Under the Microscope: A Case Report of Thoracic SMARCA4-Deficient Undifferentiated Tumor with Review of the Literature.

OBJECTIVE: SMARCA4-deficient undifferentiated tumor (SMARCA4-UT) is a highly malignant neoplasm with an undifferentiated or rhabdoid phenotype, posing a diagnostic challenge. This case report aims to create awareness about this rare neoplasm while dealing with cases presenting with undifferentiated morphology. CASE REPORT: A 55-year-old gentleman with constitutional symptoms and lymphadenopathy. Imaging revealed a mass lesion in the right upper lobe of the lung. A biopsy of the cervical lymph node showed diffusely effaced architecture replaced by sheets of undifferentiated pleomorphic cells with vesicular nuclei, prominent nucleoli, eosinophilic cytoplasm, and multiple necrotic foci. An extensive immunohistochemistry (IHC) panel was applied, which showed positivity for synaptophysin, vimentin, and focal CD34 and EMA expression. Other markers like pan-cytokeratin, p40, TTF1, CD56, INSM1, calretinin, CD45, SOX10, S100, CD30, CD117, SMA, and Desmin were negative, with INI1 retained. The IHC panel excluded the morphological differentials of carcinoma, lymphoma, rhabdomyosarcoma, melanoma, and germ cell tumor. Further literature review led to the possibility of the SMARCA4-UT entity, which had a morphology and IHC profile similar to the present case. Testing for SMARCA4 (BRG-1) by IHC showed a complete loss in the tumor cells, favoring the diagnosis of Thoracic SMARCA4-deficient undifferentiated tumor (SMARCA4-UT). CONCLUSION: SMARCA4-UTs are rare, highly aggressive, and poorly differentiated thoracic tumors. Recognizing them is vital as there is potential for therapeutic interventions such as immunotherapy and SMARCA4-targeted therapies, offering promising prospects for the future.

Cytologic Features of SMARCA4-Deficient Thoracic Sarcoma: A Case Report and Comparison with Other SWI/SNF Complex-Deficient Tumors.

BACKGROUND: SMARCA4-deficient thoracic sarcoma is a recently proposed entity of soft tissue tumors associated with an extremely poor prognosis. Its cytologic features have not been well described in the literature yet. CASE: A woman in her early 30s who presented with chest pain was found to have a tumor in the right chest wall. Cytologic smears revealed numerous atypical round-to-polygonal cells appearing singly or in loosely cohesive clusters. These cells had a well-defined cell border, scant-to-moderate cytoplasm, and enlarged vesicular nuclei with prominent nucleoli. In addition, some cells with eosinophilic globular intracytoplasmic inclusions and eccentrically located nuclei, consistent with rhabdoid cells, were observed. Immunocytochemically, the cells were at least focally positive for cytokeratin CAM5.2 and CD34 and showed a significantly reduced BRG1/SMARCA4 expression. The diagnosis was confirmed by histological, immunohistochemical, and genetic analysis of a metastatic lesion to the left axillary lymph node. CONCLUSION: Although the cytologic features of SMARCA4-deficient thoracic sarcoma are not fully unique, they are sufficiently characteristic to suspect this tumor in cases of supporting clinical and radiological features, which may promote additional immunological or molecular testing to establish a definitive diagnosis.

INSM1 Demonstrates Superior Performance to the Individual and Combined Use of Synaptophysin, Chromogranin and CD56 for Diagnosing Neuroendocrine Tumors of the Thoracic Cavity.

Despite the importance of recognizing neuroendocrine differentiation when diagnosing tumors of the thoracic cavity, the sensitivity of traditional neuroendocrine markers is suboptimal, particularly for high-grade neuroendocrine carcinomas such as small cell lung carcinoma and large cell neuroendocrine carcinoma. To increase sensitivity, neuroendocrine markers are routinely ordered as panels of multiple immunostains where any single positive marker is regarded as sufficient evidence of neuroendocrine differentiation. Insulinoma-associated protein 1 (INSM1) is a well-validated transcription factor of neuroendocrine differentiation that has only recently been evaluated for diagnostic use. We performed INSM1 immunohistochemistry on a large series of thoracic neuroendocrine and non-neuroendocrine tumors and compared its performance to synaptophysin, chromogranin, and CD56. INSM1 was positive in 94.9% of small cell lung carcinomas and 91.3% of large cell neuroendocrine carcinomas, compared with 74.4% and 78.3% with the combined panel of traditional markers. INSM1 also stained all (100%) of the atypical carcinoids, typical carcinoids and mediastinal paragangliomas, but only 3.3% of adenocarcinomas and 4.2% of squamous cell carcinomas. Overall, INSM1 demonstrated a sensitivity of 96.4% across all grades of thoracic neuroendocrine tumors, significantly more than the 87.4% using the panel of traditional markers (P=0.02). INSM1 is sufficiently sensitive and specific to serve as a standalone first-line marker of neuroendocrine differentiation. A more restrained approach to immunohistochemical analysis of small thoracic biopsies is appropriate given the expanding demand on this limited material for therapeutic biomarker analysis.

