Molecular Techniques Acta Cytologica 2014;58:495–500 DOI: 10.1159/000367961
Received: July 29, 2014 Accepted after revision: August 27, 2014 Published online: October 25, 2014
Expression of PTEN in Endometrial Liquid-Based Cytology Alessia Di Lorito a Roberta Zappacosta a Serena Capanna a Daniela Mariapia Gatta a Sandra Rosini a Fernando Carlos Schmitt b, c a Experimental and Clinical Science, G. d’Annunzio University, Chieti, Italy; b Institute of Molecular Pathology and Immunology, University of Porto, Porto, Portugal; c Department of Pathology and Medicine, Laboratoire National de Santé, Luxembourg, Luxembourg
Key Words Liquid-based cytology · PTEN · Immunocytochemistry · Endometrial sampling device
All the normal endometrium control cases were positive in cytology and histology. Conclusions: Our results suggest that endometrial devices provide useful material for the diagnosis and evaluation of PTEN expression. © 2014 S. Karger AG, Basel
© 2014 S. Karger AG, Basel 0001–5547/14/0585–0495$39.50/0 E-Mail [email protected] www.karger.com/acy
Introduction
Endometrial cancer represents the most common gynecologic malignancy in developed countries [1]. Dilatation and curettage has long been recognized as the gold standard in the diagnosis of endometrial diseases. However, over the years other endometrial sampling devices have been developed [1]. Molecular alterations in endometrial cancers were previously described using formalin-fixed, paraffin-embedded (FFPE) tissues with particular attention, in endometrioid carcinomas, to the PTEN-PI3K pathway [2–4]. PTEN (phosphatase and tensin homolog) is a tumor suppressor gene that negatively regulates the PI3K-AKT signaling pathway, controlling cell proliferation, growth and apoptosis. Inactivation of PTEN and activation of PI3K produce AKT phosphorylation, β-catenin accumulation in the nuclei and activation of gene transcription [5]. Correspondence to: Dr. Alessia Di Lorito Department of Experimental and Clinical Science Cytopathology Unit, Santissima Annunziata Hospital via dei Vestini, IT–66100 Chieti (Italy) E-Mail alessia.dilorito @ gmail.com
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Abstract Objectives: Endometrial cytology offers a reliable alternative to biopsy in endometrial cancer detection and it may be useful in obtaining material to study prognostic and predictive markers. Over the years, new sampling devices have been developed. Molecular alterations in endometrial cancers were previously described using formalin-fixed paraffinembedded tissues with particular attention, in endometrioid carcinomas, to the PTEN-PI3K pathway. PTEN evaluation could be useful in endometrial carcinomas for selecting patients for target therapies. Study Design: We studied 51 endometrial samples collected using the Endogyn device and 71 obtained with the Endoflower dispositive device, and processed using liquid-based cytology. Most of the cases were matched with a corresponding histological biopsy. The overall accuracy of Endoflower was 100%. Immunohistochemistry (IHC) and immunocytochemistry (ICC) for PTEN were performed using monoclonal antibody 6H2.1 from DAKO. Results: The IHC showed PTEN-null glands in 4 cases. The same cancers were negative in ICC. Among the 10 carcinomas on cytology, PTEN-null glands were found in 1 case.
a
b
Fig. 1. Endometrium sampling devices used in the study: Endogyn (a) and Endoflower (b).
Clinical trials have suggested that PTEN inactivation is linked to the response to mTOR, Akt and PARP inhibitors, and resistance to HER2 inhibitors. In advanced cancer, although responses to first-line treatment are frequently observed, relapses are common and effective therapeutic options for recurrent tumors are lacking [6– 9]. Some studies have suggested an important role of PTEN evaluation in endometrial carcinomas, using FFPE material [10, 11]. In endometrial disease, cytology is a simple, painless, inexpensive and accurate way to obtain material for cancer detection and for morphological diagnosis [12]. It can also be used to study preventive and predictive markers, including PTEN [13]. In this study, the first aim was to assess the applicability of a new device, the Endoflower dispositive (RI-MOS, Mirandola, Italy), in collecting cells from the endometrial cavity. We compared this method with a previous method of sampling, the Endogyn device (Biogyn, Mirandola, Italy). The second aim was to perform a direct comparison of PTEN expression in the cytology and histology of endometrial carcinomas.
