Intertumoral Heterogeneity of Primary Breast Tumors and Synchronous Axillary Lymph Node Metastases Reflected in IHC-Assessed Expression of Routine and Nonstandard Biomarkers.

Breast cancer (BC) remains a significant healthcare challenge. Routinely, the treatment strategy is determined by immunohistochemistry (IHC)-based assessment of the key proteins such as estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), and Ki-67. However, it is estimated that over 75% of deaths result from metastatic tumors, indicating a need to develop more accurate protocols for intertumoral heterogeneity assessment and their consequences on prognosis. Therefore, the aim of this preliminary study was the identification of the expression profiles of routinely used biomarkers (ER, PR, HER2, Ki-67) and additional relevant proteins [Bcl-2, cyclin D1, E-cadherin, Snail+Slug, gross cystic disease fluid protein 15 (GCDFP-15), programmed death receptor 1 (PD-L1), and phosphatase of regenerating liver 3 (PRL-3)] in breast primary tumors (PTs) and paired synchronous axillary lymph node (ALN) metastases. A total of 67 tissue samples met the inclusion criteria for the study. The expression status of biomarkers was assessed in PTs and ALN metastases using tissue microarrays followed by IHC. In 11 cases, the shift of intrinsic molecular BC subtype was noticed between PTs and paired ALN metastases. Moreover, a significant disproportion in E-cadherin presence (p = 0.0002) was noted in both foci, and the expression status of all proteins except for HER2 demonstrated considerable variance (k = 1, p < 0.0001). Importantly, in around 30% of cases, the ALN metastases demonstrated discordance, i.e., loss/gain of expression, compared to the PTs. Intertumoral synchronous heterogeneity in both foci (primary tumor and node metastasis) is an essential phenomenon affecting the clinical subtype and characteristics of BC. Furthermore, a greater understanding of this event could potentially improve therapeutic efficacy.

Tissue and serum mRNA profile of MMPs-2/9 as a potential novel biomarker for the most individual approach in infantile hemangiomas and cancer disease.

Propranolol is a widely-known beta-blocker approved for treating infantile hemangiomas (IH). The mechanisms behind the spectacular IH involution after propranolol treatment remain unclear. Recently, there is strong evidence of overexpression of numerous angiogenic factors in IH tissues, and it is reported that propranolol influences their pathways. However, a number of MMPs studies is highly limited. Here, for the first time, we propose a comprehensive approach by analyzing the expression levels of metalloproteinases-2/9 (MMPs-2/9) and tissue metalloproteinase inhibitor-2 (TIMP-2) in vivo on both, molecular and immunohistochemical levels, and in both, IH tissues and in the serum of IH patients, and relates the obtained results to the tumor's biology and systemic propranolol treatment. MATERIAL AND METHODS: MMPs-2/9 and TIMP-2 were analyzed in 71 IH tissue samples using immunohistochemistry and real-time PCR, and in 50 serum samples of IH patients by ELISA. RESULTS: Significantly lower MMPs-2/9 and higher TIMP-2 levels were observed in IH tissues on the mRNA level as well as lower serum MMP-2 concentration among the treated individuals. CONCLUSION: MMPs-2/9 and TIMP-2 are both involved in the biology of IH and the propranolol pathways enabling their antiangiogenic properties. The most reliable method of IH examination appears to be direct MMPs-2/9 mRNA evaluation in tumor tissue; and MMP-2 evaluation in patients' serum is a valuable complement to it. Tissue and serum mRNA MMPs assessment may represent a suitable novel biomarker identifying tumor progression and involution processes with potential clinical impact in IH as well as in cancer disease.

Molecular and immunohistochemical expression of apoptotic proteins Bax, Bcl-2 and Caspase 3 in infantile hemangioma tissues as an effect of propranolol treatment.