SMARCA4-deficient thoracic sarcoma: report of a case and insights into how to reach the diagnosis using limited samples and resources.

SMARCA4-deficient thoracic sarcoma is a recently proposed new entity of soft tissue sarcomas with an undifferentiated round cell morphology that is diagnostically challenging. Here we report a case of this tumor where the diagnosis was established using limited samples and resources. A woman in her early 30s developed two intrathoracic masses. Biopsies for these lesions showed sheets of undifferentiated round/rhabdoid cells that retained SMARCB1 expression. Further analysis revealed a reduced SMARCA4 expression and a complete loss of SMARCA2 expression in tumor cells. Subsequent Sanger sequencing identified a nonsense c.1546A>T (p.516Lys>Ter) mutation in SMARCA4 and confirmed the diagnosis. Our case highlighted clinicopathological correlation and rational use of tissue sections for immunohistochemistry may enable to diagnose this tumor even when only limited samples are available. Recognition of this new entity is important for further understanding of the disease and the future development of specific therapies.

NUT carcinoma in children and adults: A multicenter retrospective study.

BACKGROUND: Nuclear protein of the testis (NUT) carcinoma (formerly NUT midline carcinoma) is an aggressive tumor defined by the presence of NUT rearrangement with a poor prognosis. This rare cancer is underdiagnosed and poorly treated. OBJECTIVE: The primary objective of this study was to describe the clinical, radiologic, and biological features of NUT carcinoma. The secondary objective was to describe the various treatments and assess their efficacy. METHODS: This retrospective multicenter study was based on review of the medical records of children and adults with NUT carcinoma with specific rearrangement or positive anti-NUT nuclear staining (>50%). RESULTS: This series of 12 patients had a median age of 18.1 years (ranges: 12.3-49.7 years). The primary tumor was located in the chest in eight patients, the head and neck in three patients, and one patient had a multifocal tumor. Nine patients presented regional lymph node involvement and eight distant metastases. One-half of patients were initially misdiagnosed. Specific NUT antibody was positive in all cases tested. A transient response to chemotherapy was observed in four of 11 patients. Only two patients were treated by surgery and five received radiotherapy with curative intent. At the end of follow-up, only one patient was still in remission more than 12 years after the diagnosis. Median overall survival was 4.7 months (95% confidence interval [CI]: 2.1-17.7). CONCLUSION: NUT carcinoma is an aggressive disease refractory to conventional therapy. Early diagnosis by NUT-specific antibody immunostaining in cases of undifferentiated or poorly differentiated carcinoma to identify the specific rearrangement of NUT gene is useful to propose the optimal therapeutic strategy.

[Efficacy of PD-1/PD-L1 immune checkpoint inhibitors and PD-L1 testing in thoracic cancers]. / Efficacité des inhibiteurs du checkpoint immunitaire PD-1/PD-L1 et testing PD-L1 dans les cancers thoraciques.

