Mean Nuclear Area Research Articles

Small sized centroblasts as poor prognostic factor in follicular lymphoma - Based on artificial intelligence analysis

Histological assessment of centroblasts is an important evaluation in the diagnosis of follicular lymphoma, but there is substantial observer variation in assessment among hematopathologists.We aimed to perform quantitative morphological analysis of centroblasts in follicular lymphoma using new artificial intelligence technology in relation to the clinical prognosis. Hematoxylin and eosin slides of lesions were prepared from 36 cases of follicular lymphoma before initial chemotherapy. Cases were classified into three groups by clinical course after initial treatment. The ‘excellent prognosis’ group were without recurrence or progression of follicular lymphoma within 60 months, the ‘poor prognosis’ group were those that had relapse, exacerbation, or who died due to the follicular lymphoma within 60 months, and the ‘indeterminate prognosis’ group were those without recurrence or progression but before the passage of 60 months. We created whole slide images and image patches of hematoxylin and eosin sections for all cases. We designed an object detection model specialized for centroblasts by fine-tuning YOLOv5 and segmented all centroblasts in whole slide images. The morphological characteristics of centroblasts in relation to the clinical prognosis of follicular lymphoma were analyzed. Centroblasts in follicular lymphoma of the poor prognosis group were significantly smaller in nuclear size than those in follicular lymphoma of the excellent prognosis group in the following points: median of nuclear area (p = 0.013), long length (p = 0.042), short length (p = 0.007), nuclear area of top 10 % cells (p = 0.024) and short length of top 10 % cells (p = 0.020). Cases with a mean nuclear area of <55 μm2 had poorer event-free survival than those with a mean nuclear area of ≥55 μm2 (p < 0.0123). AI methodology is suggested to be able to surpass pathologist's observation in capturing morphological features. Small-sized centroblasts will likely become a new prognostic factor of follicular lymphoma.

Correlation between 18F-FDG PET-derived parameters and quantitative pathological characteristics of soft tissue sarcoma.

18F-fluorodeoxyglucose (18F-FDG) positron emission tomography (PET) has been widely used for evaluating patients with soft tissue sarcoma (STS). However, uncertainties and overlap among individuals may be observed, and the relevance of these findings remains to be further explored. The present study was aimed at investigating the correlation between PET metabolic parameters and quantitative pathological characteristics in STS. We retrospectively collected 39 patients with STS who underwent 18F-FDG PET/computed tomography (CT) examination before treatment. Metabolic parameters including the maximum standardized uptake value (SUVmax), metabolic tumor volume (MTV), total lesion glycolysis (TLG), and intratumoral FDG uptake heterogeneity (IFH) were measured. Histological grading was performed according to the French Federation of Cancer Centers grading system. Continuous staining of tissue sections and digital quantitative analysis methods were used, the characteristics of tumor nucleated cells were observed through hematoxylin-eosin staining, and the expression of CD163, CD68, CD8, and CD4 in tumor tissues was determined by immunohistochemistry (IHC), then the correlation between FDG metabolic parameters and the above quantitative pathological characteristics in patients with STS were evaluated. The SUVmax of 18F-FDG PET/CT in STS was positively correlated with the total nuclear area (r=0.355, P=0.027). SUVmax was also positively correlated with the expression levels of CD163, CD68, CD8, and CD4 (r=0.582, 0.485, 0.343, and 0.324, with P<0.001, 0.002, 0.032, and 0.044, respectively), but was not significantly correlated with cell count and mean nuclear area (all P>0.05). However, MTV, TLG, and IFH were not significantly correlated with the above quantitative pathological characteristics. Further multivariate logistic regression analysis indicated that only CD163 expression and histological grade were independently correlated with SUVmax. Moreover, SUVmax remained positively correlated with CD163 expression in the low-grade STS (r=0.820, P=0.001) and high-grade STS groups (r=0.430, P=0.028). 18F-FDG uptake was positively correlated with the quantitative pathological features of soft tissue tumors. SUVmax may be a meaningful method reflecting the level of M2 macrophage infiltration and may provide additional valuable information for preclinical evaluation of STS.

