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Multicentric study coordinated by THE SPANISH ASSOCIATION OF SURGEONS
INTRODUCTION AND OBJECTIVES
Colorectal cancer (CRC) is a leading health
problem being the most common cause of death in men after bronchial
carcinoma, and in women is surpassed only by breast cancer (1).
In Spain, it is considered as a standard risk as in western countries
with the appearance of 15,000 new cases during 1994.
One of the reasons why the pathogeny of CRC
is of primary interest results from the complex interaction between
environmental and genetic factors. Despite many misunderstood
aspects of inherited factors of CRC , a group of patients in whom
inheritance plays an important role can nowadays be defined. Thus,
taking hereditary aspects into account, CRC is classified, into
the following groups: 1) sporadic colon cancer with no genetic
association; 2) inherited syndrome of polyps and cancer; 3) hereditary
nonpolyposis colorectal cancer and 4) family adenomatous polyposis.
Family adenomatous polyposis (FAP) is a syndrome
transmitted with autosomal dominant character occurring with the
appearance of multiple polyps which in the course of the disease
are bound to degenerate into a CRC, unless a prophylactic cholecistectomy
is performed. The APC gene accounting for FAP has been located
in the 5q21-22 region by deletions found in this region in certain
patients. The ultimate cause of the syndrome is the loss of the
APC gene or its mutation, circumstances which are unnoticed at
the cytological level so that the ADN of the patient must be studied
to assess the probable loss of heterocigosity (LOH); that is,
if a copy of the gene has been lost. The finding of this gene
has involvement ranging from genetic counselling, prenatal diagnosis
and follow-up of asymptomatic individuals.
At present, a rather large number of CRC
patients is recognized, which seems sporadic and has increased
susceptibility to developing adenocarcinoma sequence. This supposed
gene is transmitted from one generation to another in an autosomal
dominant manner although with low penetration so that in this
inherited syndrome of polyps and cancer patients may have or not
first-degree relatives with this lesion.
The syndrome of hereditary nonpolyposis colorectal
cancer (HNPCC) or Lynch's syndrome is characterized by: 1) age
of onset below the diagnosis median in the general population,
usually below age 45 years old. 2) the tumour being usually located
in the upper half of the colon, i.e., from the caecum, to the
splenic flexure; 3) increased occurrence of primary multiples
and 4) vertical transmission and twin aggregation following the
dominant autosomic pattern.
Although its frequency may be variable, Lynch's
syndrome is estimated to occur from 2% to 5% (in some populations
even 10-15%) of all the CRC, i.e., one from 20 to 30 sporadic
cancers (4). This accounts for about 300 new cases every year
in Spain and several thousands all over the world. If the incidence
is so high, the HNPCC may be the most common inherited disease
in western society. The International Collaborative Group on HNPCC
has established a series of minimal diagnostic criteria (5) as
follows:
At least three relatives should have been histologically diagnosed as CRC with no adenomatous polyposis, two of whom should have a first-degree relative.
Two consecutive generations should be involved.
One of the patients should be under 50 years when diagnosed of CRC.
The building of the genealogical tree is essential
for HNPCC diagnosis and is, at present, the only possible approach.
Another interesting point is the increased frequency rate in these
patients of tumors involving other organs: endometrium, stomach,
kidney, skin, urothelium, liver, ovary, larynx and pancreas. Due
to the increased prevalence of endometrium cancer, Lynch classified
families having colon and endometrium cancer in the subgroup II
of its syndrome. Nevertheless, it is now known that the appearance
of other tumours in any of the locations mentioned above, is common,
above all when family trees are built extending to second and
third-degree relatives. In fact, in 30 Finnish families with HNPCC,
cases of tumours, other than colon tumors, were detected in all
of them (6).
A remarkable number of genetic and biomolecular
studies has attempted to detect the underlying genetic alteration
in HNPCC. The research of all the genome of patients with HNPCC
has been performed by means of the binding analysis with DNA markers
called "microsatellites". These markers are a short
sequence of di or trinucleotides which are scattered in all the
genome and show great variability among healthy individuals. In
1993, two genes which account for susceptibility to HNPCC in some
families were detected and are located in both chromosome 2p 16
(hMSH2) (40-50%) and chromosome 3p21-23 (hMLH1) (30-40%) (7, 8).
