Aim: Neuroendocrine (NE) tumours of the female genital tract are quite rare. Diagnosis of such tumours is based on histological findings, although this does not allow discriminating between primary and secondary tumours. In this paper the authors present a rare case of a patient presenting with a bilateral large cell NE carcinoma of the ovary and a well-differentiated NE carcinoma of the appendix. The aim of the study was to understand whether the two tumours were distinct or rather it was a metastatic cancer.
Methods: Array-CGH (aCGH) was used to assess clonality of these two tumours. By this approach it was possible to demonstrate that these two components showed patterns of monoclonality.
Results: The NE nature of both tumours was confirmed by histological examination, based on the expression of NE markers in both tumours. aCGH confirmed a common origin of both tumours as they shared several common molecular lesions. These results suggest that the invasive NE ovarian cancer may have arisen from the well-differentiated NE tumour of the appendix.
Conclusion: Although diagnosis of NE tumours is made on histological ground, distinction of primary vs. metastatic cancers is not possible. Molecular techniques as aCGH may be key to assess clonality of tumours as they allow classification of tumours from the same patients as either independent primary cancers or metastases.
Keywords: Neuroendocrine tumours, clonality, array-CGH
The term neuroendocrine (NE) tumour encompasses different neoplasms originating from the diffuse NE cell system. These tumours more commonly arise in the gastrointestinal (GI) tract, pancreas, lung and thymus, whereas a gynaecological presentation, either as a primary or a secondary tumour, is quite uncommon.
Currently NE tumours are broadly classified in two groups, as poorly differentiated NE carcinomas (NECs) and well-differentiated NE tumours (NETs). Small cell carcinoma and large cell NE carcinoma are categorised as NECs, whereas typical and atypical carcinoids fall into the group of NETs.[1-4]
Gynaecological NE tumours are most commonly cervical small cell carcinoma and ovarian carcinoids, whereas very rarely NECs or NETs are found in other regions of the female genital tract.
It is noteworthy that NE tumours may also be secondary to a different primary tumour, therefore it is important to differentiate primary NE from metastatic disease and other carcinomas with a NE component.
NE tumours are classified according to the World Health Organisation (WHO) classification of NE tumours. Histologically, tumours are diagnosed as NE when they express at least two NE markers such as chromogranin A, synaptophysin or neuron-specific enolase, although other peptides and hormones may also be found, including calcitonin, gastrin, serotonin, substance P, vasoactive intestinal peptide, pancreatic polypeptide, somatostatin and adrenocorticotrophic hormone.[1-3] In some cases, NE tumours may also display some non-NE features, usually either glandular or squamous components.
Diagnosis of NE tumours may be challenging and can sometimes be achieved only following hysterectomy, as a cervical biopsy may be insufficient to allow the identification of the NE component, thus leading to diagnosis of a poorly differentiated cervical cancer. As NE tumours may differ from other types of cervical cancer in prognosis and treatment a proper diagnosis is mandatory.
Management of NE tumours
Different therapeutic approaches are available to treat NE tumours. Usually such tumours are targeted using a multimodal approach. In 2011 the Society of Gynaecologic Oncology published a document summarising available literature on NET of the female reproductive tract on which current therapeutic algorithms are based. Treatment of NECs is based on the guidelines of the North American Neuroendocrine Tumour Society. The choice of treatment depends on the stage of the tumour and whether or not the tumour has spread. Generally, chemoradiation is suggested for early stage disease whereas the recommended approach for more advanced disease includes a combination of chemotherapy and radiation.