BACKGROUND: Infantile hemangiomas (IHs) are the most common benign tumors of childhood. They are characterized by a unique clinical course with two phases, proliferation and involution, which are followed by regression. The therapy of infantile hemangiomas was revolutionized in 2008 by the introduction of propranolol, however, the mechanism of its influence on hemangiomas remains unclear. METHODS: The study included 71 patients with IHs, 27 of whom were treated with propranolol while the remaining 44 were used as a comparative group. The expression of Bcl-2, Bax and Caspase3 was determined with immunohistochemistry and mRNA of Bax, Bcl-2 and Caspase3 were assessed with the use of RT-PCR. RESULTS: Both methods revealed a statistically significant decrease in Bcl-2 expression and an increase in Bax in IHs tissues after propranolol treatment. CONCLUSIONS: The results obtained for Bax and Bcl-2 proteins may indicate a link between the effect of propranolol and apoptosis. Higher Bax and lower Bcl-2 expression in the propranolol treated group indicates a strong pro- apoptotic action countering any anti-apoptotic activity; apoptosis was indicted in IH tissue as a potential result of propranolol treatment, with potential clinical impact in other tumors.

The first investigation of Wilms' tumour atomic structure-nitrogen and carbon isotopic composition as a novel biomarker for the most individual approach in cancer disease.

The paper describes a novel approach to investigating Wilms' tumour (nephroblastoma) biology at the atomic level. Isotope Ratio Mass Spectrometry (IRMS) was used to directly assess the isotope ratios of nitrogen and carbon in 84 Wilms' tumour tissue samples from 28 cases representing the histological spectrum of nephroblastoma. Marked differences in nitrogen and carbon isotope ratios were found between nephroblastoma histological types and along the course of cancer disease, with a breakout in isotope ratio of the examined elements in tumour tissue found between stages 2 and 3. Different isotopic compositions with regard to nitrogen and carbon content were observed in blastemal Wilms' tumour, with and without focal anaplasia, and in poorly- and well-differentiated epithelial nephroblastoma. This first assessment of nitrogen and carbon isotope ratio reveals the previously unknown part of Wilms' tumour biology and represents a potential novel biomarker, allowing for a highly individual approach to treating cancer. Furthermore, this method of estimating isotopic composition appears to be the most sensitive tool yet for cancer tissue evaluation, and a valuable complement to established cancer study methods with prospective clinical impact.

Hepatoblastoma Biology Using Isotope Ratio Mass Spectrometry: Utility of a Unique Technique for the Analysis of Oncological Specimens.

INTRODUCTION: Hepatoblastoma is the most common primary liver tumor in children. However, it occurs rarely, with an incidence of 0.5-1.5 cases per million children. There is no clear explanation of the relationship between clinicopathologic features, therapy, and outcome in hepatoblastoma cases, so far. One of the most widely accepted prognostic factors in hepatoblastoma is histology of the tumor. The aim of the study was to determine the potential differences in biology of hepatoblastoma histological subtypes at the atomic level using the unique method of isotope ratio mass spectrometry, which is especially valuable in examination of small groups of biological samples. MATERIAL/METHODS: Twenty-four measurements of nitrogen stable isotope ratio, carbon stable isotope ratio and total carbon to nitrogen mass ratio in fetal and embryonal hepatoblastoma tissue were performed using a Sercon 20-22 Continuous Flow Isotope Ratio Mass Spectrometer (CF-IRMS) coupled with a Sercon SL elemental analyzer for simultaneous carbon-nitrogen-sulfur (NCS) analysis. RESULTS: A difference of about 1.781‰ in stable nitrogen isotope 15N/14N ratio was found between examined hepatoblastoma histological subtypes. CONCLUSIONS: The prognosis in liver tumors cases in children may be challenging particularly because of the lack of versatile methods of its evaluation. Isotope ratio mass spectrometry allows one to determine the difference between hepatoblastoma histological subtypes and clearly indicates the cases with the best outcome.

Serum and tissue profile of VEGF and its receptors VGFR1/R2 in children with infantile hemangiomas on systemic propranolol treatment.

