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Original Article
Establishment of a Lung Cancer Biobank of a Southern Chinese Population
From Guangzhou Institute of Respiratory Disease, State Key Laboratory of Respiratory Disease, the First A ffiliated Hospital of Guangzhou Medical College,
Guangzhou 510120, Guangdong Province, China
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Abstract
There is an increasing need for establishment of biobanks for human cancer, which may facilitate basic and clinical researches as well as the
development of novel approaches to early diagnostics, prevention and treatment, including personalized medicine. Herein we report the establishment of
a lung cancer biobanks using biological samples from a lung cancer patient population in Southern China. Since 2007, we
have collected lung cancer tissue from 1,054 patients with lung cancer, from whom 11,895 frozen tumor and normal matched tissues, 4,899
tissue paraffin blocks, and 3,562 blood serum samples were accumulated. The information on clinical manifestations, laboratory tests, and
follow-up was maintained by an independent information management system, including three data sets. The primary data set included
frozen tissue specimens, formalin-fixed paraffin-embedded tissues, blood specimens and clinical and long-term follow-up data. The secondary data set
included DNA, RNA and proteins extracted from corresponding tissue specimens. And the tertiary data set contained improved genome, RNA and proteomics correlation analysis with relevant clinical and long-term follow-up data. The lung cancer biobanks is
accessible to academic research and public services. To our best knowledge, this biobanks represents the first lung cancer tissue reservoir in
China and should facilitate the basic and clinical research of this disease and developing diagnostic markers and novel therapeutic modalities.
Key words
lung cancer; biobanks; tissue bank; southern Chinese population
J Thorac Dis 2009;1:17-22. DOI: 10.3978/j.issn.2072-1439.2009.12.01.016
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Introduction
The incidence of lung cancer in 2007 is estimated to be 213,380
with 160,390 deaths in the United States. It will contribute to 31%
of male and 26% of female cancer-related deaths and is the largest
cause of cancer-related mortality in both men and women (1). According to
histopathologic and biological perspectives, lung cancer
is a highly complex neoplasm (2). More than 99% of lung cancers
are carcinomas. However, within that broad category exist several
histological subtypes, including small cell lung carcinoma (SCLC;
20% of lung cancers) and the non-small cell lung cancer (NSCLC)
subtypes squamous cell carcinoma (30% ); adenocarcinoma, including the noninvasive subtype
bronchioloalveolar carcinoma
(40%); and large cell carcinoma (9%) (3). The frequency of lung
cancer histological subtypes has shifted in the past decades: adenocarcinoma
has surpassed squamous cell carcinoma as the most
common type of lung cancer, and the incidence of SCLC is steadily
decreasing.
Advances in understanding the basic science of cancer are dependent upon, and to great extent limited by, the availability of
high quality appropriate tissue samples for research. Increasingly,
the introduction of new therapeutic agents is also dependent on
demonstrated efficacy in human tumour tissue or cell cultures. Recent acceleration in the availability of new technological platforms,
dubbed 'the omics revolution', also makes great demands on any
tissue available for research at the DNA, mRNA and expressed
protein levels (4). Development of a tissue and blood (serum, plasma,
and circulating cells) bank with specimens obtained from patients with lung cancer,
including detailed clinical data, is of the utmost importance. As we can achieve potential research goals (1): identify molecular tissue and blood (e.g., genetic polymorphism and
serum proteomic) markers predictive of survival, recurrence, and
metastasis development in patients with lung cancer; (2) establish
characteristics of precursor lesions and the field cancerization phenomenon in lung cancer pathogenesis by smoking status, gender,
and, very importantly, ethnic background; (3) identify molecular
tissue and blood markers to predict response to and survival benefit
from radiotherapy, chemotherapeutic or targeted therapeutic agents
at time of recurrence (5).
Different responses in the treatment of lung cancer are
attributable to racial differences (6). The large geographical span
between northern and southern China leads to large differences in
terms of climate and dietary habits. In the north of China where an
arid climate prevails, the diet is meat-based and the northern
Chinese population is closer to having an East Asian ethnic origin.
Whereas, in the south of China, the climate is humid encouraging
more vegetable-based diet, and the population mostly has South
Asian ethnic origins. The investigation of the hemorheological
reference value in healthy people of different cities of China
(Beijing, Qiqihaer, Yantai, Yibin and Guangzhou) indicated that
the discrepancy of hemorheological indices was different between
northern and southern China (7,8). On the other hand, in northern
China, air pollution caused by burning coal indoors and/or
outdoors during the long heating season may play an important role
in lung cancer etiology. Coal consumption in northern China is
much higher than southern China (9). So the establishment of lung
cancer biobanks in southern China will help researchers find
features and special therapeutic targets for lung cancer in the
southern Chinese population.
