Pro: "Is Spread Through Air Spaces an In Vivo Phenomenon or an Inducible Artifact?"

In this PRO-CON debate, you will read very different perspectives about a simple question regarding an observation under the microscope: What is the significance of tumor cells in the air spaces of the lung parenchyma beyond the tumor edge of a resected lung cancer? An important underlying question is whether this entire PRO-CON debate is a mere academic exercise or whether spread through air spaces (STAS), as currently defined, describes a clinically useful phenomenon. The journey of STAS began with a complete paradigm shift to reverse the thinking that all air space tumor cells beyond the edge of lung cancers are an artifact. This led to a new concept where STAS could be separated from artifacts with a definition that has proven to be clinically useful. As with any major change in thinking, it is understandable that there would be some disagreement with this paradigm shift. Nevertheless, after a decade since it was described, many pathologists and clinicians around the world have found STAS to provide important information about the behavior of lung cancer. Numerous PRO-STAS articles supporting the usefulness of STAS have been published with clinical data on many thousands of patients from numerous institutions all over the world. In contrast, for the CON-STAS articles, widespread international representation and data are limited. It is now difficult to ignore the numerous reports and is reasonable to consider how to use the presence of STAS in clinical decisions. Hopefully, this PRO-CON debate will further stimulate clinical and scientific investigations aimed at a better understanding of STAS.

Malodour from oral malignant fungating wound: sprayed metronidazole - case report.

Metronidazole gel or ointment is recommended for the treatment of malodour from malignant fungating wounds. However, this medication may not settle adequately in oral lesions because its texture causes discomfort and it tends to be washed out by saliva. We report a case of malodour due to an oral lesion that was well controlled with sprayed metronidazole.

Color Standardization and Stain Intensity Calibration for Whole Slide Image-Based Immunohistochemistry Assessment.

Automated quantification of human epidermal growth factor receptor 2 (HER2) immunohistochemistry (IHC) using whole slide imaging (WSI) is expected to eliminate subjectivity in visual assessment. However, the color intensity in WSI varies depending on the staining process and scanner device. Such variations affect the image analysis results. This paper presents methods to diminish the influence of color variation produced in the staining process using a calibrator slide consisting of peptide-coated microbeads. The calibrator slide is stained along with tissue sample slides, and the 3,3'-diaminobenzidine (DAB) color intensities of the microbeads are used for calibrating the color variation of the sample slides. An off-the-shelf image analysis tool is employed for the automated assessment, in which cells are classified by the thresholds for the membrane staining. We have adopted two methods for calibrating the color variation based on the DAB color intensities obtained from the calibrator slide: (1) thresholds for classifying the DAB membranous intensity are adjusted, and (2) the color intensity of WSI is corrected. In the experiment, the calibrator slides and tissue of breast cancer slides were stained together on different days and used to test our protocol. With the proposed protocol, the discordance in the HER2 evaluation was reduced to one slide out of 120 slides.

Standardizing HER2 immunohistochemistry assessment: calibration of color and intensity variation in whole slide imaging caused by staining and scanning.

In the evaluation of human epidermal growth factor receptor 2 (HER2) immunohistochemistry (IHC) - one of the standard biomarkers for breast cancer- visual assessment is laborious and subjective. Image analysis using whole slide image (WSI) could produce more consistent results; however, color variability in WSIs due to the choice of stain and scanning processes may impact image analysis. We therefore developed a calibration protocol to diminish the staining and scanning variations of WSI using two calibrator slides. The IHC calibrator slide (IHC-CS) contains peptide-coated microbeads with different concentrations. The color distribution obtained from the WSI of stained IHC-CS reflects the staining process and scanner characteristics. A color chart slide (CCS) is also useful for calibrating the color variation due to the scanner. The results of the automated HER2 assessment were compared to confirm the effectiveness of two calibration slides. The IHC-CS and HER2 breast cancer cases were stained on different days. All stained slides and CCS were digitized by two different WSI scanners. Results revealed 100% concordance between automated evaluation and the pathologist's assessment with both the scanner and staining calibration. The proposed method may enable consistent evaluation of HER2.

Yttrium-90 Activity Quantification in PET/CT-Guided Biopsy Specimens from Colorectal Hepatic Metastases Immediately after Transarterial Radioembolization Using Micro-CT and Autoradiography.

