Abstract
Leiomyomatoid angiomatous neuroendocrine tumour (LANT) is possibly a new disease entity that was reported as a dimorphic neurosecretory tumour with a leiomyomatous vascular component; it was found in the pituitary. We describe uterine LANT-like malignant tumour in a 45-year-old woman with uterine mesenchymal tumour, diagnosed clinically as uterine leiomyoma. She underwent laparoscopic myomectomy. The tumour consisted of hyalinized vasculature, containing factor VIII-positive endothelium and α-smooth muscle actin-positive vascular smooth muscle cells, and stromal cells, expressing neuroadhesion molecules. Both vascular and stromal components diffusely expressed chromogranin A. Histopathological examinations of uterine LANT-like malignant tumour revealed the common characteristic abnormalities of malignant uterine mesenchymal tumours, i.e. leiomyosarcomas. From our research, defective expression of calponin H1 and proteasome β9 (PSMB9)/β1i is observed in uterine LANT-like malignant tumour similarly to immunopathological findings of uterine leiomyosarcoma. These findings meet the definition of uterine LANT-like malignant tumour, and the research findings of our clinical case suggest that LANT is a special type of neuroendocrine neoplasm and is not organ specific.
Leiomyomatoid angiomatous neuroendocrine tumour (LANT) was firstly reported as a new neoplastic category (1). The histopathological concept of LANT is that of a dimorphic neoplasm consisting of an admixture of neurosecretory cells and leiomyomatous stroma surrounding intratumoural vessels (1). The original clinical case was reported as a pituitary neoplasm. Histopathological studies with LANT revealed there to be two major tumour components (1). The first cell population comprised of cytokeratin-negative neuroendocrine cells positive for neuroadhesion molecules and chromogranin A as a serum marker of neuroendocrine tumours (1). The second constituent was a smooth muscle actin (SMA)-positive leiomyomatous vascular component associated with positivity for a cell–cell adhesion molecule, CD34, in endothelia (1). On the basis of this particular histopathological pattern and immunophenotype, our research group proposed the descriptive diagnosis of LANT and suggested that it may be a variant of a dimorphic pituitary neoplasm, possibly related to null cell adenoma. Because no additional reports of this entity have since been published, whether LANT is a pituitary-specific tumour or a type of soft-tissue tumour is unclear. Our research group reports here uterine LANT-like malignant tumour, but this one occurring in the uterus, and histopathological examinations of LANT revealed the common characteristic abnormalities of uterine mesenchymal malignant tumours, i.e. leiomyosarcoma (LMS). This dimorphic tumour contained both vascular and stromal components. We suggest that uterine LANT-like malignant tumour may be derived from neuroendocrine cells that may have differentiated into uterine smooth muscle cells under the influence of transforming growth factor-β (TGFβ).
Materials and Methods
Tissue collection. A total of 51 patients aged between 32 and 83 years who were diagnosed with smooth muscle tumours in the uterus were selected from pathological files. Serial sections were cut from at least two tissue blocks from each patient for haematoxylin and eosin staining and immunostaining. All tissues were used with the approval of the Ethical Committee of Shinshu University (approval no.4737, no.150, no.M192) after obtaining written consent from each patient. The pathological diagnosis of human uterine mesenchymal tumours was performed using established criteria with some modifications (2,3). Briefly, usual leiomyoma (LMA) was defined as a tumour exhibiting typical histological features with a mitotic index (MI) [obtained by counting the total number of mitotic figures (MFs) in 10 high-power fields (HPFs)] of <5 MFs per 10 HPFs. Cellular LMA was defined as a tumour with significantly increased cellularity (>2,000 myoma cells/HPF) and a MI<5, but without cytological atypia. Bizarre LMA was defined as a tumour either with diffuse nuclear atypia and a MI<2, or with focal nuclear atypia and a MI<5 without coagulative tumour cell necrosis. Smooth muscle tumour of uncertain malignant potential was defined as a tumour with no mild atypia and a MI<10, but with coagulative tumour cell necrosis. LMS was diagnosed in the presence of a MI>10 with either diffuse cytological atypia, coagulative tumour cell necrosis, or both. Of the 113 uterine mesenchymal tumours, 52 cases were diagnosed as uterine LMA, three cases were bizarre LMA, 58 cases were uterine LMS, and one case was uterine LANT-like malignant tumour, which was obtained from Tokushima University School of Medicine. Protein expression studies with cervical epithelium and carcinoma tissues were performed using tissue arrays (uterine cancer tissues; AccuMax Array, Seoul, Korea). Details regarding tissue sections are indicated in the manufacturer's literature (AccuMax Array).
