Comparative Genomic Hybridization(CGH) of Granulosa Cell Tumor (GCT)
Experiment Report by Siegfried Hong
Summary
Objective. I learn the basic knowledge of pathology of granulosa cell tumor and how to make
a CGH process.
Methods. I use CGH to detect the patient’s DNA to see whether it gains or loses.
Result. The patient’s DNA gains 14q, 12q; loses 22q.
Introduction
We can divide ovarian cancer roughly into three kinds: epithelial tumor, germ cell tumor,
and sex cord stromal tumor. Granulosa cell tumor belongs to sex cord stromal tumor. It is
separated into adult type (AGCT; abbreviated from adult granulosa cell tumor) for 95% of all
sex cord stromal tumor and juvenile type (JGCT; abbreviated from juvenile granulosa cell
tumor) for 5% of all sex cord stromal tumor. AGCT usually happens in postmenopausal
women, and with less and late recurrence. JGCT usually happening in women younger than
30 years old and often recurring in three years is more malignant than AGCT.
Generally speaking, most granulosa cell tumor is often low malignat potencial. This is
because GCT causes endocrine manifestation (e.g. high estrogen level makes endometrial
hyperplasia, endometrial carcinoma, breast cancer, etc.), it can be diagnosed before the tumor
cell metastases to other organs outside the ovary. There are no difference in the incidence rate
between different race.
Because GCT grows in the ovary, it would cause symptoms in the abdomen and pelvis (e.g.
enlarged abdomen, palpable mass, abdomen discomfort, abdomen pain, dysuria, urinary
frequency, constipation). If the GCT ruptures or is partially cystic or hemorrhage, it would
cause acute abdominal pain, nausea, vomiting, dizziness, shoulder pain. Other manifestations
include: (1) in prepubetal girl: precocious pseudopuberty, second male sexal character
because of the secretion of testosterone by GCT. (2) in premenopausal women: secondary
amenorrhea, oligomenorrhea, menorrhagia. (3) postmenopausal women: abnormal
endometrial bleeding because of endometrial hyperplasia, breast tenderness, increasing
vaginal secretion, secondary male sex character.
The secretion of GCT is estradiol, inhibin, and MIS (abbreviated from Müllerian-Inhibiting
Substance). MIS is a kind of antimüllerian hormone (AMH), which can be used for judging if
tumor recurs. MIS is more specific than estradiol and inhibin because MIS only occurs by
tumors originated from ovary.
In gross, GCT is often round to ovoid mass around 2~50 cm in diameter, may be solid or
multicystic. In microscope, AGCT can be divided into well-differenciated group and less
well-differenciated group. There are several pattern in well-differenciated group, including
microfollicular pattern, macrofollicular pattern, trabecular pattern, and insular pattern. The
most common pattern is microfollicular pattern with Call-Exner body. Less welldifferenciated group includes diffuse and watered-silk (moiré) or gyriform pattern. The
nuclear appearance is the same in well-differenciated group and less well-differenciated
group: coffee-bean appearance. Because GCT is low malignant potencial, mitotic figures is
often few and with mild nuclear atypia. The microscopic morphology of JGCT is different
from AGCT. It consists of round hyperchromatic nuclei without coffee-bean appearance of
nuclear. There are much more mitotic figures and nuclear atypia in JGCT. It appears that
JGCT is more malignant than AGCT.
The staging system for surgery of ovarian carcinoma is based on 1987 International
Federation of Gynecology and Obstetrics nomenclature. This system divides ovarian
carcinoma into four stages according to the site of the tumor. Stage І, stage П, and stage Ш
can be separated into a, b, and c individually.
Stage І: Tumor is confined to the ovaries.
Stage П: Tumor involves one or both ovaries, with pelvic extension.
Stage Ш: Tumor involves one or both ovaries, with peritoneal implants outsideof the pelvies
and/or positive retroperitoneal lymph nodes. Superficial liver metestases are also included in
this stage.
Stage ІV: Distant metastases are present.
The primary treatment of GCT is surgery, accompanied with chemotherapy and/or
radiotherapy. Chemotherapy and radiotherapy are especially for those patients who have
higher stage or recurrence disease. After therapy, patients should be long term followed up.
Comparative genomic hybridization (CGH) is a powerful molecular cytogenetic tool for
detecting chromosome imbalances from the whole genome screening of the test sample. It can
mark chromosome subregions with gain or losing. I try to use this method to detect the
chromosome imbalances of granulosa cell tumor specimen.
Materials and Methods
The number of the patient’s specimen is 01-P00847.
The Protocol for First Day
1. Tube A: Add 6 μl patient’s DNA with 17 μl buffer and 77 μl water into tube A.
2. Tube B: Add 3.4 μl normal DNA with 17 μl buffer and 79.6 water into tube B for
reference.
3. Add 1 μl dTTP into both tube A and tube B.
4. Add 2 μl dATP, dCTP, dGTP mix solution into both tube A and tube B.
5. Add 1 μl biotin-16-dUTP into tube A.
6. Add 1 μl digoxigenin-11-dUTP into tube B.
7. Add 2 μl DNA polymerase І into both tube A and tube B.
8. Add 1 μl TRIS into both tube A and tube B.
9. Mix tube A and tube B for few moment.
10. Centrifuge tube A and tube B at 15°C. If the DNA comes from paraffin-embedded
tissue, centrifuge this DNA for 50 minutes. If the DNA comes from fresh tissue,
centrifuge this DNA for 60 minutes. This process can decrease the activity of
DNAase.
11. During centrifuge, make gel which is prepared for electrophoresis.
12. After centrifuge, take 5 μl DNA solution out from tube A. Add 3 μl loading buffer
(EDTA) into the solution taken out.