A Novel Algorithm for Evaluating Bone Metastatic Potential of Breast Cancer through Morphometry and Computational Mathematics

Bone metastases represent about 70% of breast cancer metastases and are associated with worse prognosis as the tumor cells acquire more aggressive features. The selection and investigation of patients with a high risk of developing bone metastasis would have a significant impact on patients’ management and survival. The patients were selected from the database of Carol Davila Clinical Nephrology Hospital of Bucharest. Their tumor specimens were pathologically processed, and a representative area was selected. This area was scanned using an Olympus VS200 slide scanner and further analyzed using QuPath software v0.4.4. A representative group of approximately 60–100 tumor cells was selected from each section, for which the following parameters were analyzed: nuclear area, nuclear perimeter, long axis and cell surface. Starting from these measurements, the following were calculated: the mean nuclear area and mean nuclear volume, the nucleus to cytoplasm ratio, the length of the two axes, the long axis to short axis ratio, the acyclicity and anellipticity grade and the mean internuclear distance. The tumor cells belonging to patients known to have bone metastasis seemed to have a lower nuclear area (&lt;55 µm2, p = 0.0035), smaller long axis (&lt;9 µm, p = 0.0015), smaller values for the small axis (&lt;7 µm, p = 0.0008), smaller mean nuclear volume (&lt;200 µm3, p = 0.0146) and lower mean internuclear distance (&lt;10.5 µm, p = 0.0007) but a higher nucleus to cytoplasm ratio (&gt;1.1, p = 0.0418), higher axis ratio (&gt;1.2, p = 0.088), higher acyclicity grade (&gt;1.145, p = 0.0857) and higher anellipticity grade (&gt;1.14, p = 0.1362). These parameters can be used for the evaluation of risk category of developing bone metastases. These results can be useful for the evaluation of bone metastatic potential of breast cancer and for the selection of high-risk patients whose molecular profiles would require further investigations and evaluation.

Evaluation of Nuclear Morphometry and Ki-67 Proliferative Marker in Astrocytomas: A Ambispective Study

Introduction: Astrocytomas are the commonest primary Central Nervous System (CNS) tumours. Its diagnosis is based on histopathological criteria defined by the World Health Organization (WHO) 2016 that grades astrocytoma into four grades. The subjective nature of WHO grading has prompted for more objective methods to evaluate nuclear features. Furthermore, Ki-67, a marker of cellular proliferation is a useful diagnostic tool that also helps in prognostic evaluation and to plan adjuvant therapy in astrocytomas. Aim: To evaluate the nuclear morphometry and Ki-67 proliferative marker in astrocytomas and to assess the relationship of WHO grade with proliferative activity using Ki-67 immunostaining. Materials and Methods: This ambispective study was conducted in the Department of Pathology, JSS Academy of Higher Education and Research, Mysuru, Karnataka, India, with a total duration of four years (October 2017 to September 2021). From 35 astrocytoma cases, Haematoxylin and Eosin (H&amp;E) stained slides were retrieved and reviewed appropriately by two pathologists and graded using the WHO criteria. Nuclear morphometric analysis was performed on the H&amp;E slides using Olympus BX-41 research microscope. The parameters evaluated were mean nuclear length, mean nuclear diameter, mean nuclear perimeter, mean nuclear area, Mean Nuclear Roundness Factor (MNRF) and mean nuclear ellipse form. The cases were then stained with Ki-67 antibody. The relationships between WHO grade of astrocytoma and nuclear morphometry and WHO grade with proliferative index was analysed. Results: According to WHO grading, four cases were Grade 1 (11.4%), 12 grade 2 (34.3%), six grade 3 (17.1%) and 13 grade 4 (37.1%). Significant correlation was seen between WHO grading and mean nuclear length, diameter, perimeter, area and Ki-67 with p-value of less than 0.001. Conclusion: Astrocytoma is an extremely heterogeneous disease with unpredictable outcome. The widely used WHO grading is subjective, while nuclear morphometry, using computer assisted image analysis, can ensure more objective assessment. The Ki-67 index could provide valuable information and may compliment the other parameters.

Comparative Evaluation of Buccal Exfoliated Cells in Individuals with Diabetes Mellitus and Healthy Controls: A Cytomorphometric Analysis.

Diabetes mellitus is the third most frequent cause of mortality and morbidity worldwide. Patients with diabetes exhibit a variety of oral symptoms, and hence the early detection of this condition can be addressed by a dentist. The current study aimed to study the cytomorphometric alterations of buccal exfoliated cells in individuals with type II diabetes mellitus. The study included thirty diabetics and thirty healthy controls. The smears were obtained from the buccal mucosa and stained with Papanicolaou stain and hematoxylin and eosin stain. The presence of inflammatory cells, microbial carriage, nuclear enlargement, and perinuclear halo and binucleation were examined on the slides. Cellular and nuclear parameters were quantitatively measured using Image J software. Statistical analysis was done using SPSS software, and the Student's t-test was employed. No inflammatory cells or microbes were observed in Group I individuals; however, the perinuclear halo was observed in 16.6% and binucleated cells in 3.3% of the controls. Inflammatory cells, consisting mainly of neutrophils and lymphocytes were seen in 40%, microbial carriage in 26.6%, perinuclear halo in 73.3%, and binucleated cells in 36.6% of the diabetic patients. The mean nuclear diameter, area, and nuclear-cytoplasmic ratio were significantly high in diabetic patients when compared to healthy controls. Oral exfoliated mucosal cells of patients with diabetes mellitus exhibit distinct cytomorphometric alterations such as increased nuclear diameter, nuclear area, and nuclear-cytoplasmic ratio.