Immunohistochemistry. Our research group evaluated the characteristics of uterine LANT-like malignant tumour and uterine mesenchymal tumours by means of immunohistochemical (IHC) staining. IHC staining for calponin H1, caveolin 1, cyclin B1, cyclin E1, cytokeratin, desmin, endoglin, epidermal growth factor receptor (EGFR), oestrogen receptor (ER), Ki-67/MIB1, proteasome β9 (PSMB9)/β1i, progesterone receptor (PR), αSMA, TP53, and vimentin was performed on serial tumour sections. Anti-calponin H1 was purchased from Bioss Inc. (Woburn, MA, USA). Antibodies for caveolin1, ER (ER1D5), Ki-67 (MIB-1), PR (PR10A), and TP53 (DO-1) were purchased from Immunotech (Marseille, France). Anti-αSMA was purchased from Dako (Agilent Technologies Glostrup Denmark). Antibodies for endoglin, and EGFR were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Antibodies for cyclin B1, cyclin E1, and vimentin, were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Antibodies for cytokeratin, and desmin were purchased from VLVbio (Hästholmsvägen Nacka, Sweden). Anti-human PSMB9/β1i was produced by SIGMA-Aldrich collaboration Laboratory (SIGMA-Aldrich, Japan Science and Technology Agency and Shinshu University). IHC was performed using the avidin-biotin complex method previously described. Briefly, one representative 5-μm tissue section was cut from a paraffin-embedded sample of the radical hysterectomy specimen from patients with human uterine LMS. Sections were deparaffinized and rehydrated in graded alcohol series. After the samples were washed with phosphate-buffered saline (pH 7.4), the samples were incubated with normal mouse serum for 20 min. Sections were then incubated at room temperature for 1 h with primary antibody. After extensive washing, the samples were reacted for 60 min with adequate peroxidase-labeled secondary antibody (Histofine Simple Stain MAX-PO; Nichirei Bioscience, Tokyo, Japan). The immunoreaction was visualized, after removal of unbound secondary antibodies via incubation with diaminobenzidine (DAB)-hydrogen peroxidase solution. Immunostained samples were counterstained with hematoxylin and mounted with SUPER Mount (Matsunami Glass Industry Co, Osaka, Japan). Normal mesenchymal portions in the specimens were used as positive controls. Negative controls consisted of tissue sections incubated with normal rabbit IgG instead of the primary antibody. These studies were registered at Shinshu University in accordance with local guidelines (approval no. M192).
Sequencing of the catalytic domains of the Janus kinase 1 (JAK1), JAK2 and signal transducer and activator of transcription 1 (STAT1) and genes and the promoter region of PSMB9/β1i gene. To determine whether somatic mutations exist in the ATP-binding region or kinase activation domain of JAK1 and JAK2, in the promoter region of PSMB9/β1i gene at Tyr701, or Ser727 of STAT1 in human uterine LMS, genomic DNA was isolated and direct sequencing was carried out. Genomic DNA was extracted from consecutive paraffin-embedded human uterine LMS tissue and normal myometrial tissue sections using microwave-based DNA extraction method for PCR amplification (4). To avoid contamination with normal myometrium or inflammatory cells, the tumour areas were confirmed using a haematoxylin and eosin-stained glass slide as a template. The tumour tissues were scraped by razor-microdissection from consecutive paraffin-embedded tissue sections. The genomic DNA was subjected to polymerase chain reaction (PCR), and restricted DNA fragments for direct sequencing analysis were amplified using published oligonucleotide primers. PCR products were directly sequenced using a DYEnamic Terminator Cycle Sequencing Kit (Amersham-Biosciences, Piscataway, NJ, USA) with an ABI Prism 3100 Genetic Analyzer (Applied Biosystem, Foster City, CA, USA). The sequences of mutant JAK1, JAK2, STAT1, and the promoter region of PSMB9/β1i gene derived from individual uterine LMS tissue sections are registered in the DNA Data Bank of Japan (Accession: AB219242, DJ055380, DJ055379, DJ055378, DJ055377, DJ055376). Details of primer sets for direct sequence analysis are indicated in Table I.
Results
During the course of an annual health examination, a healthy 45-year-old woman was found to be anaemic, and a gynaecologist found a uterine tumour causing hyperme-norrhoea. Magnetic resonance imaging examination disclosed a myometrial mass of 60×57×59 mm with heterogeneous intratumoural signals. The radiological diagnosis was uterine LMA with partial degeneration. At the patient's request, laparoscopic myomectomy was performed. The soft, white tumour had a cauliflower-like surface, which was incompatible with the radiological diagnosis. Histologically, the tumour possessed two major components: The first, prominent vasculature with small lumina and hyalinized walls, and, the second, a cellular stromal component. Specifically, we noted oval- or spindle shaped nuclei with fine chromatin and faint nucleoli, obscure cytoplasm, and very poor mitotic activity. All randomly obtained histopathological samples from five different paraffin blocks showed the same histopathological pattern.
