Predict Tumor Behavior Research Articles

Microvascular tumour invasion in renal cell carcinoma: the most important prognostic factor

To evaluate the role of microvascular invasion (MVI) in the primary lesion for predicting tumour behaviour in patients with renal cell carcinoma (RCC), as reliable clinical prognostic factors would be very valuable. MVI was assessed in 230 patients with clinically localized RCC (stages T1-4NxM0) who had a radical nephrectomy and/or nephron-sparing surgery. The median (range) follow-up was 48 (3-130) months. The impact of MVI on disease progression and its correlation with clinical and histopathological factors was analysed, including whether patients were symptomatic or not at presentation, Fuhrman nuclear grade, tumour size, pathological stage and lymph node metastasis. Regression analyses and survival curves were used to determine if MVI was associated with the prognosis of RCC. There was MVI in 59 patients (26%); of these, 46% developed disease recurrence. Among the 171 patients with no MVI, only 11 (6%) had tumour recurrence. MVI was associated with tumour diameter, nuclear grade, pathological stage, lymph node metastasis and the presence of sarcomatous elements in the tumour. Multivariate analysis showed that MVI was an independent predictor of disease recurrence and the most important factor related to death. MVI is an independent predictor of prognosis in patients with RCC.

Pulmonary glomus tumor

Glomus tumor (GT) is an infrequent but distinct neoplasm. Pulmonary GT is a rare neoplasm with only a few cases reported in the literature. These tumors are usually benign and, although rare, tumors with aggressive behavior have been reported. The tumor size, location, and histomorphological features may be useful in predicting tumor behavior. We present here a case of pulmonary GT that was initially diagnosed as a typical carcinoid tumor. The differential diagnosis as well as the recent classification of GTs is discussed along with a review of literature.

The expression of mismatched repair genes and their correlation with clinicopathological parameters and response to neo-adjuvant chemotherapy in breast cancer.

BackgroundThe DNA mismatch repair (MMR) pathway is an important post-replicative repair process. It is involved in the maintenance of genomic stability and MMR genes have therefore been named the proofreaders of replicating DNA. These genes repair the replicative errors of DNA and are thus imperative for genomic stability.The MMR genes have been found to be involved in promoting cytotoxicity, apoptosis, p53 phosphorylation and cell cycle arrest following exposure to exogenous DNA damaging agents. Loss of MMR function prevents the correction of replicative errors leading to instability of the genome, and can be detected by polymorphisms in micro satellites (1–6 nucleotide repeat sequences scattered in whole of the genome). This phenomenon, known as micro satellite instability (MSI), is a hallmark of MMR dysfunction and can be used as a marker of MMR dysfunction in colorectal and other malignancies. An alternative method for detection of MMR dysfunction is to test the expression of protein products of the MMR genes by immunohistochemistry (IHC), as mutations in these genes lead to reduced or absent expression of their gene products.Correlation between loss of MMR function and clinical, histopathological, behavioral parameters of the tumor and its response to chemotherapy in breast cancers may be of value in predicting tumor behavior and response to neoadjuvant chemotherapy (NACT). Neoadjuvant chemotherapy is an integral part of multimodal therapy for locally advanced breast cancer and predicting response may help in tailoring regimens in patients for optimum response.Materials After approval by the IRB(Institutional Review Board) and ethical committee of the hospital, 31 cases of locally advanced breast carcinoma (LABC) were studied to assess the correlation between MMR dysfunction, clinicopathological parameters and objective clinical response to neoadjuvant chemotherapy using immunohistochemistry. The immunohistochemical analysis for four MMR protein products -MLH1, MSH2, MSH6 and PMS2 was done in the pre NACT trucut biopsy specimen and after three cycles of NACT with C AF (cyclophosphamide, adriamycin, 5-fluorouracil) regimen, in the modified radical mastectomy specimen.Results and conclusionThere was no significant correlation observed between expression of MMR proteins and age, family history, tumor size or histological type. However there was a statistically significant negative correlation between MLH1, MSH2 expression and histological grade. There was also a negative correlation observed between PMS2 expression after neo-adjuvant chemotherapy and clinical response. Cases with high post NACT expression of PMS2 were poor responders to chemotherapy. MSH6 was the most frequently altered MMR gene, with a negativity rate of 48% and the patients with high expression responded poorly to NACT. The study highlights the possible role of MMR expression in predicting aggressive tumor behavior (histological grade) and response to neoadjuvant chemotherapy in patients with LABC.