Assessment of megakaryocytes’ size and morphology in selected myeloproliferative neoplasms

&lt;br&gt;&lt;b&gt;Introduction:&lt;/b&gt; Contemporary technological development make the cytological analysis of megakaryocytes (MKs) a valuable tool for supporting differential diagnosis of myeloproliferative neoplasms’ (MPNs) early stages.&lt;/br&gt; &lt;br&gt;&lt;b&gt;Aim:&lt;/b&gt; The aim of the study was to identify megakaryocyte’s cytological differences between chronic myelogenous leukemia (CML), polycythemia vera (PV) and essential thrombocythemia (ET), as well as to assess the usefulness of this analysis in differential diagnosis of the aforementioned MPNs.&lt;/br&gt; &lt;br&gt;&lt;b&gt;Materials and methods:&lt;/b&gt; Bone marrow aspirate smears were obtained from 20 patients with CML, 15 patients with PV and 12 patients with ET. The control included bone marrow biopsies from 15 patients without any hematological disorder. The following megakaryocyte’s parameters were examined: cell diameter, cytoplasmic area, nuclear area, nuclear-cytoplasmic ratio and nuclear lobulation.&lt;/br&gt; &lt;br&gt;&lt;b&gt;Results:&lt;/b&gt; Cytological analysis revealed a significantly decreased megakaryocytes’ size and the prevalence of cells with hypolobulated nuclei in CML patients comparing to other groups. MKs in ET were found to have characteristic staghorn-like hyperlobulated nucleus, whereas polycythemia vera showed the predominance of cells with multilobulated nuclei (4-7 lobes) and a tendency to hyperlobulation. However, there were no significant differences in mean values for cytoplasmic and nuclear area among these two groups of patients.&lt;/br&gt; &lt;br&gt;&lt;b&gt;Conclusion:&lt;/b&gt; Characteristic megakaryocytes’ cytological differences in CML patients may be useful in differential diagnosis of the CML’s early stage. MKs’ size and nuclear-cytoplasmic ratio are not useful in differentiation between polycythemia vera and essential thrombocythemia.&lt;/br&gt;

Comparative Cytomorphometric Study of Exfoliated Oral Epithelial Cells from a Population of Smokers, Tobacco Users and Gutkha Chewers

Introduction: Tobacco remains one of the most important preventable cause of addiction, sickness and mortality in the world as it affects the oral epithelium. Normal epithelium undergoes continuous exfoliation or shedding of its superficial cells, and it is replenished by new crop of cells from the basal layer. The rationale of exfoliative cytology lies in the epithelial physiology. The purpose of this study is to conduct quantitative cytomorphometric studies on squames obtained from a population of Tobacco and Gutkha users, to assess the cytomorphometric changes in Nuclear Area (NA) and Cytoplasmic Area (CA) of squames from tobacco smokers, tobacco chewers, gutkha chewers, to compare the results with control group normal healthy individuals and among themselves. Methods: Oral smears were taken from buccal mucosa of total 200 individuals. The smears were histochemically stained and cytomorphological assessment was done. Groups were compared by one way analysis of variance (ANOVA) and the significance of mean difference between the groups was done by Tukey’s post hoc test after ascertaining normality by Shapiro-Wilk test andhomogeneity of variances by Levene’s test. Results: The mean Nuclear area of tobacco chewer (C) was the highest and the normal individual (N) was the least. The mean Cytoplasmic area of normal individual (N) was the highest and the tobacco chewer(C) was the least. Conclusions: This study supports and extends the view that cytomorphometric evaluation of keratinocytes can serve as a useful diagnostic adjunct for early detection of oral cancer. Keywords: exfoliative cytology; cytomorphometric; tobacco users; gutkha users.