13. Take 1.5 μl pUC19 DNA with 3 μl loading buffer and 5 μl water as marker for gel
electrophoresis.
14. Take 5 μl DNA solution out from tube B. Add 3 μl loading buffer into the solution
taken out.
15. Electrophoresis for 45 minutes. After electrophoresis, take a photo to see if we slow
down the activity of DNAase. The best length for hybridization is between 500~1000
kb.
The Protocol for Second Day
1. Prepare fresh ethanol solution series (70%, 85%, 100%) one day before and store them
at -20°C.
2. Prepare the slides that have metaphase chromosome on them. Mark the areas of slides
with good metaphase chromosome.
3. Pepsin treatment: Add 70 ml water into the coplin jar with 20 μl pepsin, then add 700
μl 1N HCl. Put the slides in for 5 minutes. Then, wash the slides in 100 ml 2×SSC for
5 minutes. Dehydrate the slides in -20°C 70%, 85%, 100% alcohol for 5 minutes
separately in series.
4. Mix 67 μl nicktranslated tumor DNA with 67 μl nicktranslated reference DNA and 80
μl Cot-1 DNA in a tube. Then centrifuge this tube for a short moment at 4 °C.
5. Add 12 μl CH3COONa (pH 4.8, 3 M) and 570 μl 100% ethanol into the tube. Mix it
and then put this tube in the refrigerator for 1 hour at -80°C.
6. Take the tube from the -80°C refrigerator out. Centrifuge it for 30 minutes in 4°C. We
can see after centrifugation there are two layers in the tube. The upper layer is alcohol;
the lower one is DNA. Take the alcohol out from the tube and throw it away. Be
careful do not suck the DNA in the bottom of the tube.
7. Add 800 μl alcohol into the tube for washing the DNA. Mix them and then centrifuge
the tube in 45°C for 1300 rpm, 30 minutes.
8. Take ethanol away from the tube. air dry the tube for 30 minutes.
9. Prepare the DNA denaturation solution. Mix 5 ml 20×SSC, 5 ml NaH2PO4, 5 ml
water, and 35 ml deionized formamide together. Make sure the pH of the solution is at
7.
10. Put the coplin jar of denaturation solution into water bath. The temperature of the
water bath is 71.5°C (in order to keep the temperature of denaturation solution inside
the coplin jar at 69°C).
11. Critical steps: Put the slide with metaphase chromosome into the denaturation solution
for exactly 2 minutes. Then, put the slide into 70%, 85%, 100% alcohol in series for 5
minutes separately. Then, air dry the slide.
12. Add 6 μl formamide to the tube with tumor DNA to make DNA denature. Mix them
and vortex the tube for 1 or 2 hour at 37° C.
13. Add 7 μl hybridization buffer (There are 30% dextrose and fat in the hybridization
buffer.) into the tube with tumor DNA.
14. Vortex this tube at 300 rpm, 78°C for 6 minutes.
15. Then, vortex this tube at 1300 rpm, 37°C for 20 minutes. But this step
(prehybridization) can be ignored.
16. Take the DNA out of the tube and put it onto the slide. Cover with glass and fix with
fixogum. Hybridize for 3 days at 37°C.
The Protocol after Hybridization
1. prepare solution A, B, C, and blocking solution (There are bovine serum albumin in it
in order to make antibodies more specific.) and detecting solution.
2. Put solution A, B, C in water bath at 43°C.
3. Put the slide into solution B for 2 minutes to take off the fixogum and glass.
4. Put the slide into solution A for 5 minutes for 3 times.
5. Put the slide into solution B for 5 minutes for 3 times.
6. Put the slide into blocking solution for 30 minutes.
7. Prepare antibodies in the dark.
8. Shake antibodies for 10 minutes at 1000 rpm, 37°C.
9. Centrifuge antibodies for 4 minutes at 8000 rpm.
10. Use solution C to wash the slide. Then, add antibodies onto the slide in the dark. Put
the slide at 37°C for 1 hour.
11. Wash the slide in solution C for 3 times, 3 minutes each time at 43°C.
12. Put DAPI onto the slide and then cover the slide with a slip. Store it at 4°C.
13. Take images.
Result
The result is that the tumor DNA has gained chromosome 14q, 12q, and loss chromosome
22q.
Discussion
This is the first time I go the laboratory to do experiment. I have never been done any
experiment before. I found that it takes patience and carefulness in every step very much to
finish an experiment. I also learn how to analyze the image after the experiment. To separate
the chromosome needs patience and experience. It is really tired to sit before the computer in
the dark and watch it for a long time. Much thanks to Dr. Schulten and technicians Ina and
Christina, they always answer my stupid questions with detail. And also Dr. Gunawan, thanks
for him to give me a short but necessary lecture. I am glad that my result is just as similar as
they did before. That means I did not make any troubles in the protocol.
Besides comparative genomic experiments, thanks Prof. Füzesi for taking me to join his
lecture class. In fact, my image class will begin from this semester, so I feel very difficult to
understand although a kind and beautiful student called Vera translated Deutsch into English
in class for me. But I still paid attention to the class, tried to catch the key point. I was very
surprised that almost the whole class processed in Prof. Füzesi’s questions continuously. In
the pathology class in my school, students just can observe the lesion from the slides with
microscope. Here, students can also observe it from gross specimen. This is quite different
from the class in Taiwan, but I like this kind of teaching here much more.
Reference
1. C. M. Michener. Granulosa-Thecal cell tumor.
http://www.emedicine.com/med/topic928.htm
2. Yue-Shan Lin, Hock-Liew Eng. Molecular cytogenetics of ovarian granulosa cell
tumors by comparative genomic hybridization. Gynecologic Oncology 97 (2005) 6873.
3. Dr. Hans-Jürgen Schulten, Ina Duckmann, Christina Enders