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Introduction. Papillary urothelial carcinomas are currently graded as either low- or high-grade tumors based on World Health Organization (WHO) 2022 guidelines for genitourinary tumors. However, a minority of tumors are mixed-grade tumors, composed predominantly of low-grade cancer with a minor high-grade component. In the 2022 WHO these cancers are recognized as having outcomes comparable to low-grade cancers, although data to date has been limited. Methods. The pathology records of a large academic institution were searched for mixed-grade, non-muscle invasive papillary carcinomas of the bladder and ureter in order to characterize prognosis of these cancers. Results. Of 136 cancers, the majority (n = 104, 76.5%) were solitary, mixed-grade tumors, while 21 (15.4%) had a concurrent low-grade cancer and 11 (8.1%) had multiple mixed-grade tumors at the time of diagnosis. At follow-up (median 48.3 months, range = 1.3 months-18.1 years), 71 cancers recurred (52.2%): 52 (38.2%) as low- or mixed-grade cancers and 18 (13.2%) as high-grade cancers. There were no instances of stage-progression to >pT2. Conclusions. The clinical outcome of mixed-grade carcinomas was similar to what has been reported for low-grade carcinomas. Based on our results, and prior congruent studies of mixed-grade lesions, these lesions may be regarded as a distinct sub-category with a better prognosis than high-grade tumors.
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Prostate cancer prognostication largely relies on visual assessment of a few thinly sectioned biopsy specimens under a microscope to assign a Gleason grade group (GG). Unfortunately, the assigned GG is not always associated with a patient's outcome in part because of the limited sampling of spatially heterogeneous tumors achieved by 2-dimensional histopathology. In this study, open-top light-sheet microscopy was used to obtain 3-dimensional pathology data sets that were assessed by 4 human readers. Intrabiopsy variability was assessed by asking readers to perform Gleason grading of 5 different levels per biopsy for a total of 20 core needle biopsies (ie, 100 total images). Intrabiopsy variability (Cohen κ) was calculated as the worst pairwise agreement in GG between individual levels within each biopsy and found to be 0.34, 0.34, 0.38, and 0.43 for the 4 pathologists. These preliminary results reveal that even within a 1-mm-diameter needle core, GG based on 2-dimensional images can vary dramatically depending on the location within a biopsy being analyzed. We believe that morphologic assessment of whole biopsies in 3 dimension has the potential to enable more reliable and consistent tumor grading.
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Próstata , Neoplasias da Próstata , Masculino , Humanos , Próstata/patologia , Biópsia , Neoplasias da Próstata/patologia , Biópsia com Agulha de Grande Calibre , Gradação de TumoresRESUMO
Prostate cancer treatment decisions rely heavily on subjective visual interpretation [assigning Gleason patterns or International Society of Urological Pathology (ISUP) grade groups] of limited numbers of two-dimensional (2D) histology sections. Under this paradigm, interobserver variance is high, with ISUP grades not correlating well with outcome for individual patients, and this contributes to the over- and undertreatment of patients. Recent studies have demonstrated improved prognostication of prostate cancer outcomes based on computational analyses of glands and nuclei within 2D whole slide images. Our group has also shown that the computational analysis of three-dimensional (3D) glandular features, extracted from 3D pathology datasets of whole intact biopsies, can allow for improved recurrence prediction compared to corresponding 2D features. Here we seek to expand on these prior studies by exploring the prognostic value of 3D shape-based nuclear features in prostate cancer (e.g. nuclear size, sphericity). 3D pathology datasets were generated using open-top light-sheet (OTLS) microscopy of 102 cancer-containing biopsies extracted ex vivo from the prostatectomy specimens of 46 patients. A deep learning-based workflow was developed for 3D nuclear segmentation within the glandular epithelium versus stromal regions of the biopsies. 3D shape-based nuclear features were extracted, and a nested cross-validation scheme was used to train a supervised machine classifier based on 5-year biochemical recurrence (BCR) outcomes. Nuclear features of the glandular epithelium were found to be more prognostic than stromal cell nuclear features (area under the ROC curve [AUC] = 0.72 versus 0.63). 3D shape-based nuclear features of the glandular epithelium were also more strongly associated with the risk of BCR than analogous 2D features (AUC = 0.72 versus 0.62). The results of this preliminary investigation suggest that 3D shape-based nuclear features are associated with prostate cancer aggressiveness and could be of value for the development of decision-support tools. © 2023 The Pathological Society of Great Britain and Ireland.