Table 1. Comparison of cytological and histological diagnoses using Endogyn and Endoflower devices
Cytology
Histology
Suspicious/positive Negative
suspicious/positive
negative
49 2
2 70
The sensitivity, specificity, NPP and PPV obtained were 96, 97, 97 and 100%, respectively.
ical material was routinely fixed in formalin and embedded in paraffin. The slides were stained with hematoxylin and eosin and examined by two different pathologists, in order to avoid observer bias. Histological diagnoses were made according to World Health Organization criteria [14] and cytological diagnoses were given considering architectural and cytological features. However, for statistical analysis, we considered two categories: ‘negative for malignancy’, including negative findings and hyperplasia without atypia, and ‘suspicious/positive for malignancy’, including cancer and hyperplasia with atypia (table 1). Statistical analyses were based on comparison with McNemar’s test. The percent agreement was also calculated.
Material and Methods
496
Acta Cytologica 2014;58:495–500 DOI: 10.1159/000367961
Immunocytochemical Analysis For the second part of the study, we considered only cancer cases. We examined 12 endometrioid cancer LBC samples with corresponding FFPE blocks (surgical specimens) and 14 endometrioid cancer LBC samples without corresponding FFPE tissues. We used 10 benign endometrial cytological samples with the respective biopsies as controls. Immunohistochemistry (IHC) and immunocytochemistry (ICC) for PTEN were performed using monoclonal DAKO 6H2.1 antibody, as previously described [15, 16]. In particular, for ICC, cytological samples were centrifuged; cell pellets were transferred into the plastic chambers, mounted on glass slides and fixed with 95% ethanol. The slides were left at room temperature for 72 h. The ICC and the IHC were performed according to the manufacturer’s protocol and previous studies [16, 17]. PTEN IHC and ICC were scored as positive and negative. In all IHC cases, stromal cells and
Di Lorito et al.
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Endometrial Sampling and Morphological Diagnoses In the first part of this study, we made a comparison between two different methods of collecting endometrial cells: the Endogyn and the Endoflower devices (fig. 1). We examined the diagnoses of 51 liquid-based cytology (LBC) endometrial samples, obtained from 2000 to 2004 using Endogyn, comparing them with the corresponding histological diagnoses. Then, we analyzed the morphological diagnoses of 70 LBC samples, gathered using Endoflower, with the corresponding biopsies, selected from 2006 to 2013. Samples were centrifuged and the pellet with the cells was put into a PreservCyt® (Cytyc Corporation, Marlborough, Mass., USA) in order to eliminate the mucus and blood. These vials were then processed by a ThinPrep 2000 automated slide processor and the slides were stained with routine Papanicolaou stain. Histolog-
Results
Endometrial Sampling Devices In the first part of the study we considered the diagnoses of 51 LBC samples collected with Endogyn and the corresponding biopsies. In cytology, we found 13 ‘suspicious/positive for malignancy’ cytological diagnoses and 38 ‘negative for malignancy’. In the corresponding histological diagnoses we found 15 ‘suspicious/positive for malignancy’ and 36 ‘negative for malignancy’. The prevalence of ‘negative for malignancy’ obtained was 74.6% (38/51) for cytology and 70.6% (36/51) for histology. On the other hand, the prevalence of ‘suspicious/positive for malignancy’ was 25.4 (13/51) and 29.4% (15/51), respectively (table 1). Statistical analysis showed a percent agreement of 84% (p < 0.01) and Cohen’s κ of 0.46. The negative predictive value (NPP) and positive predictive value (PPV) obtained were 95 and 100%, respectively, with 2 false negative results (3.9%). Analyzing the results obtained with the Endoflower dispositive device, we found 34/70 samples ‘negative for malignancy’ both in cytology and in histology (59.6%), and 36/70 samples ‘suspicious/positive for malignancy’ (63%). The percent agreement was 100% (p = 0) and Cohen’s κ was 1. The NPP and the PPV obtained were both 100%, without false positive and false negative results. Considering the comparison between cytological and histological diagnosis, we found that 49 ‘suspicious/positive for malignancy’ and 70 ‘negative for malignancy’ cases were confirmed by histology. There were 2 discordant cases for which the cytological diagnoses were negative (table 1). The sensitivity, specificity, NPP and PPV obtained were 96, 97, 97 and 100%, respectively, with 2 false negative results (1.6%; table 1). PTEN ICC In the second part of the investigation, all 12 endometrioid carcinomas identified initially in the endometrial LBC were confirmed in histological surgical samples. We also made a diagnosis of endometrioid carcinoma in 14 LBC samples for which we do not yet have the corresponding surgical specimens. All these women, with a PTEN in Endometrial Cytology
