ON BCG INTRAVESICAL IMMUNOTHERAPY AND STUDY OF VEGF AND C-erb-B2 EXPRESSION
by
SANJAY KUMAR DEPARTMENT OF SURGERY (GENERAL) CHHATRAPATI SHAHUJI MAHARAJ MEDICAL UNIVERSITY UP, LUCKNOW 226 003 (U.P.), INDIA
C H H A T R A P A T I S H A H U JI MAHARAJ M E D I C A L U N I V E R S I T Y
PROFORMA Submitted to CSM Medical University UP, Lucknow for the degree of M.S. (GEN. SURGERY)
AUG, 2011
To, The Registrar C.S.M.MedicalUniversity, Lucknow. Through Proper channel
Subject: Submission of thesis proforma for MS. (GEN. SURGERY). Respected Sir,
With due regards I wish to state that I have been selected as a candidate for M.S. (GENERAL SURGERY) examination of C.S.M. Medical University to be held in 2013. I humbly request you to kindly register my subject of thesis entitled, EVALUATION OF CASES OF SUPERFICIAL BLADDER TUMOR ON BCG INTRAVESICAL IMMUNOTHERAPY AND STUDY OF VEGF AND C-erb-B2 EXPRESSION. The necessary particulars and proforma along with recommendations of chief supervisor and co-supervisors are being submitted herewith. Thanking you, Yours obediently,
SANJAY KUMAR Junior Resident, 2nd Year Department of General Surgery, C.S.M.MedicalUniversity, Lucknow. Forwarded and Recommended by:
PROF.A. A. SONKAR MS,FACS, FUICC Professor and Head, Department of General Surgery, C.S.M.MedicalUniversity, Lucknow.UP.
Recommendation of Chief supervisor:
Dr. HarvinderSingh Pahwa MS MCh (UROLOGY), FMAS Associate Professor Department of General Surgery, C.S.M. Medical University, Lucknow
Recommendations of the co-supervisors:
DR. AWANISH KUMAR MS Assistant Professor Department Of General Surgery C.S.M. Medical University (Erstwhile K.G.M.U), Lucknow
PROF. RAJ MEHROTRA MD Professor & Head Department of Pathology C.S.M.MedicalUniversity, Lucknow
PROF. S.M. Natu MSc, PhD Professor Department Of Pathology, C.S.M.Medicaluniversity, Lucknow
PROF. Madhu Mati Goel MD Professor Department Of Pathology, C.S.M. Medical university, Lucknow
PROF. SANJEEV MISHRA MS, Mch Professor & Head Department Of Surgical Oncology C.S.M. Medical University, Lucknow
PROF. S. N. SHANKHWAR MS MCh Professor & Head Department of Urology C.S.M.Medical University, Lucknow
DR. APUL GOEL MS ,Mch Assistant Professor, Department of Urology C.S.M. Medical University, Lucknow
DR. VISHWAJEET MS ,Mch Assistant Professor, Department of Urology C.S.M. Medical University, Lucknow
PROFORMA Name : SANJAY KUMAR Year and Month of Graduation : March, 2007 University from which graduated : C.S.M.Medical University, Lucknow Course to which admitted : M.S.( General Surgery) Date of Admission to the Course : 31 st May, 2010 Present Status : Junior Resident-2 nd Year Department of General Surgery, C.S.M.MedicalUniversity, Lucknow. Department in which subject of thesis falls : Department of General Surgery, C.S.M.MedicalUniversity, Lucknow. Title of Thesis : Evaluation Of cases of Superficial Bladder Tumor on BCG Intravesical Immunotherapy and study of VEGF & C-erb-B2 expression Brief resume of the work proposed to be undertaken for thesis : Enclosed herewith
INTRODUCTION
Carcinoma urinary bladder is a significant cause of morbidity and mortality. Bladder cancer is the fourth most common malignancy in men and the tenth most common in women. Cigarette smoking is currently the single most important risk factor contributing to the development of bladder cancer. Risk of bladder cancer increases two- to fourfold among smokers, and declines, but does not return to baseline, on smoking cessation. Other risk factors include exposure to environmental carcinogens (eg, aromatic amines), chemotherapy with cyclophosphamide, and pelvic radiation. Hematuria is the most common sign of bladder cancer, with about 80% of transitional cell carcinomas (TCCs)being diagnosed with either gross or microscopic hematuria. Some patients, particularly those with carcinoma in situ (CIS), will have symptoms of bladder irritability, including urinary frequency, urgency, and anddysuria. Patients with hematuria and those with persistent symptoms should receive a thorough evaluation for bladder cancer. Transitional cell carcinoma (TCC) accounts for over 90% of bladder cancer. Of these, 70%are superficial (nonmuscle-invasive) cancers. Superficial TCC has a high rate of recurrence (50%70%) and may progress to invasive disease, which is then associated with higher rates of morbidity and mortality. Squamous cell carcinoma (SCC) accounts for only 5%of bladder cancer cases in the United States, and it is usually associated with chronic cystitis resulting fromurinary calculi, long-term indwelling catheters, or bladder diverticula. The following stages are used for bladder cancer 2 : Stage 0 (Papillary Carcinoma and Carcinoma in Situ) In stage 0, abnormal cells are found in tissue lining the inside of the bladder. These abnormal cells may become cancer and spread into nearby normal tissue. Stage 0 is divided into stage 0a and stage 0is, depending on the type of the tumor: Stage 0a is also called papillary carcinoma, which may look like tiny mushrooms growing from the lining of the bladder. Stage 0is is also called carcinoma in situ, which is a flat tumor on the tissue lining the inside of the bladder. Stage I In stage I, cancer has formed and spread to the layer of tissue under the inner lining of the bladder. Stage II In stage II, cancer has spread to either the inner half or outer half of the muscle wall of the bladder.
