Carcinoma of unknown primary (CUP): an update for histopathologists.

Carcinoma of unknown primary (CUP) is a heterogeneous group of metastatic cancers in which the site of origin is not identifiable. These carcinomas have a poor outcome due to their late presentation with metastatic disease, difficulty in identifying the origin and delay in treatment. The aim of the pathologist is to broadly classify and subtype the cancer and, where possible, to confirm the likely primary site as this information best predicts patient outcome and guides treatment. In this review, we provide histopathologists with diagnostic practice points which contribute to identifying the primary origin in such cases. We present the current clinical evaluation and management from the point of view of the oncologist. We discuss the role of the pathologist in the diagnostic pathway including the control of pre-analytical conditions, assessment of sample adequacy, diagnosis of cancer including diagnostic pitfalls, and evaluation of prognostic and predictive markers. An integrated diagnostic report is ideal in cases of CUP, with results discussed at a forum such as a molecular tumour board and matched with targeted treatment. This highly specialized evolving area ultimately leads to personalized oncology and potentially improved outcomes for patients.

Tumour 63 protein (p63) in breast pathology: biology, immunohistochemistry, diagnostic applications, and pitfalls.

Tumour protein 63 (p63) is a transcription factor of the p53 gene family, encoded by the TP63 gene located at chromosome 3q28, which regulates the activity of genes involved in growth and development of the ectoderm and derived tissues. p63 protein is normally expressed in the nuclei of the basal cell layer of glandular organs, including breast, in squamous epithelium and in urothelium. p63 immunohistochemical (IHC) staining has several applications in diagnostic breast pathology. It is commonly used to demonstrate myoepithelial cells at the epithelial stromal interface to differentiate benign and in situ lesions from invasive carcinoma and to characterize and classify papillary lesions including the distinction of breast intraduct papilloma from skin hidradenoma. p63 IHC is also used to identify and profile lesions showing myoepithelial cell and/or squamous differentiation, e.g. adenomyoepithelioma, salivary gland-like tumours including adenoid cystic carcinoma, and metaplastic breast carcinoma including low-grade adenosquamous carcinoma. This article reviews the applications of p63 IHC in diagnostic breast pathology and outlines a practical approach to the diagnosis and characterization of breast lesions through the identification of normal and abnormal p63 protein expression. The biology of p63, the range of available antibodies with emphasis on staining specificity and sensitivity, and pitfalls in interpretation are also discussed. The TP63 gene in humans, which shows a specific genomic structure, resulting in either TAp63 (p63) isoform or ΔNp63 (p40) isoform. As illustrated in the figure, both isoforms contain a DNA-binding domain (Orange box) and an oligomerization domain (Grey box). TAp63 contains an N-terminal transactivation (TA) domain (Green box), while ΔNp63 has an alternative terminus (Yellow box). Antibodies against conventional pan-p63 (TP63) bind to the DNA binding domain common to both isoforms (TAp63 and p40) and does not distinguish between them. Antibodies against TAp63 bind to the N-terminal TA domain, while antibodies specific to ΔNp63 (p40) bind to the alternative terminus. Each isoform has variant isotypes (α, ß, γ, δ, and ε).

Emerging Role of Scintigraphy Using Bone-Seeking Tracers for Diagnosis of Cardiac Amyloidosis: AJR Expert Panel Narrative Review.

Amyloidoses are a complex group of clinical diseases that result from progressive organ dysfunction due to extracellular protein misfolding and deposition. The two most common types of cardiac amyloidosis are transthyretin amyloidosis (ATTR) and light-chain (AL) amyloidosis. Diagnosis of ATTR cardiomyopathy (ATTR-CM) is challenging owing to its phenotypic similarity to other more common cardiac conditions, the perceived rarity of the disease, and unfamiliarity with its diagnostic algorithms; endomyocardial biopsy was historically required for diagnosis. However, myocardial scintigraphy using bone-seeking tracers has shown high accuracy for detection of ATTR-CM and has become a key noninvasive diagnostic test for the condition, receiving support from professional society guidelines and transforming prior diagnostic paradigms. This AJR Expert Panel Narrative Review describes the role of myocardial scintigraphy using bone-seeking tracers in the diagnosis of ATTR-CM. The article summarizes available tracers, acquisition techniques, interpretation and reporting considerations, diagnostic pitfalls, and gaps in the current literature. The critical need for monoclonal testing of patients with positive scintigraphy results to differentiate ATTR-CM from AL cardiac amyloidosis is highlighted. Recent updates in guideline recommendations that emphasize the importance of a qualitative visual assessment are also discussed.

