A comparative study of immunohistochemical staining for neuron-specific enolase, protein gene product 9.5 and S-100 protein in neuroblastoma, Ewing's sarcoma and other round cell tumours in children

A comparative study of immunohistochemical staining for neuron-specific enolase (NSE), protein-gene product 9.5 (PGP 9.5) and S-100 was made in 71 undifferentated round cell tumours from 65 children using formalin-fixed tissues and a standard alkaline phosphatase-anti-alkaline phosphatase method. All of 29 neuroblastomas marked for NSE and 27 for PGP 9.5; staining was diffuse and usually strong in all tumour elements, irrespective of the degree of differentiation. Patterns of staining remained consistent in primary, recurrent and metastatic tumours and were not modified by previous chemotherapy. S-100 staining was weak and confined to cell processes and schwannian elements in less than half of the tumours studied. Two primitive neuroectodermal tumours both stained strongly for NSE and PGP 9.5. Staining for NSE was observed in single maturing cells in 3/12 rhabdomyosarcomas and in tubular elements in 2/4 Wilms' tumours; primitive rhabdomyoblasts and undifferentiated renal blastema were negative; seven lymphomas were negative. Six of 17 skeletal Ewing's sarcomas showed light to moderate cytoplasmic staining for NSE and PGP 9.5. The site, histology and clinical course of these marker-positive Ewing's sarcomas showed no distinctive features. Staining for PGP 9.5 is a useful additional marker for neural differentiation in round cell tumours.     

N-myc amplification in neuroblastomas: histopathological, DNA ploidy, and clinical variables.

The association between tumour N-myc amplification, DNA ploidy, and various prognostic factors (patient age, tumour stage at diagnosis, primary site and histopathological differentiation) was studied in 18 children who had neuroblastoma, ganglioneuroblastoma, or ganglioneuroma. Amplification of genomic N-myc was observed in six patients who had been treated with chemotherapy before surgery (one with stage III and five with stage IV). All these tumours were located in the adrenal or upper retroperitoneum; five were neuroblastomas and one was a ganglioneuroblastoma. Three of them were aneuploid and three diploid. The degree of N-myc patients with tumour N-amplification varied from 20 to 1500 copies without relation to ploidy. All patients with tumour N-myc amplification died of their tumour. Amplification was always associated with poor prognosis, independent of tumour cell ploidy. Four patients without such amplification also died: three had diploid tumours, the fourth was aneuploid. It is suggested that genomic N-myc amplification takes place mainly in adrenal and retroperitoneal neuroblastomas and can be a feature of tumours with and without histological signs of differentiation. The precise role of N-myc in the pathogenesis of neuroblastoma remains unclear.     

N-myc gene product expression in neuroblastoma.

The presence and distribution of N-myc gene product were studied in 13 neuroblastomas and five ganglioneuroblastomas, using immunohistochemical techniques. Nine tumors (eight neuroblastomas and one ganglioneuroblastoma of composite type) contained neuroblastoma cells with positive nuclei for N-myc protein. Microscopic examination showed that most of the positive neuroblastoma cells seemed to be immature, with no apparent neuronal differentiation. Nine of 11 tumours with amplified N-myc gene copies exhibited tumour cells with positive immunostaining for the N-myc gene product, while none of the seven non-N-myc amplified cases contained immunoreactive tumour cells. The survival of the patients positive for N-myc protein was significantly low compared with that of the negative ones. It is concluded that immunohistochemical staining for the N-myc gene product will facilitate prediction of the prognosis of patients with neuroblastoma.     

High level of apoptosis and low AKT activation in mass screening as opposed to standard neuroblastoma

Sartelet H, Ohta S, Barrette S, Rougemont A‐Laure, Brevet M, Regairaz M, Harvey I, Bernard C, Fabre M, Gaboury L, Oligny L L, Bosq J, Valteau‐Couanet D & Vassal G
(2010) Histopathology 56 , 607–616 High level of apoptosis and low AKT activation in mass screening as opposed to standard neuroblastoma Aims: Neuroblastoma is a paediatric solid tumour with a poor outcome except in children <1 year old. Based on catecholamine urinary excretion, mass screening (MS) programmes have been organized but failed to decrease the mortality of this tumour. To test the hypotheses of a spontaneous maturation/differentiation or regression, the levels of poly (ADP‐ribose) polymerase (PARP)‐1, an early apoptosis marker, of PhosphoAKT, a major apoptosis inhibitor, and of maturation/differentiation were compared in standard and in MS neuroblastomas. Methods and results: We performed a case–control study of 55 primary tumours and 21 metastases of MS neuroblastomas. Matched controls were standard unscreened neuroblastomas and were paired according to age, stage, and MYCN amplification. The tumours were included in tissue microarrays. Immunohistochemical staining was performed using antibodies against, AKT, phosphoAKT, TRKB and PARP‐1. The expression of PARP‐1 and that of phosphoAKT were significantly higher in standard than in MS neuroblastomas independently of age and stage of the tumour. PhosphoAKT and PARP‐1 expression was significantly correlated in both tumours. Conclusions: These data suggest that the better prognosis of patients with MS neuroblastomas compared with classical neuroblastomas was secondary to spontaneous tumour regression mediated by higher levels of apoptosis associated with low activation of AKT.     