mean age of 66 years (range 25–88), underwent a gynecological examination for bleeding or spotting in menopause. They had one or more risk factors in anamnesis, including hypertension, obesity and hormonal therapies. All these women gave their written informed consent to participate in the study, according to the Declaration of Helsinki (1964). Morphological diagnosis showed 6 FIGO grade 2 endometrioid carcinomas, 3 grade 1 endometrioid carcinomas, and 3 FIGO grade 3 endometrioid carcinomas, with evident tumor invasion into the outer half of the myometrium in 3 cases, but without metastasis at the lymph nodes. We also selected 10 control cases, collected by the same procedure. In particular, there were 5 endometrium samples with dyshormonal features, 2 endometrial atrophic and 3 proliferative endometrium samples, both in cytology and in histology. The other 14 samples were all endometrioid carcinomas, well differentiated, for which there were no histological samples available. The IHC showed PTEN-null glands in 4 cases (fig. 2). The same cancers were also negative in ICC. In the other 8 cancers, the PTEN expression was positive both in cytology and in histology. In the 10 LBC-sampled cancers, PTEN ICC was negative in 1 case (fig. 2). Regarding the control cases, all the samples showed positivity both in cytology and in histology as expected. The results obtained were 100% concordant.
Discussion
Endometrial Sampling Devices In the past, cytological diagnosis for endometrial disease was not so common, particularly due to morphological interpretation difficulties caused by the presence of mucus and blood, and by some changes linked to the menstrual cycle and hormonal pressure. The introduction of LBC methods in addiction to new collecting and processing procedures has allowed a reevaluation of endometrial cytology. In the clinical perspective, the main and important advantages of using this technique are the low cost, the easy performance in office-based surroundings and the minimal discomfort for patients. Many studies have demonstrated its high accuracy in detecting endometrial cancer, especially with the LBC method [16– 21]. In fact, the high detection rate is due to the possibility of taking samples from the entire uterus cavity, improving the diagnostic rate of biopsy [18].
Acta Cytologica 2014;58:495–500 DOI: 10.1159/000367961
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blood vessels were intensely positive for PTEN, so they were used as positive internal controls. Tumors in cytology and in histology are considered positive with diffuse positive granular cytoplasm and nuclear staining in more than 50% of the cells in the sample. Tumors with no or rare and sparse positive cells, but in any case with less than 50% cancer cells, are considered negative [16].
a
b
c
d
Fig. 2. a, b Immunoreactivity of PTEN in
In the first part of this study, we compared two different devices for collecting endometrial cells. For years, the routine diagnostic procedure for women with irregular bleedings was dilatation and curettage with ultrasonography. Recently, a variety of endometrial sampling devices have been developed. We tested the Endogyn and the Endoflower. A previous study showed a statistically significant difference between Endogyn cytology and dilatation and curettage for the diagnoses of hyperplasia with atypia and adenocarcinoma [17]. We obtained the same results using the Endogyn. Using the Endoflower dispositive, we observed the same results in ‘negative for malignancy’ as well as in ‘suspicious/positive for malignancy’ categories, both in histology and in cytology. PTEN ICC In the second part of the study, we evaluated PTEN expression in endometrial LBC samples, comparing the results with histology. PTEN expression abnormalities have been described in many human tumors as prostate, thyroid, brain, melanoma and endometrium. Inactiva498
Acta Cytologica 2014;58:495–500 DOI: 10.1159/000367961
tion of the two alleles of PTEN by mutation or deletion is required to block its activity. However, other mechanisms are also common, like promoter hypermethylation and loss of heterozygosity. Several studies have suggested that the immunohistochemical evaluation of PTEN expression may be an effective way to demonstrate the absence of the protein in a lesion [22, 23]. According to the literature and based on our own experience, the LBC residual material, after morphological evaluation, is also suitable for ancillary techniques [19– 21]. Recently, we demonstrated that it is possible to use archived endometrial LBC samples to detect molecular alterations with a good amount and high quality of DNA extracted. We showed the same PTEN alteration both in histology and in cytology [19]. However, the high costs and the limitations of applying molecular techniques in routine practice stimulated us to study the consistency of PTEN immunostaining in LBC samples. In a previous study, investigators demonstrated PTEN immunoreactivity in endometrial cancers in thin-layer specimens. They established a cut-off value of 50% of Di Lorito et al.