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Próstata , Neoplasias da Próstata , Masculino , Humanos , Gradação de Tumores , Próstata/patologia , Neoplasias da Próstata/patologia , Prognóstico , Prostatectomia/métodos , Medição de RiscoRESUMO
BACKGROUND: Open-top light-sheet (OTLS) microscopy is a technique that allows for high-resolution 3D imaging of tissue specimens and can therefore provide a more detailed assessment of tissue architecture. Given that Gleason grading is based on tissue architecture, we hypothesized that OTLS microscopy would enable us to survey a larger amount of tissue and detect occult prostate cancers in men who had prostate core biopsy specimens initially classified as being benign-appearing who later developed clinically significant prostate cancers. METHODS: Benign appearing tissue (based on routine pathologic evaluation) from 20 patients who subsequently developed a clinically significant prostate cancer (experimental group) was evaluated with OTLS microscopy and compared to tissue from 20 patients who underwent prostate biopsy and never developed a clinically significant prostate cancer (control group). We compared the incidence of detectable prostate cancer between groups. RESULTS: Baseline clinical characteristics were similar between the experimental and control groups. Three patients (15%) in the control group and one (5%) in the experimental group had suspicious findings on low-resolution OTLS microscopy. Higher resolution OTLS imaging revealed two patients (10%) in the control group had an occult prostate cancer, while no occult cancers were found in the experimental group. CONCLUSION: In spite of a high pretest probability for the presence of an occult prostate cancer, we did not identify cancer in our experimental group. This may be due to under-sampling at the time of prostate needle biopsy.
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Próstata , Neoplasias da Próstata , Masculino , Humanos , Próstata/diagnóstico por imagem , Próstata/patologia , Microscopia , Neoplasias da Próstata/patologia , Biópsia/métodos , Biópsia por AgulhaRESUMO
Light-sheet microscopy has emerged as the preferred means for high-throughput volumetric imaging of cleared tissues. However, there is a need for a flexible system that can address imaging applications with varied requirements in terms of resolution, sample size, tissue-clearing protocol, and transparent sample-holder material. Here, we present a 'hybrid' system that combines a unique non-orthogonal dual-objective and conventional (orthogonal) open-top light-sheet (OTLS) architecture for versatile multi-scale volumetric imaging. We demonstrate efficient screening and targeted sub-micrometer imaging of sparse axons within an intact, cleared mouse brain. The same system enables high-throughput automated imaging of multiple specimens, as spotlighted by a quantitative multi-scale analysis of brain metastases. Compared with existing academic and commercial light-sheet microscopy systems, our hybrid OTLS system provides a unique combination of versatility and performance necessary to satisfy the diverse requirements of a growing number of cleared-tissue imaging applications.
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Microscopia , Animais , Camundongos , Microscopia/métodosRESUMO
Prostate cancer treatment planning is largely dependent upon examination of core-needle biopsies. The microscopic architecture of the prostate glands forms the basis for prognostic grading by pathologists. Interpretation of these convoluted three-dimensional (3D) glandular structures via visual inspection of a limited number of two-dimensional (2D) histology sections is often unreliable, which contributes to the under- and overtreatment of patients. To improve risk assessment and treatment decisions, we have developed a workflow for nondestructive 3D pathology and computational analysis of whole prostate biopsies labeled with a rapid and inexpensive fluorescent analogue of standard hematoxylin and eosin (H&E) staining. This analysis is based on interpretable glandular features and is facilitated by the development of image translation-assisted segmentation in 3D (ITAS3D). ITAS3D is a generalizable deep learning-based strategy that enables tissue microstructures to be volumetrically segmented in an annotation-free and objective (biomarker-based) manner without requiring immunolabeling. As a preliminary demonstration of the translational value of a computational 3D versus a computational 2D pathology approach, we imaged 300 ex vivo biopsies extracted from 50 archived radical prostatectomy specimens, of which, 118 biopsies contained cancer. The 3D glandular features in cancer biopsies were superior to corresponding 2D features for risk stratification of patients with low- to intermediate-risk prostate cancer based on their clinical biochemical recurrence outcomes. The results of this study support the use of computational 3D pathology for guiding the clinical management of prostate cancer. SIGNIFICANCE: An end-to-end pipeline for deep learning-assisted computational 3D histology analysis of whole prostate biopsies shows that nondestructive 3D pathology has the potential to enable superior prognostic stratification of patients with prostate cancer.