More recent studies have described new genes involved: the hMPS1
in which 1q31-33 with an incidence rate of 5-10%. These genetic
alterations involve the mechanisms controlling the reliability
of the ADN replication and, therefore, the reparation of errors
which usually occur in the ADN replication are not properly carried
out. Nevertheless, there is no universal genetic or molecular
marker so far which is definite for diagnosing all the families
with HNPCC, and thus the diagnosis must be based on the clinical
criteria mentioned above.
The detection of even a few cases of HNPCC
is indeed of great importance not only for the patient diagnosed
(type of surgery to be performed, screening studies in other organs,
follow-up, etc.) but also for the at risk relatives (9). The approach
of a suitable and effective screening and follow-up method for
both the patients and risk-relatives is another of the main challenges
in the daily clinical practice. The standarization of biomolecular
techniques and their application in clinical practice will allow
us to achieve a remarkable improvement in the diagnostic approach
to HNPCC.
The main objectives of this study are:
To identify families with this syndrome from the registry of patients with CRC diagnosis in the corresponding hospitals or services as well as the building of the genealogical tree,
To estimate its incidence in this cohort of patients, and
To design an effective screening and a follow-up programme for patients and families.
PATIENTS AND METHODS
Setting: the population admitted to the services
of General and Digestive Surgery and Medical Oncology from our
hospitals.
One of the aims of our protocol is to create
a registry of patients with Lynch's syndrome. Thus, each patient
diagnosed and treated must be included in the registry and communicated
to the coordination of the study in order to be recorded. We then
give some guidelines for the diagnosis of patients and identification
of such families:
DEVELOPMENT OF OBJECTIVE 1
According to the way in which the registry
of patients is organised in each of the services included in thisstudy, the analysis of its own registrys must be initiated so
that a first selection of patients is made before starting the
family study. This selection is based on the detection of patients
diagnosed or treated of colorectal cancer fulfilling the following
criteria:
Age under 50.
Located in the right colon.
History of multiple primary tumor.
Mucinous type histology.
Presence of family history of CRC.
Personal and family background of cancer in
other locations of the spectrum included in Lynch's syndrome.
Once the preselection has been performed,
the likely families are identified by reviewing the medical records
and mainly by personal interviews with the patient and/or relatives
since, as mentioned in the introduction, nowadays there are no
genetic or biological markers for this syndrome.
As far as possible, the diagnosis reported
by the relatives must be confirmed even requesting clinical and
pathology studies from other hospitals (Annex 1).
With the data obtained after reviewing histories
and the interview, the genealogical trees are built following
the method used by various authors (10,11) (Annex II). The study
of these trees will allow us to isolate those cases fulfilling
the criteria established for the identification of families involved
with HNPCC (4). These criteria are:
Vertical transmission: when a father or a brother has CRC involvement or one of the parents or siblings
are involved, at an early age, with cancer in the following locations: endometrium, stomach, ureter, liver,
ovary, larynx, gallbladder or kidney.
Family aggregation: in the generation of
the patient, 50% of the siblings with cancer involvement in any
location or in all the family (parents, siblings, offspring) 50%
or more of the members affected with cancer in any location.
Early onset: CRC onset in the patient or in
a first-degree-relative before the age of 50-55 years or development
of a cancer with early onset in the following locations: endometrium,
stomach, pancreas, ureter, liver, ovary, larynx, gallbladder or
kidney.
Location in the right colon: cancer located
in the ascending, transversal of the caecum or flexures.
- Multiple primary tumors: the patient or
a first-degree relative has two or more independent primary cancers
both in the large bowel and in other organs.
Mucinous adenocarcinoma hystological type.
Once the families are isolated, they will
be classified if they meet 2, 3, 4 or more criteria. The more
criteria fulfilled, the greater the probability to undergo HNPCC.
