Can Mitotic Figures in Hyperchromatic Crowded Groups be Cytodiagnostic Criteria for High-Grade Squamous Intra-epithelial Lesions?

Aims: The present study aimed to investigate whether the presence of mitoses in hyperchromatic crowded groups (HCGs) in cervical cytological specimens can serve as cytological criteria for high-grade squamous intra-epithelial lesions (HSILs). Methods and Material: Various parameters were examined, including the frequency of mitotic figures per high power field (HPF) in Pap, hematoxylin eosin (HE) samples, and PHH3 immunocytochemical (ICC) and immunohistochemical (IHC) analyses. Results: In the Pap and PHH3-ICC samples, the number of mitotic figures observed in HCGs was significantly higher in HSIL (P < 0.001) compared to other groups. Furthermore, the frequency of observing two or more mitoses was significantly higher in HSIL (Pap: P = 0.002, PHH3-ICC: P < 0.001) than in low-grade squamous intra-epithelial lesions (LSILs). Moreover, a comparison between Pap samples and PHH3-ICC showed that the frequency of two or more mitoses was significantly higher in the PHH3-ICC analysis of HSIL (P = 0.042). Regarding HE and PHH3-IHC samples, counting the number of mitoses in the lower and middle/upper layers of the squamous epithelial layer revealed that HSIL had a significantly higher value (HE: P = 0.0089, PHH3-IHC: P = 0.0002) than LSIL in the middle/upper layers. Conclusions: Hence, the presence of two or more mitotic figures in HCGs per HPF in cervical cytology indicates a suspicion of HSIL. The detection of mitoses in PHH3-ICC samples is more sensitive and easier to observe than in Pap samples, making it a valuable mitotic marker.

The 1000 Mitoses Project: A Consensus-Based International Collaborative Study on Mitotic Figures Classification.

Introduction. The identification of mitotic figures is essential for the diagnosis, grading, and classification of various different tumors. Despite its importance, there is a paucity of literature reporting the consistency in interpreting mitotic figures among pathologists. This study leverages publicly accessible datasets and social media to recruit an international group of pathologists to score an image database of more than 1000 mitotic figures collectively. Materials and Methods. Pathologists were instructed to randomly select a digital slide from The Cancer Genome Atlas (TCGA) datasets and annotate 10-20 mitotic figures within a 2 mm2 area. The first 1010 submitted mitotic figures were used to create an image dataset, with each figure transformed into an individual tile at 40x magnification. The dataset was redistributed to all pathologists to review and determine whether each tile constituted a mitotic figure. Results. Overall pathologists had a median agreement rate of 80.2% (range 42.0%-95.7%). Individual mitotic figure tiles had a median agreement rate of 87.1% and a fair inter-rater agreement across all tiles (kappa = 0.284). Mitotic figures in prometaphase had lower percentage agreement rates compared to other phases of mitosis. Conclusion. This dataset stands as the largest international consensus study for mitotic figures to date and can be utilized as a training set for future studies. The agreement range reflects a spectrum of criteria that pathologists use to decide what constitutes a mitotic figure, which may have potential implications in tumor diagnostics and clinical management.

Grading medullary thyroid carcinoma on fine-needle aspiration cytology specimens with the International Medullary Thyroid Carcinoma Grading System: A cytologic-histologic correlation.

BACKGROUND: Medullary thyroid carcinoma (MTC) is a rare cancer of parafollicular C-cell origin. The International MTC Grading System (IMTCGS) incorporates mitotic activity, the presence of necrosis, and the Ki67 proliferation rate (PR) to classify MTCs as low or high grade. The ability to predict IMTCGS grade in cytology was assessed. METHODS: MTCs with cytology and subsequent surgical follow-up were reviewed. Cytology slides were reviewed for mitotic figures, apoptoses, and necrosis, and a Ki67 PR was calculated when possible. Findings were correlated with final IMTCGS grade. RESULTS: Twenty-five MTC fine-needle aspirations (FNAs) were identified, with nine identified as high grade (36%). By using a PR cutoff of 5%, Ki67 on FNA material (Ki67FNA) showed 92% concordance (n = 22 of 24) with surgical Ki67 and a correlation coefficient (R2) of 0.72. Sensitivity and specificity of Ki67FNA for predicting high-grade MTC were 38% and 100%, respectively. Multiple mitotic figures were present in a single slide of 43% (n = 3 of 7) of evaluable high-grade MTCs, whereas only one of 16 low-grade MTCs showed a single mitotic figure. Definitive apoptoses were present in five of seven high-grade MTC FNAs but were absent in 16 low-grade MTCs. The sensitivity and specificity of apoptoses/necrosis on cytology for high-grade MTCs were 71% and 88%, respectively. CONCLUSIONS: Ki67FNA ≥5% shows low sensitivity but high specificity for predicting high-grade MTC. The presence of multiple mitotic figures in a single slide or definitive apoptotic bodies are both highly suggestive of high-grade MTC, and should warrant a close examination for necrosis and a careful Ki67 PR count.

