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From the Department of Biomedical Sciences and
Oncology,*
University of Turin, Turin, Italy; the Division
of Cell and Molecular Pathology
and the
Department of Pathology,
University of
Zürich, Zürich, Switzerland; and the Department of
Pathology and Laboratory Medicine,§
Mayo Clinic
and Foundation, Rochester, Minnesota
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Because of the variety of patterns and admixed components in MMFCs, several terms have been proposed to designate these neoplasms, including "mixed follicular–parafollicular carcinoma,"20 "compound medullary-papillary carcinoma,"8 "composite carcinomas of the thyroid,"24 "differentiated thyroid carcinoma, intermediate type,"25 and "stem cell carcinoma."9 The last term was proposed because the majority of authors believe that MMFCs are derived from neoplastically transformed uncommitted stem cells with the capacity to differentiate into tumor components with morphological and histochemical characteristics of both follicular and medullary neoplasms of the thyroid.9,10,12,13,17,18,20-22 This hypothesis was further supported by reports demonstrating Tg and CT coexpression in tumor cells of MMFCs13,26 and the development of occasional mixed tumors with Tg-positive cells in a transgenic murine model for MTC.27 However, the stem cell theory has never been proved by direct evidence. Thus the embryological background and possible molecular mechanisms leading to MMFC are still unclear.
The aim of the present study was to determine whether the two tumor
components in MMFC are derived from the same cell clone or whether they
arise independently. For this purpose, the two tumor components of each
MMFC were separated by (laser-based) microdissection and individually
analyzed for allelic losses at nine different chromosomal loci, somatic
mutations of the RET and gs
gene, as well as
clonal composition in female patients.28,29
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A total of 12 MMFCs of the thyroid were collected from the files of the Mayo Clinic (Rochester, MN) (five cases), the Department of Pathology, University of Turin (Turin, Italy) (six cases), and the Department of Pathology, University of Zürich (Zürich, Switzerland) (one case). Tumor specimens had been fixed in 10% buffered formaldehyde solution and embedded in paraffin according to standard procedures.
All tumors had previously been studied by conventional histology,
immunohistochemistry, or in situ
hybridization.5,11
Ten tumors met all histopathological
criteria for the diagnosis of MMFC as defined by the WHO
classification.7
One tumor (file number T5; Table 1
, Figure 1
), which presented as a primary tumor
with components of a MTC and follicular variant of a papillary
carcinoma, exhibited papillary features in the lymph node metastases
together with a medullary component. Another tumor (file number T2) was
included in the study because of the mixed medullary-papillary
features encountered in the lymph node metastases despite the fact that
two separate primary papillary and medullary carcinomas were identified
simultaneously in the same thyroid lobe (Figure 1)
.
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. Immunohistochemistry
Freshly cut sections from archival paraffin blocks were stained with hematoxylin and eosin (H&E) and used for light microscopic examination. Serial sections (4 µm) of all 12 tumors were cut and mounted on Superfrost Plus glass slides (Menzel Gläser, Germany). Immunoreactive sites for calcitonin (CT) (polyclonal, 1:200; Dako, Glostrup, Denmark) and thyroglobulin (Tg) (monoclonal, 1:50; Dako) were detected using the avidin-biotin-complex (ABC) systems.30
Double immunostaining of CT and Tg was performed using the APAAP system31 and naphthol phosphate/fast red TR (Dako) as a chromogen for CT, and subsequently the ABC system and diaminobenzidine (Walter, Kiel, Germany) for Tg immunohistochemistry.
Molecular Analyses
DNA Extraction
Tumor DNA was isolated from five 10-µm paraffin sections of each tumor as described.29 In 11 of the 12 cases, nontumorous tissue of the same patient was also available, and DNA was extracted as well.
Selected cases were submitted to laser-based microdissection (PALM Laser-Microbeam Systems GmbH, Germany)32,33 to obtain isolated single neoplastic follicles and single nests of medullary carcinoma. The dewaxed and hematoxylin-stained tissue was placed in 0.5-ml Eppendorf tubes (Eppendorf GmbH, Hamburg, Germany) and boiled for 10 minutes at 94°C in 18 µl 1x polymerase chain reaction (PCR) buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCl; GeneAmp, Perkin Elmer, Roche, NJ). Then 0.5 µg of proteinase K diluted in 1 µl of double-distilled water (ddH2O) was added to each sample and incubated at 55°C for 24 hours. Then samples were incubated for 10 minutes at 94°C to inactivate the proteinase K. Next 7.5 µl of the solution was added to the appropriate PCR mixture for further PCR amplification, under the conditions described below.
For clonality analysis,29 microdissected tissue samples were placed in 0.5-ml Eppendorf tubes and boiled for 10 minutes at 94°C in 18 µl 1x restriction enzyme Buffer L (Boehringer Mannheim GmbH, Mannheim, Germany). After proteinase K treatment, as described above, 10 U of HpaII (in 1 µl of ddH2O) was added, and the samples were incubated at 37°C overnight. Then inactivation of the enzyme was performed at 94°C for 10 minutes, and 7.5 µl of the final solution was used as a template for further PCR amplification.