Tumoral immune environment is a major component of cancer. Its composition and its organization represent a reproducible characteristic of tumors and a validated prognostic factor. In non-small cell lung cancer (NSCLC), cytotoxic T CD8+ lymphocyte density, associated with a Th1 environment and tertiary lymphoid structures impacts survival. Tumor cell-immune cell interaction is targeted by PD1/PD-L1 inhibitors. In advanced NSCLC, PD1/PD-L1 inhibitors are more effective than second-line chemotherapy. Pembrolizumab outperforms first-line chemotherapy in NSCLC strongly positive for PD-L1. PD1/PD-L1 inhibitors are currently tested in mesothelioma and thymic tumors. PD-L1 expression evaluated with immunochemistry is the most studied predictive biomarker of PD1/PD-L1 inhibitor efficacy. Tumor and immune cell expression of PD-L1 is still difficult to evaluate because of intra-tumoral heterogeneity and expression modulation by the microenvironment. Four commercial diagnostic antibodies are in development, with differences concerning recognized epitopes, methodology of evaluation of PD-L1 expression, positivity threshold, kit and platforms used. Clinical trials in NSCLC have shown that patients with tumors strongly positive for PD-L1 derived the best clinical benefit with PD1/PD-L1 inhibitors whereas clinical benefit is less common in tumors negative for PD-L1. PD-L1 expression is not a perfect biomarker since some PD-L1 negative NSCLC respond to PD1/PD-L1 inhibitors and some PD-L1 positive NSCLC do not. PD-L1 testing is likely to be implemented in daily practice for selection of advanced NSCLC that will be treated with pembrolizumab, underscoring the relevance of ongoing harmonization studies of the use of the different antibodies available for PD-L1 testing.

[PD1/PD-L1 immunohistochemistry in thoracic oncology: Where are we?] / Immunohistochimie PD-1/PD-L1 en oncologie thoracique : où en sommes-nous ?

The assays for the assessment of the PD-L1 status by immunohistochemistry are available in clinical studies in thoracic oncology to predict response to immunotherapies targeting the PD-1/PD-L1 pathway. With the arrival of this new class of molecules in second line and very soon in first line of treatment for patients with advanced or metastatic non-small cell lung cancer, these tests will certainly be required in routine once these new drugs will be granted marketing authorization. The rapid introduction of these "companion" or "complementary" tests seems essential to select patients to benefit from these effective but also expensive and sometimes toxic therapies. Although challenged by some oncologists (as some patients not expressing PD-L1 may sometimes respond to PD-1/PD-L1 blockade), the anti-PD-L1 immunohistochemically approach seems inevitable in 2017. This new activity developed in the pathology laboratories raises several questions: which anti-PD-L1 clone should be used? On which device? What threshold of positivity should be considered? Should PD-L1 expression be assessed on tumor cells as well as on the immune cells? What controls should be used? Comparative studies are underway or have been already implemented in order to answer some of these questions. This review addresses the different evaluation criteria for immunohistochemistry using the main anti-PD-L1 antibodies used to date as well the recently published studies using these antibodies in thoracic oncology.

Epithelioid sarcoma-like hemangioendothelioma on the chest wall.

Epithelioid sarcoma-like hemangioendothelioma is a rare vascular neoplasm that usually occurs in the distal extremities of young adults. Although the overall behavior of this neoplasm is usually indolent with a low risk of distant metastasis, the risk of local recurrence is significant. Therefore, initial surgical treatment with an adequate margin is important to improve the prognosis. However, epithelioid sarcoma-like hemangioendothelioma is frequently misdiagnosed. A preoperative misdiagnosis could result in resection without a sufficient margin. Herein, we describe a 68-year-old man with epithelioid sarcoma-like hemangioendothelioma on the chest wall, which was treated by wide resection despite difficulties with the preoperative diagnosis.

Ectopic thymic carcinoma presenting as an intrathoracic mass.

An asymptomatic 83-year-old man was found to have a right intrathoracic tumor. Computed tomography demonstrated a soft-tissue density mass measuring 55 × 25 × 22 mm adjacent to the right anterior chest wall. At surgery, the tumor was found to adhere to the diaphragm and right lung, contiguous with the mediastinal fat tissue. Histology of the resected specimen demonstrated proliferation of spindle and sarcomatous cells with multinucleated giant cells. Thus the tumor was diagnosed as undifferentiated thymic carcinoma and was considered to have arisen from ectopic thymic tissue. At 2 years postoperatively, the patient had no evidence of recurrence.

Solitary fibrous tumour of the chest wall.