UNLABELLED: In the last few years propranolol has revolutionized infantile hemangioma therapy. This nonselective ß bloker has been proven to be safe and effective but the molecular bases of its actions remain unclear. One of debated theories holds that propranolol may inhibit angiogenesis and induce apoptosis. To investigate this claim, this study aims to analyze the serum and tissue profiles of VEGF and VEGRR1/2 in patients treated with propranolol. MATERIALS AND METHODS: To assess the expression if VEGF and VEGRR1/2 we used three independent methods. First we analyzed serum VEGF levels in 50 children with IH before and 3 months after the therapy using ELISA test (I.). Then we used immunohistochemistry to evaluate tissue expression of VEGF and VEGFR1/2 in IH treated (n=27) and not treated (n=45) with propranolol (II.). Finally we assessed mRNA of VEGF and VEGFR1/2 in the same patients as in part II (III.). RESULTS: (I) There was no distinct decrease of VEGF level in children with IH after propranolol treatment. (II) We found no significant difference in VEGFR1 and VEGFR2 expression in hemangiomas from the study and control group. The expression of VEGF was even higher than before therapy. (III) VEGF and VEGFR1 mRNA expression was significantly lower in IH tissue after propranolol treatment compared to those without treatment. VEGFR2 demonstrated no differences in expression between the two groups. CONCLUSIONS: The obtained results show distinct discrepancies between in vitro and clinical studies as well as among different methods used for analyzing the same phenomenon. Only VEGF and VEGFR1 expression in mRNA studies may prove the proposed theory of antiangiogenic properties of propranolol. Other results do not confirm it and remain inconsistent with the fantastic clinical response to this medication.

Evaluation of potential prognostic value of Bmi-1 gene product and selected markers of proliferation (Ki-67) and apoptosis (p53) in the neuroblastoma group of tumors.

INTRODUCTION: Cancer in children is a very important issue in pediatrics. The least satisfactory treatment outcome occurs among patients with clinically advanced neuroblastomas. Despite much research, the biology of this tumor still remains unclear, and new prognostic factors are sought. The Bmi-1 gene product is a currently highly investigated protein which belongs to the Polycomb group (PcG) and has been identified as a regulator of primary neural crest cells. It is believed that Bmi­1 and N-myc act together and are both involved in the pathogenesis of neuroblastoma. The aim of the study was to assess the potential prognostic value of Bmi-1 protein and its relations with mechanisms of proliferation and apoptosis in the neuroblastoma group of tumors. MATERIAL/METHODS: 29 formalin-fixed and paraffin-embedded neuroblastoma tissue sections were examined using mouse monoclonal antibodies anti-Bmi-1, anti-p53 and anti-Ki-67 according to the manufacturer's instructions. RESULTS: There were found statistically significant correlations between Bmi-1 expression and tumor histology and age of patients. CONCLUSIONS: Bmi-1 seems to be a promising marker in the neuroblastoma group of tumors whose expression correlates with widely accepted prognostic parameters. The pattern of BMI-1 expression may indicate that the examined protein is also involved in maturation processes in tumor tissue.

The first protocol of stable isotope ratio assessment in tumor tissues based on original research.

Thanks to proteomics and metabolomics, for the past several years there has been a real explosion of information on the biology of cancer, which has been achieved by spectroscopic methods, including mass spectrometry. These modern techniques can provide answers to key questions about tissue structure and mechanisms of its pathological changes. However, despite the thousands of spectroscopic studies in medicine, there is no consensus on issues ranging from the choice of research tools, acquisition and preparation of test material to the interpretation and validation of the results, which greatly reduces the possibility of transforming the achieved knowledge to progress in the treatment of individual patients. The aim of this study was to verify the utility of isotope ratio mass spectrometry in the evaluation of tumor tissues. Based on experimentation on animal tissues and human neoplasms, the first protocol of stable isotope ratio assessment of carbon and nitrogen isotopes in tumor tissues was established.

Prognostic significance of MCM 2 and Ki-67 in neuroblastic tumors in children.

INTRODUCTION: Neuroblastic tumors can be characterized by three features: spontaneous regression, maturation and aggressive proliferation. The most common and routinely used method of assessing tumor cell proliferation is to determine the Ki-67 index in the tumor tissue. Despite numerous studies, neuroblastoma biology is not fully understood, which makes treatment results unsatisfactory. MCM 2 is a potential prognostic factor in the neuroblastoma group. MATERIAL/METHODS: The study is based on retrospective analysis of 35 patients treated for neuroblastic tumors in the Department of Pediatric Surgery and Oncology of the Medical University of Lodz, during the period 2001-2011. The material comprised tissues of 16 tumors excised during the operation and 19 biopsy specimens. Immunohistochemical examinations were performed with immunoperoxidase using mouse monoclonal anti-MCM 2 and anti-Ki-67 antibodies. RESULTS: We observed that MCM 2 expression ranged from 2% to 98% and the Ki-67 index ranged from 0 to 95%. There was a statistically significant correlation between expression of MCM 2 and the value of the Ki-67 index and a correlation close to statistical significance between expression of MCM 2 and unfavorable histopathology. There was no statistical relationship between expression of MCM 2 and age over 1 year and N-myc amplification. DISCUSSION: The presented research shows that MCM 2 may have prognostic significance in neuroblastic pediatric tumors and as a potential prognostic factor could be the starting point of new individualized therapy.