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Aprendizado Profundo , Imageamento Tridimensional/métodos , Próstata/diagnóstico por imagem , Neoplasias da Próstata/diagnóstico por imagem , Neoplasias da Próstata/epidemiologia , Idoso , Biópsia com Agulha de Grande Calibre , Estudos de Coortes , Humanos , Masculino , Pessoa de Meia-Idade , Próstata/patologia , Prostatectomia , Neoplasias da Próstata/patologia , Neoplasias da Próstata/cirurgia , Medição de Risco , Coloração e RotulagemRESUMO
BACKGROUND: Localized prostate cancers (PCs) may resist neoadjuvant androgen receptor (AR)-targeted therapies as a result of persistent intraprostatic androgens arising through upregulation of steroidogenic enzymes. Therefore, we sought to evaluate clinical effects of neoadjuvant indomethacin (Indo), which inhibits the steroidogenic enzyme AKR1C3, in addition to combinatorial anti-androgen blockade, in men with high-risk PC undergoing radical prostatectomy (RP). METHODS: This was an open label, single-site, Phase II neoadjuvant trial in men with high to very-high-risk PC, as defined by NCCN criteria. Patients received 12 weeks of apalutamide (Apa), abiraterone acetate plus prednisone (AAP), degarelix, and Indo followed by RP. Primary objective was to determine the pathologic complete response (pCR) rate. Secondary objectives included minimal residual disease (MRD) rate, defined as residual cancer burden (RCB) ≤ 0.25cm3 (tumor volume multiplied by tumor cellularity) and elucidation of molecular features of resistance. RESULTS: Twenty patients were evaluable for the primary endpoint. Baseline median prostate-specific antigen (PSA) was 10.1 ng/ml, 4 (20%) patients had Gleason grade group (GG) 4 disease and 16 had GG 5 disease. At RP, 1 (5%) patient had pCR and 6 (30%) had MRD. Therapy was well tolerated. Over a median follow-up of 23.8 months, 1 of 7 (14%) men with pathologic response and 6 of 13 (46%) men without pathologic response had a PSA relapse. There was no association between prostate hormone levels or HSD3B1 genotype with pathologic response. CONCLUSIONS: In men with high-risk PC, pCR rates remained low even with combinatorial AR-directed therapy, although rates of MRD were higher. Ongoing follow-up is needed to validate clinical outcomes of men who achieve MRD.
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Membro C3 da Família 1 de alfa-Ceto Redutase/antagonistas & inibidores , Antagonistas de Androgênios/uso terapêutico , Antineoplásicos Hormonais/uso terapêutico , Terapia Neoadjuvante , Prostatectomia , Neoplasias da Próstata/tratamento farmacológico , Acetato de Abiraterona/uso terapêutico , Idoso , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias da Próstata/cirurgia , Tioidantoínas/uso terapêutico , Resultado do TratamentoRESUMO
High-throughput methods for slide-free three-dimensional (3D) pathological analyses of whole biopsies and surgical specimens offer the promise of modernizing traditional histology workflows and delivering improvements in diagnostic performance. Advanced optical methods now enable the interrogation of orders of magnitude more tissue than previously possible, where volumetric imaging allows for enhanced quantitative analyses of cell distributions and tissue structures that are prognostic and predictive. Non-destructive imaging processes can simplify laboratory workflows, potentially reducing costs, and can ensure that samples are available for subsequent molecular assays. However, the large size of the feature-rich datasets that they generate poses challenges for data management and computer-aided analysis. In this Perspective, we provide an overview of the imaging technologies that enable 3D pathology, and the computational tools-machine learning, in particular-for image processing and interpretation. We also discuss the integration of various other diagnostic modalities with 3D pathology, along with the challenges and opportunities for clinical adoption and regulatory approval.