Extrapleural solitary fibrous tumours (SFTs) are rare tumours characterized by patternless spindle cells with haemangiopericytoma-like vascular spaces. Previously the tumours have been classified as haemangiopericytoma, an entity that is now considered obsolete. We report a case of extrapleural SFT arising in the soft tissue of the chest wall. The patient was a 31-year-old Malay lady presenting with a mobile swelling of the right chest wall for more than five years. During excision the tumour was noted to be well-circumscribed and yellowish in colour, giving an impression of lipoma. Microscopically, the tumour had patternless architecture, characterized by hypocellular and hypercellular areas. It was composed of uniform, spindle-shaped cells displaying oval nuclei, inconspicuous nucleoli, pale cytoplasm and indistinct cell borders. The mitotic count was 2 per 10 HPF. Branching, medium-sized thin-walled blood vessels in a haemangiopericytomatous growth pattern, some with hyalinised wall were identified. The neoplastic cells were immunoreactive to CD99 and CD34 and were non-immunoreactive to Desmin, Smooth Muscle Actin, S100 protein and EMA. We elucidate the challenges in diagnosing this tumour in this unusual location.

Endobronchial-ultrasound needle aspiration and endoscopic ultrasound-fine-needle aspiration in thoracic diseases.

EBUS-TBNA and EUS-FNA are minimally invasive techniques rapidly gaining ground in the non-surgical invasive diagnostic approach to thoracic diseases due to their high accuracy and low morbidity and mortality compared to surgical techniques. Moreover, in the diagnosis and staging of lung cancer the combination of the two techniques is superior to either test alone. In this review we focus on the role of EBUS-TBNA and EUS-FNA in both malignant and non-malignant thoracic diseases.

Thoracic Myoepithelial Tumors: A Pathologic and Molecular Study of 8 Cases With Review of the Literature.

Thoracic myoepithelial tumors (MTs) are a rare group of tumors showing predominant or exclusive myoepithelial differentiation. They are poorly characterized from both a morphologic and genetic standpoint, in particular features that separate benign from malignant behavior. We examined the histologic and immunohistochemical features of 8 primary thoracic MTs and performed fluorescence in situ hybridization for EWSR1, FUS, PLAG1, and HMGA2, as well as several partner genes. Half (4/8) of the MTs occurred in large airways, and 3 had infiltrative borders. All cases showed immunoreactivity for epithelial markers, in conjunction with S100 protein or myogenic markers. MTs showed morphologic characteristics analogous to MTs at other sites, with no tumors having ductal differentiation. Necrosis and/or lymphovascular invasion was present in 5 cases, with mitotic activity ranging from 0 to 6 mitoses/2 mm² (mean 1). Metastases occurred in 2 cases, and no patients died of disease. Gene rearrangements were identified in half of the cases, with EWSR1-PBX1, EWSR1-ZNF444, and FUS-KLF17 fusions identified in 1 case each and 1 case having EWSR1 rearrangement with no partner identified. No cases were found to have HMGA2 or PLAG1 abnormalities. Compared with fusion-negative tumors, fusion-positive tumors tended to occur in patients who were younger (50 vs. 58 y), female (1:3 vs. 3:1 male:female ratio), and demonstrated predominantly spindle and clear cell morphology. Using a combined data set of our case series with 16 cases from the literature, poor prognosis was significantly correlated with metastases (P=0.003), necrosis (P=0.027), and ≥5 mitoses/2 mm²/10 high-power field (P=0.005). In summary, we identify a subset of thoracic MTs harboring rearrangements in EWSR1 or FUS, and our data suggest that necrosis and increased mitotic activity correlate with aggressive clinical behavior.

A subcutaneous pleomorphic hyalinizing angiectatic tumor of soft parts of the right chest wall: report of a rare case.

Pleomorphic hyalinizing angiectatic tumor of the soft parts is an extremely rare mesenchymal tumor consisting of spindled and pleomorphic tumor cells and clusters of ectatic, fibrin-lined vessels. It typically occurs in the subcutaneous tissues of the distal extremities, usually the ankles and feet. Here we present a case of pleomorphic hyalinizing angiectatic tumor of the soft parts of the right chest wall in a 51-year old female. The tumor was subcutaneous, nonencapsulated, and about 2.0 cm×1.0 cm. Microscopically, the tumor was composed of numerous ectatic, fibrin-filled, thin-walled blood vessels, surrounded by spindled or pleomorphic tumor cells arranged in sheet-like or fascicular architecture, or randomly. Mitotic activity of the tumor cells was low. Immunohistochemical analysis shows that the tumor cells were positive for CD34 and vimentin, but negative for CD31, CK, desmin, EMA, HMB45, Myo D1, P63 and S-100. Ki67 index was about 1%.