Pitfalls in machine learning-based assessment of tumor-infiltrating lymphocytes in breast cancer: A report of the International Immuno-Oncology Biomarker Working Group on Breast Cancer

The clinical significance of the tumor-immune interaction in breast cancer is now established, and tumor-infiltrating lymphocytes (TILs) have emerged as predictive and prognostic biomarkers for patients with triple-negative (estrogen receptor, progesterone receptor, and HER2-negative) breast cancer and HER2-positive breast cancer. How computational assessments of TILs might complement manual TIL assessment in trial and daily practices is currently debated. Recent efforts to use machine learning (ML) to automatically evaluate TILs have shown promising results. We review state-of-the-art approaches and identify pitfalls and challenges of automated TIL evaluation by studying the root cause of ML discordances in comparison to manual TIL quantification. We categorize our findings into four main topics: (1) technical slide issues, (2) ML and image analysis aspects, (3) data challenges, and (4) validation issues. The main reason for discordant assessments is the inclusion of false-positive areas or cells identified by performance on certain tissue patterns or design choices in the computational implementation. To aid the adoption of ML for TIL assessment, we provide an in-depth discussion of ML and image analysis, including validation issues that need to be considered before reliable computational reporting of TILs can be incorporated into the trial and routine clinical management of patients with triple-negative breast cancer. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Recent Advances in Pathology: the 2023 Annual Review Issue of The Journal of Pathology.

The 2023 Annual Review Issue of The Journal of Pathology, Recent Advances in Pathology, contains 12 invited reviews on topics of current interest in pathology. This year, our subjects include immuno-oncology and computational pathology approaches for diagnostic and research applications in human disease. Reviews on the tissue microenvironment include the effects of apoptotic cell-derived exosomes, how understanding the tumour microenvironment predicts prognosis, and the growing appreciation of the diverse functions of fibroblast subtypes in health and disease. We also include up-to-date reviews of modern aspects of the molecular basis of malignancies, and our final review covers new knowledge of vascular and lymphatic regeneration in cardiac disease. All of the reviews contained in this issue are written by expert groups of authors selected to discuss the recent progress in their particular fields and all articles are freely available online (https://pathsocjournals.onlinelibrary.wiley.com/journal/10969896). © 2023 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Pitfalls in soft tissue cytopathology.

Fine needle aspiration biopsy (FNAB) is a diagnostic modality for the evaluation of suspicious soft tissue masses. Despite its reasonable sensitivity, specificity and positive predictive value in differentiating benign from malignant neoplasms, the exact subtyping of the primary soft tissue tumours can be challenging. Certain tumours constitute "pitfalls" and add to the diagnostic challenge. This review provides a detailed account of the diagnostic challenges in soft tissue cytopathology, including pitfalls and, more importantly, the ways to overcome these challenges by integrating clinical details, key cytomorphological features and judicious application of ancillary techniques.

Urinary tract cytology showing variant morphology and divergent differentiation.

Urothelial carcinoma represents a diverse group of tumours with distinct histologic subtypes, each exhibiting unique cytomorphologic features, architectural growth patterns, and/or well-developed aberrant differentiation. In fact, there are more than 13 subtypes of urothelial carcinoma recognized in the 2022 WHO classification of tumours in the urinary tract. The identification of these subtypes is crucial for an accurate diagnosis of urothelial carcinoma, and many have important clinical implications. Variant/divergent features may coexist with conventional high-grade urothelial carcinoma (HGUC) or present with 100% variant morphology. In urinary tract cytology (UTC), urothelial carcinoma can display divergent differentiation, such as squamous, glandular, or small cell carcinoma differentiation. The use of cell block preparations and immunohistochemistry with available residual urine can enhance diagnostic accuracy. On the other hand, identifying urothelial carcinoma variants, including nested, micropapillary, and plasmacytoid subtypes, poses significant challenges in UTC. Many cases of these variants are only detected retrospectively after variant histology has been established from resection specimens. Moreover, some variants exhibit features inconsistent with the diagnostic criteria for HGUC according to the Paris System for Reporting Urinary Tract Cytology. Nevertheless, the rarity of pure variant morphology and the occurrence of some false negatives for these variant cases are essential to maintain the specificity of UTC overall. This review covers the histology, cytomorphology, and important clinical aspects observed in urothelial carcinoma exhibiting divergent differentiation and various urothelial carcinoma variants detected in UTC.

