Gastrointestinal cancers and neurofibromatosis type 1 features in children with a germline homozygous MLH1 mutation.

Abstract

Heterozygous germline DNA mismatch repair gene mutations are typically associated with hereditary nonpolyposis colorectal cancer. The molecular hallmark of this syndrome is high-frequency microsatellite instability in the tumors. Rare childhood cases with homozygous or compound heterozygous DNA mismatch repair gene mutations have a described predisposition to leukemia, lymphoma, and brain tumors but not to gastrointestinal cancer. We have now characterized a family in which 2 children with a homozygous germline DNA mismatch repair gene mutation developed early-onset gastrointestinal cancers. The 11-year-old proband had café-au-lait macules and developed metastatic duodenal adenocarcinoma that arose in a tubulovillous adenoma. His 9-year-old sister with café-au-lait macules and axillary freckling presented with malignant colon polyps. A 6-year-old sister with café-au-lait macules, hairy nevi, and a plexiform neurofibroma of the tongue has no malignancies to date. The family history did not fulfill the Amsterdam criteria for hereditary nonpolyposis colorectal cancer, but 2 relatives in their 60s had gastric cancer and colorectal cancer, whereas the parents, who are first cousins, remain cancer free. The proband's metastatic duodenal cancer and his sister's malignant colon polyps had high-frequency microsatellite instability but had detectable MLH1, MSH2, and MSH6 proteins by immunohistochemistry. Because some germline DNA mismatch repair gene deficiencies are associated with apparently intact immunohistochemical DNA mismatch repair gene expression in tumors, we proceeded to DNA sequencing, which showed that all 3 children had a germline homozygous MLH1 missense mutation (exon 18, codon 687, CGG-->TGG), whereas both parents were heterozygous for this mutation.

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CASE REPORT
Gastrointestinal Cancers and Neurofibromatosis Type 1
Features in Children With a Germline Homozygous
MLH1 Mutation
STEVEN GALLINGER,*,‡MELYSSA ARONSON,* KATAYOON SHAYAN,?ELYANNE M. RATCLIFFE,¶,‡‡
JUSTIN T. GERSTLE,** PATRICIA C. PARKIN,‡‡,§§HEIDI ROTHENMUND,* MARINA CROITORU,*
EWA BAUMANN,? ?PETER R. DURIE,¶,‡‡,¶¶ROSANNA WEKSBERG,¶¶,‡‡AARON POLLETT,§
ROBERT H. RIDDELL,§BO Y. NGAN,?ERNEST CUTZ,?ALAIN E. LAGARDE,? ?and
HELEN S. L. CHAN‡‡,##,***
*Familial Gastrointestinal Cancer Registry and Centre for Cancer Genetics and Departments of‡Surgery and§Pathology and Laboratory
Medicine, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, University of Toronto, Toronto, Ontario; Divisions of##Hematology/
Oncology,¶Gastroenterology and Clinical Nutrition,§§Pediatric Medicine, and Genetics and Metabolics,‡‡Department of Pediatrics,
**Division of General Surgery and Department of Surgery, and?Division of Pathology and Department of Laboratory Medicine, Research
Institute ***Cancer Research, and¶¶Integrative Biology and Genetics Programs, Hospital for Sick Children, University of Toronto, Toronto,
Ontario; and??Centre for Experimental Cancer Research and Ottawa Health Research Institute, Ottawa Regional Cancer Centre, Ottawa,
Ontario, Canada
Heterozygous germline DNA mismatch repair gene mu-
tations are typically associated with hereditary nonpol-
yposis colorectal cancer. The molecular hallmark of this
syndrome is high-frequency microsatellite instability in
the tumors. Rare childhood cases with homozygous or
compound heterozygous DNA mismatch repair gene
mutations have a described predisposition to leukemia,
lymphoma, and brain tumors but not to gastrointestinal
cancer. We have now characterized a family in which 2
children with a homozygous germline DNA mismatch
repair gene mutation developed early-onset gastrointes-
tinal cancers. The 11-year-old proband had cafe ´-au-lait
macules and developed metastatic duodenal adenocar-
cinoma that arose in a tubulovillous adenoma. His
9-year-old sister with cafe ´-au-lait macules and axillary
freckling presented with malignant colon polyps. A
6-year-old sister with cafe ´-au-lait macules, hairy nevi,
and a plexiform neurofibroma of the tongue has no
malignancies to date. The family history did not fulfill
the Amsterdam criteria for hereditary nonpolyposis colo-
rectal cancer, but 2 relatives in their 60s had gastric
cancer and colorectal cancer, whereas the parents, who
are first cousins, remain cancer free. The proband’s
metastatic duodenal cancer and his sister’s malignant
colon polyps had high-frequency microsatellite instabil-
ity but had detectable MLH1, MSH2, and MSH6 proteins
by immunohistochemistry. Because some germline DNA
mismatch repair gene deficiencies are associated with
apparently intact immunohistochemical DNA mismatch
repair gene expression in tumors, we proceeded to DNA
sequencing, which showed that all 3 children had a
germline homozygous MLH1 missense mutation (exon
18, codon 687, CGG3TGG), whereas both parents were
heterozygous for this mutation.