Rhabdomyosarcoma in children in the light of isotope ratio mass spectrometry.

Rhabdomyosarcoma is the third most common solid tumor in children and the most common soft tissue sarcoma in this age group. However, 5-year survival is only observed in approximately 70% of cases, and the prognosis for patients with progressive disease is still poor. The authors hypothesize that the still unidentified differences in embryonal and alveolar tumor biology reflect the complex chemical reactions occurring during cell growth and metabolism and may be pursued in isotopic fractionation processes. Presented herein is the first evaluation of the nitrogen and carbon isotope ratio using isotope ratio mass spectrometry in the two major rhabdomyosarcoma histologic types. 15N enrichment was found in tumor tissues of embryonal histological type. The obtained result may indicate that individual patient considerations such as isotope ratio, in addition to widely accepted prognostic factors, may facilitate patient classification in terms of risk groups.

[Evaluation of COX-2 protein expression in melanocytic nevi in children]. / Ocena odczynów z COX-2 w znamionach melanocytarnych u dzieci.

UNLABELLED: Melanocytic skin tumors can be divided into benign nevi and malignant which take the form of melanoma. Melanocytic nevi are common in both adults and children. It is widely acknowledged that they are one of the risk factors of the formation of melanoma. However, melanoma among children is rare. In addition, differentiation of benign and malignant melanocytic tumors is often challenging. Recent studies suggest that COX-2 protein maybe useful in excluding malignant transformation of melanocytic lesion. The aim of the study was to evaluate the occurrence and differences of reactions with COX-2 in groups of nevi in children and melanoma adults. MATERIAL AND METHODS: The study included 75 common nevi and 43 atypical nevi incised in children and 15 cases of melanoma removed in adults. Paraffin blocks were used to make a preparations with routine hematoxylin and eosin staining (H + E) and immunohistochemistry. The results were statistically analyzed. RESULTS: Positive reactions were observed in both melanocytic nevi and melanoma. Differences between benign and malignant melanocytic tumors were statistically significant. Differences within melanocytic nevi were not statistically significant. CONCLUSIONS: The reactions of COX-2 are present in all nevi and do not allow to differentiate between their various types. COX-2 has potential utility in the differentiation of benign and malignant melanocytic tumors.

Is the SIOP-2001 Classification of Renal Tumors of Childhood accurate with regard to prognosis? A problem revisited.

INTRODUCTION: The goal of this study was to analyze morbidity and mortality of Wilms' tumor based on the revised SIOP-2001 classification. MATERIAL AND METHODS: Sixty-four patients with unilateral Wilms' tumor, 33 girls (51.5%) and 31 boys (48.5%), aged 1 to 144 months (mean: 42.8 months) were treated between 1993 and 2009. All patients underwent multimodal therapy according to the SIOP protocols. The follow-up period ranged from 2 to 18 years (mean: 11.6 years). RESULTS: Thirty-three patients (51.6%) had intermediate-risk, 6 (9.4%) low-risk and 25 (39%) high-risk tumors. Stage I disease was diagnosed in 28 (43.7%), stage II in 19 (29.7%), stage III in 8 (12.5%) and stage IV in 9 patients (14.1%). Event-free survival (EFS) in the entire group was 78.1% and OS was 92.2%. The EFS in stage IV (44.4%) was significantly lower than in stage I (82.1%, p = 0.04), stage II (89.5%, p = 0.02) and in the entire group (78.1%, p = 0.04). Sixteen complications were observed in 14 children (21.9%); metastases in 7 cases (10.9%), 8 relapses (12.5%) and 5 deaths (7.8%). Blastemal (20/24 - 83.3%) and anaplastic (3/24 - 12.5%) subtypes were responsible for mortality in high-risk tumors (OS - 87.5%), while poorly differentiated epithelial (7/34 - 20.6%) and regressive (8/34 - 23.5%) subtypes decreased OS (94.1%) in the intermediate-risk tumors. CONCLUSIONS: The results of our study show that epithelial and regressive subtypes were responsible for mortality in the intermediate-risk Wilms' tumors.