Stage III In stage III, cancer has spread from the bladder to the fatty layer of tissue surrounding it and may have spread to the reproductive organs (prostate, seminal vesicles, uterus, or vagina). Stage IV In stage IV, cancer has spread from the bladder to the wall of the abdomen or pelvis. Cancer may have spread to one or more lymph nodes or to other parts of the body. Bladder Cancer Prognostic Biomarkers: Tumor-related antigens M344, 19A211, T138 Proliferating antigens Ki-67, PCNA Blood group antigens ABH, Lewis Oncogenes c-ras, mdm2, c-erbB2, c-myc, c-jun Cellular adhesion molecules E-cadherin, integrins DNA ploidy ICM, LSCM, FISH, FLOW Growth factors EGF, TGF, FGF, VEGF Angiogenesis and inhibitors Thrombospondin-1,angiostatin Cell cycle proteins p53, pRB, cyclins, p21 Apoptosis pathway bcl-2/BAX ratio Increase bcl-2: immortalized Increase BAX: apoptosis
Treatment option by stages: Stage 0 (Papillary Carcinoma and Carcinoma in Situ) Treatment of stage 0 may include the following: Transurethral resection with fulguration. Transurethral resection with fulguration followed by intravesical biologic therapy or chemotherapy. Segmental cystectomy. Radical cystectomy. A clinical trial of photodynamic therapy. A clinical trial of biologic therapy. A clinical trial of chemoprevention therapy given after treatment so the condition will not recur (come back). Treatment of stage I bladder cancer may include the following: Transurethral resection with fulguration.
Transurethral resection with fulguration followed by intravesical biologic therapy or chemotherapy. Segmental or radical cystectomy. Radiation implants with or without external radiation therapy. A clinical trial of chemoprevention therapy given after treatment to stop cancer from recurring (coming back). A clinical trial of intravesical therapy. Stage II Bladder Cancer Treatment of stage II bladder cancer may include the following: Radical cystectomy with or without surgery to remove pelvic lymph nodes. Combination chemotherapy followed by radical cystectomy. External radiation therapy combined with chemotherapy. Radiation implants before or after external radiation therapy. Transurethral resection with fulguration. Segmental cystectomy. Stage III Bladder Cancer Treatment of stage III bladder cancer may include the following: Radical cystectomy with or without surgery to remove pelvic lymph nodes. Combination chemotherapy followed by radical cystectomy. External radiation therapy combined with chemotherapy. External radiation therapy with radiation implants. Segmental cystectomy. Stage IV Bladder Cancer Treatment of stage IV bladder cancer may include the following: Radical cystectomy with surgery to remove pelvic lymph nodes. External radiation therapy (may be as palliative therapy to relieve symptoms and improve quality of life). Urinary diversion as palliative therapy to relieve symptoms and improve quality of life. Cystectomy as palliative therapy to relieve symptoms and improve quality of life. Chemotherapy alone or after local treatment (surgery or radiation therapy). A clinical trial of chemotherapy 3 . Intravesical (within the bladder) BCG is one of the most common and most effective immunotherapy used in superficial bladder cancer treatment.. the mechanism of action of BCG is unknown. BCG is an attenuated mycobacterium developed as a vaccine for tuberculosis that has demonstrated antitumor activity in several different cancers, including UC ( Morales et al, 1976 ). The original regimen described by Morales and colleagues included a percutaneous dose, which was discontinued after success using a similar intravesical regimen by Brosman (1982) . Bacillus Calmette-Guerin (BCG) is the vaccination used to protect against tuberculosis (TB), yet when administered into the bladder is thought to provoke an immune response. The intravesical BCG, an inactivated form of the bacterium Mycobacterium tuberculosis, is administered via catheter directly into the bladder where it remains for two hours. The immune response is successful in reducing the recurrence of bladder cancer, possibly by destroying any remaining cancer cells.