Immunohistochemical Detection of Protein Gene Product 9.5 (PGP 9.5) in Canine Epitheliotropic T-Cell Lymphoma (Mycosis Fungoides)

Protein gene product 9.5 (PGP 9.5), a ubiquitin COOH-terminal hydrolase initially considered specific for neural and neuroendocrine tissues, is expressed in a variety of epithelial and mesenchymal tumors. During immunohistochemical evaluation of a cutaneous epitheliotropic T-cell lymphoma (mycosis fungoides [MF]) in a dog, strong reactivity for PGP 9.5 was observed. This unexpected result prompted us to examine PGP 9.5 immunoreactivity in 13 additional cases of canine mycosis fungoides. All tumors were confirmed as T-cell epitheliotropic lymphoma by histopathology and immunohistochemistry for CD3. Eight of 14 cases were positive for PGP 9.5, with reactivity mainly in the cytoplasm and less commonly in the nucleus. One case had strong reactivity in the cell membrane, sometimes with concurrent paranuclear staining. Immunoreactivity did not correlate with location (epidermal, dermal, and adnexal) of tumor cells. Disease outcome did not vary between PGP 9.5-positive and negative tumors. Although PGP 9.5 immunoreactivity in MF did not predict tumor behavior in these dogs, it has had prognostic value in certain human carcinomas. This unexpected staining of lymphocytes in mycosis fungoides with an antibody to PGP 9.5 demonstrates its presence in nonneuroendocrine tumors and precludes its use as the sole diagnostic marker in discrete cell tumors in the skin.

Goblet Cell Carcinoid Tumors (Adenocarcinoid) of the Appendix

Goblet cell appendiceal carcinoids represent rare tumors that exhibit histologic features of both adenocarcinomas and neuroendocrine tumors. We present the long-term results of a series of 15 patients, focusing on clinical manifestations, diagnosis, and management. Eight male and seven female patients (median age, 52.8 years) were included. Final diagnosis was confirmed by histology. Patients were evaluated clinically, biochemically, and radiologically every four months. Median follow-up was 30 months. The majority of patients (7/15) presented with symptoms compatible with acute appendicitis. Right hemicolectomy was performed in all except one, who subsequently developed metastases. Three patients had metastases at previous diagnosis. Plasma chromogranin-A was slightly elevated in two of them, while urinary 5-hydroxy-indol-acetic acid was normal. (111)Indium-labeled octreotide scintigraphy was positive only in two of the four patients with metastases. Ki67 index was greater than 20 percent in all of them, while in only one with local tumor. Combination chemotherapy with either cisplatin plus etoposide or with 5-fluorouracil, cisplatin, and streptozotocin was administered to all patients with metastases resulting in temporary stabilization of disease. Twelve patients are alive, while three died of their disease 9, 13, and 14 months after diagnosis. The diagnostic value of chromogranin-A, urinary 5-hydroxy-indol-acetic acid, and (111)Indium-labeled octreotide scintigraphy seems to be limited in these tumors. Ki67 index appears to predict tumor behavior. Right hemicolectomy may reduce the risk of developing metastases. Chemotherapy may have efficacy in metastatic disease, however, more data are required to determine this and the optimal regimen.

