In-Depth Immunological Typization of Children with Sickle Cell Disease: A Preliminary Insight into Its Plausible Correlation with Clinical Course and Hydroxyurea Therapy.

Sickle cell disease (SCD) is a condition of functional hypo-/a-splenism in which predisposition to bacterial infections is only a facet of a wide spectrum of immune-dysregulation disorders forming the clinical expression of a peculiar immunophenotype. The objective of this study was to perform an in-depth immunophenotypical characterization of SCD pediatric patients, looking for plausible correlations between immunological biomarkers, the impact of hydroxyurea (HU) treatment and clinical course. This was an observational case−control study including 43 patients. The cohort was divided into two main groups, SCD subjects (19/43) and controls (24/43), differing in the presence/absence of an SCD diagnosis. The SCD group was split up into HU+ (12/19) and HU− (7/19) subgroups, respectively receiving or not a concomitant HU treatment. The principal outcomes measured were differences in the immunophenotyping between SCD patients and controls through chi-squared tests, t-tests, and Pearson's correlation analysis between clinical and immunological parameters. Leukocyte and neutrophil increase, T-cell depletion with prevalence of memory T-cell compartment, NK and B-naïve subset elevation with memory and CD21low B subset reduction, and IgG expansion, significantly distinguished the SCD HU− subgroup from controls, with naïve T cells, switched-memory B cells and IgG maintaining differences between the SCD HU+ group and controls (p-value of <0.05). The mean CD4+ central-memory T-cell% count was the single independent variable showing a positive correlation with vaso-occlusive crisis score in the SCD group (Pearson's R = 0.039). We report preliminary data assessing plausible clinical implications of baseline and HU-related SCD immunophenotypical alterations, which need to be validated in larger samples, but potentially affecting hypo-/a-splenism immuno-chemoprophylactic recommendations.

Persistence of a monosomic cell line in a fetus with mosaic trisomy 8.

We report on a fetus presenting with an increased nuchal translucency, in which chorionic villus sampling led to the diagnosis of mosaic trisomy 8. Ultrasound scan performed at 15(+6) weeks revealed bilateral cleft lip and palate, flat facial profile, and arrhinia. Pregnancy was terminated at 16(+6); postmortem examination showed additional findings including hypospadias, bilateral renal dysplasia, and focal portal fibrosis of the liver. In order to confirm the presence of trisomy 8, FISH analysis was performed in abnormal renal and hepatic tissue, which, unexpectedly, showed a higher fraction of cells with only one fluorescent probe signal (43% and 23%, respectively), if compared with normal fetal liver and kidney (3-10%). This finding is consistent with the survival in this fetus of a monosomic cell line after mitotic non-disjunction, which is in contrast with what is generally thought about mosaic trisomy genesis. We hypothesize that the possible persistence of the monosomic cell line, in addition to the variable distribution of aneuploid cells in the body tissues, could explain the high heterogeneity of mosaic trisomy 8 phenotype.

Gene expression profiling in glioblastoma and immunohistochemical evaluation of IGFBP-2 and CDC20.

Thirty-nine glial tumours (28 glioblastomas (GB) and 11 low-grade gliomas) were investigated with DNA microarrays to reveal a possible specific gene expression profile. Unsupervised classification through hierarchical cluster analysis identified two groups of tumours, the first composed of low-grade gliomas and the second mainly composed of GB. Nine genes were identified as most informative: seven were over-expressed in low-grade gliomas and under-expressed in GB; on the contrary, two genes, insulin-like growth factor binding protein 2 (IGFBP-2) and cell division cycle 20 homologue (CDC20), were over-expressed in GB and under-expressed in low-grade tumours. This same genetic profile was confirmed by reverse transcriptase polymerase chain reaction. Immunohistochemistry for IGFBP-2 was positive in 88.8% of the cases of GB and in only one low-grade glioma, whilst CDC20 immunostained 74.1% of the cases of GB and none low-grade glioma. This was confirmed in an additional series of cases studied with immunohistochemistry only. In conclusion, over-expression of mRNA levels of IGFBP-2 and CDC20 is highly related to GB, IGFBP-2 and CDC-20 gene and protein expressions are strongly correlated, and IGFBP-2 and CDC20 immunopositivity can be useful for the identification of GB in small biopsies.