Vascular endothelial growth factor (VEGF) is an important stimulator of angiogenesis, the development and recruitment of new blood vessels. Through angiogenesis, VEGF plays animportant role in the pathogenesis of cancer, as growing tumors require new conduits toprovide nutrients and remove waste. In addition, VEGF increases vascular permeability, whichmay facilitate metastasis and result in increased oxygen delivery to tumors. VEGF is expressed by a variety of solid and hematologic neoplasms and inhibition of VEGF is used therapeutically in the treatment of certain cancers. Abnormal expression or function of receptors forgrowth factors can enhance the proliferative capacityof malignant cells. EGF is a potent mitogen excreted inhigh concentrations in human urine in a biologicallyactive form59but is found in lower concentrations inurine of BC patients.60This may reflect increased ligand binding as EGFRs, which are normally found onlyin the basal layer of the urothelium, become abnormally expressed throughout the entire urothelium in TCC,including those superficial cells directly exposed tourine. EGFR expression as detected by immunohistochemistry correlates with increased grade and stageand with poor prognosis.61Abnormal EGFR expressionis also found in cystoscopically normal-appearingurothelium in patients with TCCs elsewhere in the bladder,62thus supporting the notions of the existence ofan urothelial field defect63and that abnormal EGFR expression may be an early event in BC tumorigenesis.Additionally, because ligands that work through EGFRsnot only induce mitogenesis but also cellular motility,stimulation of EGFRs in malignant urothelium mayencourage transepithelial motility and tumor invasionas well as proliferation. The proto-oncogene c-erb-B2 encodes a transmembrane receptor-like protein similar to the epidermal growth factor receptor (EGFR). In a subset ofTCCs, c-erb-B2 is overexpressed; this appears to be due to gene amplification. The overexpressed c-erbB2 has tyrosine kinase activity similar to that of the activated EGFR and the ability to stimulate cellular growth. Although some investigators have shown a relationship with high grade and/or stage in tumorsthatoverexpressed c-erb-B2, others found no suchcorrelation.In view of these conflicting results, further evaluation is required to determine the prognostic value of detecting c-erb-B2 in BC. Nevertheless, c-erb-B2 illustrates a potential mechanism by whichoncogenes contribute to malignant transformation. Additionally, BCs that overexpress this membrane bound oncoprotein may be the target for immunological therapeutic strategies that recognize it.
AIM AND OBJECTIVES Our study aims to:- 1. Clinical, pathological and radiological evaluation of case of superficial bladder tumor on intravesical BCG therapy. 2. Study of levels of VEGF and C-erb B2 in tissue. 3. Study of urinary VEGF level. MATERIALS AND METHODS Study design Observational study. Study duration 1 year Patients: We will study urine and bladder tissue specimens from superficial bladder cancer patients taking treatment in deptt. Of general surgery , urology department& deptt. Of surgical oncology of our institute before and after instillation of intravesical BCG immunotherapy. After written consent urine and tissue samples will be taken. Patient demographic data and medical history will be obtained and patients taken previous treatment will be excluded from the study. Diagnosed Patients (suspects of urinary bladder tumor) will undergo cystoscopy as a reference standard for detection of bladder cancer and all tumors or suspicious lesions and biopsies will be taken. Biopsy specimens will be sent for histopathology to diagnose and grade the tumor. The tissue will be placed in liquid nitrogen and stored at -80C for performing estimation of VEGF and C-erb B2content. Fresh midstream urine samples will be collected from bladder carcinoma patients before cystoscopy and after procedure.Clinicopathologicalstaging will be done according to TNM classification (AJCC). Then the association of VEGFand C-erb B2 levels in urine and tissue will be studied. Inclusion criteria: only the patients who have histologically proven superficial transitional urinary bladder cancer planned for intravesical BCG instillation will be included in the study. Exclusion criteria: patients who have taken systemic chemotherapy for urinary bladder cancer and immunocompromised patients and patients having tumor other than TCC will be excluded from the study. Clinical presentation: hematuria (gross or microscopic), pain during urination, frequent urination (polyuria), feeling the need to urinate without results.