Neuron-specific enolase in the diagnosis of neuroblastoma and other small, round-cell tumors in children

Immunocytochemical staining for neuron-specific enolase (NSE) was performed in 44 round-cell tumors from children by the improved immunoglobulin-enzyme bridge method with antiserum against NSE. The tumors studied consisted of 15 neuroblastomas showing various degrees of histologic differentiation, 13 Ewing's sarcomas, ten soft tissue sarcomas of diverse origin, and six lymphomas of bone and soft tissues. Neuron-specific enolase was detected in all neuroblastomas, irrespective of the degree of histologic differentiation. None of the other round-cell tumors was positive for NSE, except one embryonal rhabdomyosarcoma that contained differentiated myoblasts. The primitive cells of this tumor were negative as well. It is concluded that immunocytochemical staining with antibodies to NSE is a practical and reliable method for distinguishing neuroblastomas from other nonneural round-cell tumors in children. This is true even for the most primitive forms of neuroblastomas, in which morphologic techniques are less reliable. Neuron-specific enolase may also be useful in delineating the neural histogenesis of other ill-defined tumors.     

Immunohistochemical localization of Trk receptor protein in pediatric small round blue cell tumors.

Expression of Trk protein has been documented by Northern analysis in neuroblastomas with good prognosis. To localize the expression of this protein at the cellular level within individual tumors, we adapted a recently characterized pan-Trk antibody for use in formalin fixed, paraffin-embedded tissue. We have examined a group of small round blue cell tumors occurring in children, including both high and low stage neuroblastomas, to assess the presence or absence of Trk expression and its cellular localization. Positive staining for Trk protein was observed in four of four low stage (good prognosis) neuroblastomas, five of five primitive neuroectodermal tumors/Ewing's sarcoma, five of five rhabdomyosarcomas, and no lymphomas. Within the neuroblastomas, expression of Trk protein was most striking in ganglion cells, in which positive cytoplasmic staining was demonstrated regardless of tumor stage. The latter observation may lend further insight into the pathobiology of this malignant childhood tumor.     

Immunohistochemical localization of neurofilaments and neuron-specific enolase in 29 cases of neuroblastoma.

Twenty-nine neuroblastomas have been examined with the use of rabbit antibodies specific for each of the three neurofilament polypeptides, with a monoclonal antibody specific for the NF-L polypeptide, and with a rabbit antibody specific for neuron-specific enolase. When frozen material was used, all neuroblastomas were positive with the neurofilaments antibodies. When alcohol-fixed paraffin-embedded material was used, neurofilament staining was weaker and the fixation procedure appeared to destroy the epitopes recognized by the NF-L antibodies preferentially. Although all neuroblastomas were positive for neurone-specific enolase, so were two rhabdomyosarcomas, suggesting that NSE is not an appropriate marker to distinguish the different small blue cell tumors of children.     

Alpha-fetoprotein production by a malignant mixed müllerian tumour of the uterus.

A case of alpha-fetoprotein production by a uterine malignant mixed müllerian tumour is described. The patient was a 68 year old woman who developed intraabdominal recurrence of a stage 1 uterine tumour which had been treated surgically seven years previously. Her serum alpha-fetoprotein was raised at 21,000 micrograms/l (normal < 10 micrograms/l) and staining with immunoperoxidase confirmed that the tumour was the site of alpha-fetoprotein production. The patient was treated with combination chemotherapy but died two weeks after the first course. This is believed to be only the second such case reported.     

Mesoblastic nephroma contains fibronectin but lacks laminin.

Non-metastatic mesoblastic nephromas from four young children were shown to contain fibronectin but not laminin using an immunoperoxidase staining procedure. In contrast, one metastasising spindle celled sarcomatous tumour from a neonate was laminin positive. During embryogenesis primitive nephrogenic mesenchyme contains only fibronectin and no laminin; metanephric blastema (permanent kidney) is positive for laminin. The staining for fibronectin and laminin may help to ascertain the histogenesis of different types of renal tumour.     