Trisomy of chromosome 6 in Merkel cell carcinoma within lymph nodes.

Merkel cell carcinoma (MCC) of the skin is a neuroendocrine tumor with characteristic histological and immunohistochemical features. Among various cytogenetic changes, trisomy of chromosome 6 has been reported in 47% of cases using in situ hybridization. Primary tumors, morphologically and immunohistochemically identical to MCCs of the skin, have been described in other organs, including lymph nodes. Here, a cytogenetic study of four cases of MCC of lymph nodes is presented. Four cases of primary MCCs of lymph nodes and ten cases of cutaneous MCCs were studied for chromosome 6 using fluorescent in situ hybridization (FISH). All cases showed typical features of MCC both at hematoxylin and eosin (H&E) and immunohistochemistry. FISH showed trisomy 6 in two out of the four cases ofMCCs of lymph node as well as in 6 out 10 cases of MCCs of skin. Lymph nodal and cutaneous MCCs share same histological and immunohistochemical features, as well as same cytogenetic alteration for chromosome 6. It seems that there are more similarities than differences between cutaneous and lymph nodal MCCs. Whether lymph nodal MCCs are primary tumors or metastases from regressed skin lesions is still questionable, although several findings indicate a primary origin.

Malignant ectomesenchymoma: genetic profile reflects rhabdomyosarcomatous differentiation.

Malignant ectomesenchymoma (MEM) represents a heterogeneous group of tumors, most likely originating from pluripotent primitive neural crest cells. In this report, we present an 8-month-old infant boy with an MEM on the left scrotum. Retrospective review of the incision biopsy showed the presence of a few ganglion cells in an otherwise classic embryonal rhabdomyosarcoma (RMS), whereas in the resection specimen after chemotherapy the combined RMS and ganglioneuroma components were very obvious. Cytogenetic analysis of the residual lesion showed an abnormal karyotype, 49, XY, +2, -6, +11, +20, +mar, with a hyperploidy in a subset of cells. By fluorescence in situ hybridization analysis, the marker chromosome was identified as originating from chromosome 6, and the tumor cells were negative for PAX3/PAX7 disrupting translocations specific for alveolar RMS. Gains of chromosomes 2, 11, and 20, found in the current case, are a common finding in embryonal RMS. These gains probably reflect the myogenic differentiation of MEM and support the genetic link between these 2 neoplasms. In addition to the conventional cytogenetics, array comparative genomic hybridization analysis was performed on the primary and residual tumors. The genomic profiles of both specimens were basically the same including the presence of 2 distinctive chromosome 6p21.32-p21.2 and 6p11.2 amplification regions in the primary tumor, which vanished in the postchemotherapy specimen. The pretreatment biopsy exhibited strong expression of HMGA1 and HMGA2 proteins in immunohistochemistry, with the shift toward the loss of expression of both genes in the posttreatment tumoral tissue. This finding supports the oncogenic properties of the HMGA family of proteins and their role in the process of malignant transformation.

Prospective study of gefitinib in epidermal growth factor receptor fluorescence in situ hybridization-positive/phospho-Akt-positive or never smoker patients with advanced non-small-cell lung cancer: the ONCOBELL trial.