Chemotherapy streptozotocin, 5-fluorouracil, doxorubicin, etoposide and cisplatin has been largely used to treat metastatic endocrine cancers, but currently is only applied for very undifferentiated forms.[8-10] With some protocols it has been possible to achieve a 24-month remission, but the drawback of this treatment is the onset of several and severe side effects. Another possibility is to use interferon, which can inhibit hormonal secretion and can control tumour growth by different mechanisms, as G0/G1 cell cycle block, 2’-5’A-synthetase induction, immune system modulation and angiogenesis inhibition, eventually resulting in tumour cell apoptosis. This protocol has an average remission of 20 months, and can be improved if it is administered in combination with somatostatin analogues. The most common side effects for such treatment are flu-like symptoms and a moderate myelodepression.[12,13] Somatostatin analogues are of particular interest for the treatment of NE tumours as 80-90% of NE tumours express somatostatin receptors. In particular, 5 different receptors, mapping on different chromosomes and with different functions in distinct organs have been identified, being types 1, 2 and 5 involved in controlling cell proliferation, 3 affecting apoptosis. Although with different mechanisms, all of these receptors bind to somatostatin. Of these analogues, octreotide acetate has the longest half-life and has sensibly improved the quality of life of neuroendocrine cancer patients, and it is widely used for the therapy of such tumours.[17,18] More recently, analogues with an even longer half-life have been obtained (octreotide acetate LAR and lanreotide) capable of a long-lasting plasmatic release up to 14-28 days.[16,19] Somatostatin analogues side effects are generally modest, mainly related to the GI tract.
Radiotherapy can also be used for the treatment of these tumours. So far, traditional radiotherapy is barely used for the treatment of such cases, and it is mainly used for managing symptoms in most advanced cases. On the other hand, a more tailored approach, as the administration of radiometabolic treatments, has been developed, combining a somatostatin analogue to ab-emitting isotope, as octreotride bound to the radioisotope Yttrium 90 (90Y). Such complex binds to somatostatin receptors present on cancer cells, thus destroying them. Radioisotopes of new generation are formed by the chelating agent DOTA bound to Yttrium 90 (90Y, DOTATOC) or to Lutetium 177 (177Lu) DOTATATE. So far, no side effects have been reported for such treatments. Iodine-131-meta-iodobenzylguanidine, a radiopharmaceutical agent that is taken up by tumour cells and eventually destroys them, has also been applied to management of these tumours.
When multiple tumours are found synchronously it is important to establish whether these tumours have arisen as independent primary lesions or whether they are genetically similar. Also, assessing clonality may be relevant from the clinical point of view especially when this information impacts on treatment. In this study, we used array-CGH (aCGH) to analyse the molecular bases of two synchronous tumours arisen in the same patients, to assess the possibility of a common origin.
A 58-year-old woman underwent laparohisterectomy and bilateral annessectomy, appendicectomy and omentectomy for ovarian cancer. Macroscopically the right ovary presented a solid whitish 2.5 cm × 2 cm neoplasm, spreading to the capsule and presenting nodules on the outer surface. The left ovary presented a solid whitish mass of 2 cm × 1.5 cm not affecting the capsule.
Several leiomyoma formations ranging from 1.5 cm to 2.5 cm were observed throughout the uterus. Whitish nodules were also present in the uterus serosa in connection with the Fallopian tubes. A second tumour of 7 mm was found in the appendix.
From the available data, it was not possible to establish whether the patient was presenting with distinct primary tumours or with a metastatic cancer. After three years the woman developed brain metastases and deceased after a few months.
Formalin-fixed paraffin embedded tumour biopsies from this patient have been obtained by the University Hospital of Siena (Italy). This study was approved by the ethics committee of the University of Siena, Italy. Written informed consent was also obtained.
Diagnosis was confirmed by morphology on histological slides stained with HE, Giemsa and by immunophenotyping, according to the WHO. Histological examination showed uniform cells which have a round to oval stippled nucleus and scant, pink granular cytoplasm. High power examination shows bland cytopathology [Figure 1A-C]. Immunohistochemical studies were performed on representative paraffin sections from each case using microwave pre-treatment of slides for antigen retrieval, as previously reported. A large panel of antibodies recognising formalin-resistant epitopes of the various antigens was applied [Table 1].