Neoplasia—all you wanted to know but were afraid to ask

This presentation will give a brief outline of three important aspects of neoplasia: the role of genetic defects, prediction of tumour behaviour and how tumours cause morbidity and mortality. Neoplasia is a process of uncontrolled new growth of tissue resulting in a space-occupying neoplasm (or tumour). All cells within a neoplasm are thought to be clones of a single cell, which developed genetic defects that are then passed on to further generations of cells. Neoplasia can therefore be seen as a genetic disease. Particularly in human diagnostic pathology, prediction of the behaviour of a tumour is critical for providing a correct prognosis and treatment options. This has traditionally been performed by assessing the morphological characteristics of the tumour in a biopsy specimen. Combined with other clinico-pathological information, this allows staging or grading of the tumour. Whilst this is still an important part in the overall assessment of the likely behaviour of a tumour, many additional laboratory tests (on the neoplasm itself or on serum/plasma samples) are now also available as an adjunct. The ways in which tumours cause clinical signs are diverse; as well as acting as space-occupying lesions, they can release a number of factors that can lead to adverse effects in sites well removed from the mass.

MicroRNA BIOGENESIS, FUNCTIONALITY AND CANCER RELEVANCE

MicroRNAs (miRNA) are small non-coding RNAs that negatively regulate gene expression in a sequence- specific manner. Post-transcriptional silencing of target genes by miRNA occurs either by specific cleavage of homologous mRNA or by specific inhibition of protein synthesis. MiRNAs are essential regulators of various processes such as proliferation, differentiation, development, cell death and interaction between virus and host cell. The aim of this paper is to summarize the main findings from research on miRNA biogenesis, functionality and cancer relevance. A narrative literature review of all of the relevant papers known to the authors was conducted. Several human diseases including cancer are associated with aberrant regulation of miRNAs expression or deficiency in miRNA biogenesis. Analysis of miRNA expression signatures can serve as a valuable tool for cancer classification, diagnostics and prediction of tumor behavior. There has been demonstrated a possibility to use these microRNA signatures for a specific cancer classification with potential predictive and therapeutic value. The known data provide evidence that microRNAs may open new ways for cancer diagnosis, prognosis estimation and therapy.

Proteomic Strategies for the Characterization and the Early Detection of Lung Cancer

Biochemical techniques focused on changes in the proteome in benign and malignant conditions have been applied to the study of lung cancer. Although relatively little information is currently available about the human lung proteome, the major goals of the analysis of lung cancer are to better understand tumor biology, to define early detection biomarkers and predictors of tumor behavior, and to identify potential new therapeutic targets. In this review, we summarized the last 10 years of research in this area with emphasis on its application to the early detection of lung cancer. Basic analytical tools, such as two-dimensional gel electrophoresis, mass spectrometry, and protein arrays, are described and placed in the context of lung cancer research.

Expression of cyclo-oxygenase-2 in ependymal tumors

Up-regulation of cyclo-oxygenase-2 (COX-2), a cytokine-induced enzyme that metabolizes arachidonic acid into prostaglandins, has been described in some brain tumors, including astrocytomas. Little is known about its expression in ependymal neoplasms. The objective of the present study was to assess COX-2 immunostaining of ependymal tumors. Retrospective COX-2 immunohistochemical analysis was conducted on 117 ependymal tumors. Statistical analysis was performed using Student t-test. The study group (56 men and 44 women, mean age, 30.8 years) was comprised of 48 low-grade ependymomas (WHO grade II), 12 anaplastic ependymomas (WHO grade III), 27 myxopapillary ependymomas (WHO grade I) and 13 subependymomas (WHO grade I). At last known follow-up (range, 12-226 months; mean, 74 months), 52 patients were alive with no evidence of tumor, 16 patients were alive with residual tumor, nine patients died with tumor, one patient died with no tumor and three died with tumor status unknown. Nineteen patients had less than 12 months of follow-up. Thirty-six (36%) patients had tumors, which demonstrated positive COX-2 staining, including 16/27 (59%) myxopapillary ependymomas, 3/13 (23%) subependymomas, 14/48 (29%) ependymomas and 3/12 (25%) anaplastic ependymomas. Statistically significant COX-2 positive immunostaining was observed in myxopapillary ependymomas versus WHO grade II (P = 0.03) and grade III (P = 0.02) tumors. Increased COX-2 expression in myxopapillary ependymoma as compared to the WHO grade II and II ependymoma was observed. The reason for this apparent increased immunoexpression in these low-grade tumors is uncertain. COX-2 inhibitors may play a role in treatment of the subset of ependymal tumors that demonstrate increased expression. COX-2 staining did not reliably predict tumor behavior.