PURPOSE: To evaluate the yttrium-90 (90Y) activity distribution in biopsy tissue samples of the treated liver to quantify the dose with higher spatial resolution than positron emission tomography (PET) for accurate investigation of correlations with microscopic biological effects and to evaluate the radiation safety of this procedure. MATERIALS AND METHODS: Eighty-six core biopsy specimens were obtained from 18 colorectal liver metastases (CLMs) immediately after 90Y transarterial radioembolization (TARE) with either resin or glass microspheres using real-time 90Y PET/CT guidance in 17 patients. A high-resolution micro-computed tomography (micro-CT) scanner was used to image the microspheres in part of the specimens and allow quantification of 90Y activity directly or by calibrating autoradiography (ARG) images. The mean doses to the specimens were derived from the measured specimens' activity concentrations and from the PET/CT scan at the location of the biopsy needle tip for all cases. Staff exposures were monitored. RESULTS: The mean measured 90Y activity concentration in the CLM specimens at time of infusion was 2.4 ± 4.0 MBq/mL. The biopsies revealed higher activity heterogeneity than PET. Radiation exposure to the interventional radiologists during post-TARE biopsy procedures was minimal. CONCLUSIONS: Counting the microspheres and measuring the activity in biopsy specimens obtained after TARE are safe and feasible and can be used to determine the administered activity and its distribution in the treated and biopsied liver tissue with high spatial resolution. Complementing 90Y PET/CT imaging with this approach promises to yield more accurate direct correlation of histopathological changes and absorbed dose in the examined specimens.

Anticholinergic drugs for death rattle in dying patients with cancer: multicentre prospective cohort study.

BACKGROUND: This study aimed to investigate the effectiveness of anticholinergics (AC) for death rattle in dying patients with cancer. METHODS: This is a prospective cohort study enrolled Terminally ill adult (20 years or older) patients with cancer who developed substantial death rattle (Back score ≥2) from 23 palliative care units in Japan. AC treatment for death rattle was prescribed according to primary physician's decision. The primary outcome was the proportion of patients whose death rattle improved, which was defined as a Back score of ≤1. We compared the proportion of improved cases in patients treated with (AC group) and without (non-AC group) AC, controlling potential confounders by employing propensity score weighting. RESULTS: Of the 1896 patients enrolled, we included 196 who developed a substantial death rattle. Of these, 81 received AC. 56.8% in the AC group and 35.4% in the non-AC group had an improved death rattle at 8 hours after baseline. In the weighted analysis, AC group showed significant improvements in death rattle, with an adjusted OR of 4.47 (95% CI 2.04 to 9.78; p=0.0024). All sensitivity analyses achieved essentially the same results. In the subgroup analysis, ACs were strongly associated with death rattle improvement in men, patients with lung cancer, and type 1 death rattle (adjusted OR 5.81, 8.38 and 9.32, respectively). CONCLUSIONS: In this propensity score-weighted analysis, ACs were associated with death rattle improvement in terminally ill patients with cancer who developed substantial death rattle. TRIAL REGISTRATION NUMBER: UMIN-CTR (UMIN00002545).

A pilot study of micro-CT-based whole tissue imaging (WTI) on endoscopic submucosal dissection (ESD) specimens.

Endoscopic submucosal dissection can remove large superficial gastrointestinal lesions in en bloc. A detailed pathological evaluation of the resected specimen is required to assess the risk of recurrence after treatment. However, the current method of sectioning specimens to a thickness of a few millimeters does not provide information between the sections that are lost during the preparation. In this study, we have produced three-dimensional images of the entire dissected lesion for nine samples by using micro-CT imaging system. Although it was difficult to diagnose histological type on micro-CT images, it successfully evaluates the extent of the lesion and its surgical margins. Micro-CT images can depict sites that cannot be observed by the conventional pathological diagnostic process, suggesting that it may be useful to use in a complementary manner.

Pathological Evaluation of Rectal Cancer Specimens Using Micro-Computed Tomography.