Figure 1: Hematoxylin and eosin (HE) staining (A-C) and immunohistochemistry (D-F) of both tumours. (A) HE of neuroendocrine carcinoma of the appendix, magnification ×1; (B) HE of neuroendocrine carcinoma of the appendix, magnification ×4; (C) HE of neuroendocrine carcinoma of the ovary, magnification ×20; (D) immunohistochemical analysis for CD56, magnification ×40; (E) immunohistochemical analysis for chromogranin, magnification ×20; and (F) immunohistochemistry for synaptophysin, magnification ×40. Both tumours were positive by immunohistochemistry to all the three markers, thus suggesting a neuroendocrine derivation. Images in (D-F) are representative sections of both tumoursClick here to view
Table 1: List of the antibodies used for immunohistochemistryClick here to view
Formalin fixed paraffin embedded section (10 µm) of tumour biopsies were deparaffinised with xylene and DNA extracted with the NucleoSpin kit (Macherey-Nagel), according to manufacturer’s instructions. Amount and quality of DNA were assessed by Nanodrop by UV reading at 260 nm, 260/230 and 260/280 ratios (Celbio, Italy).
For aCGH analysis 500 ng of genomic DNA extracted from multiple sections of representative blocks were used, using the Agilent 4 × 44 K platform following manufacturer’s instructions, as previously described. Briefly, DNA was labelled using the Agilent Genomic DNA Labelling Kit Plus according to the manufacturer’s protocol (Oligonucleotide Array-Based CGH for Genomic DNA Analysis 2.0v). Genomic DNA was mixed with 5 μL of 2.5× random primer solution (Agilent Technologies) and nuclease-free water to a total volume of 31 μL. Denaturation of the mix was carried out at 95 °C for 3 min followed by a 5 min incubation in ice. To each sample were then added 10 μL of 5× buffer, 5 μL of 10× dNTP nucleotide mix, 1 μL of Klenow fragment (Agilent Technologies), and 3 μL of Cy5-dNTP (well-differentiated appendix tumour, referred to as first tumour) or 3 μL of Cy3-dNTP (large cell ovarian cancer, referred to as second tumour). A 3-h incubation at 37 °C was then performed. Purification of labelled samples was performed using the CyScribe GFX Purification Kit (Amersham Biosciences) according to the manufacturer’s instruction. The two different samples were then pooled and mixed with 25 μg Human CotI DNA (Invitrogen), 26 μL blocking buffer (Agilent Technologies), and 130 μL hybridisation buffer (Agilent Technologies). The mix was then denatured at 95 °C for 5 min followed by a pre-association at 37 °C for 1 h, before hybridisation to the array. Hybridisation was carried out for 40 h at 65 °C in a rotating oven (0.040 × g), followed by several washes in wash buffers supplied with the Agilent 105 A kit. Slides were dried and scanned using an Agilent G2565BA DNA microarray scanner.
Image and data analysis
Image analysis was carried out using CGH Analytics Software v.3.4.40 (Agilent Technologies) with default settings. Genes residing in the loci of gains/losses were analysed by the Decipher software (DatabasE of genomiC varIation and Phenotype in Humans using Ensembl Resources). Function of the genes of interest was then classified according to the Gene Ontology (GO), according to the ontologies visualized by the Gene Ontology Consortium using the Amigo2 software (amigo.geneontology.org). Enrichment in expression of genes involved in specific cellular functions was evaluated using gene set enrichment analysis (GSEA, www.broadinstitute.org/gsea/msigdb), as previously described. Specifically, GSEA was performed in the terms of Gene Ontology Biological Process, positional gene sets, hallmark gene set, and curated gene using default options (displaying top 50 gene sets with false discovery rate Q-value below 0.05).
Histopathological characterisation of both tumours
A panel of antibodies was analysed by immunohistochemistry on representative sections of both tumours. The bilateral ovarian mass resulted to be a large cell NEC (synaptophysin+, chromogranin-, CD56+, CK7+, WT1+, PTEN+, α-inhibin-, vimentin-, ER-, PR-), with a Ki67 rate of about 40%. The tumour was found to infiltrate the capsule, the Fallopian tubes and the periaxial stroma and was staged as a pT2aNX.