Preparation of samples: All steps of sample preparation will be carried out at 4C. Tissue pieces weighing 50 mg will homogenized with homogenizer in 1 ml of ice-cold 50mM tris extraction buffer, pH 7.4 ( contains 5m EDTA, 10 ml/l triton-X100 and the following protease inhibitors: 1g/ml leupeptin and 0.2 M henylmethylsupfonylfluoride). The resulting homogenate will be centrifuged for 5 min at 20000x gravity. Supernatant (cell lysate) will be frozen at -80C. Fresh midstream, urine samples (20ml) obtained before and after surgery will be centrifuged at 1000x gravity for 10 min. The supernatant will be separated and stored at -80C. VEGFand C-erb B2 enzyme immunoassay (EIA). Human VEGF and C-erb B2 level will be measured using an enzyme immunoassay quantikine kit. This assay uses a quantitative sandwich technique. This system uses a solid phase monoclonal antibody and an enzyme linked polyclonal antibody raised against recombinant human VEGFand C-erb B2. In brief, both VEGFand C-erb B2 standards and samples (cell lysate and urine supernatant) are placed in the wells pre-coated with murine monoclonal antibody against VEGFand C-erb B2; the wells are incubated for 2 hrs. and allowed to react at room temperature. The resulting immune complexes are bound onto the plate and any unbound reactants are removed by a washing step, followed by addition of horse-radish peroxidase conjugated murine antibody directed to another epitope of VEGFand C-erb B2 molecule to sandwich the immunobolized reaction complex, the wells are incubated for 2hourson a rotator. Substrate chromogen solution is used to develop the color after removal of unbounded components. The optical density is read using spectrophotometer set at 490 nm. The concentrations of VEGFand C-erb B2 will be determined by comparing the optical density of test samples to the standard curve. Each sample will be analyzed in triplicate and the mean values will be used as final concentration. The control for differences; cell lysate concentration VEGF expression is standardized to the total protein content (pg/mg protein) using Bradford method with bovine serum albumin as the calibrator, while urine samples are normalized by creatinine content (pg/mg creatinine), the urinary creatinine levels will be determined based on jaffe reaction. Bradford assay The Bradford assay, a colorimetric protein assay, is based on an absorbance shift in the dye Coomassie when the previously red form coomassie reagent changes and stabilizes into coomassie blue by the binding of protein. During the formation of this complex, two types of bond interaction take place: the red form of coomassie dye first donates its free electron to the ionizable groups on the protein, which causes a disruption of the protein's native state, consequently exposing its hydrophobic pockets. These pockets on the protein's tertiary structure
bind non-covalently to the non-polar region of the dye via van der Waals forces, positioning the positive amine groups in proximity with the negative charge of the dye. The bond is further strengthened by the ionic interaction between the two. Binding of the protein stabilizes the blue form of coomassie dye, thus the amount of complex present in solution is a measure for the protein concentration by use of an absorbance reading. The (bound) form of the dye has an absorption spectrum maximum historically held to be at 595 nm. The cationic (unbound) forms are green or red while binding of the dye to protein stabilizes the blue anionic form. The increase of absorbance at 595 nm is proportional to the amount of bound dye, and thus to the amount (concentration) of protein present in the sample. Jaffe reaction Creatinine in serum or urine is determined by Jaffe's reaction where creatinine produces quantitatively an orange color with picric acid in alkaline medium. After allowing incubation time of 15 min at room temperature for color development the color is measured at 520nm. Without taking into consideration the acidic nature of standard, protein free filtrate (PFF) of serum and urine, 1% picric acid and 0.75N NaOH are used in this reaction for color development in standard, PFF of serum and urine. An investigation was thought to be necessary to determine the optimum alkali concentration required in standard, PFF of serum and urine. The results show that 0.25, 0.75 and IN NaOH give maximum color in urine, standard and PFF of serum respectively. A standard solution of creatinine is prepared in 0.1N HCl and the PFF of serum is obtained by addition of fresh tungstic acid . Alkali is consumed to neutralise the acids in both these cases. For urine creatinine measurement, a direct diluted urine sample is used .The difference in the requirement of NaOH is conceivable. The routine use of 0.75N NaOH irrespective of the nature of specimen as is done in all biochemical laboratories, for creatinine measurement needs modification in the light of this investigation. Statistical analysis: the relations will be studied using linear regression analyses by kraskal-wallis and mann-whiney U tests. All p values will be 2 sided. Statistical significance in the study will set up as p<0.05.