T
deficiency and neurofibromatosis type 1 (NF1) features
who developed early-onset gastrointestinal cancers. Af-
fected subjects from families with hereditary nonpolypo-
sis colorectal cancer (HNPCC) typically present in the
fourth or fifth decades of life with colorectal cancer or
other specific extracolonic malignancies such as endome-
trial adenocarcinoma.1–4HNPCC is caused by germline
mutations of MMR genes, most commonly MSH2 or
MLH1, with an autosomal dominant pattern of trans-
mission.2,5–8A heterozygous MMR gene mutation is
detectable in approximately 50% of cases among families
that satisfy the Amsterdam criteria.2,5–10
HNPCC is uncommon, the union of 2 heterozygous
MMR gene mutation carriers is rare. One quarter of the
offspring from 2 heterozygous MMR gene carriers are
predicted to be compound heterozygotes or true homozy-
gotes. On the basis of current knowledge of genomic
instability and cancer, an individual with germline mu-
tations of both copies of an MMR gene would be pre-
dicted to have a strong predisposition to cancer. Indeed,
children with germline homozygous or compound het-
his is the first reported family of children with
homozygous DNA mismatch repair (MMR) gene
Because
Abbreviations used in this paper: APC, adenomatous polyposis coli;
CK, cytokeratin; HNPCC, hereditary nonpolyposis colorectal cancer;
MMR, DNA mismatch repair; MSI-H, high-frequency microsatellite in-
stability; MSS, microsatellite stable; NF1, neurofibromatosis type 1;
PCR, polymerase chain reaction.
© 2004 by the American Gastroenterological Association
0016-5085/04/$30.00
doi:10.1053/j.gastro.2003.11.008
GASTROENTEROLOGY 2004;126:576–585

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erozygous MMR mutations in 5 previously reported
families consistently developed hematologic malignan-
cies, brain tumors, or both.11–15Perhaps surprisingly,
gastrointestinal cancers have not been reported in these
subjects.
We now report a kindred in which 2 of 3 siblings
proven to have a germline homozygous MLH1 mutation
developed early-onset gastrointestinal cancers. Similar to
previously described homozygous MMR gene mutation
cases, these children showed features of NF1. However,
unlike the previous reports, 2 of our subjects developed
gastrointestinal cancers in the first 2 decades of life. One
presented at age 11 with widely metastatic adenocarci-
noma of the duodenum, and the 9-year-old sibling de-
veloped 3 malignant colonic adenomatous polyps. Gas-
trointestinal adenocarcinomas are extremely rare in
children, and this is only the third case reported in the
literature of a child with duodenal adenocarcinoma. The
young ages of these children and the rarity of their
gastrointestinal cancers led to the search for a heritable
genetic mechanism. The demonstration of high-fre-
quency microsatellite instability (MSI-H) in gastrointes-
tinal tumors from the children, as well as microsatellite
instability in lymphoblast DNA, provided confirmatory
biological evidence for MMR deficiency as causative for
the development of cancer in these children.