Minichromosome maintenance 2 (MCM2) is a new prognostic proliferative marker in Wilms tumour.

OBJECTIVE: We examined expression of minichromosome maintenance 2 (MCM2) by immunohistochemistry in nephrectomy specimens of children with nephroblastoma treated according to the Society International d'Oncologie Pediatrique (SIOP) scheme to determine its potential prognostic significance. MATERIAL AND METHODS: 18 children with nephroblastoma, 9 females and 9 males, 2 months to 7 years of age, treated in the Department of Oncology and Paediatric Surgery, Medical University of Lodz, during the period 1994-2006 were analysed. Children were treated by neoadjuvant chemotherapy and subsequent nephrectomy according to SIOP protocols -93 and 2001 and followed up from 2 to 11 years. The tumour stage and classification in nephrectomy specimens were established according to the revised 2001 SIOP working classification of renal tumours of childhood. RESULTS: In low risk nephroblastoma MCM2 expression was low, ranging from 0% in two cases of completely necrotic nephroblastoma to 5% in one child with cystic partially differentiated nephroblastoma. In mesoblastic nephroma, which is a distinct type of neoplasm with a low malignant potential and the most common congenital renal neoplasm, MCM2 expression was variable ranging from 2-5% in 2 children with stage I disease to 70% in one child with stage III disease In intermediate risk nephroblastoma MCM2 expression was low (10%) in one case of regressive type nephroblastoma and stage II disease and intermediate to high in children with epithelial type nephroblastoma, ranging from 40-50% in one case with stage I disease to 70% and 70-100% in 2 children with stage I and stage IV disease, respectively. In high risk nephroblastoma (7 children with nephroblastoma blastemal type) MCM2 expression was intermediate to high, ranging from 40 to 90%. MCM2 expression tends to be lower in children with less advanced stage of disease (stage II) and higher in more advanced disease (stage III and IV). Two children with blastemal type and high (> 60% MCM2) died of disease within 2-4 years from diagnosis and one child was lost to follow-up. Both children who died were older 8.5 yo M and 7 yo M and presented with advanced disease stage IV or III with anaplasia. CONCLUSION: MCM2 is a promising prognostic factor in WT treated according to the SIOP scheme.

The use of computer image analyses in the assessment of chosen histoclinical features of nephroblastoma cells in children.

The goal of the research was to assess the nuclear morphometric discriminant function (MV(f)) in the analyzed group of nephroblastomas and to evaluate its potential prognostic value. We found a statistically important correlation between favourable histology of Wilms' tumours (according to SIOP 93-01 and SIOP 2001 classifications of kidney tumours of childhood) and low values of MV(f) discriminant function, which was also associated with the best prognosis for the patients. We believe that MV(f) assessment may be a helpful tool in making prognosis in doubtful cases of nephroblastomas in children.

Histoclinical study of nephroblastoma in relation to current and previous SIOP Classification of Renal Tumors of Childhood.

Histological classifications are essential for prognosis in children malignancies. Currently, the histological type of tumor is one of the main prognostic factors in this group. We investigated histoclinical features of nephroblastoma in relation to SIOP 93-01 and SIOP 2001 Classifications of Renal Tumors of Childhood. We examined all the routinely available histological features and histological nephroblastoma types and investigated their influence on patients' survival with the use of log-rank test and Kaplan-Meier method. The results of statistical analysis indicated that SIOP 93-01 more precisely separated nephroblastoma types according to their biology and malignant potential. We also observed that epithelial type of nephroblastoma showed a mixture of results typical for both intermediate and high risk tumors. What is more, we noticed statistically important correlations between developmental defects found in patients with nephroblastoma and tumor volume and the course of disease.

Examination of expression of WT1 gene product and CD44 adhesive molecule in nephroblastoma histologic types.