Abstract P5-09-01: Using isogenic model systems to determine mechanisms regulating mutant p53 protein stability in breast cancer cells

Abstract Two of the most common events in triple-negative breast cancers (TNBC) are mutation of the tumor suppressor gene TP53 and development of aneuploidy. This is often the case in aggressive and molecularly heterogeneous cancer subtypes such as TNBC, where ~80% of cases harbor a mutation in TP53. In addition to losing wild-type (WT) tumor-suppressive function, mutant p53 proteins are proposed to acquire gain-of-function (GOF) activity, leading to novel oncogenic phenotypes. Mutant p53 proteins can accumulate to high levels in tumors, and this stabilization is thought to be a pre-requisite for mutant p53 to exert its GOF effects. Mechanisms underlying the accumulation of mutant p53 are not well understood. To study mutant p53 GOF mechanisms and phenotypes, we used CRISPR/Cas9-mediated genome editing and developed two isogenic TNBC cell line models (one non-transformed and one tumor-derived) that express the most frequently occurring p53 missense mutations (R175H and R273H), are deficient for functional p53 protein (null), or retain the wild-type (WT) protein. In these engineered models, endogenous p53 expression is regulated by the native p53 promoter, thus providing a controlled system for rigorous functional experimentation across different p53 states. Through functional genomics analyses comparing isogenic TNBC cell lines, which initially differed only by the TP53 genotype, we have evaluated the relationship between mutant p53 and development of aneuploidy and determined that development of aneuploidy and not TP53 genotype contributed to several previously reported mutant p53 GOF phenotypes in vitro and in vivo1. Immunoblotting, immunofluorescence, and immunohistochemistry experiments revealed that mutant p53-expressing cell lines display a range of p53 protein levels, with several maintaining low levels of p53, similar to the cell lines which contain WT p53. In addition, mutant p53 levels correlate with aneuploidy, but not mean nuclear area or whole-genome doubling status, and protein levels increase over time in cell lines that become aneuploid. Using our various clonal cell lines that exhibit differing p53 protein stability, we are currently determining if p53 stability is dysregulated in aneuploid cell lines after copy number gain or loss of genes encoding critical p53 regulators, leading to the accumulation of the mutant protein. Analysis of cancer cell line genetic dependencies and human tumor data from The Cancer Genome Atlas (TCGA) has identified genetic and clinical correlates with increased mutant p53 protein levels. Utilizing biochemical and bioinformatic approaches, we aim to uncover mechanisms for increased mutant p53 stability and identify novel regulators of both WT and mutant p53 proteins. The dissection of mechanisms that contribute to stabilization of p53 protein will not only give insight into the regulation and tumor suppressive function of WT p53, but it has the potential for clinical translation in human cancers that have high-frequency p53 mutation and high levels of mutant p53 protein, as targeting mutant p53 through pharmacologic inhibition or antibody-based therapies is an active area of investigation. 1Article in press in Nature Communications Citation Format: Lindsay Redman-Rivera, Hailing Jin, Brian D Lehmann, Jennifer A Pietenpol. Using isogenic model systems to determine mechanisms regulating mutant p53 protein stability in breast cancer cells [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-09-01.

Correlation of Gleason Grading System and Nuclear Parameters by Nuclear Morphometry in Patients of Prostatic Adenocarcinoma: A Study Protocol

Background: Adenocarcinoma prostate is one of the significant causes of death in men. The Gleason Grading System is the most commonly used mortality to assess the degree, yet, identical results among blinded pathologists are challenging to obtain and hence come down to an unobjectionable result.&#x0D; Aim and Objectives: To evaluate the relationship between histopathologically obtained Gleason’s Grading Score and various nuclear morphometric parameters using a computer-aided system in Prostatic Adenocarcinomas.&#x0D; Methods: A series of 31 new and histopathologically diagnosed cases of adenocarcinoma prostate will be taken over one year, and the following nuclear morphometric parameters will be studied: mean nuclear area, mean nuclear length, mean nuclear perimeter, mean nuclear roundness factor, mean nuclear area factor and mean nuclear form ellipse. These individual parameters will be correlated with the Gleason Score of the individual cases.&#x0D; Expected Outcome: The present study expects the nuclear atypia to be more in cases with a higher Gleason Score and avoid the inter-observer contradictions in diagnosis. It can be used as a tool to quantify the aggression of the malignancy and, thus, assess disease progression and prescribe a justifiable management protocol. Nuclear morphometrical analysis can be a more accurate, objective, and effective method in the diagnostic and prognostic significance of prostate adenocarcinoma.

Nuclear morphology in breast lesions: refining its assessment to improve diagnostic concordance.