Case Report
Proband
The proband (Figure 1; subject IV-1), an 11-year-
old boy, presented with an 8-month history of fatigue,
abdominal pain, weight loss, and iron-deficiency anemia.
Physical examination showed a cachectic-appearing pre-
pubertal boy with occult blood positive stools but no
perianal disease and no extraintestinal manifestations of
inflammatory bowel disease. On his lower back, there
were several cafe ´-au-lait macules, each measuring 5–10
mm. Eye examination showed no Lisch nodules, and
dilated fundus examination results were normal. An up-
per gastrointestinal contrast study was reported as nor-
mal but was limited by suboptimal ingestion of barium.
Barium enema results were normal. Multiple hepatic
lesions were identified on abdominal ultrasound.
Abdominal computerized tomography and magnetic
resonance imaging confirmed the liver lesions and
showed a persistent abnormality suggestive of thickening
and infiltration of the duodenojejunal junction, a mass in
the pelvis outside the bowel, and bilateral lung lesions.
Needle biopsy of the liver lesions showed focal replace-
ment of the hepatic parenchyma with metastatic tumor
composed of glandular structures with focal cribriform
changes. Special staining showed mucin secretion by the
neoplastic cells. These cells were positive for pan-cytok-
eratin (CK), CK20, CK8, CK18, epithelial membrane
antigen, and carcinoembryonic antigen but were nega-
tive for CK7, CD30, S100, ?-fetoprotein, and ?1antit-
rypsin. Side-viewing endoscopy showed a normal stom-
ach and proximal duodenum with no visible polyps, but
the scope could not be passed beyond the second part of
the duodenum. A pediatric colonoscope was successfully
passed into the third part of the duodenum and showed
an annular constricting tumor that almost completely
occluded the lumen. Duodenal biopsy samples showed a
tubulovillous adenoma with foci of high-grade dysplasia
in continuity with intramucosal and invasive adenocar-
cinoma (Figure 2). On colonoscopy, there were no visible
Figure
HNPCC family showing 4 gener-
ations (I–IV), with relevant indi-
viduals in each generation indi-
cated by numbers on the top
right corner of the symbols.
Males are indicated by squares
and females by circles. Double
lines indicate a consanguine-
ous union. Family members
with reported or confirmed can-
cers are shown in black. Their
ages at diagnosis are indicated
below the symbols. Diagonal
lines indicate deceased individ-
uals with age of death (d)
shown. Numbers within the
symbols indicate the numbers
of unaffected siblings. NF1,
features of neurofibromatosis.
1. Pedigreeof this
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MLH1 HOMOZYGOSITY AND CHILDHOOD GI CANCERS 577

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polyps or other lesions in the rectum and descending
colon, and there was no colonic involvement from the
pelvic mass.
The patient underwent palliative gastrojejunostomy
and received gemcitabine/5-fluorouracil chemotherapy
and, eventually, home total parenteral nutrition. He died
10 months after the diagnosis with widespread lung and
abdominal metastases, ascites, and malignant pleural
effusions.
Proband’s Sister (Child 2)
The proband’s sister (Figure 1; subject IV-2), a
9-year-old girl, presented shortly thereafter with a his-
tory of mild abdominal pain without any other symp-
toms. She was extensively investigated because of her
brother’s history. She was a healthy-looking prepubertal
girl who had minimal axillary freckling and 3 cafe ´-au-
lait macules measuring 5–10 mm on her neck and torso.
Eye examination showed a right-sided iris lesion at 3
o’clock that was suggestive of a Lisch nodule. Dilated
fundus examination showed a superionasal lesion in the
right eye that was suggestive of a hamartoma. Abdom-
inal sonography and computerized tomography showed a
1.6 ? 1.4 ? 2 cm left upper quadrant mesenteric mass.