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grade 2 endometrioid carcinomas: cytology viewing loss of staining in more than 50% of cancer cells (a); relative histopathology exhibits the same lack of staining with positivity in the stroma and blood vessels, considered as a positive internal control (b). c, d Immunoreactivity of PTEN in grade 1 endometrioid carcinomas: cytology showing brown granules in the cytoplasm and brown nuclei, considered as positive (c); corresponding histopathology displays the same positivity in the glands as in the stroma (d). Original magnification: a, c ×400; b, d ×100.
PTEN expression, which is useful for the correct diagnosis of endometrioid carcinomas in endometrial cytology [16]. According to this previous report, we applied the same cut-off value of 50% for PTEN-negative immunoreactivity. In our series, we had 4 cases with PTEN-null glands, corresponding in 1 case to high-grade endometrioid carcinomas and in 3 samples to low-grade endometrioid carcinomas. Other authors reported that the loss of PTEN protein expression in their series was present in 61 and 63% of endometrioid carcinomas [21, 23]. In our study, the rate of PTEN-negative immunoreactivity was 20% in the 26 endometrioid carcinomas examined. Our results also showed that the negative PTEN expression by immunostaining was not linked to increased grade. In fact, in the literature it has been confirmed that PTEN loss of expression is an early event in tumorigenesis and it correlates with the prognosis and response to target therapies [23–26]. A huge number of studies have also demonstrated the potential use of PI3K inhibitors in endometrial advanced cancer patients who had also experienced failure with standard therapies. Clinical data demonstrated a good response to treatment that includes PI3K/AKT/mTOR inhibitors [7]. In fact, some studies are looking to use a combination, rather than single-agent therapies [27, 28]. This is in agreement with preclinical data which suggested that single-agent PI3K/AKT/mTOR pathway inhibition may not always be sufficient to induce a response because PIK3CA mutations often coexist with other molecular alterations [9]. In a recent work, investigators have demonstrated that IHC identified partial or complete PTEN protein loss in 44% of the tumors in which no PTEN sequencing abnormalities were identified. In other words, IHC outper-
formed gene sequencing, with the identification of more endometrial carcinoma with PTEN loss. This correlated with the activation of the PI3K/AKT pathway [22]. However, the accurate identification of these abnormalities is an important part of the patient triage and it will also be necessary for the selection of patients who could benefit from targeted therapies [27, 28]. Considering the limited life expectancy of women with advanced endometrial cancer, LBC with ICC could be an accessible, simple and reproducible tool. It is also important because it can be performed within a narrow time frame.
Conclusions
Our results suggest that it is possible to use endometrial LBC samples collected with an office-based painless cytological dispositive to diagnose endometrial diseases. In our work, in spite of a limited number of cases, LBC in fact proved to be a good tool not only for morphological diagnosis, comparing with histological diagnosis, but also for the detection of biomarker expression. In the future, pathologists will be called to determine the status of genes and target proteins before the application of targeted drugs, in particular for women with advanced or recurrent endometrial cancer. We demonstrated that evaluation of PTEN loss by ICC is possible with the application of standard techniques, with concordant IHC results.
Disclosure Statement The authors have no connection with any companies or products mentioned in this article, and no competing interests.
