Image-based multiplex immune profiling of cancer tissues: translational implications. A report of the International Immuno-oncology Biomarker Working Group on Breast Cancer.

Recent advances in the field of immuno-oncology have brought transformative changes in the management of cancer patients. The immune profile of tumours has been found to have key value in predicting disease prognosis and treatment response in various cancers. Multiplex immunohistochemistry and immunofluorescence have emerged as potent tools for the simultaneous detection of multiple protein biomarkers in a single tissue section, thereby expanding opportunities for molecular and immune profiling while preserving tissue samples. By establishing the phenotype of individual tumour cells when distributed within a mixed cell population, the identification of clinically relevant biomarkers with high-throughput multiplex immunophenotyping of tumour samples has great potential to guide appropriate treatment choices. Moreover, the emergence of novel multi-marker imaging approaches can now provide unprecedented insights into the tumour microenvironment, including the potential interplay between various cell types. However, there are significant challenges to widespread integration of these technologies in daily research and clinical practice. This review addresses the challenges and potential solutions within a structured framework of action from a regulatory and clinical trial perspective. New developments within the field of immunophenotyping using multiplexed tissue imaging platforms and associated digital pathology are also described, with a specific focus on translational implications across different subtypes of cancer. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Reproducible Reporting of the Collection and Evaluation of Annotations for Artificial Intelligence Models.

This work puts forth and demonstrates the utility of a reporting framework for collecting and evaluating annotations of medical images used for training and testing artificial intelligence (AI) models in assisting detection and diagnosis. AI has unique reporting requirements, as shown by the AI extensions to the Consolidated Standards of Reporting Trials (CONSORT) and Standard Protocol Items: Recommendations for Interventional Trials (SPIRIT) checklists and the proposed AI extensions to the Standards for Reporting Diagnostic Accuracy (STARD) and Transparent Reporting of a Multivariable Prediction model for Individual Prognosis or Diagnosis (TRIPOD) checklists. AI for detection and/or diagnostic image analysis requires complete, reproducible, and transparent reporting of the annotations and metadata used in training and testing data sets. In an earlier work by other researchers, an annotation workflow and quality checklist for computational pathology annotations were proposed. In this manuscript, we operationalize this workflow into an evaluable quality checklist that applies to any reader-interpreted medical images, and we demonstrate its use for an annotation effort in digital pathology. We refer to this quality framework as the Collection and Evaluation of Annotations for Reproducible Reporting of Artificial Intelligence (CLEARR-AI).

INTEGRATE-Circ and INTEGRATE-Vis: unbiased detection and visualization of fusion-derived circular RNA.

MOTIVATION: Backsplicing of RNA results in circularized rather than linear transcripts, known as circular RNA (circRNA). A recently discovered and poorly understood subset of circRNAs that are composed of multiple genes, termed fusion-derived circular RNAs (fcircRNAs), represent a class of potential biomarkers shown to have oncogenic potential. Detection of fcircRNAs eludes existing analytical tools, making it difficult to more comprehensively assess their prevalence and function. Improved detection methods may lead to additional biological and clinical insights related to fcircRNAs. RESULTS: We developed the first unbiased tool for detecting fcircRNAs (INTEGRATE-Circ) and visualizing fcircRNAs (INTEGRATE-Vis) from RNA-Seq data. We found that INTEGRATE-Circ was more sensitive, precise and accurate than other tools based on our analysis of simulated RNA-Seq data and our tool was able to outperform other tools in an analysis of public lymphoblast cell line data. Finally, we were able to validate in vitro three novel fcircRNAs detected by INTEGRATE-Circ in a well-characterized breast cancer cell line. AVAILABILITY AND IMPLEMENTATION: Open source code for INTEGRATE-Circ and INTEGRATE-Vis is available at https://www.github.com/ChrisMaherLab/INTEGRATE-CIRC and https://www.github.com/ChrisMaherLab/INTEGRATE-Vis.

Training pathologists to assess stromal tumour-infiltrating lymphocytes in breast cancer synergises efforts in clinical care and scientific research.