Although evaluation of nuclear morphology is important for the diagnosis and categorisation of breast lesions, the criteria used to assess nuclear atypia rely upon the subjective evaluation of several features that may result in inter- and intraobserver variation. This study aims to refine the definitions of cytonuclear features in various breast lesions. ImageJ was used to assess the nuclear morphological features including nuclear diameter, axis length, perimeter, area, circularity and roundness in 160 breast lesions comprising ductal carcinoma in situ (DCIS), invasive breast carcinoma of no special type (IBC-NST), tubular carcinoma, usual ductal hyperplasia (UDH), columnar cell change (CCC) and flat epithelial atypia (FEA). Reference cells included normal epithelial cells, red blood cells (RBCs) and lymphocytes. Reference cells showed size differences not only between normal epithelial cells and RBCs but also between RBCs in varied-sized blood vessels. Nottingham grade nuclear pleomorphism scores 1 and 3 cut-offs in IBC-NST, compared to normal epithelial cells, were < ×1.2 and > ×1.4 that of mean maximum Feret's diameter and < ×1.6 and > ×2.4 that of mean nuclear area, respectively. Nuclear morphometrics were significantly different in low-grade IBC-NST versus tubular carcinoma, low-grade DCIS versus UDH and CCC versus FEA. No differences in the nuclear features between grade-matched DCIS and IBC-NST were identified. This study provides a guide for the assessment of nuclear atypia in breast lesions, refines the comparison with reference cells and highlights the potential diagnostic value of image analysis tools in the era of digital pathology.

Role of Lamin A and emerin in maintaining nuclear morphology in different subtypes of ovarian epithelial cancer.

The nuclear lamina protein, Lamin A and inner nuclear membrane protein, emerin participate in maintaining nuclear morphology. However, their correlations with the nuclear shape in the four representative ovarian epithelial cancer subtypes, high-grade serous carcinoma (HGSCa), clear cell carcinoma (CCCa), endometrioid carcinoma (EMCa) and mucinous carcinoma (MUCa), remains unclear. The present study aimed to investigate the association between nuclear morphology and nuclear membrane protein expression in four histological subtypes of ovarian epithelial cancer. A total of 140 surgically resected ovarian cancer specimens were subjected to Feulgen staining to evaluate nuclear morphology, and immunohistochemistry analysis to assess Lamin A and emerin expression. The histological images were analyzed via computer-assisted image analysis (CAIA). The results demonstrated that the mean nuclear area of EMCa was significantly smaller compared with CCCa (P=0.0009). The standard deviation of the mean nuclear area was used to assess nuclear size variation, and the results indicated that EMCa lesions were significantly smaller than CCCa lesions (P=0.0006). Regarding the correlation between the Lamin A-positive rate and nuclear morphological factors, positive correlations were observed with nuclear area in CCCa and EMCa (R=0.2855 and R=0.2858, respectively) and nuclear perimeter in CCCa, EMCa and MUCa (R=0.2409, R=0.4054 and R=0.2370, respectively); however, a negative correlation with nuclear shape factor was observed in HGSCa and EMCa (R=−0.2079 and R=−0.3707, respectively). With regards to the correlation between emerin positivity and nuclear morphological factors, positive correlations were observed with nuclear shape factor in HGSCa (R=0.2673) and nuclear area in CCCa (R=0.3310). It is well-known that HGSCa and CCCa have conspicuous nuclear size variation, and EMCa has small nuclei without strong atypia. These findings were verified in the present study via CAIA. Taken together, the results of the present study suggest that Lamin A strongly contributes to the maintenance of nuclear morphology in ovarian epithelial cancer compared with emerin, although their contributions differ based on tumor subtype.

The importance of morphometric parameters in differentiating benign/reactive urothelial cells from low-grade urothelial carcinoma: computer-assisted study on urine specimens

Background. Urine cytology is deemed a sensitive method in detection of high-grade urothelial carcinoma. In contrast, detection of low-grade urothelial carcinoma (LGUC) and its differentiation from reactive lesions is difficult with urinary cytology. Objective. Our study aims to determine the effectiveness of morphometric parameters in differentiating reactive urothelial cells from LGUC by cytological examination of urine specimens. Methods. Voided urine samples were used for the study, while the cases were randomized into two groups: those diagnosed with LGUC (first group; N=10) and those which were not diagnosed with LGUC (second group; N=10). The morphometric parameters of major nuclear diameter (MaND), minor nuclear diameter (MiND), mean nuclear area (MNA), cell diameter (CD), mean cell area (MCA), as well as MaND/CD, MiND/CD, MiND/MaND and MNA/MCA ratios were measured on 100 urothelial cells for each case through ScopeImage® 9.0 software. Results. A statistically significant difference was found between the mean values of MiND/CD (p=0.017) and MNA/MCA (p=0.002) ratios of groups. The mean value of both parameters in the first group constituted 0.2 and higher, and below 0.2 in the second group. Conclusion. The ratios of MiND/CD and MNA/MCA in urothelial cells proved significantly higher in patients with LGUC than benign/reactive cases. The reliability of these findings in differentiating LGUC from benign/reactive lesions needs to be verified through studies examining a large number of cases. These parameters can be assessed much faster through a special software enabling an automatic measurement and thus can be used in routine cytological examination.