PURPOSE: In non-small-cell lung cancer (NSCLC), clinical and biologic predictors for epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor sensitivity have been identified in retrospective studies, and there is urgent need to validate these results in prospective trials. The ONCOBELL trial is a prospective phase II study evaluating gefitinib sensitivity in NSCLC patients who never smoked or have increased EGFR gene copy number or activation of the antiapoptotic protein Akt. PATIENTS AND METHODS: EGFR gene copy number was evaluated using fluorescence in situ hybridization (FISH), and presence of phospho-Akt was evaluated using immunohistochemistry. Additional tests included immunohistochemistry analysis of EGFR, FISH analysis of HER2, and mutation analysis of EGFR, HER2, and K-ras. RESULTS: From November 2004 to February 2006, 183 patients were screened, and 42 patients were enrolled onto the trial. We observed one complete and 19 partial responses, for an overall response rate (RR) of 47.6% (95% CI, 32.5% to 62.7%). Median duration of response was 6.1 months, median time to progression (TTP) was 6.4 months, 1-year survival rate was 64.3%, and median survival time was not reached. EGFR FISH-positive patients, compared with negative patients, had higher RR (68.0% v 9.1%, respectively; P < .001), longer TTP (7.6 v 2.7 months, respectively; P = .02), and a trend for longer survival (median survival not reached v 7.4 months, respectively; P = .3). Therapy was well tolerated, and there were no drug-related deaths. Median follow-up time was too short for significance tests of differences in survival outcomes. CONCLUSION: Gefitinib is active and well tolerated in patients with trial characteristics, and EGFR FISH analysis is an accurate predictor for such therapy.

EGFR and HER2 gene copy number and response to first-line chemotherapy in patients with advanced non-small cell lung cancer (NSCLC).

BACKGROUND: A critical point in designing clinical trials comparing chemotherapy with epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) in patients with non-small cell lung cancer (NSCLC) is the expected benefit with standard chemotherapy in presence of biological features indicative of TKI sensitivity. The aim of this study was to assess whether EGFR and HER2 gene copy number and Akt activation are associated with response to first-line chemotherapy. METHODS: Tumor samples from 190 patients with NSCLC were analyzed. EGFR and HER2 gene copy number were evaluated by fluorescence in situ hybridization in 185 and 184 cases, respectively. Akt activation was assessed by immunohistochemistry (n = 176). Additional biomarkers included EGFR DNA sequencing (n = 65), and EGFR immunohistochemistry (n = 185). RESULTS: Response rate was not associated with EGFR, HER2, and P-Akt status, irrespective of the method used for biomarker assessment. Among patients with EGFR gene mutations, response to chemotherapy was observed only in individuals with exon 19 deletion (response rate: 46.6% versus 0%, p = 0.02). Among the 190 patients analyzed, 123 received a treatment with a TKI as second- or third-line therapy. When assessed by fluorescence in situ hybridization or DNA sequencing, EGFR-positive patients seemed to be more sensitive to TKIs than to chemotherapy in terms of response rate and time to progression, whereas in EGFR-negative patients, response rate and time to progression favored chemotherapy. CONCLUSION: This study suggested that EGFR expression and gene copy number, HER2 gene copy number, and P-Akt expression are not associated with response to first-line chemotherapy in NSCLC. Prospective phase III trials should compare standard chemotherapy with a TKI in selected NSCLC.

Epithelioid hemangioendothelioma of the spinal cord: Description of a case with cytogenetic analysis.

A case of epithelioid hemangioendothelioma of the cauda equina is reported. The patient presented with rapidly worsening low back pain. Magnetic resonance imaging revealed a sharply demarcated intradural lumbar lesion. A bluish-red lesion, attached to the filum terminale, was removed. The patient is alive without evidence of recurrence 18 months after surgery. The tumor was composed of variously sized vessels lined by epithelioid endothelial cells with clear cytoplasm and centrally located, moderately atypical nuclei. These cells were immunoreactive for CD31 and factor VIII antibodies. Cytogenetic analysis disclosed two clones: 44-45X, - Y [cp3]/46XY[11]. Epithelioid hemangioendothelioma may arise in several sites, the most common being soft tissues. It is a borderline tumor that may recur, may metastasize, and rarely causes death. The present case appears to be the first example of epithelioid hemangioendothelioma of the spinal cord.