Whole-block imaging (WBI) using micro-computed tomography (micro-CT) allows the nondestructive reconstruction of a three-dimensional view of tissues, implying that WBI may be used for accurate pathological evaluation of patients with rectal cancer. HOWEVER, the clinical impact of this approach is unclear. We aimed to clarify the efficacy of WBI in the whole-mount specimens of locally advanced rectal cancer. A total of 237 whole-mount formalin-fixed paraffin-embedded blocks from 13 patients with rectal cancer who underwent surgical treatment were enrolled and scanned with micro-CT to generate three-dimensional images. WBI was evaluated following the conventional pathological review of the corresponding whole-slide imaging (WSI). WBI identified all tumor sites detected using WSI. Furthermore, WBI revealed one additional tumor site, which was not detected using WSI. Tumor resection margin was significantly closer to the soft-tissue edge when measured using WBI (7.7 mm vs. 6.6 mm, p < 0.01). Seventy-six percent of tumor deposits on WSI were changed according to the evidence of tumor interaction with the surrounding tissues confirmed using WBI. Furthermore, WBI revealed 25 additional lymph nodes, six of which were metastatic. The combination of conventional hematoxylin and eosin-stained imaging and WBI may contribute to an accurate pathological assessment.

The impact of death rattle on bereaved families: not the sound itself, but the resonance with their feelings.

BACKGROUND: This study aimed to explore (i) the consistency between physician-rated and bereaved family-perceived intensity of death rattle, (ii) the relationship between intensity of death rattle and the bereaved family's distress and (iii) the bereaved family's experience and feelings related to suctioning for death rattle. METHODS: We used matched data for deceased patients from a prospective cohort study of cancer patients admitted to a palliative care unit, and their bereaved families from a nationwide questionnaire survey in Japan. The intensity of death rattle using Back's score was evaluated prospectively by physicians and retrospectively by bereaved families. RESULTS: In total, 1122 bereaved families answered (response rate: 66.7%). Of these, 297 reported the development of death rattle. The maximum intensity of death rattle evaluated by physicians and perceived by bereaved families was poorly correlated (Spearman correlation coefficient 0.188, P = 0.082). The optimal cut-off point of Back's score for detecting high-level distress was 1/2, with a low accuracy of prediction (area under the curve 0.62). More than 70% of bereaved families indicated suctioning reduced the intensity of death rattle, made patients comfortable and themselves relieved, whereas a similar proportion felt patients were in distress during suctioning. Families who felt suctioning was gently performed and discussed well whether to do suctioning with health care providers felt less needs for improvement. CONCLUSIONS: Bereaved family-perceived intensity of death rattle did not correlate to physician-evaluated intensity, and the intensity of death rattle itself seemed to poorly correlate to family distress. Gently performed suctioning based on sufficient discussion with families can help reduce family-perceived patient discomfort.

Micro-computed tomography: A novel diagnostic technique for the evaluation of gastrointestinal specimens.

Micro-computed tomography (micro-CT) is a non-destructive modality that can be used to obtain high-resolution three-dimensional (3 D) images of the whole sample tissue; the usefulness of micro-CT has been reported for evaluation of breast cancer and lung cancer. However, this novel diagnostic technique has never been used for evaluating endoscopically resected gastrointestinal specimens. In the present study, we scanned 13 formalin-fixed paraffin-embedded (FFPE) tissue blocks of a normal human colon and gastric tissue samples using micro-CT. The evaluation comprised a comparison of the acquired whole block images with the images of the corresponding cross-sectional slice of the hematoxylin and eosin-stained slide. Micro-CT was able to produce images of the whole sample and clearly depict tissues such as glandular structures, muscularis mucosae, and blood vessels in the FFPE tissue blocks of normal gastrointestinal samples. Furthermore, the 3 D reconstructed could be used to create a cross-sectional image and reflected the surface structure of samples obtained from any site. Micro-CT has the potential to become a highly promising pathological diagnostic assistance tool for endoscopically resected gastrointestinal specimens in combination with conventional microscopic examination.

Integrated digital pathology at scale: A solution for clinical diagnostics and cancer research at a large academic medical center.