REVIEW OF LITERATURE Bladder cancer refers to any of several types of malignant growths of the urinary bladder. It is a disease in which abnormal cells multiply without control in the bladder. The most common type of bladder cancer begins in cells lining the inside of the bladder and is called transitional cell carcinoma (sometimes urothelial cell carcinoma). IMMUNOTHERAPY Intravesical immunotherapy results in a massive local immune response characterized by induced expression of cytokines in the urine and bladder wall and by an influx of granulocytes and mononuclear cells. The mechanism of action has been intensively investigated. The initial step appears to be direct binding to fibronectin within the bladder wall, subsequently leading to direct stimulation of cell-based immunologic response. Numerous cytokines involved in the initiation or maintenance of inflammatory processes, including tumor necrosis factor-, granulocyte-macrophage colony-stimulating factor, interferon (IFN)-, and interleukin (IL)-1, IL-2, IL-5, IL-6, IL-8, IL-10, IL-12, and IL-18, have been detected in the urine of patients treated with intravesical BCG. The observed pattern of cytokine induction with preferential up regulation of interferon- and IL-2 reflects induction of a Th1 response. This immunologic response activates cell-mediated cytotoxic mechanisms that are believed to underlie the efficacy of BCG in the prevention of recurrence and progression (Bohle and Brandau, 2003). Bacillus Calmette-Guerin BCG is an attenuated mycobacterium developed as a vaccine for tuberculosis that has demonstrated antitumor activity in several different cancers, including UC (Morales et al, 1976). The original regimen described by Morales and colleagues included a percutaneous dose, which was discontinued after success using a similar intravesical regimen by Brosman (1982).
BCG is stored in refrigeration and reconstituted from a lyophilized powder. Connaught, Tice, Armand Frappier, Pasteur, Tokyo, and RIVM strains all arise from a common original strain developed at the Pasteur Institute. The vaccine is reconstituted with 50 mL of saline and should be administered through a urethral catheter under gravity drainage soon thereafter because aggregation occurs (Ratliff et al, 1994). Treatments are generally begun 2 to 4 weeks after tumor resection, allowing time for reepithelialization to minimize the potential for intravasation of live bacteria (Lamm, 1992). In the event of a traumatic catheterization, the treatment should be delayed for several days. After instillation, the patient should retain the solution for 2 hours. Some
clinicians have advocated the patient turn from side to side to bathe the entire urothelium, but there is no scientific support for this practice. Fluid, diuretic, and caffeine restriction has been recommended to limit dilution of the agent and to facilitate retention of the agent for 2 hours. Oral desmopressin, 200 g, given 1 hour before administration has also been suggested (Cliff et al, 2000). Patients are instructed to clean the toilet with bleach, although there is no demonstrable risk of close contact infection. BCG Treatment of Carcinoma in Situ (CIS) American urologists use BCG by a 2:1 margin compared with intravesical chemotherapy, whereas European urologists favor chemotherapy. BCG is approved for this indication by the U.S. Food and Drug Administration (FDA). The initial tumor-free response rate is as high as 80% (Brosman, 1982 ; DeJager et al, 1991 ; Lamm 1992a, 1992 Hudson and Herr, 1995). Approximately 50% of patients experience a durable response for a median period of 4 years. Over a 10-year period, approximately 30% of patients remain free of tumor progression or recurrence, so close follow-up is mandatory. The majority of these occur within the first 5 years (Herr et al, 1992 ). Herr (1989) reported progression in 19% of initial responders at 5 years but found the rate to be 95% in nonrespondersfindings confirmed by other investigators (Coplen et al, 1990; Harland et al, 1992). The American Urological Association (AUA) Guidelines Panel supported BCG as the preferred initial treatment option for CIS (Smith et al, 1999). BCG has gained a preeminent role in North America based on higher efficacy reports compared with intravesical chemotherapy. In over 600 patients, there was a 68% complete response rate to BCG and a 49% complete response rate to chemotherapy. In responders, 68% of patients treated with BCG remained disease-free as compared with 47% of patients receiving chemotherapy, based on a median follow-up of 3.75 years. The overall disease-free rates were 51% and 27%, respectively (Sylvester et al, 2005 ). BCG Treatment of Residual Tumor Intravesical BCG can effectively treat residual papillary lesions but should not be used as a substitute for surgical resection. Investigators have demonstrated a nearly 60% response by residual tumor with intravesical BCG alone (Brosman, 1982; Schellhammer et al, 1986 ; Coplen et al, 1990). Carcinoma of the mucosa or the superficial ducts of the prostate can be adequately treated by BCG with a 50% tumor-free rate. A limited TURP can be effective in decreasing tumor burden and facilitating exposure of the prostate surface to BCG administration. Tumor-free rates of 50% can be attained (Bretton et al, 1990 ).