In order to diagnose HNPCC, the following criteria established
by the International Collaborative Group of Study on hereditary
nonpolyposis colorectal cancer (5) must be met:
At least three relatives must have histological
confirmation of CRC, one of whom must be a first-degree relative
from the other two. FAP must be excluded.
Two successive generations must be affected.
One of the relatives must be younger than 50 years at the time of CRC diagnosis.
As soon as the HNPCC families have been identified,
they are classified as to they have extracholic lesions as follows:
A file must be completed including all the
cases and their relatives (Annex III) and submitted to the Coordination
Board of the study so as to include it in the Registry.
DEVELOPMENT OF OBJECTIVE II
Diagnostic and therapeutic involvement
Following HNPCC diagnosis, three different
situations may occur:
A patient with HNPCC diagnosed de novo.
In this case and due to the high rate of synchronic and metachronic
cancers, a subtotal colectomy with ileorectal anastomosis is recommended
(12). In the case of a Lynch's syndrome II, the need of a hysterectomy
with double prophylactic anexectomy must be considered in each
case individually according to the data obtained from studying
the genealogical tree (increased number of neoplasm cases in the
female genital tract). After surgery, the follow-up of these patients
must necessarily include a biannual endoscopy control of the residual
rectum stump; in those cases with Lynch's Syndrome II, a yearly
endometrium biopsy, yearly ovary ultrasonography and a bi-yearly
pelvic study. If the extracolonic cancer is also extragenital,
the follow-up must be individualized for each location since prophylactic
treatment on these organs cannot be performed.
Other members of the family not yet involved.
The recommendations for this group of patients include: biannual
detection of occult blood in stools from the age of 20 years;
yearly colonoscopy from age 25 or 5 years before the onset of
CRC in the youngest patient of the family. The recommendations
for patients with Lynch's Syndrome II are as follows: pelvic study
with transvaginal ultrasonography and endometrial suction biopsies
every 3 years, starting at the age of 35.
- A patient affected and treated of CRC before being diagnosed as HNPCC.
An HNPCC patient non-recognised
and carrier of a conventional partial resection for CRC has a
40% risk for developing a metachronic tumor within 10 years. Therefore,
a thorough programme for controlling residual colon and extracolonic
target organs must be indicated in Lynch's Syndrome II cases.
Some authors (12) consider that in these patients, a subtotal
colectomy with ileorectal anastomosis must also be completed.
Follow-up indications are the same as the ones of the first case,
including those specific for Lynch's Syndrome II.
Some authors (2, 13) have pointed out the
rather good prognosis of HNPCC with higher survival rates within
5 years as compared to sporadic CRC. Due to this fact and the
early onset age, all the efforts to reach an early diagnosis and
suitable treatment are warranted.
If the use of a sensitive molecular diagnostic
and specific test for HNPCC becomes common, it would be very useful
to preselect patients and relatives suspected of having this syndrome.
The implementation of genetic techniques for the clinical diagnosis
of HNPCC is an achievement which is foreseeable at short term
so that the identification of relatives is of great interest to
facilitate its development.
MOLECULAR BIOLOGY and/or GENETIC STUDIES
Instability analysis of microsatellites
The study proposed by us is a complement for
the registry and we consider it of great interest since it will
enable us to establish a molecular diagnosis and genetic counselling
in the most developed countries and it will also be very helpful
for the surgeon and the scientific community in general. Patients
and relatives included in the registry and who have undergone
these studies will undergo a follow-up for several years without
further disturbances for the patients because once the DNA samples
are obtained these may be processed every time a new marker appears
so that the security margin regarding the prediction of genetic
risk is increased.
Samples.
The following samples are required for this study:
Patient with cancer (HNPCC):
Blood samples: 10 ml of
blood to obtain constitutional DNA. Samples are collected before
the patient undergoes any chemotherapy or radiotherapy treatment
or is perfused. In the cases in which this is not possible, blood
samples must be obtained after surgery and the adjuvant treatments
but when their effects on blood have disappeared.
Tissue samples: two tissue samples must be
taken: one from the tumor and another one of healthy mucosa
located as far as possible from the tumor.