Immunohistochemical expression of epidermal growth factor receptor in salivary gland tumours

Summary Immunohistochemical localization of epidermal growth factor receptor (EGFR) in normal salivary glands and tumours (108 cases) was studied using a monoclonal antibody. In the normal salivary glands, EGFR was occasionally detected in ductal segments of intercalated, striated, and excretory ducts, but not in acinar cells. The frequency of positive EGFR staining in salivary gland tumours was not high: pleomorphic adenoma, 33.8%; mucoepidermoid tumour, 25.0%; adenolymphoma, 44.4%; and sialoadenocarcinoma, 66.6%. Pleomorphic adenomas showed positive staining for EGFR on the luminal side of luminal cells and in squamous metaplastic cells of tumour tissue. Some modified myoepithelial cells were also reactive whereas outer spindle tumour cells were unstained. Adenolymphomas regularly exhibited positive EGFR staining in the cell membrane; mucoepidermoid carcinoma displayed positive staining in cell membranes in epidermoid tumour cells and cytoplasmic staining in mucous-secreting tumour cells. Sialocarcinomas revealed cell membrane staining and whole cytoplasmic staining for EGFR. The immunohistochemical localization of EGFR could be classified into two types, one the cell membrane-positive type found in epithelial tumour cells, and the second the cytoplasmic positive type seen in normal ductal cells, the luminal tumour cells of pleomorphic adenomas and mucous-secreting tumour cells.     

Urinary bladder carcinoma producing granulocyte colony stimulating factor (G-CSF): A case report with immunohistochemistry

A rare case of urinary bladder carcinoma with granulocyte colony stimulating factor (G-CSF) production was reported. In an 83-year-old female, marked neutrophilia in the peripheral blood decreased from 132,500/mm³ to 3,300/mm³ after tumour resection. The tumour was a transitional cell carcinoma. The serum G-CSF level reduced from 238 pg/ml pre-operatively to normal (60 pg/ml) after the operation. Immunohistochemical investigation of the resected tumour with monoclonal antibody specific for G-CSF revealed positive staining in the carcinoma cells, confirming G-CSF secretion.     

Distribution of ferritin, transferrin and lactoferrin in breast carcinoma tissue.

An immunoperoxidase staining technique was used for detecting three major iron binding proteins (ferritin, transferrin and lactoferrin) in 40 breast carcinoma cases and six benign breast proliferative lesions. Ferritin staining was detected mainly in connectival stroma and in histiocytes surrounding neoplastic cells. Few and faint ferritin positivities were also detected in neoplastic cells of 20 carcinoma cases. Transferrin was found inconsistently in myoepithelial cells surrounding normal ductules, or around neoplastic ducts of ductal in situ carcinoma. In eight carcinoma cases, transferrin staining was also positive in neoplastic cells. Lactoferrin was detected only in normal breast epithelial cells and in benign breast proliferative lesions. These immunohistochemical findings may suggest that raised serum ferritin concentrations in breast carcinoma patients might be attributed to stromal reaction rather than to tumour synthesis. Transferrin staining of neoplastic cells in these carcinoma cases appears to be very intriguing, particularly since transferrin is considered an obligate requirement for growing cells, and transferrin receptors have been demonstrated only in dividing cells. On the basis of the immunohistochemical data, lactoferrin might be used as a pointer to benign lesions.     

Expression of pan-neuroendocrine proteins in 53 neuroblastic tumors. An immunohistochemical study with neuron-specific enolase, chromogranin, and synaptophysin

The presence and distribution of pan-neuroendocrine markers such as neuron-specific enolase (NSE), chromogranin (CG), and synaptophysin (SYP) were investigated by immunohistochemistry in 53 cases of neuroblastic tumors, including three cases of ganglioneuromas, 17 ganglioneuroblastomas, and 33 neuroblastomas. In ganglioneuromas, all three markers were observed both in ganglion cells and in neurofibrils. All cases of ganglioneuroblastoma were positive for these markers, however, some variability of staining intensity was noted. Of the 33 cases of neuroblastomas, all were positive for NSE, 23 (70%) for CG, and 31 (94%) for SYP. Neuron-specific enolase was detected not only in the majority of the neuroblasts showing signs of differentiation, but also in some undifferentiated neuroblasts. Chromogranin was found mainly in differentiated neuroblasts with enlarged cytoplasm and nuclei, but was scarcely found in undifferentiated cells. Synaptophysin was detected in some undifferentiated neuroblasts, as well as in differentiated neuroblasts. Two cases without SYP-positive cells were also negative for CG. Our observations conclude that antibodies against NSE and SYP are helpful as a diagnostic aid for neuroblastic tumors.     

