—  SPECIALTY CONFERENCE  —

Gastrointestinal Pathology

Case 2 - Ileal Neuroendocrine Tumor (WHO Grade 1, ENETS/AJCC pT3, pN1, M0) with Surrounding Active Schistosoma Mansoni Infection

Tomas Slavik, Ampath Pathology, Pretoria, South Africa





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Pathological/Microscopic Findings and any Immunohistochemical or Other Studies:
Macroscopy revealed ileum obstructed by a firm white tumor with vague focal yellow discoloration and a mural kink. Microscopy demonstrated nests, acini, cords and single cell strands of neoplastic cells embedded in a densely fibrous stroma. Tumor cells appeared monotonous with a moderate amount of light eosinophilic granular cytoplasm. Nuclei were round with focal mild pleomorphism and demonstrated inconspicuous nucleoli. A maximum of 1 mitosis/10 high power fields and Ki67 index of 1% were noted. Immunoperoxidase stains for pancytokeratin (CAM5.2), synaptophysin, chromogranin A, CD56 and NSE were strongly positive. S-100 was negative. No PAS-d staining was found in the neoplastic acinar component, which revealed luminal CEA positivity. The tumor extended into the subserosal fat without involving the serosal surface and two of fifteen mesenteric lymph nodes demonstrated metastatic tumor. The patient had no evidence of other metastatic disease. Surrounding the tumor was a prominent eosinophil-rich granulomatous infiltrate. This was directed at partly preserved elongated parasitic ova (approximately 120 µm in length and half as wide). An isolated preserved spine was identified. The ova and spine were positive for PAS-d and Ziehl-Neelsen stains. Isolated adult worms were also noted (intravenous and displaced into the bowel lumen).


Case 2 - Figure 1
Macro - Macroscopy of obstructing ileal tumor with mural kink
Case 2 - Figure 2
HE x4 - Low power view of tumor extending into intact overlying mucosa
Case 2 - Figure 3
HE x10 - Tumor showing insular/nested growth pattern
Case 2 - Figure 4
HE x10 - Areas of acinar tumor growth

Case 2 - Figure 5
HE x40 - Monomorphic tumor cells with round nuclei and inconspicuous nucleoli
Case 2 - Figure 6
HE x4 - Extension of tumor into subserosal fat
Case 2 - Figure 7
Ki-67 x10 - Ki67 immunoperoxidase stain showing low proliferative index (1%)
Case 2 - Figure 8
HE x4 - Pronounced granulomatous inflammation directly adjacent to tumor

Case 2 - Figure 9
HE x10 - Granulomas with central degenerated parasite ova
Case 2 - Figure 10
HE x40 - Eosinophil-rich granuloma with central elongated parasite ovum
Case 2 - Figure 11
HE x4 - Mesenteric lymph node with metastatic tumor
Case 2 - Figure 12
ZN x40 - Acid-fast parasite ova and spine (Ziehl-Neelsen stain)

Case 2 - Figure 13
HE x10 - Adult parasite worm in small subserosal vein
Case 2 - Figure 14
HE x10 - Pair of parasite worms displaced into bowel lumen

Differential Diagnoses:
1. Neuroendocrine tumor

2. Combined neuroendocrine and glandular epithelial tumor

3. Gangliocytic paraganglioma with surrounding active schistosomiasis.

Final Diagnosis
Ileal Neuroendocrine Tumor (WHO Grade 1, ENETS/AJCC pT3, pN1, M0) with Surrounding Active Schistosoma Mansoni Infection.

Case Discussion

Introduction
Gastrointestinal (GI) neuroendocrine tumors (NETs) are a heterogeneous group of neoplasms arising from the diffuse neuroendocrine system of the gut. Although these tumors have an endocrine phenotype and characteristics, they also express neural markers, hence the term "neuroendocrine". GI NETs are rare, but their incidence has been increasing steadily and significantly over the last four decades, at least partly due to an improvement in the sensitivity of screening techniques [1]. Lower jejunal and ileal tumors now make up 23–28% of GI NETs and have an age-adjusted incidence rate of 0.28–0.88/100 000 population per year [2, 3].

Macroscopic pathology
Lower jejunal and ileal NETs often reveal macroscopic clues which aid in their distinction from adenocarcinoma and other malignant small bowel tumors. Even large jejuno-ileal NETs usually demonstrate an intact overlying mucosa. Extensive mural fibrosis (due to fibroblast growth factor production) leads to muscularis propria thickening and frequent characteristic mural kinking/buckling. Formalin fixation typically elicits a yellow tumor discoloration and hypervascularity may impart a red hue to the directly adjacent bowel wall. Approximately 30% of jejuno-ileal NETs are multiple (often present as sessile polyps) and may be associated with malignant tumors elsewhere in the GI tract [4].