Clinical experience with gefitinib: an update.

Gefitinib is an orally active, selective epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) that blocks signal transduction pathways involved in cell proliferation. This drug demonstrated impressive and durable responses in patients with heavily pretreated non-small cell lung cancer (NSCLC). In two large phase II trials, responses were observed in 9-19% of unselected patients, along with symptom improvement and benefit in quality of life. Biological mechanisms underlying TKI sensitivity have recently been discovered. There is evidence that specific EGFR gene mutations and/or amplification confer a particularly sensitive phenotype, especially in individuals with tumors demonstrating activation of the anti-apoptotic protein Akt. However, in all so far conducted clinical trials, no patient selection has been made, providing a logical explanation for the negative results observed in large phase III studies. In the present review, we will summarize the results observed in clinical trials with gefitinib. We will present results obtained in NSCLC and in other solid tumor, focusing on biological and clinical markers predicting drug sensitivity.

Epidermal growth factor receptor (EGFR) targeted therapies in non-small cell lung cancer (NSCLC).

The Epidermal Growth Factor Receptor (EGFR) family, including EGFR, HER2, HER3, and HER4, is implicated in the development and progression of cancer, and is expressed in many human epithelial malignancies, including Non-Small Cell Lung Cancer (NSCLC). Several molecules were synthesized to inhibit the extracellular domain of EGFR, such as cetuximab (Erbitux), the extracellular domain of HER2, such as trastuzumab (Herceptin) or the EGFR tyrosine kinase domain, such as gefitinib (Iressa) and erlotinib (Tarceva). Gefitinib and erlotinib are orally active, selective EGFR tyrosine-kinase inhibitors (EGFR-TKI) that produce objective response rates in about 10% of advanced NSCLC. More recently, erlotinib produced a significant improvement in survival when compared to placebo in pretreated NSCLCs. Among clinical characteristics, although female gender, and adenocarcinoma histology, showed to be significantly associated to TKI sensitivity, never smoking history is probably the most relevant factor. Presence of specific EGFR gene mutations or EGFR gene amplification confer a particularly sensitive phenotype, and patients with activation of the anti-apoptotic protein Akt are more sensitive, when Akt activation is sustained by a EGFR dependent mechanism. Cetuximab is a human-murine chimeric anti-EGFR IgG monoclonal antibody that has demonstrated both in vitro and in vivo antitumor activity in tumor cell lines expressing EGFR. It has shown impressive activity when combined with radiation by increasing the antitumor effect of radiation therapy. Cetuximab has a synergistic effect with cisplatin and may play a role in reversing resistance to chemotherapy. Cetuximab demonstrated to be active in pretreated NSCLCs, and its activity as first-line therapy in combination with chemotherapy is currently under evaluation. Efforts should be made for the identification of biological mechanism underlying cetuximab sensitivity and emerging data suggest that the drugs is more active in patients with EGFR gene amplification. In NSCLC, trastuzumab produced disappointing results when combined with chemotherapy, but probably patients were not properly selected. Recent findings in gefitinib treated patients support HER2 analysis by fluorescence in situ hybridization as a complementary test for selection of patient candidate for EGFR targeted therapies. Combination of EGFR targeting agents with other biological drugs is under investigation.

The fast-track biopsy (FTB): description of a rapid histology and immunohistochemistry method for evaluation of preoperative breast core biopsies.