Genetic Differentiation of Appendiceal Tumor Malignancy

To use differential gene expression of candidate markers to discriminate benign appendiceal carcinoids (APCs) from malignant and mixed cell APCs. Controversy exists in regard to the appropriate surgical management of APCs since it is sometimes difficult to predict tumor behavior using traditional pathologic criteria. We have identified 5 differentially expressed genes (a mitosis-regulatory gene NAP1L1, an adhesin MAGE-D2, an estrogen-antagonist, the metastasis marker MTA1, the apoptotic marker NALP, and chromogranin A) that define gut neuroendocrine cell behavior. Total RNA was isolated using TRIzol reagent from 42 appendiceal samples, including appendiceal carcinoids identified at exploration for appendicitis (no evidence of metastasis; n = 16), appendicitis specimens (n = 11), malignant appendiceal tumors (> 1.5 cm, evidence of metastatic invasion; n = 7), and mixed (goblet) cell appendiceal adenocarcinoids (n = 3), normal appendiceal tissue (n = 5), and 5 colorectal cancers. Gene expression (CgA, NAP1L1, MAGE-D2, MTA1, and NALP1) was examined by Q-RT PCR (Applied Biosystems) and quantified against GAPDH. CgA message was elevated (> 1000-fold, P < 0.05) in all tumor types. NAP1L1 was elevated (> 10-fold, P < 0.03) in both malignant and goblet cell adenocarcinoids compared with normal and incidental lesions (P < 0.006). MAGE-D2 and MTA1 message were significantly elevated (> 10-fold, P < 0.01) in the malignant and goblet cell adenocarcinoid tumors but not in the appendicitis-associated carcinoids or normal mucosa. The apoptotic marker, NALP1, was overexpressed (> 50-fold, P < 0.05) in the appendicitis-associated and malignant appendiceal carcinoids but was significantly decreased (> 10-fold, P < 0.05) in the goblet cell adenocarcinoids. Elevated CgA transcript and protein levels indicative of a carcinoid tumor were identified in one acute appendicitis sample with no histologic evidence of a tumor. These data demonstrate that malignant APCs and goblet cell adenocarcinoids have elevated expression of NAP1L1, MAGE-D2, and MTA1 compared with appendiceal carcinoids identified at surgery for appendicitis. This and the differences in NALP1 gene expression (decreased in goblet cell adenocarcinoids) provide a series of molecular signatures that differentiate carcinoids of the appendix. CgA identified all appendiceal tumors as well as covert lesions, which may be more prevalent than previously recognized. The molecular delineation of malignant appendiceal tumor potential provides a scientific basis to define the appropriate surgical management as opposed to morphologic assessment alone.

P27 downregulation and metallothionein overexpression in gastric cancer patients are associated with a poor survival rate

As a significant number of curatively treated gastric cancer patients will ultimately relapse, there is an urgent need to investigate new prognostic markers for identification of high-risk patients. In this study, we investigated the possible role of molecular markers involved in cell cycle regulation (B1 and D3 cyclins, and p27) and cell protection (metallothionein, MT) in predicting tumor behavior and clinical outcome in gastric cancer patients. Analysis of the above indicators was performed by immunohistochemistry on 73 gastric cancer patient samples and 25 normal gastric mucosa specimens. Normal gastric mucosa cells displayed low expressions of B1 and D3 cyclins and MT, and intense p27 staining. Conversely, gastric tumor cells showed higher cyclin D3 and MT, and lower p27 expressions. B1 cyclin expressions were not different between normal and tumor tissue. p27 and MT expressions were altered in almost all cancer samples, and were strongly correlated with tumor progression. Advanced extent of the primary tumor, nodal metastasis, low p27, and high MT expressions were the best combination of variables for prediction of poor clinical outcome. Each marker predicted outcome better than staging based on tumor-node (TNM) system. Survival and recurrence rates decreased as molecular alterations increased. Finally, molecular profile determination correctly predicted the prognosis in patients with same TNM stage. p27 and MT expressions strongly correlated with clinical outcome allowing to identify an unfavorable group of patients that may benefit from tailored treatments. The role of B1 and D3 cyclins in gastric cancer remains to be elucidated.