Low-grade sinonasal non-intestinal-type adenocarcinoma: a rare case report and literature review.

Sinonasal non-intestinal-type adenocarcinoma is a rare but important differential diagnosis in patients presenting with recurrent, unexplained epistaxis. Low-grade types have a more favourable prognosis as opposed to the more aggressive high-grade. Symptoms include nasal obstruction and epistaxis that can last up to 5 years. We report a case of a rare low-grade sinonasal non-intestinal-type adenocarcinoma in a 43-year-old male who is frequently exposed to wood and dust particles. Endoscopy revealed right nasal mass occupying the entire nasal cavity as well as inferior turbinate hypertrophy and mass attached to the nasal septum on computed tomography. Biopsy confirmed the diagnosis and was classified as pT1NX with the presence of mitotic figures, which are more commonly present in the high-grade subtype.

Inclusion of fibroblasts and collagen fibrils in the cytologic grading of canine cutaneous mast cell tumors.

BACKGROUND: Fibroblasts and/or collagen fibrils have not been included in previous cytologic grading schemes of canine mast cell tumors (MCTs), and their association with biological behavior is broadly debated. OBJECTIVES: This study aimed to evaluate the cytologic findings of canine MCT, with emphasis on the microenvironment, and propose a novel cytologic grading system correlated with mortality and histologic grade. MATERIAL AND METHODS: Cytology smears of canine cutaneous MCTs were retrospectively reviewed and compared with their histopathologic counterparts using Cohen´s Kappa test. One-year survival rates were also compared with the cytologic and histopathologic variables using Pearson´s correlation test. RESULTS: From 92 first-occurrence canine cutaneous MCTs, the five features most associated with mortality were selected for a new grading system. The five features were cytoplasmic granulation, fibroblast and/or collagen fibril concentrations, and the presence of mitotic figures, multinucleation, and karyomegaly. Among concordant histopathologic and cytologic cases (ie, the same grades using both systems), mortality rates were 2.6% (1/38) for low-grade and 71.4% (10/14) for high-grade cases (P < 0.001, chi-square). For false-negative and false-positive results, mortality rates were 33% (1/3) and 45% (5/11), respectively (P = 0.707). CONCLUSIONS: Unlike the Camus cytologic grading system, the present amendment excluded binucleation and included fibroblasts and/ or collagen fibrils, which in higher concentrations were associated with increased survival and a low histopathologic grade. Cytologic grading with the inclusion of fibroblast and collagen fibril concentrations correlated with survival, as did the Camus cytologic and Kiupel histopathologic grades; however, further studies are needed to confirm the prognostic value of this novel cytologic grading scheme.

Computer-assisted mitotic count using a deep learning-based algorithm improves interobserver reproducibility and accuracy.

The mitotic count (MC) is an important histological parameter for prognostication of malignant neoplasms. However, it has inter- and intraobserver discrepancies due to difficulties in selecting the region of interest (MC-ROI) and in identifying or classifying mitotic figures (MFs). Recent progress in the field of artificial intelligence has allowed the development of high-performance algorithms that may improve standardization of the MC. As algorithmic predictions are not flawless, computer-assisted review by pathologists may ensure reliability. In the present study, we compared partial (MC-ROI preselection) and full (additional visualization of MF candidates and display of algorithmic confidence values) computer-assisted MC analysis to the routine (unaided) MC analysis by 23 pathologists for whole-slide images of 50 canine cutaneous mast cell tumors (ccMCTs). Algorithmic predictions aimed to assist pathologists in detecting mitotic hotspot locations, reducing omission of MFs, and improving classification against imposters. The interobserver consistency for the MC significantly increased with computer assistance (interobserver correlation coefficient, ICC = 0.92) compared to the unaided approach (ICC = 0.70). Classification into prognostic stratifications had a higher accuracy with computer assistance. The algorithmically preselected hotspot MC-ROIs had a consistently higher MCs than the manually selected MC-ROIs. Compared to a ground truth (developed with immunohistochemistry for phosphohistone H3), pathologist performance in detecting individual MF was augmented when using computer assistance (F1-score of 0.68 increased to 0.79) with a reduction in false negatives by 38%. The results of this study demonstrate that computer assistance may lead to more reproducible and accurate MCs in ccMCTs.