A growing body of research supports stromal tumour-infiltrating lymphocyte (TIL) density in breast cancer to be a robust prognostic and predicive biomarker. The gold standard for stromal TIL density quantitation in breast cancer is pathologist visual assessment using haematoxylin and eosin-stained slides. Artificial intelligence/machine-learning algorithms are in development to automate the stromal TIL scoring process, and must be validated against a reference standard such as pathologist visual assessment. Visual TIL assessment may suffer from significant interobserver variability. To improve interobserver agreement, regulatory science experts at the US Food and Drug Administration partnered with academic pathologists internationally to create a freely available online continuing medical education (CME) course to train pathologists in assessing breast cancer stromal TILs using an interactive format with expert commentary. Here we describe and provide a user guide to this CME course, whose content was designed to improve pathologist accuracy in scoring breast cancer TILs. We also suggest subsequent steps to translate knowledge into clinical practice with proficiency testing.

Initial interactions with the FDA on developing a validation dataset as a medical device development tool.

Quantifying tumor-infiltrating lymphocytes (TILs) in breast cancer tumors is a challenging task for pathologists. With the advent of whole slide imaging that digitizes glass slides, it is possible to apply computational models to quantify TILs for pathologists. Development of computational models requires significant time, expertise, consensus, and investment. To reduce this burden, we are preparing a dataset for developers to validate their models and a proposal to the Medical Device Development Tool (MDDT) program in the Center for Devices and Radiological Health of the U.S. Food and Drug Administration (FDA). If the FDA qualifies the dataset for its submitted context of use, model developers can use it in a regulatory submission within the qualified context of use without additional documentation. Our dataset aims at reducing the regulatory burden placed on developers of models that estimate the density of TILs and will allow head-to-head comparison of multiple computational models on the same data. In this paper, we discuss the MDDT preparation and submission process, including the feedback we received from our initial interactions with the FDA and propose how a qualified MDDT validation dataset could be a mechanism for open, fair, and consistent measures of computational model performance. Our experiences will help the community understand what the FDA considers relevant and appropriate (from the perspective of the submitter), at the early stages of the MDDT submission process, for validating stromal TIL density estimation models and other potential computational models. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Uses and limitations of small-volume biopsies for the diagnosis of lymphoma.

Although surgical biopsy remains the gold standard for the diagnosis of lymphoma, small-volume biopsies including fine-needle aspiration and core needle biopsy are increasingly being used as a first line diagnostic tool. Small-volume biopsies are safe, rapid and cost effective; however, diagnostic utility varies by lymphoma subtype. It is important for pathologists and clinicians to recognize both the strengths and limitations of such biopsies.

Multi-institutional Assessment of Pathologist Scoring HER2 Immunohistochemistry.

The HercepTest was approved 20+ years ago as the companion diagnostic test for trastuzumab in human epidermal growth factor 2 (HER2) or ERBB2 gene-amplified/overexpressing breast cancers. Subsequent HER2 immunohistochemistry (IHC) assays followed, including the now most common Ventana 4B5 assay. Although this IHC assay has become the clinical standard, its reliability, reproducibility, and accuracy have largely been approved and accepted on the basis of concordance among small numbers of pathologists without validation in a real-world setting. In this study, we evaluated the concordance and interrater reliability of scoring HER2 IHC in 170 breast cancer biopsies by 18 breast cancer-specialized pathologists from 15 institutions. We used the Observers Needed to Evaluate Subjective Tests method to determine the plateau of concordance and the minimum number of pathologists needed to estimate interrater agreement values for large numbers of raters, as seen in the real-world setting. We report substantial discordance within the intermediate categories (<1% agreement for 1+ and 3.6% agreement for 2+) in the 4-category HER2 IHC scoring system. The discordance within the IHC 0 cases is also substantial with an overall percent agreement (OPA) of only 25% and poor interrater reliability metrics (0.49 Fleiss' kappa, 0.55 intraclass correlation coefficient). This discordance can be partially reduced by using a 3-category system (28.8% vs 46.5% OPA for 4-category and 3-category scoring systems, respectively). Observers Needed to Evaluate Subjective Tests plots suggest that the OPA for the task of determining a HER2 IHC score 0 from not 0 plateaus statistically around 59.4% at 10 raters. Conversely, at the task of scoring HER2 IHC as 3+ or not 3+ pathologists' concordance was much higher with an OPA that plateaus at 87.1% with 6 raters. This suggests that legacy HER2 IHC remains valuable for finding the patients in whom the ERBB2 gene is amplified but unacceptably discordant in assigning HER2-low or HER2-negative status for the emerging HER2-low therapies.