Demonstration of apoptosis in neuroblastoma and its relationship to tumour regression

The in vivo occurrence of apoptosis in neuroblastomas was investigated. Histologically, a number of tumour cells showed typical apoptotic changes, including cell shrinkage, condensed and fragmented nuclei, eosinophilic cytoplasm, and absence of the inflammatory response. These cells coincided closely with the so-called karyorrhectic cells. An electrophoretic DNA ladder, a functional hallmark of apoptosis, was demonstrated in four of six tumours, and DNA fragmentation was detected in situ by terminal deoxytransferase-mediated nick end-labelling in 26 of 35 tumour specimens (74%). The labelled cell counts ranged from 5 to 62 per 5000 tumour cells (mean±SD: 15.0±14.5). Immunoperoxidase staining revealed that an apoptosis-suppressing protein, bcl-2, was expressed abundantly in advanced-stage tumours, whereas it was absent from karyorrhectic-apoptotic cells. Several tumours with the potential for spontaneous regression were bcl-2-deficient. Immunostaining of the Fas receptor for apoptosis demonstrated that the tumour cells expressed this molecule on their cell surfaces. Our results provide evidence of apoptosis in neuroblastomas and suggest that bcl-2 and the Fas receptor may play a role in its regulatory mechanisms.     

Central neurocytoma. Immunohistochemical study: MIB1, p53 and bcl-2. Report of 5 cases

To further characterize central neurocytoma, a rare intraventricular tumour described in 1982, we analyzed six tumours by immunohistochemistry for MIB1, p53 and bcl-2. bcl-2, an inhibitor of p53-mediated apoptosis is frequently expressed in gliomas, especially in tumors with wild-type p53. Its expression in peripheral neuroblastomas suggests a down-regulation during final terminal differentiation. Six tumors from five patients (one female/four males, age ranged from 18 to 63 years) were examined. All patients were alive from 2 to 88 months after initial surgical resection. On histological sections, tumours demonstrated a typical pattern. Synaptophysin staining was seen in all cases. Proliferation index was low (< 4.5%). bcl-2 was never expressed. p53 expression varied but within low values (< 10% of cells). These latter antibodies were rarely analyzed until now in this usually benign neoplasm which represents a well differentiated variant of neuron derived tumors.     

Non-transferrin bound iron and neutropenia after cytotoxic chemotherapy.

AIMS--To investigate and characterise the appearance of non-transferrin bound iron (NTBI) in the serum of patients after cytotoxic chemotherapy and to compare this with the onset and duration of neutropenia. METHOD--Non-transferrin bound iron was measured by a bleomycin assay in patients undergoing intensive chemotherapy for treatment of acute leukaemia or lymphoma. RESULTS--NTBI was detected after 26 of 27 courses of chemotherapy and lasted for a mean of 14.5 days. The presence of NTBI correlated with the serum iron binding saturation, but not with serum ferritin. Neutropenia occurred after all courses of chemotherapy and lasted for a mean of 20.0 days. NTBI and neutropenia occurred concurrently after 23 courses of chemotherapy, and had a mean joint duration of 9.5 days. CONCLUSIONS--NTBI is consistently present in the serum of patients after cytotoxic chemotherapy, often at the same time as the patient is neutropenic. This may be an additional risk factor for the development of infective episodes after chemotherapy.     

Molecular genetic evidence supporting the neoplastic nature of stromal cells in 'fibrosis' after chemotherapy for testicular germ cell tumours

A residual retroperitoneal mass containing only fibrosis and necrosis is present in 40-52% of patients with advanced testicular germ cell tumours after chemotherapy. The biological nature and genetic characteristics of the stromal cells in these residual masses have not been adequately investigated. Laser-microdissected stromal cells from 27 patients who underwent retroperitoneal lymph node dissection after chemotherapy for metastatic testicular germ cell tumour were analysed. Allelic loss in the stromal cells of fibrosis was present at one or more of the ten microsatellite DNA loci examined in 23 (85%) of the cases. Chromosome arm 12p anomalies, the hallmark of germ cell neoplasia, were present in nine (33%) cases. The high frequency of allelic losses and chromosome arm 12p anomalies in the stromal cells from residual retroperitoneal fibrous masses after chemotherapy for testicular germ cell tumours suggests that the stromal cells are derived from the same tumour progenitor cells as the pre-existing metastatic germ cell tumour.     