OBJECTIVE: Broad adoption of digital pathology (DP) is still lacking, and examples for DP connecting diagnostic, research, and educational use cases are missing. We blueprint a holistic DP solution at a large academic medical center ubiquitously integrated into clinical workflows; researchapplications including molecular, genetic, and tissue databases; and educational processes. MATERIALS AND METHODS: We built a vendor-agnostic, integrated viewer for reviewing, annotating, sharing, and quality assurance of digital slides in a clinical or research context. It is the first homegrown viewer cleared by New York State provisional approval in 2020 for primary diagnosis and remote sign-out during the COVID-19 (coronavirus disease 2019) pandemic. We further introduce an interconnected Honest Broker for BioInformatics Technology (HoBBIT) to systematically compile and share large-scale DP research datasets including anonymized images, redacted pathology reports, and clinical data of patients with consent. RESULTS: The solution has been operationally used over 3 years by 926 pathologists and researchers evaluating 288 903 digital slides. A total of 51% of these were reviewed within 1 month after scanning. Seamless integration of the viewer into 4 hospital systems clearly increases the adoption of DP. HoBBIT directly impacts the translation of knowledge in pathology into effective new health measures, including artificial intelligence-driven detection models for prostate cancer, basal cell carcinoma, and breast cancer metastases, developed and validated on thousands of cases. CONCLUSIONS: We highlight major challenges and lessons learned when going digital to provide orientation for other pathologists. Building interconnected solutions will not only increase adoption of DP, but also facilitate next-generation computational pathology at scale for enhanced cancer research.

Conditions, possibility and priority for admission into inpatient hospice/palliative care units in Japan: a nationwide survey.

BACKGROUND: Known barriers to admission into inpatient hospice/palliative care units (PCUs) include poor accessibility and stringent conditions for admission. However, the exact criteria are unclear. The aim of this study was to clarify the actual conditions, possibilities and priorities for admission to PCU in Japan. METHODS: We conducted a nationwide, anonymous, self-administered questionnaire survey to the responsible physicians of all 251 PCUs in 2014. RESULTS: Responses were received from 190 institutions (response rate 76%). The most frequent condition for admission was 'either the patient or the family knows the diagnosis' [86%, 95% confidence interval (CI): 80-90]. For the conditions for admission to PCU, 10-40% fewer facilities answered that the patient's consent or understanding was required compared with those that answered the patient or family's consent was sufficient. Seventy-one percent (95% CI: 64-77) of PCUs answered that either the patient or a family member needed to agree to a do-not-resuscitate (DNR) policy. The factors most likely to result in refusal of admission to a PCU varied greatly. Ninety-four percent (95% CI: 90-97) of PCUs answered that patients who had undergone a long waiting time after applying for admission would be given higher priority, and approximately 50% of PCUs answered they gave priority to their outpatients and inpatients. CONCLUSIONS: The findings of this study should be used to modify the system so that appropriate palliative care can be provided to patients who wish to be admitted to PCU.

Counting mitoses: SI(ze) matters!

Mitoses are often assessed by pathologists to assist the diagnosis of cancer, and to grade malignancy, informing prognosis. Historically, this has been done by expressing the number of mitoses per n high power fields (HPFs), ignoring the fact that microscope fields may differ substantially, even at the same high power (×400) magnification. Despite a requirement to define HPF size in scientific papers, many authors fail to address this issue adequately. The problem is compounded by the switch to digital pathology systems, where ×400 equivalent fields are rectangular and also vary in the area displayed. The potential for error is considerable, and at times this may affect patient care. This is easily solved by the use of standardized international (SI) units. We, therefore, recommend that features such as mitoses are always counted per mm2, with an indication of the area to be counted and the method used (usually "hotspot" or "average") to obtain the results.

Automated 3D scoring of fluorescence in situ hybridization (FISH) using a confocal whole slide imaging scanner.