Bone densitometry in Thalassemia major: a closer look at pitfalls and operator-related errors in a 10-year follow-up population.

PURPOSE: Dual-energy X-ray absorptiometry (DXA) is the gold standard for measuring bone mineral density (BMD) with tolerable error rate, high precision, and excellent consistency. Our objective was to investigate the frequency and distribution of errors in a cohort of patients with Thalassemia major (TM). METHODS: We reviewed the DXA examinations of 340 patients with ß-TM followed by our institution, acquired in different imaging centers between 2009 and 2019. We collected sex and age at the time of the first examination and at the last visit, as well as BMD, T-score, and Z-score values. Errors were analyzed by anatomical site (lumbar spine, total hip, femoral neck). RESULTS: Out of 5099 total DXA scans, 11.85% presented one or more errors. Specifically, the incorrect examinations were 315 out of 1707 (18.45%) at the lumbar spine level, 113 out of 1697 (6.66%) at the total hip, 176 out of 1695 (10.38%) at the femoral neck. Errors in vertebral inclusion were the most frequently registered (45.86%). A significant difference resulted from the comparison of the T-score and Z-score median values of all the lumbar spine DXA examinations and the correct ones (p value 0.037 and 0.0003, respectively). CONCLUSION: Although not directly involved in the performance and interpretation of DXA, physicians interested in osteoporosis management should be familiar with the protocols to minimize errors and allow the proper use of bone densitometry. DXA obtained at the spine level is more frequently affected by errors in patients with TM, potentially influencing the diagnostic assessment of bone health status.

The Clinical and Biological Significance of Estrogen Receptor-Low Positive Breast Cancer.

Estrogen receptor (ER) status in breast cancer (BC) is determined using immunohistochemistry (IHC) with nuclear expression in ≥1% of cells defined as ER-positive. BC with 1%-9% expression (ER-low-positive), is a clinically and biologically unique subgroup. In this study, we hypothesized that ER-low-positive BC represents a heterogeneous group with a mixture of ER-positive and ER-negative tumor, which may explain their divergent clinical behavior. A large BC cohort (n = 8171) was investigated and categorized into 3 groups: ER-low-positive (1%-9%), ER-positive (≥10%), and ER-negative (<1%) where clinicopathological and outcome characteristics were compared. A subset of ER-low-positive cases was further evaluated using IHC, RNAscope, and RT-qPCR. PAM50 subtyping and ESR1 mRNA expression levels were assessed in ER-low-positive cases within The Cancer Genome Atlas data set. The reliability of image analysis software in assessment of ER expression in the ER-low-positive category was also assessed. ER-low-positive tumors constituted <2% of BC cases examined and showed significant clinicopathological similarity to ER-negative tumors. Most of these tumors were nonluminal types showing low ESR1 mRNA expression. Further validation of ER status revealed that 45% of these tumors were ER-negative with repeated IHC staining and confirmed by RNAscope and RT-qPCR. ER-low-positive tumors diagnosed on needle core biopsy were enriched with false-positive ER staining. BCs with 10% ER behaved similar to ER-positive, rather than ER-negative or low-positive BCs. Moderate concordance was found in assessment of ER-low-positive tumors, and this was not improved by image analysis. Routinely diagnosed ER-low-positive BC includes a proportion of ER-negative cases. We recommend repeat testing of BC showing 1%-9% ER expression and using a cutoff ≥10% expression to define ER positivity to help better inform treatment decisions.