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Biópsia/métodos , Interpretação de Imagem Assistida por Computador/métodos , Processamento de Imagem Assistida por Computador/métodos , Animais , Humanos , Aprendizado de Máquina , PrognósticoRESUMO
SIGNIFICANCE: Processing and diagnosing a set of 12 prostate biopsies using conventional histology methods typically take at least one day. A rapid and accurate process performed while the patient is still on-site could significantly improve the patient's quality of life. AIM: We develop and assess the feasibility of a one-hour-to-diagnosis (1Hr2Dx) method for processing and providing a preliminary diagnosis of a set of 12 prostate biopsies. APPROACH: We developed a fluorescence staining, optical clearing, and 3D open-top light-sheet microscopy workflow to enable 12 prostate needle core biopsies to be processed and diagnosed within an hour of receipt. We analyzed 44 biopsies by the 1Hr2Dx method, which does not consume tissue. The biopsies were then processed for routine, slide-based 2D histology. Three pathologists independently evaluated the 3D 1Hr2Dx and 2D slide-based datasets in a blinded, randomized fashion. Turnaround times were recorded, and the accuracy of our method was compared with gold-standard slide-based histology. RESULTS: The average turnaround time for tissue processing, imaging, and diagnosis was 44.5 min. The sensitivity and specificity of 1Hr2Dx in diagnosing cancer were both >90 % . CONCLUSIONS: The 1Hr2Dx method has the potential to improve patient care by providing an accurate preliminary diagnosis within an hour of biopsy.
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Próstata , Neoplasias da Próstata , Biópsia , Biópsia por Agulha , Humanos , Masculino , Microscopia , Próstata/diagnóstico por imagem , Neoplasias da Próstata/diagnóstico por imagem , Qualidade de VidaRESUMO
Intraepithelial fallopian tube neoplasia is thought to be a precursor lesion to high-grade serous carcinoma of the Müllerian adnexae, particularly in women with BRCA1 or BRCA2 mutations. This association has led to recommendations to assess fallopian tubes for intraepithelial atypia. However, the diagnostic reproducibility of a diagnosis of intraepithelial neoplasia is unclear. In this study, 2 gynecologic pathologists independently evaluated sections of fallopian tubes from a sample of women (N=198, 623 slides) undergoing salpingectomy. A total of 101 (54%) women were undergoing risk-reducing salpingo-oophorectomy. Pathologists were blinded to patient histories and prior diagnoses. Pathologists rendered one of three diagnoses for each slide: "negative for fallopian tube intraepithelial neoplasia (FTIN)," "indeterminate for FTIN," or "definite for FTIN." Cases that were considered by histology definite for FTIN or suspicious for FTIN were stained with p53 and Ki67. Pathologists agreed on the diagnosis of "definite for FTIN" 61.5% of the time. There was no agreement on any cases for the diagnosis of "indeterminate for FTIN." Fifteen "indeterminate for FTIN" and 12 "definite for FTIN" cases were stained with p53 and Ki67. Two of the "indeterminate" cases (13%) had p53-positive foci. Five of the "definite" cases had p53-positive foci. In 3 of the other 8 "definite" cases, there was obvious carcinoma present, but the carcinoma did not stain with p53, suggesting a possible null phenotype. We propose that immunostains should only be used to aid in the diagnosis of FTIN in cases with indeterminate histology. The use of p53 immunohistochemistry in cases that were considered "definite for FTIN" by histology was minimally helpful, and in fact often served to further confuse the diagnosis.
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Algoritmos , Biomarcadores Tumorais/análise , Carcinoma in Situ/diagnóstico , Neoplasias das Tubas Uterinas/diagnóstico , Adulto , Idoso , Estudos Transversais , Feminino , Humanos , Imuno-Histoquímica , Antígeno Ki-67/análise , Pessoa de Meia-Idade , Proteína Supressora de Tumor p53/análiseRESUMO
Prostate cancer (PCa) is a major cause of cancer death among men. The histopathological examination of post-surgical prostate specimens and manual annotation of PCa not only allow for detailed assessment of disease characteristics and extent, but also supply the ground truth for developing of computer-aided diagnosis (CAD) systems for PCa detection before definitive treatment. As manual cancer annotation is tedious and subjective, there have been a number of publications describing methods for automating the procedure via the analysis of digitized whole-slide images (WSIs). However, these studies have focused only on the analysis of WSIs stained with hematoxylin and eosin (H&E), even though there is additional information that could be obtained from immunohistochemical (IHC) staining. In this work, we propose a framework for automating the annotation of PCa that is based on automated colorimetric analysis of both H&E and IHC WSIs stained with a triple-antibody cocktail against high-molecular weight cytokeratin (HMWCK), p63, and α-methylacyl CoA racemase (AMACR). The analysis outputs were then used to train a regression model to estimate the distribution of cancerous epithelium within slides. The approach yielded an AUC of 0.951, sensitivity of 87.1%, and specificity of 90.7% as compared to slide-level annotations, and generalized well to cancers of all grades.