Fluorescence in situ hybridization (FISH) is a technique to visualize specific DNA/RNA sequences within the cell nuclei and provide the presence, location and structural integrity of genes on chromosomes. A confocal Whole Slide Imaging (WSI) scanner technology has superior depth resolution compared to wide-field fluorescence imaging. Confocal WSI has the ability to perform serial optical sections with specimen imaging, which is critical for 3D tissue reconstruction for volumetric spatial analysis. The standard clinical manual scoring for FISH is labor-intensive, time-consuming and subjective. Application of multi-gene FISH analysis alongside 3D imaging, significantly increase the level of complexity required for an accurate 3D analysis. Therefore, the purpose of this study is to establish automated 3D FISH scoring for z-stack images from confocal WSI scanner. The algorithm and the application we developed, SHIMARIS PAFQ, successfully employs 3D calculations for clear individual cell nuclei segmentation, gene signals detection and distribution of break-apart probes signal patterns, including standard break-apart, and variant patterns due to truncation, and deletion, etc. The analysis was accurate and precise when compared with ground truth clinical manual counting and scoring reported in ten lymphoma and solid tumors cases. The algorithm and the application we developed, SHIMARIS PAFQ, is objective and more efficient than the conventional procedure. It enables the automated counting of more nuclei, precisely detecting additional abnormal signal variations in nuclei patterns and analyzes gigabyte multi-layer stacking imaging data of tissue samples from patients. Currently, we are developing a deep learning algorithm for automated tumor area detection to be integrated with SHIMARIS PAFQ.

Three-Dimensional Vessel Segmentation in Whole-Tissue and Whole-Block Imaging Using a Deep Neural Network: Proof-of-Concept Study.

In the field of pathology, micro-computed tomography (micro-CT) has become an attractive imaging modality because it enables full analysis of the three-dimensional characteristics of a tissue sample or organ in a noninvasive manner. However, because of the complexity of the three-dimensional information, understanding would be improved by development of analytical methods and software such as those implemented for clinical CT. As the accurate identification of tissue components is critical for this purpose, we have developed a deep neural network (DNN) to analyze whole-tissue images (WTIs) and whole-block images (WBIs) of neoplastic cancer tissue using micro-CT. The aim of this study was to segment vessels from WTIs and WBIs in a volumetric segmentation method using DNN. To accelerate the segmentation process while retaining accuracy, a convolutional block in DNN was improved by introducing a residual inception block. Three colorectal tissue samples were collected and one WTI and 70 WBIs were acquired by a micro-CT scanner. The implemented segmentation method was then tested on the WTI and WBIs. As a proof-of-concept study, our method successfully segmented the vessels on all WTI and WBIs of the colorectal tissue sample. In addition, despite the large size of the images for analysis, all segmentation processes were completed in 10 minutes.

Detection and assessment of capsular invasion, vascular invasion and lymph node metastasis volume in thyroid carcinoma using microCT scanning of paraffin tissue blocks (3D whole block imaging): a proof of concept.

In the modern era, detailed pathologic characteristics of a thyroid tumor are crucial to achieve accurate diagnosis and guide treatment. The presence of capsular invasion (CI) is diagnostic for carcinoma, whereas vascular invasion (VI) and nodal metastasis (NM) are included in risk stratification. However, the very definition of CI and VI is surrounded by controversies and an accurate assessment of NM is lacking. Whole Block Imaging (WBI) by microCT is a new imaging modality to create 3D reconstruction of whole tissue block with microscopic level resolution without the need for tissue sectioning. In this study, we aimed to define CI, VI, and NM volume using WBI by microCT. Twenty-eight paraffin blocks (PBs) from 26 thyroid tumors were scanned. Ten PBs contained CI, whereas 7 had VI. 3D microCT images were compared with whole slide images (WSI) of corresponding H&E slides. In 2 cases with VI and/or CI, WSI of serial H&E slides were obtained and underwent 3D-reconstruction to be compared with the WBI. Satellite tumor nodules beyond tumor capsule were shown to be CI by demonstrating the point of penetration using microCT and 3D reconstruction. Additional foci of CI were detected using microCT. VI was seen using microCT. Fibrin associated with tumor thrombus was not always present on serially sectioned H&E slides. WBI by microCT scanner was able to assess the volume of NM. In conclusion, WBI is able to detect CI, VI, and assess the volume of NM in thyroid carcinoma without tissue sectioning. It is the ultimate method for the complete sampling of the tumor capsule. It has the potential to increase the detection rate of CI, better define criteria for CI and VI, and provide an accurate assessment of the volume of nodal disease. This technology may impact the future practice of surgical pathology.

Banff Digital Pathology Working Group: Going digital in transplant pathology.