Vasculopathy and Increased Vascular Congestion in Fatal COVID-19 and Acute Respiratory Distress Syndrome.

Rationale: The leading cause of death in coronavirus disease 2019 (COVID-19) is severe pneumonia, with many patients developing acute respiratory distress syndrome (ARDS) and diffuse alveolar damage (DAD). Whether DAD in fatal COVID-19 is distinct from other causes of DAD remains unknown. Objective: To compare lung parenchymal and vascular alterations between patients with fatal COVID-19 pneumonia and other DAD-causing etiologies using a multidimensional approach. Methods: This autopsy cohort consisted of consecutive patients with COVID-19 pneumonia (n = 20) and with respiratory failure and histologic DAD (n = 21; non-COVID-19 viral and nonviral etiologies). Premortem chest computed tomography (CT) scans were evaluated for vascular changes. Postmortem lung tissues were compared using histopathological and computational analyses. Machine-learning-derived morphometric analysis of the microvasculature was performed, with a random forest classifier quantifying vascular congestion (CVasc) in different microscopic compartments. Respiratory mechanics and gas-exchange parameters were evaluated longitudinally in patients with ARDS. Measurements and Main Results: In premortem CT, patients with COVID-19 showed more dilated vasculature when all lung segments were evaluated (P = 0.001) compared with controls with DAD. Histopathology revealed vasculopathic changes, including hemangiomatosis-like changes (P = 0.043), thromboemboli (P = 0.0038), pulmonary infarcts (P = 0.047), and perivascular inflammation (P < 0.001). Generalized estimating equations revealed significant regional differences in the lung microarchitecture among all DAD-causing entities. COVID-19 showed a larger overall CVasc range (P = 0.002). Alveolar-septal congestion was associated with a significantly shorter time to death from symptom onset (P = 0.03), length of hospital stay (P = 0.02), and increased ventilatory ratio [an estimate for pulmonary dead space fraction (Vd); p = 0.043] in all cases of ARDS. Conclusions: Severe COVID-19 pneumonia is characterized by significant vasculopathy and aberrant alveolar-septal congestion. Our findings also highlight the role that vascular alterations may play in Vd and clinical outcomes in ARDS in general.

Implementation and Clinical Adoption of Precision Oncology Workflows Across a Healthcare Network.

BACKGROUND: Precision oncology relies on molecular diagnostics, and the value-proposition of modern healthcare networks promises a higher standard of care across partner sites. We present the results of a clinical pilot to standardize precision oncology workflows. METHODS: Workflows are defined as the development, roll-out, and updating of disease-specific molecular order sets. We tracked the timeline, composition, and effort of consensus meetings to define the combination of molecular tests. To assess clinical impact, we examined order set adoption over a two-year period (before and after roll-out) across all gastrointestinal and hepatopancreatobiliary (GI) malignancies, and by provider location within the network. RESULTS: Development of 12 disease center-specific order sets took ~9 months, and the average number of tests per indication changed from 2.9 to 2.8 (P = .74). After roll-out, we identified significant increases in requests for GI patients (17%; P < .001), compliance with testing recommendations (9%; P < .001), and the fraction of "abnormal" results (6%; P < .001). Of 1088 GI patients, only 3 received targeted agents based on findings derived from non-recommended orders (1 before and 2 after roll-out); indicating that our practice did not negatively affect patient treatments. Preliminary analysis showed 99% compliance by providers in network sites, confirming the adoption of the order sets across the network. CONCLUSION: Our study details the effort of establishing precision oncology workflows, the adoption pattern, and the absence of harm from the reduction of non-recommended orders. Establishing a modifiable communication tool for molecular testing is an essential component to optimize patient care via precision oncology.