Evaluation of argyrophilic nucleolar organizer region staining in predicting the behavior of meningiomas.

BACKGROUNDThe number of argyrophilic nucleolar organizer regions (AgNOR) correlates with cellular proliferative activity. Comparing non-recurrent, recurrent, atypical and malignant meningiomas we studied the value of the routine applicability of the AgNOR count in the prognostication of this tumor.PATIENTS AND METHODSTwo hundred and thirty-eight meningiomas were reviewed blindly and graded using WHO grading schema. Eighty-one cases were selected and arranged in six groups according to clinical data and grading: 14 benign non-recurrent meningiomas; 14 primary benign recurrent meningiomas and their subsequent benign recurrences; 14 atypical; 11 malignant and 14 spinal meningiomas. Silver-stained slides were prepared and mean, median and standard deviation of AgNOR dots determined.RESULTSThere was a proportionate increase of AgNOR dots with increasing tumor grade. There was a significant difference between benign non-recurrent tumors versus benign recurrent (P<0.0001) and atypical or malignant (P<0.0001) tumors. A difference was also noted between the recurring tumors versus malignant ones (P= 0.002) but no significant difference was seen between recurrent and atypical; atypical and malignant; intracranial and intraspinal; and primary of recurring meningiomas with their subsequent recurrences. A mean AgNOR count of <2.3 could separate benign tumors from atypical or malignant meningiomas with 93% specificity; and 93% of tumors with benign histology had no recurrence potential if their mean AgNOR count was less than 1.8.CONCLUSIONThis study indicates that in meningioma, the AgNOR count has a good correlation with tumor grading and recurrence, which may aid pathologists and clinicians in predicting tumor behavior.

Correlation of spectral imaging and visual grading for the quantification of thymidylate synthase protein expression in rectal cancer

Quantification of protein expression in tissue sections stained by immunohistochemistry has traditionally involved visual grading techniques. However, if these results are to be used to predict tumor behavior and permit targeted therapy, there is a need for more accurate, objective, and reproducible methods. This study investigated the utility of spectral imaging as a method of quantifying thymidylate synthase protein expression in immunohistochemically stained sections of primary rectal cancer and normal rectal mucosa by comparing it with the current gold standard of manual visual grading. There was good correlation between estimates of thymidylate synthase stain intensity and area derived by spectral imaging and visual grading in both tumor and normal mucosal sections, suggesting that spectral imaging is a valid way of quantifying biologic sections stained by immunohistochemistry.

Multifocal and Multicentric Breast Cancer: Does Each Focus Matter?

The identification of multiple tumors in the breast is associated with increased nodal involvement when compared with similar staged unifocal disease. This study compares two methods of tumor size assessment to predict tumor behavior in the relationship between size and axillary node involvement for patients with multifocal and multicentric breast cancer. The histologic reports of every patient with multifocal breast cancer treated in New South Wales between April 1995 and September 1995 were examined. Tumors were assessed using two size estimates: (1) largest tumor focus diameter and (2) the aggregate diameters of all tumor foci. The dimensions were compared with unifocal tumors and against node positivity. Ninety-four (11.1%) of 848 women had multifocal breast cancer and of these 49 women (52.1%) had axillary node involvement compared with 37.5% with unifocal breast cancer (P =.007). The use of aggregate dimension reclassified significant numbers of multifocal tumors at a more advanced stage. Use of this method to stage cancers, rather than the largest tumor size, removed the excess node positivity when compared with unifocal, stage-matched breast carcinomas. The tendency of breast tumors to metastasize is a reflection of the total tumor load. Failure to measure the additional tumor burden provided by multiple small foci may understage a woman's disease. This may deny patients the opportunity of adjuvant therapies if the contribution of the smaller foci to the incidence of node positivity and survival is ignored.