The appendix tumour was diagnosed as a well-differentiated NE tumour (synaptophysin+, chromogranin+, CD56+) infiltrating the sub-serosa. The tumour was graded as G1 (Ki67 < 1%) and staged as a pTNM-ENETS: pT2.
In both cases, a NE derivation of the tumours was established by positivity to NE markers (CD56, synaptophysin, chromogranin) [Figure 1D-F].
Molecular analysis of both tumours by aCGH
To assess whether the two tumours have arisen independently or the second was resulting from the first one, clonality was assessed by high-density aCGH, to gain a mutational landscape of these tumours. Both tumours showed a few identical chromosomal abnormalities, as a deletion within the small arm of chromosome 5 and an amplification of part of the long arm of chromosome 14 [Figure 2], which may suggest a common origin for both neoplasms.
Figure 2: Common alterations were found in both tumours as a deletion of the small arm of chromosome 5 (A) and a duplication of the long arm of chromosome 14 (B), pointing at a common origin of both neoplasmsClick here to view
However, additional gains and losses were also identified in the ovarian tumour, which showed a more complex karyotype, with respect to the appendix one. This may suggest that novel additional mutations have occurred to the original clone and have contributed to the different phenotype of the second tumour. In particular, several losses on chromosomes 1, 4, 6, 7, 8, 9, 10, 11, 12, 13, 14, 17 and 22 and gains of chromosomes 1, 3, 7, 10, 11, 15, 18, 19, 20 and 21 were detected in the ovarian tumour [Table 2 and Figure 3].
Table 2: A summary of the additional genetic lesions identified by array-CGH in the ovarian tumourClick here to view
Figure 3: Additional genetic lesions were found in the ovarian tumour, which may account for its different phenotype. Among the many alterations detected, deletion of the long arm of chromosome 6 (A), duplication of small arm of chromosome 10 (B), duplication of the long arms of chromosome 15 (C) and 22 (D) are shown in figure as an exampleClick here to view
The chromosomal loci results altered by aCGH were then analysed to identify genes affected by these lesions and understand their functional role, in order to figure out whether their dysregulation, due to chromosomal gains or losses, may be relevant for malignant transformation. The genes of interest were studied using the Decipher software and their function was then investigated and categorised according to GO. First of all we analysed chromosomal alterations present in both cases. The deleted region of chromosome 5 involved loci where TPPP, which is involved in microtubule and tubulin binding, and ZDHHC11 that is a zinc finger protein involved in metabolic processes, mapped. On the other hand, gains of chromosomal 14 loci involved genes as CHD8, SUPT16H, SALL2 and METTL3 that are involved in transcription regulation and epigenetic control of gene expression; TOX4, which is involved in lymphocyte signalling, RAB2B that is a member of the RAS oncogene family and is involved in guanosine triphosphate binding [Table 3]. At GSEA, such genes turned out to significantly overlap with genes previously found to be involved in breast cancer carrying 5p15 abnormalities [Table 4].
Table 3: Functional categories of the genes of interest according to the GOClick here to view
Table 4: Gene set enrichment analysis performed on genes associated with the genetic lesions identified by array-CGH in the ovarian tumourClick here to view
GO analysis of the genes mapping on loci rearranged only in the ovarian tumour revealed that the genes of interest played several functions as nucleic acid binding and transcriptional regulation (i.e. LHX9, CPEB1, FARS2, PRKRIR, RNF34, ZNF221), protein binding (i.e. SPG7, SUMF1), ATP and cAMP binding (i.e. SPG7, GPHN, PDE4B), receptor activity (SEMA6D), growth factors activity and receptor binding platelet derived growth factor D among the others [Table 3], whose dysregulation may likely have an impact on a more complex phenotype exhibited by the ovarian tumour. Particularly, at GSEA these genes were found to be significantly enriched in FOXP3 and MAPK8 targets, as well as in genes dysregulated in acute promyelocytic leukaemia as a consequence of PML/RARA rearrangement [Table 4].