Evaluation and Quantification of Cytomorphometry in Conjunction with Visual Examination and Exfoliative Cytology in Early Diagnosis of Oral Premalignant and Malignant Lesions

Introduction: Incidence of oral epithelial dysplasia and oral squamous cell carcinoma (SCC) is very high in south Asian countries as compared to western population owing to a greater use of tobacco in these regions. While visual examination and exfoliative cytology are the most common screening and diagnostic modalities at present, it is a subjective analysis. Quantitative analyses such as nuclear size, cell size, and nuclear-to-cytoplasmic ratio may provide an accurate diagnosis and improve reproducibility. The aim of the study was to evaluate the role of morphometry as a diagnostic adjunct to exfoliative cytology and to derive a significant cutoff to identify the population at risk for development of SCC among chronic tobacco users. Material and Methods: This was an outpatient-based prospective study done in a tertiary hospital over a period of 2 years. Hundred and fifty cases with a history of chronic tobacco use for a minimum period of 5 years were evaluated. Visual inspection using acetic acid was done. Oral scrapes were taken for cytological and morphometric analysis followed by incision biopsy for histopathological evaluation, wherever possible. Results: On morphometrical analysis, mean nuclear area and nuclear:cytoplasmic (N:C) ratio increased, while the cytoplasmic area decreased from smears with normal cytology to oral dysplasia to SCC. Analysis of variance and post hoc Tukey’s honest significant difference test showed a statistically significant difference among the 3 groups (p value <0.05). A cutoff value for a significant N:C ratio was calculated as ≥0.08 using Youden’s index with a sensitivity and a specificity of 88% and 94%, respectively. Conclusion: In high-risk cases, morphometry can be a useful adjunct to exfoliative cytology and visual examination for an early and accurate diagnosis and timely intervention in oral potentially malignant and malignant lesions, thereby improving the prognosis.

Comparing World Health Organization/International Society of Urological Pathology Grading and Fuhrman Grading with the Prognostic Value of Nuclear Area in Patients with Renal Cell Carcinoma

This study was undertaken to compare Fuhrman grading with World Health Organization/International Society of Urological Pathology (WHO/ISUP) grading and stereologically measured nuclear area in patients with Clear Cell Renal Cell Carcinoma (ccRCC) or Papillary Renal Cell Carcinoma (PRCC) and to evaluate the independent predictive value of Fuhrman, WHO/ISUP and stereologically measured nuclear area combined with necrosis in a series of patients with ccRCC in relation to cancer-specific survival. In all, 124 cases of ccRCC and PRCC were included. All slides were blindly scored by two trained pathologists according to the Fuhrman and WHO/ISUP grading systems. Nuclear measurements were performed on digitally scanned slides in Visiopharm® and correlated to survival. Analysis of ccRCC and PRCC cases showed that application of WHO/ISUP grading resulted in a significant downgrading of cases from G2 to G1, when comparing with Fuhrman grading. Neither of these patients experienced progression. Cancer specific survival estimates in 101 ccRCC patients showed that WHO/ISUP grading was slightly superior in predicting cancer-specific survival. Novel models included WHO/ISUP grading and mean nuclear area (MNA) each of which combined with necrosis. Both demonstrated an increased ability to predict cancer-specific survival. The study demonstrates that WHO/ISUP grading provides superior prognostic information compared to Fuhrman grading and stereologically measured nuclear area. Necrosis in combination with either WHO/ISUP grading or MNA adds additional prognostic information.

Effect of Inhalational Therapy on Buccal Mucosal Cells in Asthmatic Patients: A Cytological Study.

ObjectiveInhalational drugs used in treating asthma have several side effects including those on oral tissues. We therefore designed a study to analyze the effects of inhalational drugs on the buccal mucosal cells of the oral cavity.MethodsSmears were obtained from clinically normal buccal mucosa of 20 randomly selected asthmatic patients who had been under inhalational therapy for at least 6 months. The Papanicolaou-stained smears were then analyzed for average nuclear area, average cytoplasmic area, and average nuclear area:cytoplasmic area ratio for each patient, and the values were compared with those of 10 healthy controls.ResultsA statistically significant decrease in cytoplasmic area (P<0.001) was found in asthmatic patients compared to controls. A significant increase in mean nuclear area:cytoplasmic area ratio (P<0.001) was noted in asthmatic patients when compared to controls.ConclusionProlonged use of inhalational drugs in patients diagnosed with asthma is associated with changes in oral epithelial cells. There is a need to assess whether these are the direct adverse effects of such drugs and whether they have any long-term impact on oral tissues.