Altered Expression of Cyclins and Cell Cycle Inhibitors in Papillary Thyroid Cancer: Prognostic Implications

Currently we lack biochemical or molecular markers that predict recurrence and metastases in thyroid cancer. Recent studies in a number of other human malignancies indicate that expression and/or subcellular localization of certain cell cycle regulators has prognostic utility. We have investigated the expression of cyclins D1 and E and of cyclin-dependent kinase inhibitor's p21 and p27 in papillary thyroid cancer (PTC) and correlated this with clinical/histological stage at diagnosis and with clinical outcome. PTCs were compared to normal thyroid, adenomas, and undifferentiated thyroid cancers (UTCs). Our studies indicate that PTCs and UTCs demonstrate low nuclear expression of cyclin E and p27, allowing a clear distinction between adenomas and these carcinomas (p < 0.004). A pattern of low nuclear expression of all four markers was observed in stage IV PTCs and UTCs, while stage I PTCs had low D1 and E accompanied by high p21 or p27. Expression of cytoplasmic cyclin D1 was significantly lower in stage IV PTCs and UTCs than in stage I-III PTC's (p </= 0.020), and appeared to correlate inversely with poor outcome in PTCs (p = 0.010). These studies suggest that evaluation of a panel of these markers and attention to their subcellular localization may be a useful adjunct in differentiating benign from malignant thyroid neoplasms and in predicting tumor behavior.

Assessment of the tumor microenvironment in cervix cancer using dynamic contrast enhanced CT, interstitial fluid pressure and oxygen measurements

Interstitial fluid pressure (IFP) and oxygen (pO(2)) measurements are prognostic factors in cervical cancer. The purpose of this study was to determine the relationship between IFP and oxygenation and parameters derived from dynamic contrast-enhanced computed tomography (DCE-CT). Dynamic contrast-enhanced computed tomography was performed in 32 patients with cervical cancer before radiation therapy. Images were acquired during intravenous contrast injection at 1 per s for 120 s and 1 per 15 s for 60 s. DCE-CT was analyzed using CT Perfusion 3 software (GE Medical Systems) to derive tumor blood flow (BF), permeability surface area product, blood volume, and mean transit time. Further analysis was performed to obtain relative peak enhancement, residual enhancement at 3 min after contrast injection (RE), time to peak and initial slope. Nodal status and tumor size were assessed with MRI. From in vivo IFP (n = 31) and pO(2) (n = 31) tumor measurements median pO(2) (mO(2)), percentage measurements less than 5 mm Hg (HP5) and mean IFP values were calculated. There was a positive correlation between BF and mO(2) (r = 0.47, p = 0.007) and between RE and HP5 (r = 0.39, p = 0.03). There was no correlation between IFP and DCE-CT parameters. There is a moderately positive, correlation between tumor oxygenation and BF as well as RE and HP5. Further study is required to determine if DCE-CT parameters are useful predictors of tumor behavior in cervical cancer.

Loss of Expression of the Adipocyte-type Fatty Acid-binding Protein (A-FABP) Is Associated with Progression of Human Urothelial Carcinomas