Thirty-six core breast biopsies from 32 patients were paraffin embedded by use of an automated microwave processor. In addition, a quick immunohistochemical method was used in selected cases. The quality of the hematoxylin and eosin (H&E) slides was very satisfactory, as were also the immunohistochemical stains for ER, PR, and Ki67 when compared to those obtained with the use of a conventional automated immunostainer. The time required to process the tissue to the final H&E stage averaged 2 hours 52 minutes, and the immunohistochemical method required 90 to 100 minutes. This procedure, which we named "fast-track biopsy'' (FTB), is quick enough to be competitive with FNAC (fine-needle aspiration biopsy) in terms of turnaround-times. The superiority of core biopsy over FNA in terms of the morphologic information provided is widely acknowledged, the only major argument currently mentioned in favor of FNAC being the shorter duration of the procedure. With the advent of FTB, it would appear that even this last remaining advantage has been erased.

Epidermal growth factor receptor gene and protein and gefitinib sensitivity in non-small-cell lung cancer.

BACKGROUND: Gefitinib is a selective inhibitor of the epidermal growth factor (EGFR) tyrosine kinase, which is overexpressed in many cancers, including non-small-cell lung cancer (NSCLC). We carried out a clinical study to compare the relationship between EGFR gene copy number, EGFR protein expression, EGFR mutations, and Akt activation status as predictive markers for gefitinib therapy in advanced NSCLC. METHODS: Tumors from 102 NSCLC patients treated daily with 250 mg of gefitinib were evaluated for EGFR status by fluorescence in situ hybridization (FISH), DNA sequencing, and immunohistochemistry and for Akt activation status (phospho-Akt [P-Akt]) by immunohistochemistry. Time to progression, overall survival, and 95% confidence intervals (CIs) were calculated and evaluated by the Kaplan-Meier method; groups were compared using the log-rank test. Risk factors associated with survival were evaluated using Cox proportional hazards regression modeling and multivariable analysis. All statistical tests were two-sided. RESULTS: Amplification or high polysomy of the EGFR gene (seen in 33 of 102 patients) and high protein expression (seen in 58 of 98 patients) were statistically significantly associated with better response (36% versus 3%, mean difference = 34%, 95% CI = 16.6 to 50.3; P<.001), disease control rate (67% versus 26%, mean difference = 40.6%, 95% CI = 21.5 to 59.7; P<.001), time to progression (9.0 versus 2.5 months, mean difference = 6.5 months, 95% CI = 2.8 to 10.3; P<.001), and survival (18.7 versus 7.0 months, mean difference = 11.7 months, 95% CI = 2.1 to 21.4; P = .03). EGFR mutations (seen in 15 of 89 patients) were also statistically significantly related to response and time to progression, but the association with survival was not statistically significant, and 40% of the patients with mutation had progressive disease. In multivariable analysis, only high EGFR gene copy number remained statistically significantly associated with better survival (hazard ratio = 0.44, 95% CI = 0.23 to 0.82). Independent of EGFR assessment method, EGFR+/P-Akt+ patients had a statistically significantly better outcome than EGFR-, P-Akt-, or EGFR+/P-Akt- patients. CONCLUSIONS: High EGFR gene copy number identified by FISH may be an effective molecular predictor for gefitinib efficacy in advanced NSCLC.

Acquisition of i(8q) as an early event in malignant triton tumors.

Malignant triton tumors (MTT) are rare soft-tissue tumors characterized by a mixture of cells with nerve sheath and skeletal muscle differentiation. MTT is a histological variant of malignant peripheral nerve sheath tumors (MPNST). No characteristic cytogenetic anomaly has been detected in MPNST or MTT. In this paper, we report on the cytogenetic findings of an MTT from a 20-year old male with neurofibromatosis (NF1). The tumoral karyotype showed the modal number to be near-diploid and an abnormal karyotype with a Robertsonian translocation and 4 markers: 49,XY,der(14;15)(q10;q10),+4mar. Spectral karyotyping revealed the karyotype: 49,XY, der(14;15)(q10;q10),+i(8)(q10)x4. Fluorescence in situ hybridization analysis of the tissue confirmed the presence of the additional i(8)(q10) in all tumoral cells. The sequence analysis of p53 revealed a polymorphism in exon 9, codon 329. The two alleles, TTC and TCC, codify for phenylalanine and serine, respectively. Our results indicate that all neoplastic cells have the same cytogenetic pattern, suggesting that both cell lines, which show nerve sheath and skeletal muscle differentiation, are derived from a unique stem cell. The acquired Robertsonian chromosomal recombinants might represent an event in the tumorigenesis of MTT, and the present data suggest that genes located on 8q can be involved in the development of MTT.