Reduced expression of p21WAF1 is an indicator of malignant behaviour in anal carcinomas

AIMS: p21 and p27 protein expression were examined in a comparatively large series of patients with squamous cell carcinoma of the anal canal and compared with clinical and histopathological data (tumour stage, nodal status and differentiation). METHODS AND RESULTS: We analysed the expression of p21 and p27 protein in 94 anal carcinomas by immunohistochemistry. Nuclear p21 and p27 staining were detected in 71% (67/94) and 75% (71/94) of the cases, respectively. There was no significant association between p27 staining and tumour stage, nodal status or overall survival. We observed that negative p21 immunoreactivity was significantly associated with poorly differentiated anal carcinomas. Furthermore, a shorter overall survival for patients with no p21 protein expression was seen. CONCLUSIONS: Our data indicate that p21 levels, but not p27 expression, may be a useful predictor of survival in patients with anal carcinomas.     

Oestrogen, progesterone, and androgen receptors in ovarian neoplasia: correlation between immunohistochemical and biochemical receptor analyses

AIM: To investigate the correlation between immunohistochemical and biochemical steroid receptor analyses by measurement of oestrogen, progesterone, and androgen receptor status in ovarian neoplasia. METHODS: Tissue samples were obtained from 27 ovarian neoplasms, including two borderline tumours. Immunohistochemical staining of the tissue slides was scored semiquantitatively, incorporating the intensity and percentage of positive staining (histo-score). Tumours with a histo-score of 10 or more were considered steroid receptor positive. The epithelial and stromal fractions of the tumours were analysed separately. To study the uniformity of receptor expression throughout a tumour, up to four samples were analysed. RESULTS: Immunohistochemical histo-scores of the oestrogen receptor in the epithelial fractions were significantly correlated with the biochemical oestrogen receptor values (r = 0.408). Androgen receptor status in the epithelial fraction was correlated with that in the stromal fraction (r = 0.741), while androgen receptor histo-scores in the epithelial fraction correlated with the biochemical assay values (r = 0.463). On biochemical analysis, 17 of the 27 ovarian tumours were oestrogen receptor positive and seven were progesterone receptor positive. On immunohistochemical analysis, eight tumours were oestrogen receptor positive and two were progesterone receptor positive. Biochemical analysis showed that 14 of the 26 tumours were slightly androgen receptor positive (10-50 fmol/mg protein), while all the others were negative. On immunohistochemical analysis, seven of the 26 tumours were androgen receptor positive. When two or more specimens from one tumour were analysed, marked differences in steroid status were found, especially in progesterone receptor and androgen receptor expression. Some parts of a tumour were steroid receptor positive, while other parts were negative owing to heterogeneity of expression. CONCLUSIONS: Immunohistochemical and biochemical analysis of steroid receptors in ovarian tumours correlated weakly or not at all. Heterogeneity of expression within a tumour and the presence of progesterone and androgen receptors in the stromal fraction partly accounted for this observation. Biochemical and immunohistochemical androgen receptor status was much lower than in previous reports.     

Nucleolar size indicates the rapidity of cell proliferation in cancer tissues

In order to define the importance of the nucleolus in tumour pathology, the relationship between nucleolar size and function and tumour mass growth rate was studied in vivo. Ten established human cancer cell lines from colon carcinomas and neuroblastomas were inoculated subcutaneously in athymic mice and the doubling time (DT) of the xenograft tumour mass was calculated. The tumour DTs ranged from 3.2 to 15.7 days. Nucleolar size was evaluated in sections from formalin-fixed and paraffin-embedded tumour samples after silver staining for AgNOR proteins, using a specific image analysis system. The nucleolar area values were inversely related to the xenograft tumour mass DTs (r=-0.90; p<0.001). Nucleolar functional activity was also evaluated using rapid, intermediate, and slow growing tumours (one each). The values of RNA polymerase I activity measured in vitro were strongly related to the corresponding tumour DTs (r=-0. 99; p=0.03). The labelling indices (LIs) of three proliferation markers, MIB1, PCNA, and bromodeoxyuridine (BrdU), were also evaluated. As revealed by the MIB1 and PCNA LIs, almost all the cells of the xenograft tumours were cycling (86.6+/-5.6 SD and 95. 5+/-2.0 SD, respectively). Neither the MIB1, PCNA or BrdU LIs were related to the xenograft tumour mass DT, showing that the different growth rates of tumour xenografts were not due to different growth fractions, but were mainly related to different cell proliferation rates. The present data demonstrate that the size and function of the nucleolus are related to the cell proliferation rate of cancer tissue. Evaluation of nucleolar size after silver staining of AgNOR proteins represents a unique parameter for the histological assessment of rapidity of cell proliferation in tumour lesions.