BCG Prophylaxis to Prevent Recurrence Early single-center studies demonstrated an advantage in decreased tumor recurrence of approximately 30% when a 6-week course of BCG was administered after recovery from TURBT (Brosman, 1982 ; Morales et al, 1992). In several larger series, tumor recurrence after TURBT was reduced by 20% to 65%, for an average of approximately 40% (Pagano et al, 1991a, 1991b; Herr et al, 1992 ; Melekos et al, 1993 ; Krege et al, 1996 ). The efficacy of BCG after TURBT for high-risk papillary disease has been demonstrated in several series of T1 lesions, with recurrence rates of 16% to 40% and progression rates of 4.4% to 40%, a substantial improvement compared with TUR alone (Cookson and Sarosdy, 1992 ; Pansadoro et al, 1995 ; Herr, 1997 ; Jimenez-Cruz et al, 1997 ; Gohji et al, 1999 ; Hurle et al, 1999a, 1999). Tumor multiplicity and associated CIS were associated with increased risk of progression. Sub staging lesions based on the presence or absence of muscularis mucosae invasion in a series of 49 patients did not improve prediction of recurrence (69% vs. 65%) or progression (22% vs. 29%) after BCG therapy ( Kondylis et al, 2000 ). Vascular endothelial growth factor (VEGF) is a chemical signal produced by cells that stimulates the growth of new blood vessels. It is part of the system that restores the oxygen supply to tissues when blood circulation is inadequate.VEGF's normal function is to create new blood vessels during embryonic development, new blood vessels after injury, and new vessels (collateral circulation) to bypass blocked vessels. When VEGF is overexpressed, it can contribute to disease. Solid cancers cannot grow beyond a limited size without an adequate blood supply; cancers that can express VEGF are able to grow and metastasize. Overexpression of VEGF can cause vascular disease in the retina of the eye and other parts of the body. Drugs such as bevacizumab can inhibit VEGF and control or slow those diseases. VEGF is a sub-family of growth factors, specifically the platelet-derived growth factor family of cystine-knot growth factors. They are important signaling proteins involved in both vasculogenesis (the de novo formation of the embryonic circulatory system) and angiogenesis (the growth of blood vessels from pre-existing vasculature). The most important member is VEGF-A. Other members are Placenta growth factor (PlGF), VEGF-B, VEGF-C and VEGF-D. The latter ones were discovered later than VEGF-A, and, before their discovery, VEGF-A was called just VEGF. A number of VEGF-related proteins have also been discovered encoded by viruses (VEGF-E) and in the venom of some snakes (VEGF-F).
