So far there are 500 patients included in this study. Again we will test DNA-ploidy and nucleotyping as diagnostic/predictive value. In addition we will make a TMA using all of the samples in the study.
We work on four studies with totally 1400 patients included.
1. Cancer in IBD patients. 2. Sporadic colon cancer 3. Dukes B colon cancer, Quasar study - Leeds, England 4. Molecular classification of cancer in colon and rectum. Prognostic and predictive value of new biomarkers.
Histological subtype: Serous 66, mucinous 80, endometroid 58, clear celled 69, mixed 10, unclassified 20 and small celled 2.
Differentiation: Well 131; moderate 47; poor 58 and not graded 69.
Conclusions DNA ploidy by high-resolution image cytometry is the best predictor of outcome for patients with early stage carcinomas of the ovary, as compared to FIGO staging, histologic subtype, age, dense adhesions and differensiation. High-resolution image cytometry is superior to flow cytometry in prediction of outcome for patients with early stage carcinomas of the ovary.
FCM \ ICM
Diploid
Tetraploid
Polyploid
Aneuploid
Sum
Diploid
85
29
4
12
130
Tetraploid
12
4
15
31
Polyploid
1
1
Aneuploid
9
4
1
54
68
Sum
94
45
10
81
230
Table 1. Concordance between DNA ploidy classification performed by image and flow cytometry in 230 of the cases of early ovarian carcinoma.
Germ cell tumors of the ovary arises from the germ cells. They are infrequent, accounting for less the 5 % of ovarian malignancies. They are heterogenous, and the most common types are dysgerminomas, endodermal sinus tumor, and immature teratomas. Choriocarcinomas, embryonal carcinomas and gonadoblastomas are also described. Generally good prognosis with >85 % 5-year survival. Average age at diagnosis about 25
Material
Retrospective study
Paraffin embedded tissue sections
49 cases of malignant ovarian germ cell tumors
20 dysgerminomas
17 immature teratomas
10 endodermal sinus tumor
1 embryonal carcinoma
1 chorio carcinoma
A subgroup of 25 cases from this material are also included in a planned study on comparison of DNA ploidy and number of chromosomal copy numbers performed by comparative genomic hybridization
FIGO stage I: 33, II: 6, III: 7 and IV: 3 Follow up > 10 years
This male organ is built up of smooth muscles and glandular tissue, and has the size of a 20-gram walnut. It is situated around the urethra, at the basis of the bladder. The function of the prostate is to produce liquids that help the sperm cells to survive (citrate and prostatic acid phosphates- PAP) and to make the semen more viscous (a glycoprotein called PSA).
The size of the prostate usually increases after the man has reached the age of 45, and it can be very large in older men (almost 60 grams). In benign prostate hyperplasia, the urethra is compressed and this leads to urination problems. Size and number of ducts and glands increases, and so do the size of the supporting tissues. In prostate cancer (prostate adenocarcinoma), the disease is difficult to discover because the tumour arise in the peripheral glands of the prostate.
We are and have been doing research on tissues of very different kind. Some projects we cooperate with other groups with, some projects we do ourselves.
Some of the projects on these materials have not yet been ended, so there are no articles published. Methods we have been using are ploidy analysis, texture analysis and immunohistochemistry methods.
The methods are based on computer assisted image analysis, where we transfer images of cell and tissue samples from microscope (light, laser scan and electron microscopy) to the computer. In the computer we can digitalise the images and analyse the heritage materials structure and organisation bit by bi. These kinds of examinations of interphase nuclei are called nucleotyping.
Nucleotyping are among other an objective measure of nuclei atypi, one of the most important parameters for pathological diagnostics and prognosis. A qualitative analysis of chomatin structure is measured simultaneously with a measure of total amount of chromatin or DNA (depending on staining method) in nuclei. The method give information on the DNA ploidy distribution in addition to traditional nuclei morphometry, and it is sensitive to larger chormosomal aberrations. Of even greater importance is the methods ability to map and quantify functional changes in DNA organisation, that may be induced of larger or smaller mutations. Such changes is to al large ectent subvisual, and will not be discovered using ordinary microscopy. Nucletyping can be described as interphase cytogenetics, that is an interphase variant of chomosomal analysis where we map and describe organisatory and functionally domenes of DNA.
Nucleotyping has a great potential as diagnostic and prognostic/predictive marker in cancer. In opposite to DNA ploidy this method has also a predictive value in cancer at advanced stages.
Nucleotyping is a main priority area at Imaging programme, and we have a number of large studies going on prostate cancer, breast cancer (DCIS and Stage I) and colon cancer, both at The Norwegian Radium Hospital and at institutions in England.
We have performed analysis of DNA ploidy with two different methods: Image cytometry and flow cytometry.
IMAGE CYTOMETRY
Equipment required for measuring DNA ploidy using image cytometry method.