Evaluating mismatch repair deficiency in colorectal cancer biopsy specimens.

Mismatch repair (MMR) testing on all new cases of colorectal cancer (CRC) has customarily been preferably performed on surgical specimens, as more tissue is available; however, new clinical trials for the use of immune checkpoint inhibitors in the neoadjuvant setting require MMR testing on biopsy samples. This study aims at identifying advantages, disadvantages and any potential pitfalls in MMR evaluation on biopsy tissue and how to cope with them. The study is prospective-retrospective, recruiting 141 biopsies (86 proficient (p)MMR and 55 deficient (d)MMR) and 97 paired surgical specimens (48 pMMR; 49 dMMR). In biopsy specimens, a high number of indeterminate stains was observed, in particular for MLH1 (31 cases, 56.4%). The main reasons were a punctate nuclear expression of MLH1, relatively weak MLH1 nuclear expression compared to internal controls, or both (making MLH1 loss difficult to interpret), which was solved by reducing primary incubation times for MLH1. A mean of  ≥ 5 biopsies had adequate immunostains, compared to ≤ 3 biopsies in inadequate cases. Conversely, surgical specimens rarely suffered from indeterminate reactions, while weaker staining intensity (p < 0.007) for MLH1 and PMS2 and increased patchiness grade (p < 0.0001) were seen. Central artefacts were almost exclusive to surgical specimens. MMR status classification was possible in 92/97 matched biopsy/resection specimen cases, and all of these were concordant (47 pMMR and 45 dMMR). Evaluation of MMR status on CRC biopsy samples is feasible, if pitfalls in interpretation are known, making laboratory-specific appropriate staining protocols fundamental for high-quality diagnoses.

Pitfalls in breast pathology.

Accurate pathological diagnosis is the cornerstone of optimal clinical management for patients with breast disease. As non-operative diagnosis has now become the standard of care, histopathologists encounter the daily challenge of making definitive diagnoses on limited breast core needle biopsy (CNB) material. CNB samples are carefully evaluated using microscopic examination of haematoxylin and eosin (H&E)-stained slides and supportive immunohistochemistry (IHC), providing the necessary information to inform the next steps in the patient care pathway. Some entities may be difficult to distinguish on small tissue samples, and if there is uncertainty a diagnostic excision biopsy should be recommended. This review discusses (1) benign breast lesions that may mimic malignancy, (2) malignant conditions that may be misinterpreted as benign, (3) malignant conditions that may be incorrectly diagnosed as primary breast carcinoma, and (4) some IHC pitfalls. The aim of the review is to raise awareness of potential pitfalls in the interpretation of breast lesions that may lead to underdiagnosis, overdiagnosis, or incorrect classification of malignancy with potential adverse outcomes for individual patients.

Second opinion (external specialist referral) practice of breast pathology: the Nottingham experience.

AIMS: Breast pathology is a challenging field, and discrepancies in diagnoses exist and can affect patient management. This study aims to review a breast referral practice and assess the pattern and frequency of breast lesions sent for an external expert review and evaluate potential impacts on patients' care. METHODS AND RESULTS: Seven hundred and forty cases that were referred to Nottingham City Hospital for a second opinion between 2019 and 2022 which have slides and reports were retrieved and reviewed. Reasons for referral, initial diagnosis, proffered specialist opinion and any discrepancy or potential impacts of management were assessed. The most frequent entities were papillary lesions (19%), fibroepithelial lesions (17%), invasive carcinomas that were sent for confirmation of the invasive diagnosis or subtyping of the invasive tumour (17%), intraductal epithelial proliferation with atypia (9%) and spindle cell lesions (8%). Other entities included biphasic tumours such as adenomyoepithelioma, as well as vascular and nipple lesions. Few cases were sent for prognostic classification or comments on the management, and in occasional cases no initial diagnosis was offered. After reviewing the cases by the expert pathologists, the initial diagnosis was confirmed or one of the suggested diagnoses was preferred in 79% of cases, including 129 cases (17%) in which the opinion resulted minor changes in the management. Significant changes in the classification of lesions were made in 132 cases (18%) which resulted in significant change in the patient management recommendation. In 14 cases (2%) a final classification was not possible, and further specialist opinion was obtained. Comments on the differential diagnosis and advice on further patient management were provided in most cases. CONCLUSIONS: This study demonstrates the value of external referral of challenging, rare and difficult to classify breast lesions. It also highlights the most common breast lesions that are likely to be challenging, and specialist opinion can refine their classification to improve patient care.