Magnetic resonance imaging confirmed that this heter-
ogeneous mass was just below the left kidney, lateral to
the left psoas muscle, and in close relation to the adjacent
bowel loops. Small hypodense lesions were noted in the
left and right lobes of the liver on the computerized
tomographic scan but not on magnetic resonance imag-
ing. On image-guided needle biopsy, the left upper
quadrant mass contained metastatic adenocarcinoma con-
sistent with an intestinal primary tumor. Upper endos-
copy and small-bowel enema results were normal.
Colonoscopy and biopsy showed 3 adenomatous polyps
in the cecum and ascending and sigmoid colon.
At exploratory laparotomy, push enteroscopy con-
firmed that the entire small bowel was normal. Four
lesions were seen on the surface of each lobe of the liver,
and intraoperative sonography showed many more le-
sions in both lobes of the liver. A wedge biopsy of 1 of
the larger liver lesions was performed. A subtotal colec-
tomy and ileorectal anastomosis were performed.
Pathologic examination showed that the cecal polyp was
a tubulovillous adenoma with foci of high-grade dysplasia
and small areas of necrosis consistent with adenocarcinoma-
in-situ. The 2-cm polyp in the ascending colon was a
moderately well differentiated adenocarcinoma. The 1.5-cm
polyp in the sigmoid colon contained foci of moderately
well differentiated adenocarcinoma (Figure 3A and B). A
mesenteric lymph node that drained the latter polyp was
necrotic, with areas of dystrophic calcification. This lymph
node presumably had contained the metastatic adenocarci-
noma found on the prior needle biopsy. Another 41 mes-
enteric lymph nodes were negative for malignancy.
The colon adenocarcinoma cells were positive for
pan-CK and high-molecular-weight CK8 and CK18,
epithelial membrane antigen, and carcinoembryonic an-
tigen but were negative for CK7, CD30, S100, ?-feto-
protein, and ?1antitrypsin. The liver showed a loss of
normal architecture with steatosis and fibrous septa di-
viding the parenchyma into small lobules, consistent
with focal nodular hyperplasia. There was no atypia. The
fibrous septa contained arterioles, small immature bile
ductules, venular anomaly, and a chronic inflammatory
infiltrate. The child was administered a 6-month course
of adjuvant irinotecan/5-fluorouracil/leucovorin chemo-
therapy and remains well at 11 months after surgery and
2 months off chemotherapy.
Proband’s Sister (Child 3)
The proband’s 6-year-old sister (Figure 1; subject
IV-3) had a plexiform neurofibroma measuring 3.5 ?
2 ? 1.5 cm removed from her tongue. The neurofibroma
had multiple well-circumscribed nests of myxoid mate-
rial that contained neural-appearing elements and fibrous
tissues. There was no nuclear pleomorphism and no
Figure 2. Low-power view of the proband’s duodenal biopsy sample;
the infiltrating adenocarcinoma forms small back-to-back glands (ar-
row). Glands close to the surface show adenomatous changes (ar-
rowhead; stain, hematoxylin and eosin; magnification, 100?). The
inset shows a close-up of stromal invasion (stain, hematoxylin and
eosin; magnification, 400?).
578 GALLINGER ET AL.GASTROENTEROLOGY Vol. 126, No. 2

Page 4

increased mitotic rate. She was a healthy-looking prepu-
bertal girl with 8 cafe ´-au-lait spots measuring 5–10 mm
distributed over her body and 2 hairy nevi on the neck.
There was no freckling in the intertriginous areas. Eye
examination showed a right-sided iris lesion at 3 o’clock
that was suggestive of a Lisch nodule. Dilated fundus
examination results were normal. Total body magnetic
resonance imaging, abdominal computerized tomo-
graphic studies with extraoral contrast for visualization
of the small and large intestines, and upper and lower
endoscopy results were unremarkable. She remains well
after 23 months of follow-up.
Family History of Cancer
The parents were first cousins from Afghanistan
(Figure 1). The maternal grandfather and the paternal
grandmother were brother and sister. The maternal great
grandfather, who was also the paternal great grandfather
(subject I-1), was reported to have had colorectal carci-
noma in his 60s. The maternal grandfather, who was also
the paternal great uncle (subject II-2), was reported to
have had gastric carcinoma at age 58. The paternal
grandmother remains unaffected at age 56. No other
known relatives with cancer were identified on the at-risk
side of the family.