Microscopic pathology
Jejuno-ileal NETs demonstrate a characteristic architecture, as originally documented by Soga and Tazawa [5]. Most tumors are of enterochromaffin (EC) cell-type (serotonin-producing) and demonstrate type A (nested or insular) growth, sometimes with admixed type C (acinar, tubular or rosette-like) areas The much less common L-cell (glucagon-like peptide and PP/PYY-producing) NET of the lower jejunum and ileum usually demonstrates type B (trabecular or ribbon-like) growth, with minimal fibrosis. This tumor revealed a type E (mixed) pattern, comprising type A, C as well as focal type B growth. Pure type D (undifferentiated or sheet-like) growth has not been reported in lower jejunal or ileal neoplasms [6]. Jejuno-ileal NETs have a bland cytology and low mitotic activity/proliferation index.

Secondary features include pronounced stromal fibrosis, which often leads to tumor retraction artifact. Characteristic vascular mural thickening and intimal elastic sclerosis, combined with stromal fibrosis, may cause secondary bowel ischemia. Tumor cells are often intimately associated with nerves, possibly reflecting a histogenetic relationship.

Immunoperoxidase staining reveals positivity for pancytokeratin markers (particularly CAM5.2); CK7 and CK20 are expressed in <25% of cases [7]. Neuroendocrine markers such as synaptophysin, chromogranin A/B, CD56, PGP9.5 and the much-derided NSE are positive. Intestinal differentiation is usually manifested by strong CDX-2 nuclear staining. CEA positivity is present in up to 70% of tumors, most often as apical/luminal staining in type C areas [8]. Interestingly, approximately 30% of jejuno-ileal NETs express prostatic acid phosphatase (PAP) [9].

Differential diagnoses
Prominent type C growth in a jejuno-ileal NET may raise the possibility of a combined neuroendocrine and glandular tumor/mixed adenoneuroendocrine carcinoma (MANEC). Both components of the latter (by definition each comprising > 30% of the tumor), however, usually have a malignant or high grade histology [6]. The monotonous cellular morphology, scarcity of mitoses, low proliferation index, absence of necrosis, as well as diffusely positive neuroendocrine markers and absence of intracellular mucin in areas of acinar growth exclude MANEC/a non-neuroendocrine glandular component.

Another diagnostic consideration is gangliocytic paraganglioma (paraganglioneuroma), a rare but distinctive triphasic GI neoplasm which demonstrates an endocrine component. Although uncommonly seen outside the duodenum, it has been documented in the jejunum and ileum [10, 11]. Admixed ganglion cells and a prominent S-100 protein positive spindle cell stroma distinguish this tumor from a typical NET.

Nomenclature, grading and staging of lower jejunal and ileal neuroendocrine neoplasms
This ileal neuroendocrine tumor highlights the paradox of GI NETs: innocuous histology associated with a potential for aggressive biologic behavior. Although Oberndorfer initially considered these proliferations benign when he first coined the term "karzinoide tumoren" (carcinoma-like tumors) in 1907 [12], it is now widely accepted that they represent low grade malignancies. In contrast to other GI NETs, tumors of the lower jejunum and ileum of comparable size demonstrate a more aggressive behavior [2]. Even small (<1 cm) jejuno-ileal NETs demonstrate metastatic spread in 30% of cases [13], with non-localized disease present in 70% of patients at diagnosis [14].

Whereas the histopathologic diagnosis of a jejuno-ileal NET is seldom challenging, the application of standardized nomenclature, grading and staging is. Terminology in the literature remains confusing, with the words "endocrine" and "neuroendocrine", as well as "neoplasm", "tumor", "carcinoma" and "carcinoid" often used interchangeably [15]. Various grading schemes are in use, and until recently no dedicated staging system existed.

The previous World Health Organization (WHO 2000) classification of GI neuroendocrine neoplasms attempted to develop a coherent terminology and advance prognostic stratification [16]. The inclusion of clinical and stage-related information in a hybrid grading scheme, and the creation of an "uncertain malignant potential" category were problematic, however. Based on recommendations by the European Neuroendocrine Tumor Society (ENETS) [17], the revised 2010 WHO classification has attempted to rectify these problems (although it has again changed the nomenclature). The term "neuroendocrine tumor (NET)" has now been adopted for low and intermediate grade neoplasms, whilst the term "neuroendocrine carcinoma (NEC)" is used for high grade neoplasms. A laudable change has been the separation of histological grading and staging parameters [6], which has led to a more rational terminology and enabled grading of advanced tumors for therapeutic decision making. Additionally, the current scheme recognizes the role of tumor heterogeneity (with respect to site and cell type), and the low grade malignant potential of virtually all GI NETs.