Subcutaneous metastasis from an atypical pulmonary carcinoid tumor.

Pulmonary carcinoid tumors are uncommon neuroendocrine tumors that rarely metastasize to the skin. We report the case of a 71-year-old woman with a longstanding history of primary atypical pulmonary carcinoid tumor who presented with a new tender cutaneous nodule. Immunostaining of the nodule was consistent with metastatic atypical carcinoid tumor of the skin including positive staining for neuroendocrine markers chromogranin and synaptophysin. Dermatologists should consider cutaneous neuroendocrine metastasis when evaluating new nodules in patients with stable pulmonary carcinoid tumors or in those with concomitant concerning respiratory symptoms.

Prospective multi-institutional evaluation of pathologist assessment of PD-L1 assays for patient selection in triple negative breast cancer.

The US Food and Drug Administration (FDA) approved the PD-L1 immunohistochemical assay, SP142, as a companion test to determine eligibility for atezolizumab therapy in patients with advanced triple negative breast cancer (TNBC) but data in lung cancer studies suggest the assay suffers from poor reproducibility. We sought to evaluate reproducibility and concordance in PD-L1 scoring across multiple pathologists. Full TNBC sections were stained with SP142 and SP263 assays and interpreted for percentage (%) immune cell (IC) staining by 19 pathologists from 14 academic institutions. Proportion of PD-L1 positive cases (defined as ≥1% IC) was determined for each assay as well as concordance across observers. We utilized a new method we call Observers Needed to Evaluate Subjective Tests (ONEST) to determine the minimum number of evaluators needed to estimate concordance between large numbers of readers, as occurs in the real-world setting. PD-L1 was interpreted as positive with the SP142 assay in an average 58% of cases compared with 78% with SP263 (p < 0.0001). IC positive continuous scores ranged from 1 to 95% (mean = 20%) and 1 to 90% (mean = 10%) for SP263 and SP142, respectively. With SP142, 26 cases (38%) showed complete two category (<1% vs. ≥1%) concordance; with SP263, 38 cases (50%) showed complete agreement. The intraclass correlation coefficient (ICC) for two category scoring of SP263 and SP142 was 0.513 and 0.560. ONEST plots showed decreasing overall percent agreement (OPA) as observer number increased, reaching a low plateau of 0.46 at ten observers for SP263 and 0.41 at eight observers for SP142. IC scoring with both assays showed poor reproducibility across multiple pathologists with ONEST analysis suggesting more than half of pathologists will disagree about IC scores. This could lead to many patients either receiving atezolizumab when they are unlikely to benefit, or not receiving atezolizumab when they may benefit.

A fast, simple, and cost-effective method of expanding patient-derived xenograft mouse models of pancreatic ductal adenocarcinoma.

BACKGROUND: Patient-derived xenograft (PDX) mouse models of cancer have been recognized as better mouse models that recapitulate the characteristics of original malignancies including preserved tumor heterogeneity, lineage hierarchy, and tumor microenvironment. However, common challenges of PDX models are the significant time required for tumor expansion, reduced tumor take rates, and higher costs. Here, we describe a fast, simple, and cost-effective method of expanding PDX of pancreatic ductal adenocarcinoma (PDAC) in mice. METHODS: We used two established frozen PDAC PDX tissues (derived from two different patients) and implanted them subcutaneously into SCID mice. After tissues reached 10-20 mm in diameter, we performed survival surgery on each mouse to harvest 90-95% of subcutaneous PDX (incomplete resection), allowing the remaining 5-10% of PDX to continue growing in the same mouse. RESULTS: We expanded three consecutive passages (P1, P2, and P3) of PDX in the same mouse. Comparing the times required for in vivo expansion, P2 and P3 (expanded through incomplete resection) grew 26-60% faster than P1. Moreover, such expanded PDX tissues were successfully implanted orthotopically into mouse pancreases. Within 20 weeks using only 14 mice, we generated sufficient PDX tissue for future implantation of 200 mice. Our histology study confirmed that the morphologies of cancer cells and stromal structures were similar across all three passages of subcutaneous PDX and the orthotopic PDX and were reflective of the original patient tumors. CONCLUSIONS: Taking advantage of incomplete resection of tumors associated with high local recurrence, we established a fast method of PDAC PDX expansion in mice.