Mechanism of action of VEGF- All members of the VEGF family stimulate cellular responses by binding to tyrosine kinase receptors (the VEGFRs) on the cell surface, causing them to dimerize and become activated through transphosphorylation, although to different sites, times and extents. The VEGF receptors have an extracellular portion consisting of 7 immunoglobulin-like domains, a single transmembrane spanning region, and an intracellular portion containing a split tyrosine-kinase domain. VEGF-A binds to VEGFR-1 (Flt-1) and VEGFR-2 (KDR/Flk-1). VEGFR-2 appears to mediate almost all of the known cellular responses to VEGF. The function of VEGFR-1 is less well-defined, although it is thought to modulate VEGFR-2 signaling. Another function of VEGFR-1 may be to act as a dummy/decoy receptor, sequestering VEGF from VEGFR-2 binding (this appears to be particularly important during vasculogenesis in the embryo). VEGF-C and VEGF-D, but not VEGF-A, are ligands for a third receptor (VEGFR-3), which mediates lymphangiogenesis. VEGF production- VEGF production can be induced in cells that are not receiving enough oxygen. When a cell is deficient in oxygen, it produces HIF, hypoxia-inducible factor, a transcription factor. HIF stimulates the release of VEGF, among other functions (including modulation of erythropoiesis). Circulating VEGF then binds to VEGF Receptors on endothelial cells, triggering a Tyrosine Kinase Pathway leading to angiogenesis. Anti-VEGF therapies are important in the treatment of certain cancers and in age-related macular degeneration. They can involve monoclonal antibodies such as bevacizumab (Avastin), antibody derivatives such as ranibizumab (Lucentis), or orally-available small molecules that inhibit the tyrosine kinases stimulated by VEGF: sunitinib (Sutent), sorafenib (Nexavar), axitinib, and pazopanib. Recent few studies have shown some relations between VEGF and urinary bladder carcinoma. These studies have been trying to establish relationships between VEGF with urinary bladder for diagnostic and therapeutic advancements in bladder carcinoma as VEGF plays an important role in carcinogenesis of bladder carcinoma. lel,n deMM,lan ahbMM,n llehn ahn eM - , (2005) studied Correlation Between Tissue Andreleased VEGF levels in urine of bladder cancer patients. They concluded a consistent association between increased tissue levels of VEGF and its released levels in urine of urinary bladder carcinoma patients using EIA method strengthening the evidence that urinary VEGF levels may be valuable marker to monitor bladder carcinoma patients.
B. Voss et al, published an article on VEGF-m RNA in patients suffering from urinary bladder carcinoma, indicated no positive correlation between serum VEGF and staging of urinary bladder tumors, but since VEGF-m RNA is expressed in many tumor cells and VEGF protein occurs in high concentration in the serum, it could be a good target for an anti-tumor therapy. Wu et al. 2003 suggested that, in addition to its pro-angiogenic properties, recent in vitro experiments also suggest a role for VEGF signaling as an autocrine and paracrine growth factor to directly promote bladder cancer growth. Bernardini et al. 2001did a retrospective evaluation of serum VEGF levels in the metastatic setting suggests a correlation of high levels with poor disease-free survival. Inoue et al. 2002 studied that baseline VEGF mRNA expression levels and micro vessel density were found to be independent prognostic factors for recurrence and metastasis in 51 patients treated with neoadjuvant MVAC chemotherapy preceding cystectomy. In addition to its pro-angiogenic role, elevated levels of VEGF in tumors lead to abnormal microvasculature. Excessive angiogenic factors recruit endothelial and perivascular cells to form tortuous and dilated blood vessels with poor rheological characteristics,abnormal tumor blood flow and increased vascular permeability [Dvorak et al 1995]. These changes lead to increased interstitial fluid pressure, which impairs the delivery of chemotherapy to tumor cells due to a decrease in the pressure gradient [Gutmannet al. 1992; Boucher et al. 1991]. By reducing VEGF levels, the aberrant tumor-associated blood vessels are eliminated and the microvasculature also appears to be remodeled, leading to more normal blood vessel architecture. This leads to improved transvascular drug delivery directly to tumor cells, which has been demonstrated in other settings [Willett et al., 2004]. Recent evidence demonstrates that VEGFR2 is expressed in urothelial carcinoma and its level of expression correlates with pathologic stage [Wu et al. 2003, Xia et al. 2006]. Targeting VEGFR2 therefore has the potential to suppress both tumor cells and blood vessels.