Correlation between cytologic features and histologic grades in cutaneous and subcutaneous soft tissue sarcomas in dogs-A pilot study.

Currently, canine soft tissue sarcoma (STS) grading is based on histopathology. In humans, several studies have demonstrated concordance between cytologic grading systems for STS and histologic grade. The aim of this study was to correlate several cytologic parameters (smear cellularity, anisokaryosis, nucleolar malignancy score, multinucleation, and the number of mitotic figures per 200 cells) that form part of a human STS cytologic grading system, with histologic grades of canine cutaneous and subcutaneous STS. Three observers (blinded) reviewed the cytologic preparations independently from cases with confirmed histologic diagnoses of STS. A cytologic grading score was assigned for each parameter. Correlations between cytologic grading scores (averaged between observers) and histologic grades were assessed using Spearman's correlation coefficient, with statistical significance defined as P < .05. Twenty-one cases were included in the study (10 Grade I STS, nine Grade II STS, and two Grade III STS). The number of mitotic figures (≥3) per 200 cells was the only parameter that showed a significant but weak, positive correlation with histologic grade (rs  = .469; P = .032). No Grade I tumors had ≥3 mitotic figures per 200 cells; however, ≥3 mitotic figures per 200 cells were only observed in 33% of Grade II tumors and 50% (one out of two) of the Grade III tumors. This pilot study suggests that an increased number of mitotic figures seen on cytology might correlate with higher grade STS; however, the sensitivity of this parameter for grading STS appears to be low.

Epithelial Distribution of E-Cadherin, p63, and Mitotic Figures in ApoTome Images to Determine the Oncogenic Potentiality of Oral Submucous Fibrosis.

The exact process of the malignant conversion of oral submucous fibrosis (OSF) to oral cancer is not fully understood. This study aimed to detect and analyze E-cadherin expression, p63 expression, and number of mitotic figures, all correlated to cancer development, in ApoTome images of oral tissues to determine the oncogenic potentiality of OSF. ApoTome images of the study groups (6 normal, 16 OSF with dysplasia, and 10 OSF without dysplasia) were recorded. Cytoplasmic and membranous E-cadherin expression, breakages of the cell membrane, and p63 expression were detected in MATLAB 2016b. The number of mitotic figures detected by MATLAB was correlated with the number of chromosomes detected by ImageJ. A Mann­Whitney U test was done to determine a significant difference between the study groups for cytoplasmic and membranous E-cadherin distribution points. Statistical significant differences were found for cytoplasmic E-cadherin distribution between normal and OSF (with dysplasia) (p = 0.0278). There was an increase in mitotic figures, p63 expression, and cytoplasmic E-cadherin expression and a decrease in membranous E-cadherin expression from normal to diseased condition. Hence, automated detection and quantification of E-cadherin, p63, and mitotic figures in ApoTome images of oral biopsies can help in determining the oncogenic potentiality of OSF.

Giant cell-rich osteosarcoma - A rare case.

Giant cell-rich osteosarcoma (GCRO) is an exceedingly rare histological variant of conventional primary osteosarcoma. It constitutes about 1%-3% of all osteosarcomas, and is extremely uncommon in the maxillofacial region. The unusual histopathological appearance and the rarity of the lesion poses a great diagnostic challenge. This article aims to present a rare case of GCRO involving the mandible in a 52-year-old male patient.

Efficiency of Crystal Violet Stain to Study Mitotic Figures in Oral Epithelial Dysplasia.