Giant tubular adenoma of the accessory breast in the anterior chest wall occurred in a pregnant woman.

BACKGROUND: Tubular adenoma of the breast is a rare benign epithelial tumor and only a few literatures have been reported; so far, no cases of tubular adenoma occurred in the accessory breast have been reported in the English literature. Clinical presentation and management of our patient are discussed along with a review of the literature on accessory mammary and tubular adenoma. CASE PRESENTATION: We present a case of 26-year-old woman (gravid 4, para 1) at 37 weeks of pregnancy with rapid enlargement in left anterior chest wall during pregnancy. Physical examination showed the left accessory breast was obviously bigger than the right one that only had a light areola around a small nipple. An elastic, mobile well-circumscribed mass measuring approximately 15 cm × 15 cm was palpated. Moreover, it was edematous and congestive with an increase in local temperature. The breast ultrasound further demonstrated the mass was a relatively homogeneous solid with short stripe blood flow signal. A single live fetus of 37 weeks gestation was observed by abdominal ultrasound scan. After a 2850 g male neonate was delivered, the right accessory breast and the mass in left accessory breast were removed. The resected specimen appeared as a solid white elastic mass with a smooth surface and the cut surface was red-grayish. Microscopically, the lesion consisted of tightly packed homogenous glandular structures which are supported by a single layer of myoepithelial cells with sparse intervening stroma. CONCLUSIONS: We describe a very rare case of giant tubular adenoma arising within an accessory breast in the anterior chest wall in a late pregnancy woman. The high concentrations of estrogen, progesterone and prolactin might account for the significant tumor enlargement during pregnancy. To our knowledge, this is the first case of giant tubular adenoma occurred within the accessory breast in the anterior chest wall. VIRTUAL SLIDES: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/6210811191552106 .

The diagnosis of thoracic malignant mesothelioma: practical considerations and recent developments.

Pleural biopsies for the evaluation of malignant mesothelioma can be some of the most challenging cases faced by the practicing surgical pathologist. We review the epidemiology, clinical presentation, and imaging studies in patients with malignant mesothelioma, and then present a practical approach to the diagnosis using the cytologic features for malignancy and whether there is an epithelioid or spindled morphology; four main scenarios are discussed. The pertinent immunohistochemical work-up is reviewed for each scenario. Following this general overview, more unusual histologic patterns are compared and unusual presentations are discussed. Brief mention of grading systems for malignant mesothelioma and the use of electron microscopy and molecular studies is made. Practical considerations in the diagnosis of malignant mesothelioma are made throughout.

[Extracardial rhabdomyoma: a clinicopathologic analysis of 9 cases].

OBJECTIVE: To investigate the clinicopathologic characteristics, differential diagnosis and biological behavior of extracardiac rhabdomyoma. METHODS: Nine cases of extracardiac rhabdomyoma diagnosed between January of 1997 and July of 2014 were reviewed. The clinical, pathologic and immunohistochemical profiles were evaluated. RESULTS: There were 5 males and 4 females at diagnosis with age ranging from 2 years and three months to 59 years (mean, 37.6 years). Sites included the head and neck region (7 cases), chest (1 case ) and vagina wall (1 case). Clinically, most cases manifested as a subcutaneous nodule or as a submucosal polypoid lesion with a mean diameter of 3.2 cm. Histologically, 4 were adult-type rhabdomyoma characterized by tightly packed large round or polygonal rhabdomyoblasts with abundant eosinophilic to clear cytoplasm; 3 were myxoid variant of fetal rhabdomyoma composed of immature myofibrils, spindled and primitive mesenchymal cells embedded in a myxoid background, 1 was an intermediate form of fetal rhabdomyoma consisting of densely arranged differentiated myoblasts with little myxoid stroma; 1 was a genital rhabdomyoma composed of elongated or strap-like myoblasts scattered in loose fibrous connective tissue. By immunohistochemistry, they showed diffuse and strong positivity for desmin, MSA and myoglobin with variable expression of myogenin. A case of intermediate type also stained for α-smooth muscle actin. Follow up data (2 months ~ 17 years) showed local recurrence in one patient 6 months after surgery. CONCLUSIONS: Rhabdomyoma is a distinctively rare benign mesenchymal tumor showing skeletal muscle differentiation, which may occassionally recur if incompletely excised. Familiarity with its clinical and morphological variants is essential to avoid misdiagnosing this benign lesion as embryonal rhabdomyosarcoma.