Primer sets for direct sequence analysis.
Smooth muscle cells in a benign mesenchymal tumour, uterine LMA, markedly expressed αSMA and neuroadhesion molecule (CD56); however, no cell was positive for neuron-specific enolase, chromogranin A. Most cells of uterine LANT-like malignant tumour diffusely express CD56 and chromogranin A (5). In addition, the cytological features of each major component were quite similar. Our research group demonstrated differential expression of several proteins in human uterine mesenchymal tumours and normal myometrium (Tables II, III and IV). IHC examination of uterine mesenchymal tumours demonstrated that although calponin H1 and PSMB9)/β1i were found to be markedly expressed in three types of uterine mesenchymal tumors and normal myometrium, loss of expression of calponin H1 and PSMB9/β1i was observed in human uterine malignant mesenchymal tumour, i.e. uterine LMS (Figure 1, Tables II and III). It is likely that calponin H1 and PSMB9/β1i are potential biomarkers which can distinguish LMS from other uterine mesenchymal tumours. Our research group therefore examined expression patterns of both proteins, calponin H1 and PSMB9/β1i in uterine LANT-like malignant tumour. IHC examination demonstrated that although calponin H1 and PSMB9/β1i were markedly expressed in normal myometrium, their loss of expression PSMB9/β1i was observed in uterine LANT-like malignant tumour similarly to immunopathological findings of uterine LMS (Figure 1, Tables II and III). In sections of uterine LANT-like malignant tumour, the vascular structure was reportedly composed of factor VIII-, CD31-, and CD34-positive endothelial cells, and αSMA- and sarcomeric muscle actin-positive vascular smooth muscle cells (5, 6). Results for D2-40, a lymphatic endothelial marker, were reportedly negative, which suggests that the vascular structure was not a lymphatic tumour such as lymphangiomyoma (7).
Differential expressions of proteasome subunit beta 9 (PSMB9/β1i) and calponin H1 in human uterine mesenchymal tumours and uterine leiomyomatoid angiomatous neuroendocrine tumour (U. LANT)-like tumour. Staining scores for PSMB9/β1i expression and calponin H1 expression from results of immunohistochemical studies.
Discussion
Here, our research group presented histological and immunopathological characteristics of a complex, dimorphic neurosecretory tumour possessing a smooth muscle cell-rich vascular component and a population of stromal cells expressing neuronal differentiation molecules. On the basis of its particular histological aspects and immune-pathological observations, we first ruled-out the possibility of a uterine LMA with a heterologous paraganglioma element (8). We also ruled out the possibility that the tumour was a glomangioma, perivascular epithelioid cell tumour, paraganglioma, solitary fibrous tumour, extragastrointestinal stromal tumour, or extragastrointestinal stromal tumour with neuroectodermal differentiation (9-14). Our histological examinations of the uterine LANT-like malignant tumour revealed the common characteristic abnormalities of LANT, which was reported as a pituitary neoplasm (1). Although the characteristics of the uterine tumour were indeed those of a LANT, we found two significant differences between the original LANT reported by Vajtai et al. (1) and our clinical case. Firstly, the original LANT was identified as a pituitary neoplasm, but our clinical case was of a uterine mesenchymal tumour. Soft-tissue tumours are known to appear in various organs, including the pituitary and uterus. Instead, our case here may have been a soft-tissue tumour with a neurosecretory phenotype. Therefore, we accepted the possibility that the tumour described here was a second clinical case of LANT. Secondly, in the original LANT report, chromogranin A and synaptophysin were predominantly detected in a “neurosecretory” component of the tumour. However, most tumour cells in uterine LANT-like malignant tumour expressed chromogranin A but not synaptophysin. Thus, tumour cells in the present clinical case were neuroendocrine regardless of their location, whether vascular or stromal. In the initial report, a few stromal “leiomyomatoid” cells appeared to express chromogranin A (1). Synaptophysin and chromogranin A, both of which are neuroendocrine markers, are localized in distinctive neurosecretory vesicles, the former predominantly in small transparent-looking vesicles and the latter in large dense-cored granules (15). It seems that part of the leiomyomatous stromal component of the pituitary LANT may have contained small, immature neuroendocrine vesicles. Recent study with electron microscopy demonstrated that the tumour cells in uterine LANT-like malignant tumour contained predominantly chromogranin A-positive, dense-cored neurosecretory granules (5). This finding suggests that the diffuse distribution of tumour cells with a neurosecretory phenotype in uterine LANT-like malignant tumour is consistent with the features of the first reported LANT. In addition, other research facilities reported that significantly elevated serum chromogranin A level was detected in two of 12 patients with uterine LMA without any histological observations (16, 17).