Hepatitis C infection and chemotherapy toxicity.

There exists little guidance on chemotherapy toxicity management in patients with a history of or active hepatitis C viral infection. We report four cases of patients with solid organ tumors and hepatitis C viral infection, who have experienced severe or unexpected toxicities with chemotherapy. Based on the four case reports, we recommend increased laboratory monitoring for toxicities, initial dose reductions for chemotherapy given with palliative intent, or pre-emptive use of growth factor support, even if the patient presents with normal liver function tests. In this patient population, we also recommend treating active hepatitis C viral infection prior to chemotherapy treatment when possible.

Compartmentalization of functions and predicted miRNA regulation among contiguous regions of the nematode intestine.

The intestine of parasitic nematodes has proven an important target for therapies aimed at prevention and treatment of diseases caused by these pathogens in humans, animals and plants. We have developed a unique research model with the intestine of Ascaris suum, the large round worm of swine and humans, that will enhance biological research on this tissue. To expand utility of this model, we quantitatively compared expression of 15,382 coding RNAs and 277 noncoding, micro RNAs (miRNAs) among 3 contiguous regions of the adult A. suum intestine. Differentially expressed transcripts were identified among regions, with the largest number expressed at significantly higher levels in the anterior region, identifying this region as the most functionally unique compared to middle and posterior regions. We further identified 64 exon splice variants (from 47 genes) that are differentially expressed among these regions. A total of 2,063 intestinal mRNA transcripts were predicted to be targeted by intestinal miRNA, and negative correlation coefficients for miRNA:mRNA abundances predicted 22 likely influential miRNAs and 503 likely associated miRNA:mRNA pairs. A. suum intestinal miRNAs were identified that are conserved with intestinal miRNAs from C. elegans (10 mature sequences and 13 seed sequences conserved), and prospective intestinal miRNAs from the murine gastrointestinal nematode, Heligmosomoides polygyrus (5 mature and 11 seeds). Most of the conserved intestinal miRNAs were also high abundance miRNAs. The data provide the most comprehensive compilation of constitutively and differentially expressed genes along the length of the intestine for any nematode species. The information will guide prospective development of many hypotheses on nematode intestinal functions encoded by mRNAs, miRNAs and interactions between these RNA populations.

A Study of the Growth Patterns of Breast Carcinoma Using 3D Reconstruction: A Pilot Study.

Lumpectomy with microscopically clear margins is a safe and effective approach for surgical management of breast carcinoma. Margins are positive for tumor in 18-50% of lumpectomies, as it is not possible to accurately determine the shape or microscopic borders of a tumor preoperatively or intraoperatively. We examined the 3D microanatomy and growth patterns of common breast carcinoma subtypes to provide guidance for lumpectomy surgery. Prospective consent was obtained for the use of excess tissue from patients undergoing lumpectomy or mastectomy for breast carcinoma. Tissue blocks from nine breast carcinomas were serially sectioned. Hematoxylin and eosin-stained slides at 100 µm intervals were scanned using a Nanozoomer (Hamamatsu, Japan) microscopic-resolution scanner. Three-dimensional reconstructions of tumors were created from scanned images using Reconstruct, open-access software. Breast carcinoma subtypes demonstrated characteristic growth patterns within breast tissue, which may have implications for lumpectomy surgery. Invasive ductal carcinomas showed a spherical shape, with a spiculated surface representing tumor cells infiltrating into surrounding parenchyma. Ductal carcinoma in situ appeared to spread along the duct system, creating dilated, tortuous, tumor-filled ducts. The invasive lobular carcinomas examined had a haphazard, linear, infiltrative growth pattern, different from the shape seen in ductal carcinomas. Our preliminary work suggests that invasive ductal and invasive lobular carcinomas appear to have distinct growth patterns in three dimensions and ductal carcinoma in situ appears to grow in a linear fashion along the duct network. The microanatomy studies described have the potential to guide refinements in breast lumpectomy technique.

