MOLECULAR CYTPATHOLOGY Section Editors: Dara Aisner, M.D. and Anders Hjerpe, M.D.
Analysis of Morphological Markers of Chromosomal Instability in Ascitic Fluid Ruchita Tyagi, MBBS, MD, DNB, PDCC,1 Pranab Dey, MBBS, MD, MIAC, FRCPATh,2* Radha Uppal, MSC,2 and Arvind Rajwanshi, MBBS, MD, MIAC, FRCPATh2
Chromosomal instability (CI) plays a major role in the carcinogenesis. Micronuclei, nuclear budding, chromatin bridges,and multipolar mitoses are the morphological markers of CI and have never been studied in routine cytological specimens. Aims of the study is to analyze the significance of morphological markers of CI in malignant and benign ascitic fluid smears. A total of sixty benign and 40 malignant ascitic fluid samples were selected for this study. All the cases with malignant ascitic fluid showed histopathological evidence of malignancy in ovary and omentum. Chromatin bridges, multipolar mitosis (MPM), micronuclei and nuclear budding were counted in 1000 cells in representative May Grunwald Giemsa (MGG) stained smears. The CI markers were correlated with the cytological diagnosis of effusion. The mean number of micronuclei, nuclear budding, chromatin bridge and multipolar mitoses found in malignant effusions were 13.2611.79, 10.1067.07, 2.5362.67, 1.964.5, respectively. The mean number of micronuclei, nuclear budding, anaphase bridges, and MPM found in benign effusion cases were 0.566761.07934, 0.516761.33, 0.66760.25, and 0, respectively. The student t test showed significant differences between malignant and benign ascitic fluid samples for each marker of CI. This is the first comprehensive study of morphological markers of CI in ascitic fluid smears. This study has shown strong correlation between markers of CI and cytological diagnosis of malignancy. In future, the knowledge of these markers can be applied to diagnose malignancy
1 Department of Pathology, Postgraduate Institute of Medical Education and Research, Chandigarh, India 2 Department of Cytopathology and Gynecological Pathology, Postgraduate Institute of Medical Education and Research, Chandigarh, India *Correspondence to: Dr Pranab Dey, MD, MIAC, FRCPath, Professor, Department of Cytopathology and Gynecological Pathology, Postgraduate Institute of Medical Education and Research, Chandigarh, India. E-mail: [email protected] Conflict of interest: There is no conflict of interest in this article Received 8 November 2013; Revised 12 November 2014; Accepted 17 December 2014 DOI: 10.1002/dc.23249 Published online 00 Month 2015 in Wiley Online Library (wileyonlinelibrary.com).
C 2015 WILEY PERIODICALS, INC. V
in suspected cases of effusion in difficult situations. Diagn. Cytopathol. 2015;00:000–000. VC 2015 Wiley Periodicals, Inc. Key Words: chromosomal instability; carcinoma; ascitic fluid; chromosomal bridge; micronucleus
Chromosomal instability (CI) plays a major role in carcinogenesis.1 The various sophisticated and expensive techniques like cytokinesis block, immunostaining with specific antibodies against centromeres, telomeres, DNA double stranded breaks, fluorescent and time lapse microscopy, flow cytometry, fluorescent in situ hybridization and comparative genomic hybridization have been used to study CI.2 The presence of chromatin bridges (CB), multipolar mitosis (MPM), nuclear budding and micronuclei on morphology has been proven to be an indicator of CI.3 These markers of CI can help to distinguish between benign and malignant effusions in difficult situations. There have been very few studies on the significance of these morphological parameters as markers of CI. Dey et al. have studied the significance of micronuclei as marker of CI in effusions, urine smears and cervical smears.4–10 There is no comprehensive study on the significance of combined morphological markers of CI in ascitic fluids. The aim of this study is to analyze the various morphological markers of CI including CB, MPM, nuclear budding, and micronuclei in ascitic fluid smears and compare their significance in malignant and benign cases. To the best of our knowledge, this is the first comprehensive cytological study to analyze morphological markers of CI in the ascitic fluid smears.
Materials and Methods Ethical justification This is a retrospective study and the work was done on archival slides. No special test was done from the Diagnostic Cytopathology, Vol. 00, No 00
1
Diagnostic Cytopathology DOI 10.1002/dc
TYAGI ET AL.