Abstract PR03: Can immunologic biomarkers (CD4/CD8/CD25/CD138) predict tumor recurrence and progression in NMIBC?

Abstract Urothelial carcinoma is the most common type of carcinoma of the urinary bladder in the Western world. Currently TNM stage and WHO grade are the most important histopathologic parameters to classify patients into low- and high-risk groups and to decide appropriate treatment. However, as differentiating between stage Ta and T1 on surgical specimens is difficult and grade has only poor to fair reproducibility, these parameters do not precisely predict tumor recurrence or stage progression. As a consequence, patients undergo an intensive follow-up regimen and as such the treatment cost per patient for bladder cancer is one of the highest among all cancer types. Therefore, better prognostic and predictive markers are warranted. The aim of the present study was to clarify whether CD8+, CD4+, CD25+, and/or CD138+ immune cell markers, as well as quantitative features Ki67, mitotic activity index (MAI), and mean nuclear area of the 10 largest nuclei (MNA), can identify non-muscle invasive bladder cancer (NMIBC) patients at high risk for tumor recurrence and stage progression. Tissue samples from 183 patients diagnosed with primary NMIBC were obtained by transurethral resection of the bladder (TURB) or from biopsies. Sections were stained by immunohistochemistry using monoclonal antibodies for T lymphocyte subsets (CD4+, CD8+, and CD25+), plasma cells (CD138+) and Ki67. MAI and MNA were assessed on HES (hematoxylin, erythrosine, and saffron) stained sections. A highly reproducible, interactive image analysis system was used for quantification of all immune cell markers and Ki67. MAI and MNA were counted manually. Out of all the investigated markers, only Ki67 showed significance with tumor recurrence (95% CI, 0.4-0.9; p=0.05) in a 10-year follow-up. The group with high Ki67 (&amp;gt;39%) had significantly longer recurrence-free survival than the group with lower values. Furthermore, out of all the examined immunologic markers, only CD25+ was significantly associated with stage progression in a survival analysis (95% CI, 1.8-106.2; p=0.001). The group of patients with high CD25+ (&amp;gt;0.24%) had significantly shorter progression-free survival than the group with lower values. The median values of MAI and CD25+ were significantly higher in pT1 tumors than pTa tumors (median 16.0, 2.5 for MAI and 0.1%, 0.95% for CD25+ respectively; p&amp;lt;0.001). In univariate analysis age, WHO73, WHO04, Stage, MAI10, and CD25 were all correlated with significant differences in progression-free survival. Furthermore, using survival analysis, the combination of MAI and CD25+ could define a group of patients with 100% (N=81/183) progression-free survival (95% CI, 1.6-4.1; p&amp;lt;0.001). In a multivariate analysis including (age, WHO73, WHO04, combination CD25/MAI10), the combination of CD25/MAI10 was the single most prognostic feature. In conclusion, our study suggests that MAI and CD25+ could be used to identify NMIBC at risk for progression of disease. In addition, Ki67 could be a candidate for assessing the risk of recurrence in NMIBC. This abstract is also being presented as Poster A06. Citation Format: Melinda Lillesand, Vebjørn Kvikstad, Ok Målfrid Mangrud, Einar G. Gudlaugsson, Bianca van Diermen-Hidle, Ivar Skaland, Jan P.A. Baak, Emiel A.M. Janssen. Can immunologic biomarkers (CD4/CD8/CD25/CD138) predict tumor recurrence and progression in NMIBC? [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2019 May 18-21; Denver, CO. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(15_Suppl):Abstract nr PR03.

Third Harmonic Generation microscopy distinguishes malignant cell grade in human breast tissue biopsies

The ability to distinguish and grade malignant cells during surgical procedures in a fast, non-invasive and staining-free manner is of high importance in tumor management. To this extend, Third Harmonic Generation (THG), Second Harmonic Generation (SHG) and Fourier-Transform Infrared (FTIR) spectroscopy were applied to discriminate malignant from healthy cells in human breast tissue biopsies. Indeed, integration of non-linear processes into a single, unified microscopy platform offered complementary structural information within individual cells at the submicron level. Using a single laser beam, label-free THG imaging techniques provided important morphological information as to the mean nuclear and cytoplasmic area, cell volume and tissue intensity, which upon quantification could not only distinguish cancerous from benign breast tissues but also define disease severity. Simultaneously, collagen fibers that could be detected by SHG imaging showed a well structured continuity in benign tumor tissues, which were gradually disoriented along with disease severity. Combination of THG imaging with FTIR spectroscopy could provide a clearer distinction among the different grades of breast cancer, since FTIR analysis showed increased lipid concentrations in malignant tissues. Thus, the use of non-linear optical microscopy can be considered as powerful and harmless tool for tumor cell diagnostics even during real time surgery procedures.

