Normal epidermal melanocytes expressed gankyrin to a lesser extent than neoplastic melanocytes. The overexpression in the in situ stage of squamous cell carcinoma and in melanocytic nevi suggests that gankyrin acts as a tumor-promoting protein in the early stages of the transition from normal to neoplastic cells. The frequent overexpression of gankyrin in melanocytic neoplasms is significant because it antagonizes the tumor suppressor, p16, which is strongly expressed in melanocytic nevi and some melanomas. Full article

Best practice in the management of non-squamous, non-small-cell lung cancer patients involves somatic testing for a range of molecular markers. Actionable oncogenic drivers of malignancy are increasingly being detected using RNA-based next-generation sequencing in the UK by referral to centralized genomic laboratory hubs. Recent audit data from the author’s case work have demonstrated an RNA sequencing failure rate of 35%. This article examines the real-world context, which may account for this failure rate, and discusses alternative options for patient care pathways. Full article

Breast carcinomas are known to metastasize to various organs of the human body. Fine needle aspiration cytology or exfoliative cytology often are the standard method for diagnosis at these metastatic sites due to ease of procurement of diagnostic material, accessibility, less complications, high sensitivity, and specificity of diagnosis and evaluation of biomarker status needed to guide future management. This comprehensive review article discusses in detail metastatic patterns, cytomorphology of metastatic breast cancer at different body sites, immunohistochemistry needed for diagnosis of breast carcinoma, sensitivity and specificity of diagnosis and breast biomarker assays in the cytology material. Full article

Background: Smoking habit is a common cause of pulmonary Langerhans cell histiocytosis (PLCH) and lung cancer and both diseases may coexist in the lung and share genetic alterations, such as V600E BRAF mutations. We collected a small series of three cases of PLCH-associated lung adenocarcinoma in order to evaluate the molecular setup in both components and underline the critical role of careful tissue selection for predictive molecular driver testing. Methods: Three cases of PLCH-associated adenocarcinoma were collected from consultation files. Clinical data from referring physicians and clinical data were obtained. The surgical biopsies were tested by immunohistochemistry and molecular analysis after separate dissection of adenocarcinoma cells and Langerhans histiocytes. Results: There were three active smoking men with a median age at diagnosis of 60.6 years. PLCH was disclosed at imaging during work-up for suspected lung cancer. Molecular analysis revealed KRAS (G12C and G13C) mutations in two cases and V600E BRAF mutation in one case of PLCH. Immunostaining with the V600E BRAF mutation specific primary antibody VE1 correctly recognized BRAF-mutated LCH. One case was wild-type in both diseases. Two similar cases were found in the literature, one of which showed a discrepant KRAS (G12D) mutation in adenocarcinoma and a V600E BRAF mutation in LCH; Conclusions: This case series of PLCH-associated adenocarcinoma underline the possibility to disclose identical genetic alterations in co-existing benign and malignant pathologies, then potentially creating erroneous interpretation of molecular analysis leading to inadequate therapeutic options in case of incorrect diagnostic recognition and inappropriate selection of both components through microdissection. Full article

There is confusion about the diagnosis, histogenesis and taxonomical efforts regarding adenosquamous carcinomas (ASCs) and mucinous adenocarcinomas (MACs), especially with calls for reconsidering the nature of high-grade mucoepidermoid carcinoma (MEC). This study aims to compare the genetic profiles of ASCs and MACs that have been previously reported in the literature and investigate if either ASC or MAC is closer in genetic mutations to high-grade MEC. Systematic searches in the NCBI, Web of Science, and Scopus databases were performed between January 2000 and August 2022. The retrieved genetic mutations were processed and annotated. Protein–protein network analysis was conducted for each neoplasm. The results were viewed and discussed in terms of molecular oncogenesis of ASCs and MACs at different topographies. Molecular profile mapping was conducted by annotating all the retrieved genes for each neoplasm using genetic network analysis (Cystoscape software program). The genetic profile of each lesion was compared to that of high-grade MEC. To conclude, both genetic profiles do not tend to intersect specifically with high-grade MEC, except for the generic mutations commonly detected in all high-grade head and neck tumors. However, the availability of data on the molecular profile of each lesion limits the generalizability of the findings of this study. Full article

This study describes the roles of laboratory information management systems (LIMS) in multi-site genetics studies in Africa. We used the HiGeneS Africa project as a case study. The study participants were recruited in six African countries between 2019 to 2021. The Baobab LIMS, a server–client-based system (an African-led innovation) was used for the coordination of the biospecimen. The development phase of the LIMS showcased the team formation, data collection, biospecimen collection, and shipment strategies. The implementation phase showcased the biospecimen registration, processing, and quality control (QC) analytics. The sample QC was done using Nanodrop, Qubit, and PicoGreen/gDNATapestation assays. The results showed that a total of 3144 study participants were recruited from Cameroon, Ghana, Mali, Rwanda, Senegal, and South Africa. The biospecimen registration provided a comprehensive registry that included patient demographics, genetic information, and clinical and blood/saliva samples from the proband and family relatives. The QC analyzes identified 30 samples that failed QC, linked to overdue storage in the freezer before DNA extraction. The LIMS components implemented in this project formed a structure that can be upscaled to artificial intelligence-based LIMS. In conclusion, this study represents the largest and the most diverse collection of biospecimens for the genetic study of hearing impairment in Africa to date. A well-characterized LIMS should be recommended for multi-site molecular studies, particularly in Africa, to enhance African participation in global genomic medicine. Full article