Materials and Methods
Immunohistochemistry
Formalin-fixed, paraffin-embedded tissues were sec-
tioned at 4 ?m, deparaffinized, and rehydrated with xylene
and alcohol. The slides underwent microwave antigen retrieval
(10 mmol/L citrate buffer, pH 6.0; 3 minutes at 115°C in
microMED T/T Mega [Hacker Instruments & Industries, Inc.,
Fairfield, NJ]). Nonspecific binding was blocked by 20%
Protein Blocker with Avidin (Signet Laboratories, Inc., Ded-
ham, MA). The slides were washed with Tris-buffered saline.
The sections were then incubated with mouse monoclonal
antibodies against MLH1 (1:40; G168-728, PharMingen, San
Diego, CA), MSH2 (1:100; FE 11, Oncogene Research Prod-
ucts, Cambridge, MA), and MSH6 (1:100; 44, BD Transduc-
tion Laboratories, Mississauga, ON, Canada) for 1 hour. The
antibodies were detected with the avidin-biotin complex
method. 3,3?-Diaminobenzidine tetrahydrochloride was used
as the chromogen, and hematoxylin was used as a counterstain.
DNA Extraction and Microsatellite Instability
Analysis
Genomic DNA was extracted from unstained paraffin
slides as described previously.16Microdissected paraffin DNA
from neoplasms used in subsequent microsatellite instability
analyses contained at least 50% tumor cells. Five mononucle-
otide and dinucleotide microsatellite markers from the Na-
tional Cancer Institute consensus panel for microsatellite in-
stability testing (BAT25, BAT26, D17S250, D5S346, and
D2S123) were amplified by polymerase chain reaction (PCR)
as described previously.16,17The presence of additional bands
in the PCR product from tumor DNA not observed in DNA
from normal tissue from the same patient was scored as insta-
bility at that particular locus. In accordance with National
Cancer Institute consensus definitions, tumor samples were
classified as MSI-H (instability at ?30% of loci screened),
Figure 3. (A) Low-power view of a colon polyp from child 2 showing
the region of invasive adenocarcinoma. The adenocarcinomas had a
cribriform architecture with loss of polarity and increased nuclear size.
The nuclei were rounded, with prominent nucleoli. Increased abnor-
mal mitotic figures and areas of necrosis were present. Most of the
glands were dysplastic with crowding large, and dilated glands had
pools of mucin in their centers (stain, hematoxylin and eosin; magni-
fication, 16?). (B) High-power view showing stromal invasion and
malignant nuclear features (stain, hematoxylin and eosin; magnifica-
tion, 400?).
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low-frequency microsatellite instability (instability at ?30%
of loci screened), or MSS (stable at all loci tested).16,17
A dilutional microsatellite instability assay using DNA
from normal cells was performed as described by Vilkki et al.13
and Parsons et al.18Briefly, Epstein–Barr virus lymphoblast
cell lines were generated from peripheral blood lymphocytes of
all 3 children and their parents. Genomic DNA was extracted
from lymphoblasts by using phenol chloroform followed by
ethanol precipitation. Samples were diluted such that the
template used in each of the multiple PCRs was approximately
1 cell equivalent. To compensate for the small amount of DNA
used in these reactions, 40 PCR cycles were performed instead
of 35 used in tumor microsatellite instability testing. PCR
products were subjected to electrophoresis by using similar
methods as for tumor microsatellite instability analysis.16
Germline Adenomatous Polyposis Coli
Gene Analysis
RNA was extracted from lymphoblasts with TRIzol
reagent (Invitrogen Life Technologies, Carlsbad, CA) by using
the manufacturer’s protocol. The protein truncation test was
used to screen for truncating germline mutations as described
previously.19
Germline DNA Mismatch Repair Gene
Analysis
Lymphoblast DNA was used to screen for alterations in
the MSH2, MLH1, and MSH6 genes by automated DNA
sequencing with a model 4000 Li-Cor sequencer (Lincoln, NE)
after PCR amplification of all exons and adjacent intronic
sequences. Templates were produced in 60-?L reaction vol-
umes (300 ng of genomic DNA) purified on Qiagen columns
and concentrated (Qiagen Inc., Canada, Mississauga, ON). A
Thermo Sequenase Primer Cycle Sequencing Kit from Amer-
sham BioSciences Corp. (Baie d’Urfe, Quebec, Canada) and
specific (IRD800) fluorescent-labeled sense/antisense sequenc-
ing primers or M13 tags (all from Li-Cor) were used in
sequencing reactions. Primer sets to amplify MSH2, MLH1,
and MSH6 exons were designed by Kolodner et al.20–22Four
overlapping fragments were sequenced to cover exon 4.