Establishing whether two tumours may have a common origin or rather have arisen independently is still challenging. Assessing tumour clonality may be of interest to understand tumour biology in order to improve patient’s management and design a more tailored therapeutic approach. Histopathological analysis of different tumours can be of help to discriminate between tumour metastases deriving from a single primary tumour or multiple, genetically unrelated primary tumours that have arisen simultaneously. However, this is not always possible and histopathological evaluation may not provide unambiguous evidence for tumour origin. Therefore, molecular analyses are nowadays more and more used to investigate the origin of different neoplasms.
Several approaches can be used in this regard, from analysis of single loci by FISH to more wide approaches, as whole-genome sequencing, which provides a complete picture of the entire genome of the patient. However, the choice of the appropriate technique must also take into consideration more practical aspects, as costs, ease of data interpretation and response timing, which may be crucial for cancer patients when an appropriate therapy has to be administered in a short time. Among several molecular techniques available to address clonality, aCGH offers the advantage of providing a full picture of tumour’s genome combined with relatively low costs and response timing. Using this approach it is possible to determine genomic rearrangements in a clinical sample with great mapping precision because of the high resolution and the presence of large number of data points. Application of aCGH in oncology and its clinical value is undoubted as it may help to differentiate new primary tumours from recurrent lesions, which may have significant implications for selection of optimal adjuvant treatment. In addition, aCGH has been used to establish clonality of a variety of synchronous tumours as breast cancers.[28,29]
Bearing this in mind, we have used aCGH to assess clonality of two synchronous tumours in a patient presenting with different NE tumours, a bilateral large cell ovarian carcinoma and a well-differentiated tumour of the appendix, to gain insights into their origin as either independent tumours or metastatic cancer.
Genetic analysis of both cancers revealed the existence of several chromosomal rearrangements, particularly in the ovarian tumour, which showed a more complex karyotype. Nevertheless, common karyotypic alterations were also detected in both samples, pointing at a common origin of the two neoplasms rather than an independent onset. Both tumours showed genetic alterations involving ZDHHC1 and TPPP on chromosome 5, and CHD8, RAB2B, SUPT16H, METTL3, among the others, on chromosome 14, whose consequent altered expression may have contributed to malignant transformation. Several reports in literature indicate that dysregulated expression of these genes may be linked to tumour onset as well as influence prognosis, as in the case of ZHDDC1. Dysregulated expression of the TPPP/p25 protein was initially linked to neurodegenerative disorders. but further studies revealed its involvement in cancer. In particular, chromosomal rearrangements involving the TPPP locus have been described in bladder and lung cancer. and knockdown of this gene seems to inhibit tumour growth and metastases. Interestingly, this gene has also been proposed as a potential therapeutic target for cancer. More recently, expression of TPPP has also been linked to patients’ outcome, as low expression of TPPP correlates with a worse prognosis in hepatocellular carcinoma. On the other hand, gains detected on chromosome 14 may also be relevant for transformation. CHD8 is involved in chromatin remodelling, which regulates the expression of crucial genes for cancer as MYC and BRCA1, whose role in ovarian cancer is well established. Its altered expression has been related to the onset of gastric and colorectal cancers and melanoma. In addition, low expression of CHD8 is associated with worse prognosis in gastric cancer, thus reinforcing its critical role in transformation.
The SUPT16H gene is also involved in chromatin remodelling, and genomic alterations involving this gene have been detected in nasopharyngeal angiofibroma, for which SUPT16H has been proposed as a potential therapeutic marker. Transcription is epigenetically regulated by methyltransferases, as MTTL3, whose function has been recently shown to crucially regulate the balance between pluripotency towards differentiation. Dysregulation of such a delicate balance may likely have an impact on transformation. Finally, the RAB2B gene is a member of the RAS oncogene family and its up-regulation has been reported in adenocarcinoma. Altogether these evidences suggest that the chromosome imbalances we detected in both tumours could very likely contribute to tumour onset in this patient.