Fig. 1. A: Nuclear budding. B: MN. C: CB. D: Multipolar mitotic figure. (may grunwald giemsa stain 3 oil immersion). Arrow indicates the feature mentioned above. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
patient’s material. Written consent was taken from the patient before the ascetic fluid tap. Patient’s identity was kept as confidential. In this study, we selected 40 malignant and 60 benign ascitic fluid samples. All the malignant cases had histopathological evidence of high grade ovarian adenocarcinoma in omentum. The benign ascetic fluid samples were negative for malignancy in cytology smear and also on clinical follow up. In each case a single sample was received and processed by centrifuged technique. Both May Grunwald Giemsa (May– Grunwald–Giemsa) and Papanicolaou’s stain were done in each sample. One May–Grunwald–Giemsa stained cellular smear from each case was chosen to study the CI markers. The smear was screened for CB, MPM, micronuclei, and nuclear buds per 1000 malignant cells by two independent observers (RT and PD). The significance of each morphological marker of CI was compared between benign and malignant cases applying the student t test. 2
Diagnostic Cytopathology, Vol. 00, No 00
Results Nuclear buds (NB) These were represented by sessile or pedunculated bud like protrusions of nuclear membrane (Fig. 1A) .The score of NB/1000 cells ranged from 0 to 35 (mean 10.10 6 7.07) in malignant cases and 0–8 (mean 0.5167 6 1.33) in benign cases.
Micronucleus (MN) MN is a small, separately lying additional nucleus, with size approximately one third of main nucleus, and is easily detected on light microscopy (Fig. 1B). Genetic damage causes chromosome fragments or whole chromosomes to lag behind at anaphase during nuclear division and these chromosomal fragments constitute the MN. Thus, MN score can be used as an indicator of genetic damage. The MN score ranged from 1 to 58 (mean 13.2 6 11.79) in malignant effusions and 0–5 (mean 0.576 1.079) in benign effusions. Thus, the
Diagnostic Cytopathology DOI 10.1002/dc
CHROMOSOMAL INSTABILITY IN ASCITIC FLUID Table I. Comparison Between Presence of Various Morphological Markers of CI in Benign and Malignant Cases MN (mean 6 SD/1000 cells)
NB (mean 6 SD/1000 cells)
CB (mean 6 SD/1000 cells)
MPM (mean 6 SD/1000 cells)
14.6 6 11.40 2.8 6 7.79
Analysis of Morphological Markers of Chromosomal Instability in Ascitic Fluid Ruchita Tyagi, MBBS, MD, DNB, PDCC,1 Pranab Dey, MBBS, MD, MIAC, FRCPATh,2* Radha Uppal, MSC,2 and Arvind Rajwanshi, MBBS, MD, MIAC, FRCPATh2
Chromosomal instability (CI) plays a major role in the carcinogenesis. Micronuclei, nuclear budding, chromatin bridges,and multipolar mitoses are the morphological markers of CI and have never been studied in routine cytological specimens. Aims of the study is to analyze the significance of morphological markers of CI in malignant and benign ascitic fluid smears. A total of sixty benign and 40 malignant ascitic fluid samples were selected for this study. All the cases with malignant ascitic fluid showed histopathological evidence of malignancy in ovary and omentum. Chromatin bridges, multipolar mitosis (MPM), micronuclei and nuclear budding were counted in 1000 cells in representative May Grunwald Giemsa (MGG) stained smears. The CI markers were correlated with the cytological diagnosis of effusion. The mean number of micronuclei, nuclear budding, chromatin bridge and multipolar mitoses found in malignant effusions were 13.2611.79, 10.1067.07, 2.5362.67, 1.964.5, respectively. The mean number of micronuclei, nuclear budding, anaphase bridges, and MPM found in benign effusion cases were 0.566761.07934, 0.516761.33, 0.66760.25, and 0, respectively. The student t test showed significant differences between malignant and benign ascitic fluid samples for each marker of CI. This is the first comprehensive study of morphological markers of CI in ascitic fluid smears. This study has shown strong correlation between markers of CI and cytological diagnosis of malignancy. In future, the knowledge of these markers can be applied to diagnose malignancy
1 Department of Pathology, Postgraduate Institute of Medical Education and Research, Chandigarh, India 2 Department of Cytopathology and Gynecological Pathology, Postgraduate Institute of Medical Education and Research, Chandigarh, India *Correspondence to: Dr Pranab Dey, MD, MIAC, FRCPath, Professor, Department of Cytopathology and Gynecological Pathology, Postgraduate Institute of Medical Education and Research, Chandigarh, India. E-mail: [email protected] Conflict of interest: There is no conflict of interest in this article Received 8 November 2013; Revised 12 November 2014; Accepted 17 December 2014 DOI: 10.1002/dc.23249 Published online 00 Month 2015 in Wiley Online Library (wileyonlinelibrary.com).