Sclerosing rhabdomyosarcoma of a chest wall in an adult: a case report and review of the literature.

Sclerosing rhabdomyosarcoma (SRMS) is a newly recognized and rare variant of rhabdomyosarcoma. This soft tissue tumor has not yet been reported as a thoracic lesion. We report a case of a 26-year-old woman who presented with a large chest wall tumor. The tumor originated from the right anterior chest wall and protruded into the intra- and extrapleural cavity. A transcutaneous needle biopsy revealed spindle cells in an abundant hyalinized and fibrous stroma. Although the tumor was considered as a malignant soft-tissue neoplasm, a definitive diagnosis could not be established. A wide excision of the chest wall including the second, third and fourth rib and a part of sternum was performed. Histologically, cytoplasmic cross-striations were found in a portion of the tumor cells. The tumor cells were positive for muscle markers, and the tumor was diagnosed as rhabdomyosarcoma consistent with a sclerosing type of rhabdomyosarcoma. Eighteen months after the complete resection, the patient has pleural disseminations but is alive and undergoing chemotherapy. This case highlights the histologic features of a rare form of rhabdomyosarcoma, and emphasizes the importance of awareness of its existence and the utility of skeletal muscle markers in distinguishing sclerosing rhabdomyosarcoma from its mimics.

Giant solitary fibrous tumor of the diaphragm: a case report and review of literature.

A young gentleman presented with difficulty in breathing. Computed tomography (CT) scan showed a huge mass located between the heart and stomach, which might have rooted in the diaphragm. Magnetic resonance imaging (MRI) with enhanced three dimensional construction showed a lobulated, heterogeneous soft tissue mass with short T1 weighted imaging signal and flake long T2-weighted imaging (T2WI). Tumor-enhanced scanning demonstrated heterogeneous contrast enhancement. The preliminary diagnosis was intra-abdominal huge mass and considering sarcoma. Resection was conducted where the base of the tumor was located in the diaphragm oppressing the left liver lobe and heart. The base of the tumor, together with partial surrounding of the diaphragm, pericardium base, and the left lateral hepatic segment, was resected. The defect in the diaphragm and pericardium was repaired by patching, and thoracic close drainage and abdominal drainage were placed following the surgical operation. The pathological report showed giant solitary fibrous tumor (SFT). This case report may provide a reference resource for the diagnosis and treatment of SFT located in the diaphragm.

Epithelioid angiosarcoma at chest wall which needs to be carefully distinguished from malignant mesothelioma: report of a rare case.

Angiosarcoma is a malignant soft tissue tumor the cells of which variably recapitulate the morphologic and functional features of normal endothelium. Most lesions are located in the deep muscles of the lower extremities followed by the arm, trunk and head and neck. Herein we present a case of epithelioid angiosarcoma which is a variant of angiosarcoma at chest wall in a 73-year-old female. Morphologically, the tumor cells are arranged predominantly in luminal structures which can be seen in both angiosarcoma and malignant mesothelioma. Most of the tumor cells are large rounded "epithelioid" cells with abundant eosinophilic cytoplasm which can be also seen in both tumors. The epithelioid of cytomorphology and the localization at chest wall of this case may remind of a diagnosis of malignant mesothelioma which should be carefully distinguished from epithelioid angiosarcoma from imaging and morphology. CT scanning of the patient shows a mass at her chest wall, the majority of which is around the rib but not inside the lung which indicates a tumor originates more likely from soft tissues of chest wall but not pleura. Immunohistochemical staining shows that the tumor cells are positive for cytokeratin, CD31, Vimentin and WT1, and negative for CEA, TTF-1, Calretinin, Mesothelial Cell (MC), CD56, CK19, and Hepatocyte. Thus this case is diagnosed as epithelioid angiosarcoma but not malignant mesothelioma. From this case we suggest that carefully reading and understanding of the imaging are a very important clue for appropriate diagnosis. A misdiagnosis may occur on the basis of misunderstanding of tumor localization and a consequent inappropriate immunohistochemical staining programme.