As far as chromosomal rearrangements of the ovarian tumour are concerned, several genomic rearrangements involve coding genes whose dysregulation may likely contribute to acquisition of a novel and more aggressive tumour phenotype. Among those, deletion of the long arm of chromosome 6, which have been recently described in different types of cancer, including prostate cancer and haematological malignancies, involving the TCBA1 gene.[44,45] Some authors report the rearrangement of the TCBA1 locus in both T-cell lymphoma and leukaemia cell lines. More recently, recurrent chromosome breakpoints involving the TCBA1 gene have been described in prostate cancer. Interestingly, these rearrangements result in TCBA1 down-regulation, suggesting a putative tumour suppressor role for this gene which could be consistent with what we detected in the second cancer. Duplications in different parts of the long arm of chromosome 15 were detected in correspondence to the loci of the SEMA6D and CEBP1 genes. Common variants involving the CEBP1 locus have been associated with an increased leukaemia risk. This gene belongs to a family of transcription factors that seem to play an important role in controlling senescence and cancer. Recently, overexpression of CEBP4 has been recognised as responsible for VEGF overexpression associated with pathologic angiogenesis. Also, this gene has been linked to epithelial-to-mesenchyme transition and breast cancer metastasis, thus indicating its important role in cancer, and suggesting that its amplification may contribute to the acquisition of a different and more aggressive phenotype of the second tumour. On the same chromosome maps the SEMA6D gene, whose product is semaphorin-6D, which promotes anchorage-independent growth of mesothelioma cells. This may suggest that dysregulation of this gene may likely confer metastatic potential to a pre-existing lesion. In addition, the association between SEMA6D and breast cancer prognosis has been recently described as it seems to play an important role in promoting patient survival, especially among triple negative breast cancer patients.
We detected gain of chromosome 21 within the OLIG2 locus in the ovarian tumour. OLIG2 overexpression can be considered a hallmark of oligodendrial tumours and is also a favourable prognostic factor in glioblastomas. OLIG2 has been recently described as belonging to the ZEB1 pathway, which links glioblastoma initiation, invasion and chemoresistance. Chromosomal losses were detected for the loci of SUMF1 and SPG7. Dysregulation of SUMF1 has been described in breast cancer, whereas SNPs of the SPG7 gene can determine drug resistance in prostate cancer patients. Several chromosomal rearrangements involving the GPHN gene, which was lost in the ovarian tumour, have been described in different cancers as leukaemia and prostate cancer[59,60] and very recently GPHN has been proposed as one of the few candidate genes responsible for haemangioblastoma molecular pathogenesis. Rearrangements for the PDE4B locus were also detected in the ovarian tumour. PDE4B has been recently described as a key regulator of B-cell lymphoma angiogenesis, which is associated with a poor prognosis. Dysregulation of this gene has been described in several cancers as prostate cancer, hepatocellular carcinoma driven by hepatitis B virus, of which PDE4B seems to be a specific target. Due to its important role in cancer, this gene is currently a very good candidate for tailored therapy and many studies are on-going to select new drugs targeting this gene.
Taken together the results of the molecular analysis by aCGH allowed to assess a common origin of both tumour and to identify additional genetic lesions in the ovarian one, which may derive from the appendix cancer upon the accumulation of further mutations, which may explain its different phenotype. These findings are in favour of the application of more sophisticated molecular techniques as aCGH in combination with a more traditional histopathological examination, to gain insights into the molecular bases of cancer. This may be of particular relevance for cases in which a different and more tailored therapeutic approach may be administered based on the identification of specific genetic lesions.
Designing and performing the genetic study: A.D. Videtta, G. De Falco
Assessing pathology of both cases: R.C. Santopietro, G. Margiotta
Performing gene set enrichment analyses: P.P. Piccaluga
Analysing data and wrote the manuscript: G. De Falco
Financial support and sponsorship
This work was supported by BolognAIL, AIRC IG 2013 N.14355 (Prof. Piccaluga), RFO (Prof. Piccaluga); and FIRB Futura 2011 RBFR12D1CB (Prof. Piccaluga).
Conflicts of interest
There are no conflicts of interest.
Written consent was provided by the patient for the present study.
This study has been approved by the Ethic Committee of the University of Siena, Italy.
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