Bladder cancer is the fifth most common malignancy in the world and represents the second most common cause of death among genitourinary tumors. Current prognostic parameters such as grade and stage cannot predict with certainty the long-term outcome of bladder cancer, and as a result there is a pressing need to identify markers that may predict tumor behavior. Earlier we identified the adipocyte fatty acid-binding protein (A-FABP), a small-molecular-mass fatty acid-binding protein that functions by facilitating the intracellular diffusion of fatty acids between cellular compartments as a putative marker of progression based on a limited study of fresh bladder urothelial carcinomas (UCs) (Celis, J. E., Ostergaard, M., Basse, B., Celis, A., Lauridsen, J. B., Ratz, G. P., Andersen, I., Hein, B., Wolf, H., Orntoft, T. F., and Rasmussen, H. H. (1996) Loss of adipocyte-type fatty acid binding protein and other protein biomarkers is associated with progression of human bladder transitional cell carcinomas. Cancer Res.56, 4782-4790). Here we have comprehensively examined the protein expression profiles of a much larger sample set consisting of 153 bladder specimens (46 nonmalignant biopsies, 11 pTa G1, 40 pTa G2, 10 pTa G3, 13 pT1 G3, 23 pT2-4 G3, and 10 pT2-4 G4) by gel-based proteomics in combination with immunohistochemistry (IHC) using a peptide-based rabbit polyclonal antibody that reacts specifically with this protein. Proteomic profiling showed a striking down-regulation of A-FABP in invasive lesions, a fact that correlated well with immunohistochemical analysis of the same samples. The IHC results were confirmed by using a tissue microarray (TMA) containing 2,317 samples derived from 1,849 bladder cancer patients. Moreover, we found that the altered expression of A-FABP in invasive UCs is not due to deregulated expression of peroxisome proliferator-activated receptor gamma (PPARgamma), a trans-activator of A-FABP. Taken together, these results provide evidence that deregulation of A-FABP may play a role in bladder cancer progression and suggest that A-FABP could have a significant prognostic value in combination with other biomarkers.

A spectrum of behaviour in silent corticotroph pituitary adenomas

Silent corticotroph adenomas (SCA) are pituitary tumours positive on immunohistochemical staining for ACTH but without clinical evidence of Cushing's disease in the patient. Previous reports suggest that these tumours may behave in a more aggressive way then other pituitary adenomas. We have followed the natural history of SCA and assessed whether histopathological indices predict tumour behaviour. We identified 22 patients in whom trans-sphenoidal surgery was performed for a non-functioning adenoma (NFA) with positive immunostaining for ACTH between 1990 and 2000 and examined the history of their disease. Patients were followed up for a mean of 4.8 years. A total of 86.7% of patients had documented visual deficits at presentation. In four cases hypercortisolaemia was observed later in the course of the disease. Two patients died as a result of their SCA and 33.3% of tumours recurred. Recurrence was more frequent in patients treated with adjuvant radiotherapy. Pathological indices (increased mitotic range and Ki-67) did not predict recurrence or malignant transformation. We suggest that certain ‘silent’ corticotroph tumours may have the potential for ACTH secretion leading to hypercortisolaemia at a later stage in the disease. The possibility of transformation to a more aggressive tumour needs to be considered in all SCA.

Influence of Fibronectin,(FN), Adhesion on Gene Expression Profiles, (GEP), of Isolated Patient Myeloma Cells.

Myeloma cells proliferate preferentially in the bone marrow and the latter has been shown to confer a survival advantage and drug resistance, (DR), through a process known as Cell Adhesion Mediated Drug Resistance or CAM-DR, a form of de-novo drug DR. Therefore, in studying the mechanisms of drug resistance in myeloma, the contribution of the microenvironment needs to be considered. Recently, we compared the genotypic and phenotypic profiles of CAM-DR melphalan resistance to acquired melphalan resistance. (Hazlehurst et al Cancer Res. 63: 7900–7906, 2003,)Briefly, we reported that de novo melphalan resistance associated with CAM-DR was significantly less complex, both phenotypically and genotypically, compared to acquired melphalan resistance in 8226 cells. GEP of adherent 8226 myeloma cells demonstrated fewer changes in gene expression compared to acquired melphalan resistance with no changes related to genes involved with DNA damage or cell cycle; however, changes were noted for genes involved in mitochondrial perturbation and caspase activation. Studies examining GEP profiles in myeloma patients, have been performed using isolated myeloma cells without considering the contribution of the tumor microenvironment and its influence on the phenotype or genotype of these cells. We hypothesize that the interactions of the myeloma cells with the microenvironment affects the myeloma GEP and needs to be considered when developing gene signatures that would ultimately predict level of response to treatment. In this study, we examined GEP of CD138+ myeloma cells isolated from 18 patients with relapsed, (n=8) or newly diagnosed (n=10), myeloma. The CAM-DR phenotype in CD-138+ cells was analyzed by exposing patient bone marrow specimens to Dexamethasone when cells were in suspension versus adhered to FN. We showed myeloma cells to be significantly more sensitive to Dexamethasone when kept in suspension vs adhered to FN in 5/6 specimens studied. For GEP studies, CD138+ were maintained in suspension or adhered to FN for 24 hours, then harvested for RNA extraction and hybridized to GeneChip arrays (Affymetrix HG-133A chip). Differential exression of genes was performed using a chi square test to compare observed vs expected. Only genes that were consistently differentially expressed in >50 % of patients with p value <.001 were considered for the final analysis. Among all the patients, FN adhesion of myeloma cells resulted in the overexpression of 24 genes and suppression of 45 genes.. In relapsed patients, 16 genes were found to be overexpressed and 56 suppressed when cells were adhered to fibronectin. In newly diagnosed patients, 123 genes were found to be overexpressed and 83 supressed on FN. Genes found to be overexpressed on FN vs suspension include adhesion related genes,(cathepsisn, integrin alpha 4 and 8), growth and proliferation related genes,(syndecan, TNF-induced protein), cytokines and chemokines (IL-16, CXC R4 and 2), and antiapoptotic proteins(MADS family).In summary, we have shown that fibronectin adhesion of patient myeloma cells alters gene expression. As we expand the number of patients studied, we should be able to develop a gene signature associated with adhesion and determine whether these genotypes are better predictors of tumor behavior and response to therapy. Identification of genes uniquely associated with cell adhesion may lead to the development on novel therapies for myeloma.