C-erbB2 C-erb-2 oncoprotein is the product of the HER-2 (neu) oncogene which is over expressed in a variety of adenocarcinomas arising at various sites. Over expression of c-erb-2 is present in 15-30% of cases of breast carcinoma and has been demonstrated to be a negative prognostic indicator. In order to evaluate the reliability and reproducibility of the immunohistochemical demonstration of the c-erbB2 oncoprotein and define criteria for positivity assessment in paraffin-embedded material from different tumours, we examined immunohistochemically 79 gastric, 100 colorectal and 20 bladder carcinomas using 5 different c-erbB2 antibodies. To determine criteria for the evaluation of c-erbB2 positivity, the observed immunohistochemical staining was compared with the actual protein prevalence, determined by Western blot analysis of each tumour. The comparison of protein levels obtained from Western blot with corresponding immunohistochemistry revealed that positive staining can only be considered to be specific for c-erbB2 oncoprotein if: (i) there is a definite staining along the tumour cell membrane with no predominant immunoreactivity inside the tumour cell cytoplasm, (ii) the intensity of immunoreactivity is comparable with a positive standard (e.g. in this study breast carcinoma) and (iii) the surrounding normal, non-neoplastic tissue shows no immunoreactivity. The graduation of staining intensities did not correlate to the actual amount of protein detected in Western blots. This study stresses the importance of defined and controlled criteria for evaluation of c-erbB2 oncoprotein positivity in immunohistochemistry. C-erb-2 over expression can be detected by immunohistochemical staining of formalin-fixed paraffin-embedded sections of tumor tissue. Membrane staining of the cells is the only reaction considered positive. A positive therapeutic response to Herceptin (TM) therapy with anti-C-erb-2 monoclonal antibody has been reported in association with 3+ staining intensity.
REFERENCES 1. BCG immunotherapy of bladder cancer: 20 years on. 353. 1999. pp. 168994. 2. http://www.cancer.gov/cancertopics/pdq/treatment/bladder/Patient/page2. 3. Boucher, Y., Kirkwood, J.M., Opacic, D., Desantis, M. and Jain, R.K. (1991) Interstitial Hypertension in Superficial Metastatic Melanomas in Humans.Cancer Res 51: 6691_6694. 4. Boffetta P (2008). "Tobacco smoking and risk of bladder cancer". Scand J UrolNephrol Suppl42 (S218): 4554. 5. Bradford, M.M. 1976. Rapid and sensitive method of quantitation of microgram quantities of protien utilizing the principle of protien dye binding. Anal. Biochem. 722: 248-251. 6. Crew, j.p., t. OBrien, r. Bicknell, s. fuggle, d. Cranston and a. harris, 1999. Urinary vascular endothelial growth and its correlation with bladder recurrence rates. J. urol. 161: 799-804 7. Droller, M.J.,D 1998. Vascular endothelial growth factor is a predictor of relapse and progression in superficial bladder cancer. J.urol, 160: 1932-1936. 8. Dvorak, H.F., Brown, L.F., Detmar, M. and Dvorak, A.M. (1995) Vascular Permeability Factor/Vascular Endothelial Growth Factor, Microvascular Hyper-permeability, and Angiogenesis. Am J Pathol 146: 1029_1039. 9. "Frankincense oil derived from Boswelliacarteri induces tumor cell specific cytotoxicity.". www.ncbi.nlm.nih.gov. 10. Gutmann, R., Leunig, M., Feyh, J., Goetz, A.E., Messmer, K., Kastenbauer, E. et al. (1992) InterstitialHypertension in Head and Neck Tumors in Patients: Correlation with Tumor Size. Cancer Res 52:1993_1995. 11. Menhaswellam, abdullaahmedabd el-aal. Correlation between tissue and released VEGF levels in urine of bladder cancer patients. American Journal oIBiochemistry and Biotechnology ; issue 1; pp: 37-42; Vol: 1; Year: 2005. 12. Robert M. Hayward, Melissa J. Kirk, Mary Sproull, Tamalee Scott, Sharon Smith, TheresaCooley-Zgela, Nancy S. Crouse, Deborah E. Citrin, and Kevin Camphausen*. Post-collection, pre-measurement variables affecting VEGF levels in urine biospecimens. J Cell Mol Med. 2008 ; 12(1): 343350. 13. Sato,k.,r.sasaki, y. oruga, n.shimoda, h. togashi, k. terada, t. sugiyama, h. kakinuma, o. ogawa and t.kato,1998. expression of vascular endothelial growth factor gene and its receptor (flt1) gene in urinary bladder cancer. Tohoku j. exp. Med. 185: 173-184. 14. The chemistry of jaffes reaction for creatinine. Greenwald, I, biochem.J.1926, xx, 665. 15. Use of statins and outcome of BCG treatment for bladder cancer. 355. 2006. pp. 27057. 16. Wu, W., Shu, X., Hovsepyan, H., Mosteller, R.D. and Broek, D. (2003) Vegf Receptor Expression and Signaling in Human Bladder Tumors. Oncogene 22: 3361_3370.