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Adenocarcinoma/diagnóstico , Colorimetria/estatística & dados numéricos , Imuno-Histoquímica/estatística & dados numéricos , Neoplasias da Próstata/diagnóstico , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Área Sob a Curva , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Biópsia por Agulha , Estudos de Coortes , Colorimetria/métodos , Amarelo de Eosina-(YS) , Hematoxilina , Humanos , Interpretação de Imagem Assistida por Computador , Imuno-Histoquímica/métodos , Queratinas/genética , Queratinas/metabolismo , Masculino , Estadiamento de Neoplasias , Próstata/metabolismo , Próstata/patologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Racemases e Epimerases/genética , Racemases e Epimerases/metabolismo , Sensibilidade e Especificidade , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
Open-top light-sheet microscopy is a technique that can potentially enable rapid ex vivo inspection of large tissue surfaces and volumes. Here, we have optimized an open-top light-sheet (OTLS) microscope and image-processing workflow for the comprehensive examination of surgical margin surfaces, and have also developed a novel fluorescent analog of H&E staining that is robust for staining fresh unfixed tissues. Our tissue-staining method can be achieved within 2.5 minutes followed by OTLS microscopy of lumpectomy surfaces at a rate of up to 1.5 cm2/minute. An image atlas is presented to show that OTLS image quality surpasses that of intraoperative frozen sectioning and can approximate that of gold-standard H&E histology of formalin-fixed paraffin-embedded (FFPE) tissues. Qualitative evidence indicates that these intraoperative methods do not interfere with downstream post-operative H&E histology and immunohistochemistry. These results should facilitate the translation of OTLS microscopy for intraoperative guidance of lumpectomy and other surgical oncology procedures.
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CONTEXT.: Ex vivo microscopy encompasses a range of techniques to examine fresh or fixed tissue with microscopic resolution, eliminating the need to embed the tissue in paraffin or produce a glass slide. One such technique is light-sheet microscopy, which enables rapid 3D imaging. Our pathology-engineering collaboration has resulted in an open-top light-sheet (OTLS) microscope that is specifically tailored to the needs of pathology practice. OBJECTIVE.: To present an image atlas of OTLS images of prostate core needle biopsies. DESIGN.: Core needle biopsies (N = 9) were obtained from fresh radical prostatectomy specimens. Each biopsy was fixed in formalin, dehydrated in ethanol, stained with TO-PRO3 and eosin, optically cleared, and imaged using OTLS microscopy. The biopsies were then processed, paraffin embedded, and sectioned. Hematoxylin-eosin and immunohistochemical staining for cytokeratin 5 and cytokeratin 8 was performed. RESULTS.: Benign and neoplastic histologic structures showed high fidelity between OTLS and traditional light microscopy. OTLS microscopy had no discernible effect on hematoxylin-eosin or immunohistochemical staining in this pilot study. The 3D histology information obtained using OTLS microscopy enabled new structural insights, including the observation of cribriform and well-formed gland morphologies within the same contiguous glandular structures, as well as the continuity of poorly formed glands with well-formed glands. CONCLUSIONS.: Three-dimensional OTLS microscopy images have a similar appearance to traditional hematoxylin-eosin histology images, with the added benefit of useful 3D structural information. Further studies are needed to continue to document the OTLS appearance of a wide range of tissues and to better understand 3D histologic structures.