Based on these considerations, we tried to establish the Lung
Cancer Biobanks at Guangzhou Institute of Respiratory Disease
and State Key Laboratory of Respiratory Disease, Guangzhou, PR
China. The biological samples such as lung cancer tissues, control
tissues, and blood, as well as donor-related clinical data have been
collecting from a Southern Chinese Population. The major
activities and services include collecting and banking freshly
frozen tissue specimens from excess surgical material and biopsies,
collecting formalin-fixed paraffin-embedded tissue specimens,
processing and banking blood components and maintaining a tissue
database with links to clinical and follow-up data. The collections
are going on and will last for ten years. The independent
information management system is controlled by an independent
group. It was hoped that these data can guide the selection of
clinical treatment method and can be extremely helpful in judging
the prognosis of lung cancer patients.
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Materials and methods
Tissue origins
Tissues collected in our lung cancer biobank were mainly from
Southern Chinese patients with lung cancer who received treatment
in the form of surgery, chemotherapy and radiotherapy in
Guangzhou Institute of Respiratory Disease. At the same time,
samples from bunamiodyl, inflammatory pseudotumor and pulmonary fibrosis patients are also collected as controls of benign
diseases. The collection began in 2007 and mainly includes frozen
lung cancer tissue specimens (including paired nonneoplastic tissues), formalin-fixed paraffin-embedded tissue specimens (tissue
blocks) and blood specimens. A flowchart for the set-up a lung
cancer biobank is outlined in Fig 1.
Collection of frozen tissue specimens
Surgical specimens were immediately snap-frozen in liquid nitrogen in situ after resected in the operating room in order to minimize the action of hypoxic phenomena on genetic expression. One
pathologist in operating room decides whether it is possible to separate one or more fragments for freezing without this operation af
fecting the correct pathological diagnosis of the process (10).
Those areas with massive ischemia and/or necrotic phenomena must be avoided.
This entire process is carried out in the most aseptic conditions possible.
Whenever possible, nonneoplastic tissue of the same organ (adjacent tissue: 2-5 cm to the cancer tissue
edge and distant tissue: more than 5 cm to the cancer tissue) is selected for freezing and for fixation and paraffin embedding.
Samples are then frozen by direct immersion in liquid nitrogen.
Components of such specimens can be preserved well and widely applied in DNA, RNA and protein research, especially available to
RNA research demanding strict preservation conditions (11-13).
Figure. 1 Tissues were mainly from patients with lung cancer who received treatment in the form of surgery, chemotherapy and radio-therapy. The collection mainly includes frozen lung cancer tissue
specimens (including paired nonneoplastic tissues), formalin-fixed
paraffin -embedded tissue specimens
specimens.
Conservation of frozen tissues and security systems
The blocks of frozen neoplastic and non-neoplastic tissue are
properly identified so that they are not used for diagnosis, except
when this is absolutely necessary. Tissues are stored at temperatures below -196°C in liquid nitrogen tank with the necessary security measures to avoid thawing and/or exposure to excessive
changes in temperature. They include: connection to the hospital's
emergency power supply, triple alarm system (local visual and acoustic, remote to hospital personnel, telephone to tumor bank
members, etc.) and keeping a nearby empty freezer (14-16).
Formalin -fixed paraffin -embedded tissue specimens (tissue blocks)
Formalin-fixed paraffin-embedded tissues are from patients who
received operation or biopsy. Corresponding paraffin blocks, glass
slides and immunostains are collected and researchers can request
unstained sections, H&E-stained sections, or thick slices for
RNA/DNA retrieval from paraffin blocks through the biobanks
(17).
Collection of blood samples
Blood samples were collected from patients who underwent surgical operations and/or chemotherapies in Guangzhou Institute of
Respiratory Disease. Blood from each patient before and after
surgery and/or chemotherapy is centrifuged after which separated
plasma/serum and blood cells are collected to be preserved in liquid nitrogen. These blood specimens are later provided for disease
tracking and observation of drug efficacy (18).
Identification of samples
Samples were organized by designated people and each sample
to be processed by the biobanks is immediately identified in accordance with the tissue identification protocol, with clear reference to
the tissue type (normal or pathological).