Expression of oestrogen receptor (ER), progesterone receptor (PR), Ki-67, tumor protein p53 (TP53), proteasome subunit beta 9 (PSMB9), and calponin H1 (Cal), and somatic mutations of TP53, Janus kinase 1 (JAK1), JAK2, signal transducer and activator of transcription 1 (STAT1), and PSMB9-promoter (PSMB9-pro.) in human uterine leiomyosarcoma.
The histogenesis of this newly-reported type of neoplasia is not clearly understood. Recent experimental studies shown that a group of neuroendocrine neoplasms represented florid vascular proliferation resulting from angiogenic factors produced by the tumour cells themselves (18, 19). Differently from this type of florid vascular proliferation, the vascular component of uterine LANT-like malignant tumour consisted of endothelial cells and vasculature-related smooth muscle cells with neurosecretory features. On the basis of this specific feature, our research group proposes that uterine LANT-like malignant tumour may be derived from neurosecretory cells that differentiated into smooth muscle cells in an angiogenic microenvironment. At present, the nature of a putative neurosecretory cell with the potential to differentiate into smooth muscle cells is poorly understood. However, our previous research finding of TGFβ-positive endothelial cells in uterine LANT-like malignant tumour tissue offers support for this differentiation hypothesis because this molecule is crucial for differentiation of vascular smooth muscle cells, as well as for cell proliferation during tumour angiogenesis (5, 6). The physiological significance of calponin H1 and PSMB9/β1i in sarcomagenesis of uterine malignant mesenchymal tumour, LMS, is reportedly demonstrated by our research findings. Both factors, calponin H1 and PSMB9/β1i, are useful as biomarkers to distinguish human uterine LMS from other uterine mesenchymal tumours (20-22). Our research findings highlight the possibility that calponin H1 and PSMB9/β1i are potential diagnostic biomarkers which can distinguish uterine LANT-like malignant tumour from other uterine mesenchymal tumours. These factors may play key roles in tumorigenesis of uterine LANT-like malignant tumour.
Classification of human uterine mesenchymal tumours. Protein expression was estimated by immunoblot analysis; immunohistochemistry (IHC) and/or quantitative-polymerase chain reaction as: −: no evidence of expression; −/+: partial expression; +: low expression; ++: medium expression; +++: high expression;. Cyclin E, proteasome subunit beta 9 (PSMB9), and calponin H1 are potential biomarkers for human uterine mesenchymal tumours.
Differential expression of proteasome β9 (PSMB9/β1i) and calponin H1 in human normal myometrium, several mesenchymal tumour types and uterine leiomyomatoid angiomatous neuroendocrine tumour (U.LANT)-like malignant tumour. A: Immunohistochemistry of PSMB9/β1i and calponin H1 in normal myometrium, usual leiomyoma (LMA), bizarre LMA, smooth muscle tumour of uncertain malignant potential (STUMP) and uterine leiomyosarcoma (LMS) tissues located in same tissue and uterine LANT-like malignant tumour. For all samples, 5-μm sections of tissues specimens were stained with anti-PSMB9/β1i antibody revealed by peroxidase-conjugated anti-rabbit IgG antibody. B: IHC studies individually performed at several medical facilities revealed a marked loss in the ability to induce the expression of PSMB9/β1i and calponin H1 in human uterine LMS tissues and human uterine LANT-like malignant tumour tissue compared to that in normal human myometrium located in the same tissue section, as well as to that in LMA tissues. Normal myometrium: 58 cases, usual LMA: 50 cases, bizarre LMA: three cases, LMS: 56 cases, uterine LANT-like malignant tumour: one case. The analyses were performed three times with similar results.
In summary, we report a uterine LANT-like malignant tumour arising in the myometrium. Findings for this clinical case suggest the possibility that LANT is not organ-specific and may instead be a type of soft-tissue tumour composed of neuroendocrine cells with the potential to differentiate into a leiomyomatous phenotype in a TGFβ-dependent manner. To confirm this hypothesis, additional examinations should be performed via immunohistological analyses.
Acknowledgements
The Authors appreciate the donation of sections of human uterine LANT-like tissue by Dr. Naomi Sakashita, Department of Pathology, Tokushima University School of Medicine. This work was supported by grants from the Ministry of Education, Culture, Science and Technology, the Japan Science and Technology Agency (JST), the Foundation for the Promotion of Cancer Research, Kanzawa Medical Research Foundation, and The Ichiro Kanehara Foundation.
Footnotes
Disclosure
The Authors report no conflicts of interest.
- Received December 26, 2016.
- Revision received March 7, 2017.
- Accepted March 10, 2017.
- Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved
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