Mitotic activity index and CD25+ lymphocytes predict risk of stage progression in non-muscle invasive bladder cancer.

In urothelial cell type non-muscle invasive urinary bladder carcinoma, TNM stage and WHO grade are widely used to classify patients into low and high‑risk groups for prognostic and therapeutic decision-making. However, stage and grade reproducibility and prediction accuracy are wanting. This may lead to suboptimal treatment. We evaluated whether proliferation features, nuclear area of the epithelial cancer cells and the composition of stromal and tumor infiltrating lymphocytes have independent prognostic value. In 183 primary non-muscle invasive bladder cancer patients with long follow-up (median for stage progression cohort: 119 months, range 5-173; median for tumor recurrence cohort: 82, range 3-165) proliferation features Ki67, PPH3 and Mitotic Activity Index (MAI), Mean Nuclear Area (MNA), lymphocyte subsets (CD8+, CD4+, CD25+) and plasma cells (CD138+) were assessed on consecutive sections. Post-resection instillation treatments (none, mitomycin, BCG) were strictly standardized during the intake period. Risk of recurrence was associated with expression of Ki67 (≤ 39 vs. > 39) and Multifocality (p = 0.01). Patients with low Ki67 had a higher recurrence rate than those with high Ki67. Lymphocyte composition did not predict recurrence. Stage progression was strongly associated with high values for MAI (>15) and CD25+ (>0.2%). In a multivariate analysis the combination of MAI and CD25+ was the single most prognostic feature (p<0.001). Validation of these results in additional, independent studies is warranted.

Effect of tobacco in human oral leukoplakia: a cytomorphometric analysis

ObjectivesTobacco use is one of the most critical risk factors for different oral diseases. The aim of this study is to demonstrate the effect of tobacco on oral mucosa by cytomorphometric analysis of cells with the help of exfoliative cytology and to find out the improvement in diagnostic sensitivity of exfoliative cytology in the detection of dysplastic changes and early oral malignancy.MethodsThe nuclear area (NA) and cytoplasmic area (CA) of cells were measured within cytological smear obtained from leukoplakia lesions of buccal mucosa of 90 tobacco users, 30 smokers (TS), 30 chewers (TC) and 30 with combined habit of smoking and chewing (TSC) and from normal buccal mucosa of 30 non users (NU) of tobacco. Each habit group consisted of 30 tobacco users with oral leukoplakia lesion with mild epithelial dysplasia only. The 30 non-users of tobacco served as controls. The mean values of the CA and NA were obtained for each case, and the nuclear/cytoplasmic area (NA/CA) ratio was calculated.ResultsThe results showed a statistically significant increase (P<0.001) in mean NA and a statistically significant decrease (P<0.001) in mean CA values of tobacco users with leukoplakia as compared to non-users, hence NA/CA ratio value was significantly higher in tobacco users with the lesion.ConclusionThe changes in cellular morphology caused by tobacco use can be visualized by use of exfoliative cytology with the help of cytomorphometric analysis. The evaluation of parameters (NA, CA and NA/CA ratio) may increase the sensitivity of exfoliative cytology for the early diagnosis of oral premalignant and malignant lesions.

Cytomorphometric Study of Changes in Buccal Mucosal Cells in Alcoholics.

Background:Chronic alcohol consumption carries a high risk for oral and pharyngeal cancers among persons who have never smoked. Excessive alcohol consumption displays cytogenetic changes in oral mucosa cells. Cytomorphometric analysis of oral mucosal cells helps in the early detection of cytomorphological transformations in alcoholics before and after the onset of carcinoma.Materials and Methods:A prospective, hospital-based, comparative study was done after written informed consent. Smears were obtained from the clinically normal buccal mucosa of 102 randomly selected alcoholic patients attending the medicine outpatient department aged above 25 years who consumed a minimum of 45 ml alcohol per day for at least 10 years and of 102 nonalcoholics as control. The slides were immediately fixed in absolute methanol and stained by the Papanicolaou (Pap) staining technique. PAP-stained smears were examined under the light microscope. Using the image J 1.47 image analysis software, a morphometric analysis of around 50 cells/case was done.Results:A statistically significant increase in mean cytoplasmic area (P < 0.001), mean nuclear area (P < 0.01), and cell-to-nuclear parameter ratio (P < 0.001) was seen in the alcohol group in comparison with the control group.Conclusion:Prolonged consumption of alcohol produces cytomorphometric changes in buccal mucosal cells before the onset of premalignant lesions.