Volume conduction: Extracellular waveform generation in theory and practice.

The extracellular waveform manifestations of the intracellular action potential are the quintessential diagnostic foundation of electrodiagnostic medicine, and clinical neurophysiology in general. Volume conduction is the extracellular current flow and associated voltage distributions in an ionic conducting media, such as occurs in the human body. Both surface and intramuscular electrodes, in association with contemporary digital electromyographic systems, permit very sensitive detection and visualization of this extracellular spontaneous, voluntary, and evoked nerve/muscle electrical activity. Waveform configuration, with its associated discharge rate/rhythm, permits the identification of normal and abnormal waveforms, thereby assisting in the diagnosis of nerve and muscle pathology. This monograph utilizes a simple model to explain the various waveforms that may be encountered. There are a limited number of waveforms capable of being generated in excitable tissues which conform to well-known volume conductor concepts. Using these principles, such waveforms can be quickly identified in real time during clinical studies.

Salivary gland neoplasms in small biopsies and fine needle aspirations.

Salivary gland neoplasms are rare and represent a diverse group of head and neck tumors. Their diagnosis in limited cellularity specimens can be challenging as many of these have overlapping clinical, radiological presentation, and pathologic features. Fine needle aspiration and/or core biopsies are more of a norm than rarity to be performed preoperatively to provide invaluable information that can guide clinical management including surgery. Even though these limited specimens may not always provide a definitive diagnosis; they have high sensitivity in confirming primary neoplasia, assessing the tumor grade, and ruling out non-surgical disease. An algorithmic pattern based approach can help narrow the differential diagnosis; leading to a definitive diagnosis with the help of specific ancillary studies.

COVID-19 deaths on weekends.

BACKGROUND: Mortality statistics about daily deaths might change on weekends due to delays in reporting, uneven staffing, a different mix of personnel, or decreased efficiency. We hypothesized that reported deaths for COVID-19 might increase on weekends compared to weekdays. METHODS: We collected data from the World Health Organization COVID-19 database. All deaths from March 7, 2020 to March 7, 2022 were included (two years). The primary analysis evaluated mean daily deaths on weekends compared to the preceding five workdays. Analyses were replicated in ten individual countries: United States, United Kingdom, France, Germany, Italy, Spain, Russia, India, Brazil, and Canada. RESULTS: The mean COVID-19 daily deaths was higher on weekends compared to weekdays (8,532 vs. 8,083 p < 0.001), equal to a 6% relative increase (95% confidence interval 3% to 8%). The highest absolute increase was in the United States (1,483 vs. 1,220 deaths, p < 0.001). The second highest absolute increase was in Brazil (1,061 vs. 823 deaths, p < 0.001). The increase in deaths on weekends remained significant during the earlier and later months of the pandemic, as well as during the greater and lesser weeks of the pandemic. CONCLUSIONS: The apparent increased COVID-19 deaths reported on weekends might potentially reflect patient care, confound community trends, and affect the public perception of risk.

Diagnostic pitfalls in computed tomography of the chest.

Anatomical variants and imaging artifacts on thoracic computed tomography (CT), when unrecognized as such, can lead to radiological misinterpretation and erroneous diagnosis. This is a concise review of 15 common CT diagnostic pitfalls due to anatomical variants and imaging artifacts which have potential to be misinterpreted as significant pathology, such as neoplasia, infection, traumatic injury, interstitial lung disease, pleural disease, or vascular lesions.

Pitfalls in Carotid Doppler Interpretation and How to Avoid Them.