References
PTEN in Endometrial Cytology
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Received: July 29, 2014 Accepted after revision: August 27, 2014 Published online: October 25, 2014
Expression of PTEN in Endometrial Liquid-Based Cytology Alessia Di Lorito a Roberta Zappacosta a Serena Capanna a Daniela Mariapia Gatta a Sandra Rosini a Fernando Carlos Schmitt b, c a Experimental and Clinical Science, G. d’Annunzio University, Chieti, Italy; b Institute of Molecular Pathology and Immunology, University of Porto, Porto, Portugal; c Department of Pathology and Medicine, Laboratoire National de Santé, Luxembourg, Luxembourg
Key Words Liquid-based cytology · PTEN · Immunocytochemistry · Endometrial sampling device
All the normal endometrium control cases were positive in cytology and histology. Conclusions: Our results suggest that endometrial devices provide useful material for the diagnosis and evaluation of PTEN expression. © 2014 S. Karger AG, Basel
© 2014 S. Karger AG, Basel 0001–5547/14/0585–0495$39.50/0 E-Mail [email protected] www.karger.com/acy
Introduction
Endometrial cancer represents the most common gynecologic malignancy in developed countries [1]. Dilatation and curettage has long been recognized as the gold standard in the diagnosis of endometrial diseases. However, over the years other endometrial sampling devices have been developed [1]. Molecular alterations in endometrial cancers were previously described using formalin-fixed, paraffin-embedded (FFPE) tissues with particular attention, in endometrioid carcinomas, to the PTEN-PI3K pathway [2–4]. PTEN (phosphatase and tensin homolog) is a tumor suppressor gene that negatively regulates the PI3K-AKT signaling pathway, controlling cell proliferation, growth and apoptosis. Inactivation of PTEN and activation of PI3K produce AKT phosphorylation, β-catenin accumulation in the nuclei and activation of gene transcription [5]. Correspondence to: Dr. Alessia Di Lorito Department of Experimental and Clinical Science Cytopathology Unit, Santissima Annunziata Hospital via dei Vestini, IT–66100 Chieti (Italy) E-Mail alessia.dilorito @ gmail.com
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Abstract Objectives: Endometrial cytology offers a reliable alternative to biopsy in endometrial cancer detection and it may be useful in obtaining material to study prognostic and predictive markers. Over the years, new sampling devices have been developed. Molecular alterations in endometrial cancers were previously described using formalin-fixed paraffinembedded tissues with particular attention, in endometrioid carcinomas, to the PTEN-PI3K pathway. PTEN evaluation could be useful in endometrial carcinomas for selecting patients for target therapies. Study Design: We studied 51 endometrial samples collected using the Endogyn device and 71 obtained with the Endoflower dispositive device, and processed using liquid-based cytology. Most of the cases were matched with a corresponding histological biopsy. The overall accuracy of Endoflower was 100%. Immunohistochemistry (IHC) and immunocytochemistry (ICC) for PTEN were performed using monoclonal antibody 6H2.1 from DAKO. Results: The IHC showed PTEN-null glands in 4 cases. The same cancers were negative in ICC. Among the 10 carcinomas on cytology, PTEN-null glands were found in 1 case.
a
b
Fig. 1. Endometrium sampling devices used in the study: Endogyn (a) and Endoflower (b).
Clinical trials have suggested that PTEN inactivation is linked to the response to mTOR, Akt and PARP inhibitors, and resistance to HER2 inhibitors. In advanced cancer, although responses to first-line treatment are frequently observed, relapses are common and effective therapeutic options for recurrent tumors are lacking [6– 9]. Some studies have suggested an important role of PTEN evaluation in endometrial carcinomas, using FFPE material [10, 11]. In endometrial disease, cytology is a simple, painless, inexpensive and accurate way to obtain material for cancer detection and for morphological diagnosis [12]. It can also be used to study preventive and predictive markers, including PTEN [13]. In this study, the first aim was to assess the applicability of a new device, the Endoflower dispositive (RI-MOS, Mirandola, Italy), in collecting cells from the endometrial cavity. We compared this method with a previous method of sampling, the Endogyn device (Biogyn, Mirandola, Italy). The second aim was to perform a direct comparison of PTEN expression in the cytology and histology of endometrial carcinomas.