Akt phosphorylation and gefitinib efficacy in patients with advanced non-small-cell lung cancer.

BACKGROUND: Gefitinib, a specific epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, has activity against approximately 10% of unselected non-small-cell lung cancer (NSCLC) patients. Phosphatidylinositol 3'-kinase (PI3K)/Akt and Ras/Raf/mitogen-activated protein kinase (MAPK), the two main EGFR-signaling pathways, mediate EGFR effects on proliferation and survival. Because activation of these pathways is dependent on the phosphorylation status of the components, we evaluated the association between phosphorylation status of Akt (P-Akt) and MAPK (P-MAPK) and gefitinib activity in patients with advanced NSCLC. METHODS: Consecutive patients (n = 106) with NSCLC who had progressed or relapsed on standard therapy received gefitinib (250 mg/day) until disease progression, unacceptable toxicity, or patient refusal. P-Akt and P-MAPK positivity was determined with immunohistochemistry using tumor tissues obtained before any anticancer treatment. Association of P-Akt and time to progression was determined by univariable and multivariable analyses. All statistical tests were two-sided. RESULTS: Of the 103 evaluable patients, 51 (49.5%) had tumors that were positive for P-Akt, and 23 (22.3%) had tumors that were positive for P-MAPK. P-Akt-positivity status was statistically significantly associated with being female (P<.001), with never-smoking history (P =.004), and with bronchioloalveolar carcinoma histology (P =.034). Compared with patients whose tumors were negative for P-Akt, patients whose tumors were positive for P-Akt had a better response rate (26.1% versus 3.9%; P =.003), disease control rate (60.9% versus 23.5%; P<.001), and time to progression (5.5 versus 2.8 months; P =.004). Response rate, disease control rate, and time to progression did not differ according to P-MAPK status. The multivariable analysis showed that P-Akt positivity was associated with a reduced risk of disease progression (hazard ratio = 0.58, 95% confidence interval = 0.35 to 0.94). CONCLUSIONS: Patients with P-Akt-positive tumors who received gefitinib had a better response rate, disease control rate, and time to progression than patients with P-Akt-negative tumors, suggesting that gefitinib may be most effective in patients with basal Akt activation.

Cytogenetic analysis of myoepithelial cell carcinoma of salivary gland.

Myoepithelial cell carcinoma (MCC) of the salivary gland is a rare entity. Here, we describe the karyotype of MCC. The patient was a 53-year-old man, with a rapidly growing lesion of the palate. Despite complete surgical excision, radio- and chemotherapy, the lesion rapidly harboured local and distant metastases leading to the death of the patient, 4 months after the diagnosis. On histological and ultrastructural examination, the primary tumour and the related metastases were composed of oval and spindle cells, with features of myoepithelial cell differentiation reported in the literature. Cytogenetic analysis showed a composite karyotype in the primary tumour: 45-46,XY, +3[cp3]/ 44-45,XY, -17[cp4]/ 46,XY[5]. The lymph-node metastasis was near-triploid and showed a complex karyotype. Our cytogenetic data differ from those described in benign or slowly growing salivary gland tumours showing myoepithelial cell differentiation. It is suggested that highly aggressive tumours might follow a different pathway of malignant transformation.

Mucoepidermoid carcinoma of the breast.