Imaging pitfalls commonly occur in carotid Doppler ultrasound and may lead to false positive diagnosis of stenosis, missed diagnosis of stenosis, and errors in grading stenosis severity. These pitfalls may result from suboptimal technique and/or patient-specific factors including coexisting cardiovascular pathology, contralateral high-grade stenosis/occlusion, tortuous vessels, tandem lesions, long-segment stenosis, nearly occlusive stenosis, and heavily calcified plaque. Awareness of these pitfalls and careful assessment of the extent of plaque on grayscale and color Doppler as well as analysis of the spectral Doppler waveforms can help avoid misinterpretation of the carotid Doppler examination.

Meckel diverticulum scintigraphy: technique, findings and diagnostic pitfalls.

Meckel diverticulum, the most common congenital anomaly of the gastrointestinal tract, results from the aberrant involution of the omphalomesenteric duct and accounts for more than 50% of unexplained lower gastrointestinal bleeding in the pediatric population. The most accurate imaging tool to identify a Meckel diverticulum containing ectopic gastric mucosa is the Technetium-99m pertechnetate Meckel scan, a scintigraphic study with a reported accuracy of 90% in the pediatric population. In addition to depicting a Meckel diverticulum with ectopic gastric mucosa, careful attention to the normal biodistribution of the radiotracer can lead to the identification of unexpected pathology with implications for patient management. This article serves to review the embryological origin and anatomical features of Meckel diverticulum, highlight the role of scintigraphy in evaluating Meckel diverticulum, and discuss the proper imaging technique when performing this test. We will focus on pitfalls that can lead to an erroneous diagnosis as well as incidental findings that can affect patient management.

How Can the Clinical Aptitude of AI Assistants Be Assayed?

Large language models (LLMs) are exhibiting remarkable performance in clinical contexts, with exemplar results ranging from expert-level attainment in medical examination questions to superior accuracy and relevance when responding to patient queries compared to real doctors replying to queries on social media. The deployment of LLMs in conventional health care settings is yet to be reported, and there remains an open question as to what evidence should be required before such deployment is warranted. Early validation studies use unvalidated surrogate variables to represent clinical aptitude, and it may be necessary to conduct prospective randomized controlled trials to justify the use of an LLM for clinical advice or assistance, as potential pitfalls and pain points cannot be exhaustively predicted. This viewpoint states that as LLMs continue to revolutionize the field, there is an opportunity to improve the rigor of artificial intelligence (AI) research to reward innovation, conferring real benefits to real patients.

A rare case of canalicular adenoma in the parotid gland: Highlighting diagnostic limitations of fine-needle aspiration.

BACKGROUND: Canalicular adenoma is a rare, benign tumor of primarily salivary gland origin that presents mostly in the upper lip. However, there are only six reports in the English literature detailing canalicular adenoma of the parotid gland, none of which discuss discrepancy between preoperative cytology and surgical pathology. In this report, we present a rare case of parotid gland canalicular adenoma where preoperative ultrasound-guided fine-needle aspiration (USFNA) suggested malignancy. The patient was treated with deep lobe parotidectomy due to the FNA results and her multiple comorbidities. However, her tumor may have been treated with observation alone if canalicular adenoma had been suspected prior to surgery. MAIN FINDINGS: A 59-year-old female with a history of heart and lung disease presented with a 1.6 cm well defined, enhancing lesion involving the superficial portion of the right parotid gland. This lesion was incidentally noted on CT angiography (CTA) of the neck and chest. The well-defined characteristics of this lesion on CT imaging suggested benign neoplasm. However, USFNA results were suggestive of a malignant parotid lesion. The patient subsequently underwent right deep lobe parotidectomy with facial nerve dissection and superficial musculoaponeurotic system (SMAS) rotational flap reconstruction. Surgical pathology and immunohistochemistry yielded a final diagnosis of benign canalicular adenoma. CONCLUSIONS: USFNA diagnosis of CA is extremely difficult due to its low-grade neoplastic cells mimicking neoplastic cells in other benign and malignant tumors of the head and neck. FNA remains a useful tool for assessing malignancy risk, but the results always have some level of uncertainty and do not provide sufficient detail. Therefore, FNA results should be interpreted in concert with imaging and patients' medical history. Cytopathologists can also report salivary gland FNA results in a more uniform and detailed manner by utilizing the Milan System for Reporting Salivary Gland Cytopathology (MSRSGC).