Table 1. Comparison of cytological and histological diagnoses using Endogyn and Endoflower devices
Cytology
Histology
Suspicious/positive Negative
suspicious/positive
negative
49 2
2 70
The sensitivity, specificity, NPP and PPV obtained were 96, 97, 97 and 100%, respectively.
ical material was routinely fixed in formalin and embedded in paraffin. The slides were stained with hematoxylin and eosin and examined by two different pathologists, in order to avoid observer bias. Histological diagnoses were made according to World Health Organization criteria [14] and cytological diagnoses were given considering architectural and cytological features. However, for statistical analysis, we considered two categories: ‘negative for malignancy’, including negative findings and hyperplasia without atypia, and ‘suspicious/positive for malignancy’, including cancer and hyperplasia with atypia (table 1). Statistical analyses were based on comparison with McNemar’s test. The percent agreement was also calculated.
Material and Methods
496
Acta Cytologica 2014;58:495–500 DOI: 10.1159/000367961
Immunocytochemical Analysis For the second part of the study, we considered only cancer cases. We examined 12 endometrioid cancer LBC samples with corresponding FFPE blocks (surgical specimens) and 14 endometrioid cancer LBC samples without corresponding FFPE tissues. We used 10 benign endometrial cytological samples with the respective biopsies as controls. Immunohistochemistry (IHC) and immunocytochemistry (ICC) for PTEN were performed using monoclonal DAKO 6H2.1 antibody, as previously described [15, 16]. In particular, for ICC, cytological samples were centrifuged; cell pellets were transferred into the plastic chambers, mounted on glass slides and fixed with 95% ethanol. The slides were left at room temperature for 72 h. The ICC and the IHC were performed according to the manufacturer’s protocol and previous studies [16, 17]. PTEN IHC and ICC were scored as positive and negative. In all IHC cases, stromal cells and
Di Lorito et al.
Downloaded by: Kungliga Tekniska Hogskolan 198.143.54.65 - 8/15/2015 4:05:40 PM
Endometrial Sampling and Morphological Diagnoses In the first part of this study, we made a comparison between two different methods of collecting endometrial cells: the Endogyn and the Endoflower devices (fig. 1). We examined the diagnoses of 51 liquid-based cytology (LBC) endometrial samples, obtained from 2000 to 2004 using Endogyn, comparing them with the corresponding histological diagnoses. Then, we analyzed the morphological diagnoses of 70 LBC samples, gathered using Endoflower, with the corresponding biopsies, selected from 2006 to 2013. Samples were centrifuged and the pellet with the cells was put into a PreservCyt® (Cytyc Corporation, Marlborough, Mass., USA) in order to eliminate the mucus and blood. These vials were then processed by a ThinPrep 2000 automated slide processor and the slides were stained with routine Papanicolaou stain. Histolog-
Results
Endometrial Sampling Devices In the first part of the study we considered the diagnoses of 51 LBC samples collected with Endogyn and the corresponding biopsies. In cytology, we found 13 ‘suspicious/positive for malignancy’ cytological diagnoses and 38 ‘negative for malignancy’. In the corresponding histological diagnoses we found 15 ‘suspicious/positive for malignancy’ and 36 ‘negative for malignancy’. The prevalence of ‘negative for malignancy’ obtained was 74.6% (38/51) for cytology and 70.6% (36/51) for histology. On the other hand, the prevalence of ‘suspicious/positive for malignancy’ was 25.4 (13/51) and 29.4% (15/51), respectively (table 1). Statistical analysis showed a percent agreement of 84% (p < 0.01) and Cohen’s κ of 0.46. The negative predictive value (NPP) and positive predictive value (PPV) obtained were 95 and 100%, respectively, with 2 false negative results (3.9%). Analyzing the results obtained with the Endoflower dispositive device, we found 34/70 samples ‘negative for malignancy’ both in cytology and in histology (59.6%), and 36/70 samples ‘suspicious/positive for malignancy’ (63%). The percent agreement was 100% (p = 0) and Cohen’s κ was 1. The NPP and the PPV obtained were both 100%, without false positive and false negative results. Considering the comparison between cytological and histological diagnosis, we found that 49 ‘suspicious/positive for malignancy’ and 70 ‘negative for malignancy’ cases were confirmed by histology. There were 2 discordant cases for which the cytological diagnoses were negative (table 1). The sensitivity, specificity, NPP and PPV obtained were 96, 97, 97 and 100%, respectively, with 2 false negative results (1.6%; table 1). PTEN ICC In the second part of the investigation, all 12 endometrioid carcinomas identified initially in the endometrial LBC were confirmed in histological surgical samples. We also made a diagnosis of endometrioid carcinoma in 14 LBC samples for which we do not yet have the corresponding surgical specimens. All these women, with a PTEN in Endometrial Cytology