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Biópsia com Agulha de Grande Calibre , Imageamento Tridimensional/métodos , Microscopia/métodos , Próstata/patologia , Corantes , Amarelo de Eosina-(YS) , Hematoxilina , Humanos , Imuno-Histoquímica , Masculino , Microscopia/instrumentação , Prostatectomia , Neoplasias da Próstata/patologia , Coloração e Rotulagem/métodosRESUMO
CONTEXT.: In vivo microscopy (IVM) allows direct, real-time visualization of tissue histology in living patients without the need for tissue removal, processing, or staining. The IVM technologies in clinical use include confocal microscopy and optical coherence tomography. These technologies also show promise for use with pathology specimens (ex vivo microscopy [EVM]). However, few systems designed for EVM are commercially available, at least in part because of the lack of defined minimal functional requirements (FRs). OBJECTIVE.: To develop minimal FRs for likely high-volume pathology applications of EVM. DESIGN.: The IVM Committee of the College of American Pathologists identified potential EVM pathology applications based on the published literature. A subcommittee of IVM and EVM early adopters and experts then defined FRs for the most likely EVM applications. RESULTS.: Potential EVM applications include assessment of margins, adequacy of needle biopsies and aspirates for diagnosis, and transplant tissues; selection of tissue for molecular studies or biorepository; and guidance in block selection from gross specimens. The first 3 applications were selected for development of FRs. The FRs were identified based on existing laboratory practices and guidelines and input from experts in the field and included device footprint and portability, specimen preparation, imaging time, field of view or resolution, morphologic diagnostic capability, yield, accuracy, ease of use, safety, and cost. CONCLUSIONS.: Consensus was achieved on FRs that would accommodate the selected EVM applications. Publication and dissemination of those FRs will provide guidance to engineers, researchers, and vendors on how to optimally adapt IVM technologies for EVM for widespread adoption by pathologists.
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Microscopia Intravital/instrumentação , Microscopia/instrumentação , Microscopia/métodos , Patologia/instrumentação , Patologia/métodos , Biópsia por Agulha , Custos e Análise de Custo , Secções Congeladas/economia , Secções Congeladas/instrumentação , Secções Congeladas/métodos , Humanos , Microscopia Intravital/métodos , Margens de Excisão , Microscopia/tendências , Microscopia Confocal , Patologia/economia , Guias de Prática Clínica como Assunto , Sensibilidade e Especificidade , Manejo de Espécimes/métodos , Tomografia de Coerência ÓpticaRESUMO
Intraoperative assessment of breast surgical margins will be of value for reducing the rate of re-excision surgeries for lumpectomy patients. While frozen-section histology is used for intraoperative guidance of certain cancers, it provides limited sampling of the margin surface (typically <1 % of the margin) and is inferior to gold-standard histology, especially for fatty tissues that do not freeze well, such as breast specimens. Microscopy with ultraviolet surface excitation (MUSE) is a nondestructive superficial optical-sectioning technique that has the potential to enable rapid, high-resolution examination of excised margin surfaces. Here, a MUSE system is developed with fully automated sample translation to image fresh tissue surfaces over large areas and at multiple levels of defocus, at a rate of â¼5 min / cm2. Surface extraction is used to improve the comprehensiveness of surface imaging, and 3-D deconvolution is used to improve resolution and contrast. In addition, an improved fluorescent analog of conventional H&E staining is developed to label fresh tissues within â¼5 min for MUSE imaging. We compare the image quality of our MUSE system with both frozen-section and conventional H&E histology, demonstrating the feasibility to provide microscopic visualization of breast margin surfaces at speeds that are relevant for intraoperative use.
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Neoplasias da Mama/diagnóstico por imagem , Mama/diagnóstico por imagem , Margens de Excisão , Microscopia Ultravioleta/métodos , Imagem Óptica/métodos , Animais , Mama/cirurgia , Neoplasias da Mama/cirurgia , Carcinoma/diagnóstico por imagem , Carcinoma/cirurgia , Feminino , Secções Congeladas , Humanos , Processamento de Imagem Assistida por Computador , Imageamento Tridimensional , Rim/diagnóstico por imagem , Mastectomia Segmentar , Camundongos , Microscopia de Fluorescência/métodos , Microscopia Ultravioleta/instrumentação , Imagem Óptica/instrumentação , Propriedades de SuperfícieRESUMO
In recent decades, various classes of nanoparticles have been developed for optical imaging of cancers. Many of these nanoparticles are designed to specifically target tumor sites, and specific cancer biomarkers, to facilitate the visualization of tumors. However, one challenge for accurate detection of tumors is that the molecular profiles of most cancers vary greatly between patients as well as spatially and temporally within a single tumor mass. To overcome this challenge, certain nanoparticles and imaging systems have been developed to enable multiplexed imaging of large panels of cancer biomarkers. Multiplexed molecular imaging can potentially enable sensitive tumor detection, precise delineation of tumors during interventional procedures, and the prediction/monitoring of therapy response. In this review, we summarize recent advances in systems that have been developed for the imaging of optical nanoparticles that can be heavily multiplexed, which include surface-enhanced Raman-scattering nanoparticles (SERS NPs) and quantum dots (QDs). In addition to surveying the optical properties of these various types of nanoparticles, and the most-popular multiplexed imaging approaches that have been employed, representative preclinical and clinical imaging studies are also highlighted.