AIM: To evaluate mitotic activity in the different grades of oral epithelial dysplasia using 1% crystal violet stain. MATERIAL AND METHODS: A descriptive study was conducted in the Department of Histopathology of the Post Graduate Medical Institute, Lahore on a total of thirty-three cases of the Oral Epithelial Dysplasia (OED). Fresh, frozen paraffin-embedded archival tissue blocks were collected from Lahore General Hospital, Lahore & Oral & Maxillofacial Surgery Department of Nawaz Sharif Hospital, Yakki Gate, Lahore. The representative sections were taken and, after processing, mounted on glass slides and stained with H&E and crystal violet stains. The stained slides were then examined under an optical microscope. The efficacy of 1% crystal violet stain to identify mitotic figures in the different grades of oral epithelial dysplasia was assessed with the sample t-test. A difference of p < 0.05 was considered to be significant. RESULTS: A comparison of the mitotic figure count in two categories in sections stained with both stains showed a statistically significant difference. An increase in the mean mitotic count was noted in the sections of OED stained with crystal violet in comparison to the sections of OED stained with H&E which was statistically significant (p = 0.00). CONCLUSION: Counting of mitotic cell is the rapid and simplest way of evaluating the proliferative activity of cells. Crystal violet stain can be a rationalised step in the staining of mitotic figures compared to the usual H&E staining and can be employed as a selective stain during routine histopathological procedures.

Mitotic activity in noninvasive papillary urothelial carcinoma: its value in predicting tumor recurrence and comparison with the contemporary 2-tier grading system.

Measures of mitotic activity predict behavior of noninvasive papillary urothelial carcinoma of the urinary bladder, but it is unclear what role these should have in tumor grading. In this article, we compare measures of mitotic activity to contemporary tumor grading, specifically in their association with recurrence of noninvasive papillary urothelial carcinoma. The study uses a retrospective cohort of 199 tumors from 124 patients. Mitotic activity was treated as a categorical variable (mitotic-inert, mitotic-low, or mitotic-high). Evaluating only first-occurrence tumors, recurrence was more frequent in mitotic-high (hazard ratio [HR], 8.8; P < .0001, Cox model) and mitotic-low tumors (HR, 3.7; P = .017) compared with mitotic-inert tumors, when controlling for treatment with intravesical bacillus Calmette-Guerin, age, and sex. Recurrence was likewise more frequent in high-grade tumors (HR, 3.1; P = .00019, Cox model) compared with low-grade tumors, controlling for these factors. However, mitotic group, but not tumor grade, was significantly associated with recurrence in a multivariate Cox model including mitotic group, tumor grade, and treatment status (HR, 6.5 [P = .0025] for mitotic-high versus reference; HR, 3.7 [P = .018] for mitotic-low versus reference). Frailty models including both first-occurrence and recurrent tumors showed similar results. Isolating the analysis to first occurrence, low-grade tumors, recurrence was more frequent in mitotic-high (HR, 6.8; P = .0044, Cox model) and mitotic-low (HR, 3.4; P = .027) tumors compared with mitotic-inert tumors. The findings indicate that mitotic activity is associated with behavior of noninvasive papillary urothelial carcinoma and may be valuable as an adjunct to the contemporary grading system.

Detecting Chromosome Condensation Defects in Gulf War Illness Patients.

BACKGROUND: Gulf War Illness (GWI) impacts 25-30% of gulf war veterans. Due to its heterogeneity in both etiology and symptoms, it has been challenging to establish the commonly accepted case definition for GWI. Equally challenging are the understanding of the general mechanism of GWI and the development of biomarkers useful for its clinical diagnosis and treatment. OBJECTIVE: We have observed that chromosome condensation defects can be detected in GWI patients. To document this phenomenon in GWI, we aim to describe and compare different types of chromosomal condensation defects in GWI patients, if possible. Since chromosomal condensation represents an important step of ensuring genome integrity, condensation defects could be used as a potential biomarker of GWI. METHODS: Lymphocytes from GWI patients have been used for short term cell culture followed by chromosome slide preparation. Both Giemsa staining and multiple color spectral karyotyping (SKY) were applied to study chromosome aberrations, focusing on different types of condensation defects. RESULTS: At least three subtypes of Defective Mitotic Figures (DMFs) were observed. Some individuals displayed elevated frequencies of DMFs. Another type of condensation defect identified as sticky chromosomes were also observed. CONCLUSION: Various types of condensation defects have been observed in GWI patients. It is rather surprising that some GWI patients exhibited a high level of chromosomal condensation defects. Previously, the elevated frequency of DMFs was only observed in cancer patients. Since chromosome condensation can be linked to other types of chromosome aberrations, as well as cellular stress conditions, the detailed mechanism and clinical impact should be further studied, especially with increased sample size.

Determining Image Processing Features Describing the Appearance of Challenging Mitotic Figures and Miscounted Nonmitotic Objects.