The Banff Digital Pathology Working Group (DPWG) was formed in the time leading up to and during the joint American Society for Histocompatibility and Immunogenetics/Banff Meeting, September 23-27, 2019, held in Pittsburgh, Pennsylvania. At the meeting, the 14th Banff Conference, presentations directly and peripherally related to the topic of "digital pathology" were presented; and discussions before, during, and after the meeting have resulted in a list of issues to address for the DPWG. Included are practice standardization, integrative approaches for study classification, scoring of histologic parameters (eg, interstitial fibrosis and tubular atrophy and inflammation), algorithm classification, and precision diagnosis (eg, molecular pathways and therapeutics). Since the meeting, a survey with international participation of mostly pathologists (81%) was conducted, showing that whole slide imaging is available at the majority of centers (71%) but that artificial intelligence (AI)/machine learning was only used in ≈12% of centers, with a wide variety of programs/algorithms employed. Digitalization is not just an end in itself. It also is a necessary precondition for AI and other approaches. Discussions at the meeting and the survey highlight the unmet need for a Banff DPWG and point the way toward future contributions that can be made.

Three-Dimensional Histologic, Immunohistochemical, and Multiplex Immunofluorescence Analyses of Dynamic Vessel Co-Option of Spread Through Air Spaces in Lung Adenocarcinoma.

INTRODUCTION: Spread through air spaces (STAS) is a method of invasion in lung adenocarcinoma and is associated with tumor recurrence and poor survival. The spatial orientation of STAS cells in the lung alveolar parenchyma is not known. The aim of this study was to use high-resolution and high-quality three-dimensional (3D) reconstruction of images from immunohistochemical (IHC) and multiplex immunofluorescence (IF) experiments to understand the spatial architecture of tumor cell clusters by STAS in the lung parenchyma. METHODS: Four lung adenocarcinomas, three micropapillary-predominant and one solid predominant adenocarcinoma subtypes, were investigated. A 3D reconstruction image was created from formalin-fixed, paraffin-embedded blocks. A total of 350 serial sections were obtained and subjected to hematoxylin and eosin (100 slides), IHC (200 slides), and multiplex IF staining (50 slides) with the following antibodies: cluster of differentiation 31, collagen type IV, thyroid transcription factor-1, and E-cadherin. Whole slide images were reconstructed into 3D images for evaluation. RESULTS: Serial 3D image analysis by hematoxylin and eosin, IHC, and IF staining revealed that the micropapillary clusters and solid nests of STAS are focally attached to the alveolar walls, away from the main tumor. CONCLUSIONS: Our 3D reconstructions found that STAS tumor cells can attach to the alveolar walls rather than appearing free floating, as seen on the two-dimensional sections. This suggests that the tumor cells detach from the main tumor, migrate through air spaces, and reattach to the alveolar walls through vessel co-option, allowing them to survive and grow. This may explain the higher recurrence rate and worse survival of patients with STAS-positive tumors who undergo limited resection than those who undergo lobectomy.

Automatic quantification of HER2 gene amplification in invasive breast cancer from chromogenic in situ hybridization whole slide images.

Human epidermal growth factor receptor 2 (HER2), a transmembrane tyrosine kinase receptor encoded by the ERBB2 gene on chromosome 17q12, is a predictive and prognostic biomarker in invasive breast cancer (BC). Approximately 20% of BC are HER2-positive as a result of ERBB2 gene amplification and overexpression of the HER2 protein. Quantification of HER2 is performed routinely on all invasive BCs, to assist in clinical decision making for prognosis and treatment for HER2-positive BC patients by manually counting gene signals. We propose an automated system to quantify the HER2 gene status from chromogenic in situ hybridization (CISH) whole slide images (WSI) in invasive BC. The proposed method selects untruncated and nonoverlapped singular nuclei from the cancer regions using color unmixing and machine learning techniques. Then, HER2 and chromosome enumeration probe 17 (CEP17) signals are detected based on the RGB intensity and counted per nucleus. Finally, the HER2-to-CEP17 signal ratio is calculated to determine the HER2 amplification status following the ASCO/CAP 2018 guidelines. The proposed method reduced the labor and time for the quantification. In the experiment, the correlation coefficient between the proposed automatic CISH quantification method and pathologist manual enumeration was 0.98. The p -values larger than 0.05 from the one-sided paired t -test ensured that the proposed method yields statistically indifferent results to the reference method. The method was established on WSI scanned by two different scanners. Through the experiments, the capability of the proposed system has been demonstrated.