mean age of 66 years (range 25–88), underwent a gynecological examination for bleeding or spotting in menopause. They had one or more risk factors in anamnesis, including hypertension, obesity and hormonal therapies. All these women gave their written informed consent to participate in the study, according to the Declaration of Helsinki (1964). Morphological diagnosis showed 6 FIGO grade 2 endometrioid carcinomas, 3 grade 1 endometrioid carcinomas, and 3 FIGO grade 3 endometrioid carcinomas, with evident tumor invasion into the outer half of the myometrium in 3 cases, but without metastasis at the lymph nodes. We also selected 10 control cases, collected by the same procedure. In particular, there were 5 endometrium samples with dyshormonal features, 2 endometrial atrophic and 3 proliferative endometrium samples, both in cytology and in histology. The other 14 samples were all endometrioid carcinomas, well differentiated, for which there were no histological samples available. The IHC showed PTEN-null glands in 4 cases (fig. 2). The same cancers were also negative in ICC. In the other 8 cancers, the PTEN expression was positive both in cytology and in histology. In the 10 LBC-sampled cancers, PTEN ICC was negative in 1 case (fig. 2). Regarding the control cases, all the samples showed positivity both in cytology and in histology as expected. The results obtained were 100% concordant.
Discussion
Endometrial Sampling Devices In the past, cytological diagnosis for endometrial disease was not so common, particularly due to morphological interpretation difficulties caused by the presence of mucus and blood, and by some changes linked to the menstrual cycle and hormonal pressure. The introduction of LBC methods in addiction to new collecting and processing procedures has allowed a reevaluation of endometrial cytology. In the clinical perspective, the main and important advantages of using this technique are the low cost, the easy performance in office-based surroundings and the minimal discomfort for patients. Many studies have demonstrated its high accuracy in detecting endometrial cancer, especially with the LBC method [16– 21]. In fact, the high detection rate is due to the possibility of taking samples from the entire uterus cavity, improving the diagnostic rate of biopsy [18].
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blood vessels were intensely positive for PTEN, so they were used as positive internal controls. Tumors in cytology and in histology are considered positive with diffuse positive granular cytoplasm and nuclear staining in more than 50% of the cells in the sample. Tumors with no or rare and sparse positive cells, but in any case with less than 50% cancer cells, are considered negative [16].
a
b
c
d
Fig. 2. a, b Immunoreactivity of PTEN in
In the first part of this study, we compared two different devices for collecting endometrial cells. For years, the routine diagnostic procedure for women with irregular bleedings was dilatation and curettage with ultrasonography. Recently, a variety of endometrial sampling devices have been developed. We tested the Endogyn and the Endoflower. A previous study showed a statistically significant difference between Endogyn cytology and dilatation and curettage for the diagnoses of hyperplasia with atypia and adenocarcinoma [17]. We obtained the same results using the Endogyn. Using the Endoflower dispositive, we observed the same results in ‘negative for malignancy’ as well as in ‘suspicious/positive for malignancy’ categories, both in histology and in cytology. PTEN ICC In the second part of the study, we evaluated PTEN expression in endometrial LBC samples, comparing the results with histology. PTEN expression abnormalities have been described in many human tumors as prostate, thyroid, brain, melanoma and endometrium. Inactiva498
Acta Cytologica 2014;58:495–500 DOI: 10.1159/000367961
tion of the two alleles of PTEN by mutation or deletion is required to block its activity. However, other mechanisms are also common, like promoter hypermethylation and loss of heterozygosity. Several studies have suggested that the immunohistochemical evaluation of PTEN expression may be an effective way to demonstrate the absence of the protein in a lesion [22, 23]. According to the literature and based on our own experience, the LBC residual material, after morphological evaluation, is also suitable for ancillary techniques [19– 21]. Recently, we demonstrated that it is possible to use archived endometrial LBC samples to detect molecular alterations with a good amount and high quality of DNA extracted. We showed the same PTEN alteration both in histology and in cytology [19]. However, the high costs and the limitations of applying molecular techniques in routine practice stimulated us to study the consistency of PTEN immunostaining in LBC samples. In a previous study, investigators demonstrated PTEN immunoreactivity in endometrial cancers in thin-layer specimens. They established a cut-off value of 50% of Di Lorito et al.