In spite of success of modern pediatric oncology, cases in which we are not able to reach the prospective affirmative effect of performed therapy are still observed. The purpose of our study was to examine the expression of WT1 gene product and CD44 adhesive molecule in nephroblastoma histologic types - one of the currently used prognostic marker for this group of tumor. We found correlations between CD44 expression and histologic type of tumor. We suppose that high CD44 expression in nephroblastoma group of tumors may confirm their high malignant potential. Expression of the WT1 gene product we found in all the investigated tumor tissue samples. However we did not found statistically significant correlations between WT1 expression and histologic type of the tumor and there was no correlation between CD44 and WT1 expression in blastemal nor of epithelial component of nephroblastoma in our study. Lack of this correlation also permits to suppose that the high activity is an integral feature of all Wilms tumor cells and is not only characteristic for anaplastic and blastemal nephroblastomas.

Number of mitotic figures in microscopic picture of nephroblastoma histologic types--estimation of the value of the oldest known prognostic factor.

There are two main widely accepted prognostic factors in nephroblastoma group of tumors--histological type of tumor and clinical stage of disease. However nuclear anaplasia, one of the elements of Wilms' tumor microscopic picture was found as a new prognostic marker in this group of tumors. Estimation of nuclear anaplasia is an obligatory procedure in currently used therapeutic protocols. In our work we compared another element of nephroblastoma microscopic picture--number of mitoses as the oldest known indicator of activity of tumor cells with prognostic markers estimated in routine histologic examination according SIOP Protocols (histological risk, nephroblastoma type and the presence of diffuse anaplasia) and with CD44 expression--widely known marker of discussed prognostic value. We found statistically important correlation between number of mitoses and all the examined features. We believe that mitotic figure counting may become in future a helpful tool in the qualification of prognosis for individual patients in doubtful cases.

Estimation of diagnostic value of chosen markers of neural differentiation in neuroblastoma group of tumors and pPNETs.

Neuroblastoma and peripheral PNETs both are typical examples of wide group of small round blue cell tumors of childhood. Matured neuroblastoma show typical clinical presentation and easy to interpret microscopic picture. Unfortunately in everyday practice much commonly appeared less differentiated neuroblastomas with difficult to predict clinical behavior and impossible to diagnose in routine stain histologic view. Peripheral PNETs are found as morphologically similar entities to some neuroblastoma subtypes but they are treated as separate CD99 positive group of tumors with different biology and clinical behavior. The aim of our study was to estimate the usefulness of neural markers expression (Neuroblastoma Marker, neurospecyfic enolase and neurofilaments) in routine separation between neuroblastoma tumors and pPNETs and between neuroblastoma subtypes according to currently used classification of those entities. We investigated 63 tumor tissue samples and found differences in expression of investigated markers between both neuroblastoma subgroups and neuroblastoma group of tumors and pPNETs.

Lipofibromatosis presenting as a neck mass in eight-years old boy--a case report.

Lipofibromatosis is a very rare pediatric neoplasm with a histologically distinctive fibrofatty pattern. Some of the authors believe that this term should encompass several rare soft tissue tumors of childhood and that tumors of childhood which contain fat as an integral component warrant new classification. We present a case report of a neck tumor in eight-years old boy which shares clinical and morphological features similar to those originally described by Fetch et al. with peaceable with literature results of immunohistochemical stains.

Estimation of prognostic value of CD44 expression in neuroblastic tumours in children.

Adversities observed in treatment of children with neoplastic disease based on new diagnostic markers and new prognostic factors. Both of them allow prognosis to be established for a single patient. The aim of our study was to examine the expression of CD44 adhesion molecule in different histologic types in a neuroblastoma group of tumours (35 cases of neuroblastoma from current files and archives) and to estimate the possible prognostic value of CD44 expression by comparison with widely accepted prognostic markers and chosen histoclinical parameters (9 cases of neuroblastoma with follow-up data). We did not find a statistically significant correlation between CD44 expression and histologic type of the tumour. However, we found that all relapses appeared among patients with tumours with the strongest CD44 expression, and that in none of the investigated tumours without relapses was strong CD44 expression ever observed. We noticed CD44 expression in 88.88% of examined tissue samples which underwent statistical analysis and we found the strongest CD44 expression in tumours situated in the retroperitoneal space. Results of log-rank test and Kaplan-Meier estimation showed that a correlation between CD44 expression and survival time was close to a statistically significant value (p=0.065). We conclude that lack of a clear statistically significant correlation between CD44 expression and histoclinical parameters and currently known prognostic factors in our study is due to the presence of many CD44 isoforms, which cannot be distinguished with commercially used antibodies, but they may play a different role in pathogenesis and spread of neuroblastoma.