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Intraoperative identification of carcinoma at lumpectomy margins would enable reduced re-excision rates, which are currently as high as 20% to 50%. Although imaging of disease-associated biomarkers can identify malignancies with high specificity, multiplexed imaging of such biomarkers is necessary to detect molecularly heterogeneous carcinomas with high sensitivity. We have developed a Raman-encoded molecular imaging (REMI) technique in which targeted nanoparticles are topically applied on excised tissues to enable rapid visualization of a multiplexed panel of cell surface biomarkers at surgical margin surfaces. A first-ever clinical study was performed in which 57 fresh specimens were imaged with REMI to simultaneously quantify the expression of four biomarkers HER2, ER, EGFR, and CD44. Combined detection of these biomarkers enabled REMI to achieve 89.3% sensitivity and 92.1% specificity for the detection of breast carcinoma. These results highlight the sensitivity and specificity of REMI to detect biomarkers in freshly resected tissue, which has the potential to reduce the rate of re-excision procedures in cancer patients. Cancer Res; 77(16); 4506-16. ©2017 AACR.
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Neoplasias da Mama/diagnóstico por imagem , Neoplasias da Mama/cirurgia , Mastectomia Segmentar/instrumentação , Imagem Molecular/instrumentação , Imagem Molecular/métodos , Nanopartículas/metabolismo , Neoplasias da Mama/patologia , Feminino , Humanos , Mastectomia Segmentar/métodosRESUMO
For the 1.7 million patients per year in the U.S. who receive a new cancer diagnosis, treatment decisions are largely made after a histopathology exam. Unfortunately, the gold standard of slide-based microscopic pathology suffers from high inter-observer variability and limited prognostic value due to sampling limitations and the inability to visualize tissue structures and molecular targets in their native 3D context. Here, we show that an open-top light-sheet microscope optimized for non-destructive slide-free pathology of clinical specimens enables the rapid imaging of intact tissues at high resolution over large 2D and 3D fields of view, with the same level of detail as traditional pathology. We demonstrate the utility of this technology for various applications: wide-area surface microscopy to triage surgical specimens (with ~200 µm surface irregularities), rapid intraoperative assessment of tumour-margin surfaces (12.5 sec/cm2), and volumetric assessment of optically cleared core-needle biopsies (1 mm in diameter, 2 cm in length). Light-sheet microscopy can be a versatile tool for both rapid surface microscopy and deep volumetric microscopy of human specimens.
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Biomarker-targeted surface-enhanced Raman scattering (SERS) nanoparticles (NPs) have been explored as a viable option for targeting and imaging multiple cell-surface protein biomarkers of cancer. While it has been demonstrated that this Raman-encoded molecular imaging (REMI) technology may potentially be used to guide tumor-resection procedures, the REMI strategy would benefit from further improvements in imaging speed. Previous implementations of REMI have utilized 1024 spectral channels (camera pixels) in a commercial spectroscopic CCD to detect the spectral signals from multiplexed SERS NPs, a strategy that enables accurate demultiplexing of the relative concentration of each NP "flavor" within a mixture. Here, we investigate the ability to significantly reduce the number of spectral-collection channels while maintaining accurate imaging and demultiplexing of up to five SERS NP flavors, a strategy that offers the potential for improved imaging speed and/or detection sensitivity in future systems. This strategy was optimized by analyzing the linearity of five multiplexed flavors of SERS NPs topically applied on tissues. The accuracy of this binning approach was then validated by staining tumor xenografts and human breast tumor specimens with a mixture of five NP flavors (four targeted NPs and one untargeted NP) and performing ratiometric imaging of specific vs. nonspecific NP accumulation. We demonstrate that with channel-compressed spectrometry using as few as 16 channels, it is possible to perform REMI with five NP flavors, with < 20% error, at low concentrations (< 10 pM) that are relevant for clinical applications.
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Strongyloides stercoralis is an important human parasite, especially in rural areas and developing countries. Infected immunosuppressed patients are at risk for hyperinfection, with severe clinical consequences. Here we describe the incidental detection and diagnosis of an unexpected S. stercoralis infection by methods designed to detect fungal 28S ribosomal DNA.