C 2015 WILEY PERIODICALS, INC. V
in suspected cases of effusion in difficult situations. Diagn. Cytopathol. 2015;00:000–000. VC 2015 Wiley Periodicals, Inc. Key Words: chromosomal instability; carcinoma; ascitic fluid; chromosomal bridge; micronucleus
Chromosomal instability (CI) plays a major role in carcinogenesis.1 The various sophisticated and expensive techniques like cytokinesis block, immunostaining with specific antibodies against centromeres, telomeres, DNA double stranded breaks, fluorescent and time lapse microscopy, flow cytometry, fluorescent in situ hybridization and comparative genomic hybridization have been used to study CI.2 The presence of chromatin bridges (CB), multipolar mitosis (MPM), nuclear budding and micronuclei on morphology has been proven to be an indicator of CI.3 These markers of CI can help to distinguish between benign and malignant effusions in difficult situations. There have been very few studies on the significance of these morphological parameters as markers of CI. Dey et al. have studied the significance of micronuclei as marker of CI in effusions, urine smears and cervical smears.4–10 There is no comprehensive study on the significance of combined morphological markers of CI in ascitic fluids. The aim of this study is to analyze the various morphological markers of CI including CB, MPM, nuclear budding, and micronuclei in ascitic fluid smears and compare their significance in malignant and benign cases. To the best of our knowledge, this is the first comprehensive cytological study to analyze morphological markers of CI in the ascitic fluid smears.
Materials and Methods Ethical justification This is a retrospective study and the work was done on archival slides. No special test was done from the Diagnostic Cytopathology, Vol. 00, No 00
1
Diagnostic Cytopathology DOI 10.1002/dc
TYAGI ET AL.
Fig. 1. A: Nuclear budding. B: MN. C: CB. D: Multipolar mitotic figure. (may grunwald giemsa stain 3 oil immersion). Arrow indicates the feature mentioned above. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
patient’s material. Written consent was taken from the patient before the ascetic fluid tap. Patient’s identity was kept as confidential. In this study, we selected 40 malignant and 60 benign ascitic fluid samples. All the malignant cases had histopathological evidence of high grade ovarian adenocarcinoma in omentum. The benign ascetic fluid samples were negative for malignancy in cytology smear and also on clinical follow up. In each case a single sample was received and processed by centrifuged technique. Both May Grunwald Giemsa (May– Grunwald–Giemsa) and Papanicolaou’s stain were done in each sample. One May–Grunwald–Giemsa stained cellular smear from each case was chosen to study the CI markers. The smear was screened for CB, MPM, micronuclei, and nuclear buds per 1000 malignant cells by two independent observers (RT and PD). The significance of each morphological marker of CI was compared between benign and malignant cases applying the student t test. 2
Diagnostic Cytopathology, Vol. 00, No 00
Results Nuclear buds (NB) These were represented by sessile or pedunculated bud like protrusions of nuclear membrane (Fig. 1A) .The score of NB/1000 cells ranged from 0 to 35 (mean 10.10 6 7.07) in malignant cases and 0–8 (mean 0.5167 6 1.33) in benign cases.
Micronucleus (MN) MN is a small, separately lying additional nucleus, with size approximately one third of main nucleus, and is easily detected on light microscopy (Fig. 1B). Genetic damage causes chromosome fragments or whole chromosomes to lag behind at anaphase during nuclear division and these chromosomal fragments constitute the MN. Thus, MN score can be used as an indicator of genetic damage. The MN score ranged from 1 to 58 (mean 13.2 6 11.79) in malignant effusions and 0–5 (mean 0.576 1.079) in benign effusions. Thus, the
Diagnostic Cytopathology DOI 10.1002/dc
CHROMOSOMAL INSTABILITY IN ASCITIC FLUID Table I. Comparison Between Presence of Various Morphological Markers of CI in Benign and Malignant Cases MN (mean 6 SD/1000 cells)
NB (mean 6 SD/1000 cells)
CB (mean 6 SD/1000 cells)
MPM (mean 6 SD/1000 cells)
14.6 6 11.40 2.8 6 7.79