CONTEXT: Previous studies showed that the agreement among pathologists in recognition of mitoses in breast slides is fairly modest. AIMS: Determining the significantly different quantitative features among easily identifiable mitoses, challenging mitoses, and miscounted nonmitoses within breast slides and identifying which color spaces capture the difference among groups better than others. MATERIALS AND METHODS: The dataset contained 453 mitoses and 265 miscounted objects in breast slides. The mitoses were grouped into three categories based on the confidence degree of three pathologists who annotated them. The mitoses annotated as "probably a mitosis" by the majority of pathologists were considered as the challenging category. The miscounted objects were recognized as a mitosis or probably a mitosis by only one of the pathologists. The mitoses were segmented using k-means clustering, followed by morphological operations. Morphological, intensity-based, and textural features were extracted from the segmented area and also the image patch of 63 × 63 pixels in different channels of eight color spaces. Holistic features describing the mitoses' surrounding cells of each image were also extracted. STATISTICAL ANALYSIS USED: The Kruskal-Wallis H-test followed by the Tukey-Kramer test was used to identify significantly different features. RESULTS: The results indicated that challenging mitoses were smaller and rounder compared to other mitoses. Among different features, the Gabor textural features differed more than others between challenging mitoses and the easily identifiable ones. Sizes of the non-mitoses were similar to easily identifiable mitoses, but nonmitoses were rounder. The intensity-based features from chromatin channels were the most discriminative features between the easily identifiable mitoses and the miscounted objects. CONCLUSIONS: Quantitative features can be used to describe the characteristics of challenging mitoses and miscounted nonmitotic objects.

Image analysis assisted study of mitotic figures in oral epithelial dysplasia and squamous cell carcinoma using differential stains.

BACKGROUND: Mitosis is a process of cell division resulting in two genetically equivalent daughter cells. Excessive proliferation of cells due to mitosis is the hallmark in pre cancer and cancer. AIMS: This study was conducted to count the number of mitotic figures in normal oral mucosa, oral epithelial dysplasia and squamous cell carcinoma in both Hematoxylin and Eosin (H&E) and Crystal Violet stained sections. Also the overall number of mitotic figures with both stains were compared along with the evaluation of staining efficacy of both the stains. METHODS AND MATERIAL: The present study was conducted on 20 specimens each of the three categories. These were further divided into two groups for staining with H&E and with 1% Crystal Violet respectively. Images were captured and analyzed using image analysis software Dewinter Biowizard 4.1. RESULTS: Comparison of mitotic figure count in three categories in sections stained with both stains showed statistically significant difference (p < 0.001). The mean number of mitotic figures seen in Crystal Violet reagent were significantly higher as seen in H&E stain (p < 0.001). The overall diagnostic efficacy of Crystal Violet was 87.6%. Crystal Violet scored over H&E stain and also helped to better appreciate metaphases in Squamous cell carcinoma and telophases in dysplasia. CONCLUSION: Number of mitotic figures progressively increase with the advancement of the pathology. Use of 1% Crystal Violet provides better appreciation of mitotic figures and can be employed as a selective stain in routine histopathology.

Fibrosarcomatous change in the background of dermatofibrosarcoma protuberans in male breast: Study of a rare case and review of the entity.

Dermatofibrosarcoma protuberans is a cutaneous soft tissue neoplasm with potential for local recurrence but distant metastasis is rare. Trunk and extremities are most commonly involved. This case presented as left-sided breast lump in a male patient. The patient underwent left-sided modified radical mastectomy. Tissues were subjected to histopathological and immunohistochemical test subsequently. The tumor cells showed storiform arrangement with nuclear pleomorphism and increased mitotic figures at places. They were reactive to CD34 and non-reactive to S-100, smooth muscle actin, desmin, cytokeratin and epithelial membrane antigen. The diagnosis of dermatofibrosarcoma protuberans with areas of fibrosarcomatous change was given. Though trunk is a common site for this tumor but its presentation as male breast lump has made the case unique.

Evaluation of efficacy of 1% Crystal Violet & Nuclear Fast Red stain compared to Haematoxyline & Eosin stain for assessing mitotic figures in oral premalignant and malignant lesions.