The 2010 WHO classification delineates three grades of GI neuroendocrine neoplasms (table 1). Well- and poorly-differentiated neuroendocrine neoplasms are separated on the grounds of morphology and proliferative rate. A tumor with high grade morphology and/or grade 3 proliferative activity is considered a neuroendocrine carcinoma (NEC): small or large cell type. Whilst evidence exists for the prognostic significance of the new grading scheme in foregut NETs, it remains to be validated for intestinal NETs. Furthermore, mitotic count on an adequate NET tissue specimen is generally accepted as an accurate indicator of proliferative rate, but the role of Ki67 (particularly in the US) remains contentious [18]. This marker is not routinely utilized in all laboratories, can demonstrate heterogeneous staining and disagreement exists on the optimal method to evaluate the Ki67 index. Additionally, definitive clinical data to determine the optimal proliferative threshold for each tumor grade are still lacking [15]. Despite these limitations, the Ki67 index has been shown to have prognostic significance in midgut NETs [19]. Ki67 is also of great diagnostic value in distinguishing between a well-differentiated (low and intermediate grade) NET and (poorly-differentiated, high grade) neuroendocrine carcinoma on small biopsies, as well as evaluating proliferative rate in biopsy material with insufficient neoplastic tissue for an accurate mitotic count [18].



Until recently, no dedicated staging system was available for GI NETs. There are now two widely-used site-specific TNM staging systems – one devised by ENETS [17] and the other by the American Joint Committee on Cancer (AJCC) [20], which are broadly based on the staging of conventional adenocarcinomas of the same site. For the jejuno-ileum, these two NET staging systems are virtually identical [21] (table 2). Although representing a definite step forward, these schemes may need refining, including stratification of the number of lymph nodes involved by metastasis, distinction between nodal micro- and macrometastases, and substaging of distant metastatic disease [18].



Evidence-based revision of these staging systems will likely be of value in further refining jejuno-ileal NET prognostication, as tumor stage at diagnosis remains the most important predictor of survival. Whilst overall survival at 5 and 10 years for jejuno-ileal NETs is cited as 60% and 43%, respectively [9, 22], five-year survival for patients with localized tumors is 65–75%, and 50% for those with non-localized disease [23]. Metastatic involvement of the liver, which will eventually develop in most patients with jejuno-ileal NETs [24], also significantly impacts on outcome. In the absence of hepatic metastases, 5- and 10-year survival rates for patients with a jejuno-ileal NET are 72% and 60%, respectively; this drops to 35% and 15% if liver metastases are present [22]. Much of this data is dated, however, and continuing advances in diagnosis and therapy seem to be leading to significant improvement in the outcome of patients with these tumors, particularly in specialized centers [1, 3, 25, 26].

Although the utilization of uniform and universally accepted nomenclature, grading and staging of jejuno-ileal (and other GI) NETs remains the ultimate goal, this is not likely to be attained in the foreseeable future. Pathologists should therefore adhere to a minimum pathology data set when reporting GI NET cases. This should include specific pathologic variables required for grading and staging (as well as the specific scheme used), and any other histological features of known or potential prognostic significance (including vascular/perineural invasion and non-ischemic tumor necrosis). One such international consensus data set for both biopsy and resection specimens of primary and metastatic GI NETs has recently been proposed by Klimstra et al [18]. Utilization of such a minimum data set is advised to ensure seamless and consistent translation of an individual patient's disease into any existing or revised scheme/system, thus ultimately improving prognostication and enabling optimal therapeutic management.

Schistosomiasis and GI NETs
Schistosomiasis (bilharzia) is a trematode parasitic infection affecting some 200 million people worldwide [27]. Most human infections are caused by three species (Schistosoma haematobium, japonicum and mansoni); S. intercalatum and mekongi are less common. Geographic distribution aids in the distinction between species. Parasite ova are usually identified on urine (S. haematobium) or stool (other Schistosoma spp) microscopy. Size, morphology and staining characteristics are all of value in typing bilharzia ova in histologic sections (table 3).



The causal relationship between S. haematobium and squamous cell carcinoma of the urinary tract is well documented [28]. Some data also suggest a possible association between S. japonicum and colorectal as well as hepatocellular carcinoma [29], but are not uniformly accepted. Potential mechanisms for the role of schistosomiasis in carcinogenesis include the presence of endogenously produced carcinogens, chronic immunomodulation with impairment of immunological surveillance, symbiotic action of other infective agents and the presence of schistosomal toxins. Currently, there is scant literature on an association between schistosomiasis and GI NETs. Only isolated cases of intestinal schistosomiasis associated with a rectal NET [30], as well as appendiceal mixed neuroendocrine and glandular tumors (goblet cell carcinoids) [31] have been documented. Whilst underreporting of this association in endemic areas is likely, no data exist to support a causal role for schistosomiasis in the pathogenesis of jejuno-ileal or other GI NETs. This finding, therefore, should be considered fortuitous.

Conclusions
Histopathologic diagnosis of a jejuno-ileal NET is seldom challenging, but application of standardized terminology, grading and staging is. The recent 2010 WHO classification and ENETS/AJCC TNM staging systems, whilst not without their limitations, have attempted to address problematic issues and past shortcomings. WHO grading of GI NETs is currently based on tumor morphology and proliferative rate. The introduction of dedicated GI NET staging systems is a step forward, but these will likely need refining as more data become available. In the interim, pathologists should adhere to a minimum pathology data set when reporting on these tumors. At present, no evidence exists to support a causal role for schistosomiasis in the pathogenesis of GI NETs.

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