Results
Tumor and Germline Molecular Analysis
The development of gastrointestinal cancers in
these young children prompted referral to the Familial
Gastrointestinal Cancer Registry at Mount Sinai Hospi-
tal, Toronto, for genetic counseling and molecular test-
ing. Research and service genetic testing studies were
performed according to protocols approved by the Mount
Sinai Hospital and the Ottawa Hospital Research Ethics
Boards.
Subjects IV-1 and IV-2 had a normal karyotype and
had normal chromosome fragility testing. Genetic test-
ing for a germline APC mutation in the proband (child
IV-1) was negative.
Because homozygous MMR gene germline mutations
had been reported recently in children with both cancer
and cafe ´-au-lait spots, we sought to determine whether
these genes contributed to the proband’s developing
duodenal adenocarcinoma. Immunohistochemical studies
were performed first, and the proband’s duodenal adeno-
carcinoma showed intact expression of the MSH2 and
MSH6 gene products, with weak expression of the
MLH1 protein. The malignant colonic polyps from child
2 (IV-2) also showed intact MSH2 and MSH6 proteins
and weak expression of the MLH1 protein (Figure 4A–
C). The patterns of protein expression of the 3 MMR
genes were similar in the hepatic focal nodular hyperpla-
sia from this child and in the tongue plexiform neurofi-
broma from child 3 (IV-3).
Microsatellite instability testing of genomic DNA
extracted from microdissected samples of the proband’s
duodenal adenocarcinoma liver metastasis and the ma-
lignant colon polyps from child 2 was performed by
using the first panel of 5 microsatellite markers recom-
mended by the National Cancer Institute Consensus
Conference.17The duodenal adenocarcinoma liver metas-
tasis and the colonic polyps were all MSI-H (Figure 5).
Two of the malignant colonic polyps from child 2
showed microsatellite instability at 4 of the 5 markers,
the other polyp had instability at 2 (BAT25 and BAT26)
of the 5 markers, and the proband’s metastatic duodenal
adenocarcinoma showed instability at 3 of the 5 markers.
Both the focal nodular hyperplasia from child 2 and the
tongue plexiform neurofibroma from child 3 were MSS
(not shown).
Although the immunohistochemical analysis of the
tumors showed positive expression of the MSH2 and
MSH6 proteins and weak expression of the MLH1 pro-
tein, the finding of MSI-H in the cancers and polyps
prompted analysis for germline MMR mutations. The
proband’s (IV-1) lymphoblast DNA was subjected to
molecular analysis that included sequencing of the entire
open reading frames of the MSH2, MLH1, and MSH6
genes. No mutations were found in the MSH2 or MSH6
sequences, with the exception of commonly reported
intronic and exonic polymorphisms. A single novel al-
teration in the MLH1 gene was detected in exon 18 at
codon 687 (normal allele CGG, Arg). As shown in Figure
6, which is reverse sequencing, the last nucleotide of
codon 687 (normally a G) was replaced with an A,
leading to the substitution of the Arg residue with a Trp
residue. The proband carried 2 mutant alleles and no
normal allele (homozygous). This finding is consistent
with both of his parents (III-1 and III-2) as Arg687Trp
heterozygotes, which was confirmed by sequencing. Fur-
ther sequencing studies showed that the proband’s 2
580GALLINGER ET AL.GASTROENTEROLOGY Vol. 126, No. 2