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grade 2 endometrioid carcinomas: cytology viewing loss of staining in more than 50% of cancer cells (a); relative histopathology exhibits the same lack of staining with positivity in the stroma and blood vessels, considered as a positive internal control (b). c, d Immunoreactivity of PTEN in grade 1 endometrioid carcinomas: cytology showing brown granules in the cytoplasm and brown nuclei, considered as positive (c); corresponding histopathology displays the same positivity in the glands as in the stroma (d). Original magnification: a, c ×400; b, d ×100.
PTEN expression, which is useful for the correct diagnosis of endometrioid carcinomas in endometrial cytology [16]. According to this previous report, we applied the same cut-off value of 50% for PTEN-negative immunoreactivity. In our series, we had 4 cases with PTEN-null glands, corresponding in 1 case to high-grade endometrioid carcinomas and in 3 samples to low-grade endometrioid carcinomas. Other authors reported that the loss of PTEN protein expression in their series was present in 61 and 63% of endometrioid carcinomas [21, 23]. In our study, the rate of PTEN-negative immunoreactivity was 20% in the 26 endometrioid carcinomas examined. Our results also showed that the negative PTEN expression by immunostaining was not linked to increased grade. In fact, in the literature it has been confirmed that PTEN loss of expression is an early event in tumorigenesis and it correlates with the prognosis and response to target therapies [23–26]. A huge number of studies have also demonstrated the potential use of PI3K inhibitors in endometrial advanced cancer patients who had also experienced failure with standard therapies. Clinical data demonstrated a good response to treatment that includes PI3K/AKT/mTOR inhibitors [7]. In fact, some studies are looking to use a combination, rather than single-agent therapies [27, 28]. This is in agreement with preclinical data which suggested that single-agent PI3K/AKT/mTOR pathway inhibition may not always be sufficient to induce a response because PIK3CA mutations often coexist with other molecular alterations [9]. In a recent work, investigators have demonstrated that IHC identified partial or complete PTEN protein loss in 44% of the tumors in which no PTEN sequencing abnormalities were identified. In other words, IHC outper-
formed gene sequencing, with the identification of more endometrial carcinoma with PTEN loss. This correlated with the activation of the PI3K/AKT pathway [22]. However, the accurate identification of these abnormalities is an important part of the patient triage and it will also be necessary for the selection of patients who could benefit from targeted therapies [27, 28]. Considering the limited life expectancy of women with advanced endometrial cancer, LBC with ICC could be an accessible, simple and reproducible tool. It is also important because it can be performed within a narrow time frame.
Conclusions
Our results suggest that it is possible to use endometrial LBC samples collected with an office-based painless cytological dispositive to diagnose endometrial diseases. In our work, in spite of a limited number of cases, LBC in fact proved to be a good tool not only for morphological diagnosis, comparing with histological diagnosis, but also for the detection of biomarker expression. In the future, pathologists will be called to determine the status of genes and target proteins before the application of targeted drugs, in particular for women with advanced or recurrent endometrial cancer. We demonstrated that evaluation of PTEN loss by ICC is possible with the application of standard techniques, with concordant IHC results.
Disclosure Statement The authors have no connection with any companies or products mentioned in this article, and no competing interests.
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