Mycophenolate mofetil as a successful therapy for idiopathic granulomatous mastitis.

Idiopathic granulomatous mastitis (IGM) is a benign chronic inflammatory breast disease of unknown etiology. No consensus exists as to the best therapeutic approach, though treatment choices include antibiotics, drainage, surgical excision, steroids, methotrexate, and observation. Herein we report a case of idiopathic granulomatous mastitis that was refractory to methotrexate and intralesional and systemic steroids but responded well to mycophenolate mofetil 1500mg twice daily. To our knowledge, this is the first report of the effective use of mycophenolate mofetil in idiopathic granulomatous mastitis.

Breast fine needle aspiration continues to be relevant in a large academic medical center: experience from Massachusetts General Hospital.

Fine needle aspiration (FNA) is increasingly being supplanted by core needle biopsy. However, breast surgeons continue to rely on FNA at our institution. This retrospective study evaluated breast FNA for its diagnostic accuracy and breast cancer biomarker testing utility. All breast FNAs performed at Massachusetts General Hospital 2009-2015 were reviewed. Cytology diagnoses were compared with subsequent tissue or clinical diagnoses. Immunohistochemistry and fluorescence in situ hybridization (FISH) results using formalin-fixed paraffin-embedded (FFPE) cell blocks and histologic tissue blocks were compared. 1654 consecutive breast FNAs were included. Breast FNA demonstrated the following diagnostic performance: positive predictive value of malignant cytology diagnosis 100 %, negative predictive value of benign cytology diagnosis 97.5 %, complete sensitivity 91.6 %, and specificity 95.5 %. Concordance rates for estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 (HER2) immunohistochemistry, and HER2 FISH were 98.2 % (κ = 0.95, p < 0.001), 100.0 % (κ = 1.000, p < 0.001), 83.1 % (κ = 0.69, p < 0.001), and 93.5 % (κ = 0.785, p < 0.001), respectively. Review of consecutive breast FNAs in a large cohort confirmed the excellent accuracy of this biopsy technique for breast lesion diagnosis. FNA FFPE cell blocks collected in the course of routine clinical care are adequate, practical, and reliable for breast cancer biomarker testing.

The Oxytricha trifallax macronuclear genome: a complex eukaryotic genome with 16,000 tiny chromosomes.

The macronuclear genome of the ciliate Oxytricha trifallax displays an extreme and unique eukaryotic genome architecture with extensive genomic variation. During sexual genome development, the expressed, somatic macronuclear genome is whittled down to the genic portion of a small fraction (∼5%) of its precursor "silent" germline micronuclear genome by a process of "unscrambling" and fragmentation. The tiny macronuclear "nanochromosomes" typically encode single, protein-coding genes (a small portion, 10%, encode 2-8 genes), have minimal noncoding regions, and are differentially amplified to an average of ∼2,000 copies. We report the high-quality genome assembly of ∼16,000 complete nanochromosomes (∼50 Mb haploid genome size) that vary from 469 bp to 66 kb long (mean ∼3.2 kb) and encode ∼18,500 genes. Alternative DNA fragmentation processes ∼10% of the nanochromosomes into multiple isoforms that usually encode complete genes. Nucleotide diversity in the macronucleus is very high (SNP heterozygosity is ∼4.0%), suggesting that Oxytricha trifallax may have one of the largest known effective population sizes of eukaryotes. Comparison to other ciliates with nonscrambled genomes and long macronuclear chromosomes (on the order of 100 kb) suggests several candidate proteins that could be involved in genome rearrangement, including domesticated MULE and IS1595-like DDE transposases. The assembly of the highly fragmented Oxytricha macronuclear genome is the first completed genome with such an unusual architecture. This genome sequence provides tantalizing glimpses into novel molecular biology and evolution. For example, Oxytricha maintains tens of millions of telomeres per cell and has also evolved an intriguing expansion of telomere end-binding proteins. In conjunction with the micronuclear genome in progress, the O. trifallax macronuclear genome will provide an invaluable resource for investigating programmed genome rearrangements, complementing studies of rearrangements arising during evolution and disease.