Various chromosomal arrangements in cells undergoing division are referred to as Mitotic figure (MF). The abnormal excess of mitotic figures is commonly seen in oral epithelial dysplasia (ED) and oral squamous cell carcinoma (OSCC). In present study, we compared the number of mitotic figures in normal oral mucosa, epithelial dysplasia & OSCC sections with haematoxyline & eosine (H&E) and 1%Crystal Violet & Nuclear Fast Red (CV&NFR) stain, also the efficacy of the CV&NFR stain as compared to H & E stain. We investigated the correlation between the number of mitotic figures & grades of OSCC. Study sample comprised of two serial sections of archival blocks of normal oral mucosa & diagnosed cases of epithelial dysplasia & OSCC. One slide stained with H& E & the other one with 1% CV & NFR. Mitotic figures were counted with the grid eyepiece. There was significant increase in number of MFs in oral ED and OSCC in comparison with normal oral mucosa. There was a highly significant increase in number of MFs in CV&NFR stained tissue sections when compared with H & E stain. Metaphase is the most commonly observed phase of mitosis. In summary, our study proposes the use of Crystal violet & Nuclear fast red stain as a selective stain for better contrast & easy identification MFs.

Intracystic Papillary Carcinoma in Male Breast with High Nuclear Grade: A Case Report.

Intracystic papillary carcinoma (IPC) in men is an extremely rare disease that accounts for less than 1% of all malignancies with only a few case presentations published so far. This report presents a case of 53-year-old male, who presented a painless swelling of his left breast. The left breast mass was 6 cm maximally and was found to be non-invasive IPC. The tumour is consistently positive for GCDFP-15, ER or PR and negative for HER-2. It consists of predominantly fibrovascular stromal lined by monotonous epithelial cells retaining intermediate to high histological grade with a high nuclear cytoplasmic ratio. In addition, increased numbers of mitotic figures were also seen. Chest X-ray, liver ultrasound and bone centigram showed no evidence of distant metastases. In short, this is the first case report from Saudi Arabia of a male having IPC, with increased number of mitotic figures and high nuclear grades.

Neuroendocrine carcinoma of the stomach-a case report.

Neuroendocrine carcinomas of stomach have been considered a rare neoplasm. The present case concerns with a 69 year old male, who presented with vague abdominal discomfort and history of malena. Upper alimentary tract endoscopy showed an ulcero-proliferative growth in the antrum. Computed tomography abdomen revealed thickening of the gastric antrum, a subtotal gastrectomy was performed after correction of anemia. Microscopic evaluation revealed a neuroendocrine neoplasm. Immunohistochemically positive for Chromogranin A and Synaptophysin.A diagnosis of Neuroendocrine carcinoma of the stomach was given based on recent WHO classification of Neuroendocrine carcinoma of the stomach and on mitotic index with reference to grading scale.

Mechanisms by which synthetic 6,7-annulated-4-substituted indole compounds with anti-proliferative activity disrupt mitosis and block cytokinesis in human HL-60 tumor cells in vitro.

BACKGROUND: Synthetic 6,7-annulated-4-substituted indole compounds, which elicit interesting antitumor effects in murine L1210 leukemia cells, were tested for their ability to inhibit human HL-60 tumor cell proliferation, disrupt mitosis and cytokinesis, and interfere with tubulin and actin polymerization in vitro. MATERIALS AND METHODS: Various markers of metabolic activity, mitotic disruption and cytokinesis were used to assess the effectiveness of the drugs in the HL-60 tumor cell system. The ability of annulated indoles to alter the polymerizations of purified tubulin and actin were monitored in cell-free assays and were compared to the effects of drugs known to disrupt the dynamic structures of the mitotic spindle and cleavage furrow. RESULTS: With one exception, annulated indoles inhibited the metabolic activity of HL-60 tumor cells in the low-micromolar range after two and four days in culture but these anti-proliferative effects were weaker than those of jasplakinolide, a known actin binder that blocks cytokinesis. After 24-48 h, antiproliferative concentrations of annulated indoles increased the mitotic index of HL-60 cells similarly to vincristine and stimulated the formation of many bi-nucleated cells, multi-nucleated cells and micronuclei, similarly to taxol and jasplakinolide, suggesting that these antitumor compounds might increase mitotic abnormality, induce chromosomal damage or missegregation, and block cytokinesis. Since annulated indoles mimicked the effect of vincristine on tubulin polymerization, but not that of taxol, these compounds might represent a new class of microtubule de-stabilizing agents that inhibit tubulin polymerization. Moreover, annulated indoles remarkably increased the rate and level of actin polymerization similarly to jasplakinolide, suggesting that they might also stabilize the cleavage furrow to block cytokinesis. CONCLUSION: Although novel derivatives with different substitutions must be synthesized to elucidate structure-activity relationships, identify more potent antitumor compounds and investigate different molecular targets, annulated indoles appear to interact with both tubulin to reduce microtubule assembly and actin to block cytokinesis, thereby inducing bi- and multinucleation, resulting in genomic instability and apoptosis.