Five cases of mucoepidermoid carcinoma (MEC) of the breast are reported. All patients were women ranging in age from 29 years to 80 years. As histological grading is one of the most important prognostic factors in breast invasive carcinomas, MEC was graded using the Auclair et al. [1] grading system specific for MEC of salivary glands and the Elston and Ellis [4] grading method, a widely employed grading system in breast cancer. It was found that the two different grading systems appear to be interchangeable in assessing the grade of MEC of the breast. Accordingly, three cases were regarded low grade (G. 1), one intermediate (G. 2) and one high grade (G. 3). The cases were studied with immunohistochemistry and were found to have the same keratin pattern shown by their salivary gland counterpart. It was found that there are more similarities than differences between MEC of the breast and of salivary glands.

Gefitinib in pretreated non-small-cell lung cancer (NSCLC): analysis of efficacy and correlation with HER2 and epidermal growth factor receptor expression in locally advanced or metastatic NSCLC.

PURPOSE: To evaluate the correlation between HER2 expression and gefitinib (ZD 1839, Iressa; AstraZeneca, London, United Kingdom) efficacy in terms of response rate, time to progression (TTP), and overall survival (OS) time. PATIENTS AND METHODS: Patients with pretreated advanced non-small-cell lung cancer (NSCLC) received gefitinib at a daily dose of 250 mg until disease progression. Tumor tissue specimens obtained at the time of primary diagnosis were collected to determine HER2/epidermal growth factor receptor (EGFR) status by immunohistochemistry. RESULTS: From February 2001 to June 2002, 63 consecutive patients were enrolled onto the study. The overall disease control rate was 58.7% (partial response [PR], 15.9%; stable disease [SD], 42.8%), median TTP was 3.3 months, and median OS was 4.1 months. Among the 43 patients in whom EGFR/HER2 status was determined, we observed six PRs (14%) and 18 SDs (42%). Disease control, including PR and SD, was 40% in the 15 patients overexpressing HER2 and 64.3% in the 28 patients not overexpressing HER2 (P =.126). No difference was found between the two groups in terms of TTP (3.5 v 3.7 months, respectively) and OS (5.7 v 6.8 months, respectively). In addition, we did not find any difference in TTP, OS, toxicity, and symptom outcome in the group of patients overexpressing both HER2 and EGFR compared with patients who had no overexpression of HER2 CONCLUSION: According to these data, efficacy, toxicity, and symptom outcome in patients with NSCLC treated with gefitinib do not seem to be related to HER2 expression.

Real-time RT-PCR of tyrosine hydroxylase to detect bone marrow involvement in advanced neuroblastoma.

Neuroblastoma (NB) is characterised by the secretion of catecholamines in approximately 95% of patients. Tyrosine hydroxylase is the rate-limiting enzyme in catecholamine biosynthesis pathway. Expression of the tyrosine hydroxylase gene (TH) is regulated in a tissue-specific manner during neonatal development and differentiation, therefore TH mRNA expression is a specific tumour marker for NB. Here we present a real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay using TaqMan technology for detection and quantification of TH mRNA in bone marrow (BM) NB patients. The degree of TH expression was derived from the ratio of the mRNAs of this gene and the reference gene, beta-actin. A ratio greater than 3x10(-2) was considered as positive for TH mRNA presence. Samples were also examined for TH mRNA by first and nested RT-PCR. Seventeen BM samples from 4 patients with disseminated NB (3 stage IV and 1 stage IVs) were evaluated at diagnosis and during treatment. We found a variable degree of TH expression ranging from 0.0344 to 26.3370 in 12/17 positive samples, while no TH mRNA (value lower than 3x10(-2)) was detected in 5/17 samples obtained after consolidation therapy. Our results show a moderate concordance between different qualitative RT-PCR methods and real-time RT-PCR. The real-time RT-PCR results seem to fit better with the natural short-term clinical follow-up of the evaluated patients, with respect to qualitative methods. Real-time TH RT-PCR could therefore be of clinical value for the assessment of a patient's prognosis by monitoring minimal residual disease (MRD).