Protein Profiling in Childhood Precursor B Acute Lymphoblastic Leukemia using Surface Enhanced Laser Desorption/Ionization (SELDI) Mass Spectrometry: A Study of Archival Samples.

Acute lymphoblastic leukemia (ALL) is the most common pediatric malignancy. Even though the risk-group adjusted therapies have dramatically improved the outcome, approximately 25% of ALL patients experience relapse of the disease. Recent gene microarray studies showed distinct gene expression profiles for immunophenotypically and genetically defined ALL subgroups and their association with treatment outcome. However, not only transcriptional control, but also a variety of post-transcriptional/post-translational modifications, play an important role in regulating protein expression and function, and ultimately may prove informative for predicting tumor behavior. In this pilot study, we hypothesize that in childhood precursor B ALL surface enhanced laser desorption/ionization mass spectrometry can identify proteins discriminating between age and WBC defined risk-groups. Ultimately, specific proteins may emerge that will provide predictive information beyond currently known prognostic factors. Our pilot series consisted of 78 pediatric precursor B ALL patients. The protein samples were obtained from archival Wright-Giemsa stained bone marrow aspirate smears. Initial experiments, using CM10 and IMAC30 chips demonstrated that the latter provided superior resolution and presented more numerous protein peaks. The protein mass spectra were originally generated using laser intensities of 150 and 160 arbitrary units. Data obtained at laser intensity of 160 was used for further analysis. Twenty-three protein peaks common to all samples were identified within examined range (up to 30 kDa; CiphergenExpressData Manager software, Ciphergen, Fremont, CA). These proteins span the spectrum between 1.0 to 15.2 kDa. The comparison of protein profiles between standard- and high-risk groups (SR and HR, respectively), WBC subgroups (less and more than 50 K/ul) and in the prognostic age groups was performed. Two protein peaks, 3.0 and 1.7 kDa, discriminated between SR and HR (p=0.001 and p=0.04, respectively). Both proteins showed significantly higher intensity in SR group. The protein peak of 3.0 kDa was further confirmed to discriminate between patients with low and high presentation WBC (p=0.01). Our results demonstrate that reliable quality protein profiles can be obtained using archival Wright-Giemsa stained bone marrow aspirate smears. Using this technology, specific proteins were found to discriminate between prognostic risk-groups. We are currently analyzing precursor B-cells sorted from non-neoplastic bone marrows to compare protein profiles to our pilot precursor B ALL series in a cluster analysis. The use of archival material facilitates analysis of large patient series and identification of protein panels with prognostic power, which can be validated in prospective studies.