Clinically relevant material and long-term follow-up data
Ethical aspects
All neoplastic tissues accepted by the biobanks in particular
biopsies, surgical specimens, and necropsy samples, absolutely respecting the procedures that guarantee correct ethical activity.
Data security
Specific computers were used as databases containing personal
information which permits the identification of the patient (name,
gender, age, clinical record number, pathology reference number,
member of staff in charge of the case, sample number and amount
of the samples). The collections and activity of the biobanks are
run and controlled by a specific group of people (21-23).
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Results
Specimen constituents of the biobanks
The collection is going on and will last for ten years. From January 2007 to May 2009 total cancer cases have reached 1,054 and
total sample copies have reached 20,356 (11,895 frozen tissues,
4899 tissue blocks, and 3562 blood specimens).
Constituent ratio of gender
Of the 1054 cancer cases, 821 (77.9%) were from male patients
and 233 (22.1%) were from females (Fig 2A). Constituent ratio of
gender was 3.52:1 male vs female patients.
Constituent ratio of age
The youngest patient was 14 years old and the oldest one was
89 years old. Age distribution for the lung cancer biobanks showed
a normal distribution pattern. Tumor specimens collected from patients aged 41 to 70 years accounted for 80.4% [(223+351+274)
/1054=80.4%] of the total number of cases (Fig. 2B).
Constituent ratio of lung cancer and benign lesions
We also found that about 40% of the cases were malignant tumors and 60% were benign lesions based on pathological examination. The constituent ratio of lung cancer to benign lesions was 0.66:1. (Fig. 2C).
Constituent ratio of tumor types
Lung adenocarcinoma and lung squamous cell carcinoma accounted for the majority
of the total surgical specimens: adenocarcinoma specimens represented 57.19% of the total number of
surgical resection specimens, followed by squamous cell carcinoma,
25.31% . Small cell lung cancer often loses the opportunity to
surgery because of early transfer and is often treated by radiotherapy and chemotherapy. Therefore Small cell lung cancer shares a
smaller proportion of surgical specimens and mostly just blood
specimens are collected for this type of cancer. Large cell carcinoma specimens represent 0.57% of surgical resection specimens
Others included Sarcoma, Carcinosarcoma and Lymphoepithelioma-like carcinoma and cumulatively represent 8.54% of surgical
resection specimens (Fig. 2D).
Lung cancer family data
Three lung cancer families were found during the process of
collection. Blood specimens have been collected from each lung
cancer patient and the immediate family members in the family. At
least 1-2 patients in each lung cancer family received surgery in
Guangzhou Institute of Respiratory Disease and frozen tissue specimens and formalin-fixed paraffin-embedded tissues were also acquired from them.
Database management
A relatively independent information management system has
been set up and controlled by a specific group. Frozen tissue specimens, formalin-fixed paraffin-embedded tissues, blood specimens
and clinically relevant material and long-term follow-up data construct the first grade data. Serial detection of DNA, RNA and
protein of the first grade data construct the second grade data. Improved genome, RNA and proteomics correlation analysis with
clinically relevant material and long-term follow-up data construct
the third grade data. These data can guide the selection of clinical
treatment method and can be extremely helpful in judging the
prognosis of lung cancer patients.
Service of the lung cancer biobanks
The goal of the lung cancer biobanks was to facilitate biomedical research by using tissues collected
from Southern Chinese patient populations. The following services are currently offered to
support this goal: (a) Banked frozen tissue specimens: DNA, RNA
and protein research group especially available to RNA research
demanding strict preservation conditions; (b) Formalin-fixed paraffin-embedded tissue: DNA and morphological study; (c) Banked
frozen serum/plasma specimen: DNA, RNA and protein research group especially available to RNA research demanding strict
preservation conditions.
Figure. 2
A. Constituent ratio of gender was 3.52:1 to male vs female patients;
B. The youngest patient was 14 years old and the oldest was 89 years old.
Age distribution for the lung cancer biobanks showed a normal distribution pattern. Tumor specimens collected from patients aged 41 to 70 years accounted for 80.4% total case number;
C. About 40% of cases were malignant tumors and 60% cases were benign lesions based on pathological examination and the Constituent ratio
of lung cancer to benign lesions was 0.66:1;
D. Lung adenocarcinoma and lung squamous cell carcinoma accounted for the majority of the total surgical specimens: adenocarcinoma specimens representing 57.19% of the total number of surgical resection specimens of followed by squamous cell carcinoma, 25.31%.