Image cytometry is based on the feulgen tequnique, which is a frequently used staining procedure in biology. The technique is based on Schiffs or Schiffs-similar binding to released aldehyde groups from DNA molecules after hydrolysis by HCl, and thereby enables staining of DNA in situ. The intensity of staining is proportional with the DNA concentration and the amount of DNA in the nucleus is expressed as amount of light absorbed by the feulgen-stain over the entire nucleus. The feulgen reaction is today in use to quantify the DNA ploidy distribution in tumor nuclei.
(The image of each nucleus is transferred from a microscope to a computer through a digital camera. The computer measures the amount of absorbed light as an expression of the amount of DNA.)
FLOW CYTOMETRY
Principles for the method in brief: A DNA specific fluorocrome binds to DNA. Nuclei is going by a liquid stream, through a optical measuring point. A laser beam splits of photons and the emmited light is measured and thereby expresses the amount of DNA in the tissue sample.
Preparation of samples (in brief) from paraffin embedded tissue :
Flow cytometry
Image cytometry
Sectioning (100mm)
Sectioning (50mm)
Deparaffination
Deparaffination
Enzyme treatment (pepsine, RNase)
Enzyme treatment (protease)
Cytospin centrifugation (monolayer)
Staining with ethidium bromide
Staining with feulgen-schiff
Measurements
Measurements
Advantages and disadvantages of flow cytometry and image cytometry:
Flow cytometry
Image cytometry
Advantages
Fast Abuntant nuclei Nuclei may be sorted
Morphological control No background noise Unlimited number of features may be evaluated
Immunhistochemistry is a powerful technique that allows a researcher to detect molecules within a tissue microenvironment. Protein expression can be studied in relation to tissue or cell compartment, and changes due to either normal or pathologic stimuli can be evaluated. Either direct or indirect methods can be used to detect an antibody bound to an antigen. The direct method uses a labeled reagent, such as a fluorescent tag or an enzyme-linked secondary antibody that binds to the primary antibody. A biotinylated secondary antibody can be used for better sensitivity allowing a tertiary step such as an enzyme-linked avidin or streptavidin to react. While the indirect method involves more assay steps, the amplification process at each step leads to increased sensitivity.
General procedure · Mount formalin fixed paraffin embedded sections (4 micron) onto silane-coated slides. · Air dry slides at 37ºC for 24 h. · Deparaffinize and rehydrate. · Incubate specimens for 20 minutes in 1-3 drops of serum block. Aspirate serum from slides. · Immediately add 1-3 drops of pre-diluted primary antibody. · Incubate for 2 hours. Rinse with PBS then wash in PBS twice for 2 minutes each on a stir plate. Aspirate excess liquid from slides. · Incubate for 30 minutes in 1-3 drops of biotinylated secondary antibody. Wash as above. · Incubate for 30 minutes in 1-3 drops of HRP-streptavidin complex. Wash as above. · Add 1-3 drops HRP substrate mixture. Develop for 30 seconds-10 minutes, or until desired stain intensity develops. Rinse with deionized H2O and transfer to a deionized H2O wash for 2 minutes on a stir plate. · Counterstain, dehydrate and mount slides Negative controls underwent similar staining procedure, but with the exclusion of the primary antibody. Positive controls were carcinomas that were shown to be immunoreactive for the studied antigens in preliminary studies.
Interpretation of staining results
The presence of immunostaining in carcinoma cells was evaluated by two independent researchers without knowledge of the clinical outcome. The presence and extent of staining was evaluated, using the following scale: 0, no staining; 1, staining of 1-5 % of tumor cells; 2, staining of 6-25 % of tumor cells; 3, staining of 26-50 % of tumor cells and 4, staining of > 50 % of tumor cells. Staining of both cell membrane and cytoplasm was interpreted as positive.
Background information - so that you understad more of our focus....
CANCER CAN BE VIEWED AS A GENETIC DISEASE
Tumour cells dont usually start out bad. They generally acquire their malignant properties over years or decades, going from benign to invasive to metastatic to lethal, in a process known as tumour progression (Nowell, P.C., Science 194, 23-28, 1976). This process occurs through a series of mutations and chromosomal aberrations that result in the accumulation of aggressive characteristics by the tumour cells (Hanan, D. & Weinberg, R.A., Cell 100,57-70, 2000). Cancer may therefore be viewed as a genetic disease in the sense that genetic (mutations) and epigenetic (organisational, methylations etc.) changes in cells are probable causes of both initiation and progression of cancer.
Findings of single event changes are extremely rare in cancer. In almost all studies, a multitude of genetic changes are observed.
CCIS Study Ductal Carcinoma in situ represents certain challenges in diagnostics, because around 20 % develop into invasive cancer and there are no certain markers to identify these. We have analysed a number of samples using both DNA ploidy and nucleotyping.
Mammae cancer, stadium I In this case we have a total of 480 patients with a defined prognosis (good prognosis: no relapse > 7 years after treatment. Bad prognosis: relapse or death of cancer within 4 years). The study is a pilot for DNA ploidy and nucleotyping as a prognostic marker in breast cancer. There are also planned to do a comparison of alternative methods for DNA ploidy measurements (FCM, manual ICM, automated ICM) and S-phase calculations.