Relatives susceptible of HNPCC:
10 ml of
blood. If one of these relatives undergoes a colonoscopy, as has
been mentioned in the protocol, it would be of great interest
to take a biopsy of colon for a genetic analysis.
Methodology.
Both the blood and tissue samples are submitted to the reference laboratory.
Patient:
Microsatellite instability analysis to determine
whether there are mutations in the genes hMSH2, hMLH1, hMPS1 or
hPMS2. DNA blood samples, normal colon tissue and tumor tissue,
as has been mentioned, must be analysed. Although no instability
has been found so far in blood, it appears to be suitable to use
these samples as control of the band pattern of the microsatellite
in the patient ( microsatellites are polymorphic at population
level but each patient presenting a particular band pattern) since
the tissue regarded as healthy is often infiltrated and therefore
shows a band pattern similar to the tumoral tissue and the instability
is unnoticed.
For this type of analysis, the microsatellite
study below is proposed. The selection criterion is based on the
previous experience of the group of Dr. González-Aguilera
from the Autonomous University of Madrid. On the other hand, it
has the advantage of being close to the genes involved in the
CRC and may be combined in the PCR itself, so that the size of
the alleles produced in the gene may be easily differentiated
microsatellites D2S123. Both may be produced in the same PCR.
Conditions of the four microsatellites proposed
of DNA are broadened in a reaction volume of 25 (l containing dNTPs
(0.2 mM) , 1(M of each first primer, 1 unit of polymerase Taq
and enzyme buffer with 2.5 MgCl2.
Programme.- 27 cycles: 30s at 94º C, 75s
at 55ºC, 15s at 72º C.
Final extension: 6 min at 72º C.
Patients and relatives:
Ligament analysis with markers which are in
or close the susceptible loci for HNPCC. For these analyses, a
computer programme called Linkage may be used which is usual for
this type of studies and may be provided to laboratories involved
in the study.
PROTOCOL OF COLLECTION AND SUBMISSION OF SAMPLES
There are several alternatives according to the possibilities of the service performing the sample collection.
Blood samples:
Blood must be collected before the patient
undergoes any chemotherapy or radiotherapy or is transfused. In
those cases in which this is not possible, blood samples must
be obtained after surgery or treatment, once the time elapsed
is considered to be sufficient for the blood not to be affected
by therapies.
Collect 10-20 ml of blood in heparinized tubes.
Label the tubes. It is very important to identify the sample with the name of the patient or the case history number or date.
Store in a refrigerator at approximately 4ºC.
The samples collected must arrive at the Reference Laboratory within 48 hours after extraction. The sample need not be sent frozen.
At the services where it is possible, the protocol may be continued to eliminate the serum which hinders the obtention of DNA in the case of storing the sample for longer time.
Extraction of blood serum:
4.1. Centrifuge for 15 minutes at 3000 r.p.m.
The blood centrifugators may easily keep these conditions. (Do
not shake when stopping, i.e., do not use the brake of the centrifuge
in no centrifugation and handle with care).
4.2. Remove the supernatant (serum) with
a Pasteur pipette taking care not to carry the white interphase,
between serum and blood and do not shake the tube.
4.3. Add physiological serum up to 50 ml
and turn the tube over to dissolve the pellet.
4.4. Centrifuge for 15 minutes at 3000 r.p.m.
4.5. Remove the supernatant and keep the pellet.
At this time the process may be stopped, freezing
the blood at -20º C. (The freezer of the refrigerator may
be used). These samples had better be sent frozen.
Tissue samples:
At surgery, an intraoperative biopsy will
be taken (at least 1 cc) of tumoral tissue as well as of healthy
tissue at distance of the tumor so that it will not be infiltrated.
Collect the sample in the most aseptic manner and place it in
a sterile plastic tube with physiological serum.
Label the tube. It is important to identify
the sample with the name of the patient or case history number
and date.
Freeze with liquid nitrogen, or in the freezer of the refrigerator.
Submit the sample to the Reference Laboratory, if possible frozen and by the fastest procedure.
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(HNPCC)