Small cell lung cancer often loses the opportunity to surgery because of early transfer and is often treated by radiotherapy and chemotherapy.
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Discussion
Research biobanks are organizations that collect and store tissue
samples for use in scientific research (12,15). However, there were
only formalin-fixed paraffin-embedded tissues in department of
pathology, which can't meet the requirement of current research.
Most RNA and protein studies require samples to be preserved in
profound hypothermia. In some countries such as the United
States, Italy, etc., the establishment of cryopreserved tissue sample
banks is progressing (12,15,16). Establishment of such a bank is
useful since ethnic and geographical differences lead to differences
in gene expression profiles, protein profiling and response to treatment.
As it is well known, Eastern and Western races are correlated with different responses to the treatment of lung cancer in several studies (24).
In order to be able to carry out research in cooperation with other biobanks such as banks in north China and the US, the lung cancer biobanks of Southern China set up by Guangzhou Institute of
Respiratory Disease and State Key Laboratory of Respiratory Disease in accordance with the international standards. Tissues were
quick frozen in liquid nitrogen, blood samples were collected during every therapy period (before and after surgery and chemotherapy) and paraffin specimens were documented for indispensable
morphological study. In medical tissue archives, samples are usually not directly associated with clinical data, which substantially
limits their usability for research. Samples in the tissue bank with
corresponding data from medical records greatly enhance their value for scientific use. To solve this problem, our biobanks contain
biological samples as tissues and blood, and donor-related clinical
data. The advantages of clinically relevant information and
long-term follow-up data are clear. By studying samples collected
before and after surgery or chemotherapy and with the availability
of clinical and follow-up data, we can track the complete course of
these treatments and their effects. Also, joint research of different
biobanks on patients' treatment response can help identify different
therapeutic targets.
Serving its original purpose of providing services for researchers in Oncology, our lung cancer biobank has been available
for use in various types of research since its establishment: South
Chinese proteomics research; South Chinese micro-RNA research;
South Chinese EGFR Mutation and amplification research; Smoking-related study of Ca2+ channel; etc. The quality of preservation
of specimens was in line with the requirements of RNA by electrophoresis, detection of miRNA expression profiling, as well as
proteomics experiment. (Fig 3)
Figure.3
A. Good-quality RNA by electrophoresis as evidenced by the 18S and 28S bands is seen in all specimens;
B. miRNA expression profiling,
qRT -PCR results showed that the
quality of preservation of specimens
was in line with the requirements of
detection of miRNA expression profiling;
C. 17cm -long SDS -PAGE gel two-way electrophoresis of lung adenocarcinoma, with 1312±36 protein spots, the quality of preservation of specimens was in line with the requirements of proteomics experiments;
D. 17cm -long SDS -PAGE gel two -way electrophoresis of adjacent tissues,with 1186±28 protein spots.
Collect these samples and clinical and follow-up data just the
beginning for our biobanks. In order to provide more service to
clinical and basic cancer research of lung cancer, we will continue
to expand the resource base of the specimen collection. We will also establish lung cancer biobanks of different regions, different nationalities and different tumor types. With the development of
molecular biology, tumor target therapy will make a good progress
and more individuals will benefit from it. We also look forward to
establishing databases of patients with relapse or of those who
failed to respond to treatment, which can provide researchers with
new information on treatment and can help explain the mechanisms of drug-resistance.
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Funding
This work is supported by China Natural Postdoctor Foundation (20080440742), Government Technology Agency Foundation
of Guangdong Province (2007B031515017) and Government
Technology Agency Foundation of Guangzhou City
(2007Z1-E0111and 2060402).
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Acknowledgments
The authors would like to express their gratitude to Dr. Zhidong
Xu of university of California, San Francisco for his excellent
help in providing advice on the establishment of the lung cancer
biobanks.
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References
Cite this article as: Li HL, Qiu Y, Zhang X, Xu X, Liao DJ, Wang W, Wen DP, Dong QH, Xian HF, Wang DY, Zhong NS, He JX. Palliative Hypofractionated Radiotherapy For Non-Small Cell Lung Cancer(NSCLC) Patients Previously Treated By Induction Chemotherapy: Is It ForMany, Some, All, Or None? J Thorac Dis 2009;1:17-22. doi: 10.3978/j.issn.2072-1439.2009.12.01.016
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