Nonisotopic PCR-SSCP and HDE RET Mutation Analysis
All 12 cases were screened for somatic or germline mutations of
the RET proto-oncogene in exons 10, 11, 15, and 16 by the
PCR-based single-strand conformation polymorphism (SSCP) and
heteroduplex gel electrophoresis (HDE) analysis as recently
described,34
using primers and PCR conditions as detailed
in Table 2
. Two tumors (file numbers M8
and M11) were from known MEN 2A patients.
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Restriction Analysis
Twenty microliters of PCR products from RET exons 15 and 16 was digested overnight at 37°C in 50 µl of medium and low-salt buffer containing 10 U of the restriction enzyme AluI (exon 15) and FokI (exon 16), respectively (Boehringer Mannheim). Restriction fragments were analyzed by 6% polyacrylamide gel electrophoresis and silver staining.35
Nonisotopic PCR-SSCP Gs Gene Mutation Analysis
Four MMFCs (file numbers T1–3 and T6) with a clearly distinct
neoplastic follicular or papillary component were analyzed for
mutations in exons 8 and 9 of the Gs gene.
Oligonucleotide primers flanking exons 8 and 9 (Table 2)
were added in
a concentration of 1 mmol/L in a 50-µl mixture containing 100–300 ng
of template DNA, 0.2 mmol/L dNTPs, 1.5 mM MgCl2, 10 mM
Tris-HCl (pH 8.3), 50 mM KCl, and 1 U of Taq DNA polymerase
(Boehringer Mannheim). Thirty-five cycles of amplification were
performed with a programmable thermal cycler (GeneAmp PCR System
9600), and the SSCP method was performed as described for the
RET analysis. Nested primers were used for an additional
amplification in cases with low DNA yield.
Loss of Heterozygosity Analysis
Eleven MMFC (T2–Z12), from which additional nontumorous tissue
from the same patient was available, were examined for loss of
heterozygosity (LOH) in microsatellite regions of chromosomes 1, 3, 7,
10, 11, and 22, using a recently described PCR-based
approach.36,37
The primer pairs D1S188, D3S1110, D3S1100,
D7S480, D7S490, D22S257, and D22S1043 were purchased from Research
Genetics (Huntsville, AL). The chromosomal location, the annealing
temperatures, and the sequence of the other two primers, D10S564 and
D11S4936, are detailed in Table 3
. For
LOH analysis, only patients heterozygous for a given DNA sequence were
considered informative, whereas the presence of either homozygosity or
an unclear distinction between paternal and maternal alleles was
considered uninformative. A twofold difference in relative allele
intensity ratios between tumor DNA and normal DNA was considered as
allelic loss.
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The clonal composition of the tumors from the four female patients (T2, M7, M8, M9) was studied by analyzing the inactivation patterns of a polymorphic X-linked region encoding the androgen receptor (AR) gene as recently described.29 Patients were considered heterozygous if PCR amplification of digested and undigested DNA from nonneoplastic tissue showed two major PCR products. Genomic DNA from paraffin-embedded tissue of a monoclonal non-Hodgkin lymphoma (female) patient and of a healthy male individual were used as controls for HpaII digestion.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and are summarized in
Table 1
.
The ratio of the two tumor components varied from 10% to 80% for the
medullary portion either separate from (two cases) or intermingled with
the follicular component (eight cases) or both (two cases). When
clearly distinct from the medullary portion, the follicular component
presented various morphological features, ranging from
well-differentiated papillary and follicular carcinoma to oxyphilic and
poorly differentiated (solid) carcinoma. In five cases, mixed features
were found in the lymph node metastases (T1, T2, T5, M11, Z12). In
patient T2, a mixed papillary/medullary lymph node metastasis was
present, although the primary tumors were topographically separated
(Figure 1)
. In patient T5, a lymph node metastasis with features of a
cystic papillary carcinoma associated with a MTC in the cyst wall
developed from a MTC with prominent follicular features (follicular
variant of papillary carcinoma) in the primary tumor (not shown). The
other three tumors (T1, M11, and Z12) with lymph node metastases
exhibited similar histological features in the primary tumors and their
metastases (eg, Z12, Figure 1
).
All tumors stained positively for calcitonin in the medullary but not in the follicular component, whereas thyroglobulin immunostaining was observed both in follicular (all tumors) and in solid tumor areas (7/12 cases).
Mutation Analysis
The results are summarized in Table 4
, and representative examples are shown
in Figures 2 and 3
. The sequence of RET exons
10, 11, 15, and 16 was studied in tumor DNA of all tumors and in the
related germline DNA from normal tissue of 11 patients. Two of our
patients (M8 and M11) were known MEN2A disease gene carriers.
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, T5). Sequence analysis revealed the presence of a mutation
at codon 883 (A883F), which was confirmed by AluI
restriction enzyme digestion (not shown). Microdissected follicles from
the positive MMFC regions, however, showed no SSCP alterations.
The heteroduplex assay for exon 16 showed abnormal patterns in tumor
DNA of two patients (T2 and T12). In one tumor (T2) aberrant band
patterns (heteroduplices) were present in PCR products from DNA of the
MTC but not of the papillary component. The same result was obtained
for the other tumor (Z12), in which follicles were microdissected from
the lymph node metastasis (Figure 3)
. The presence of a M918T mutation
at codon 918 in the positive samples was confirmed by FokI
restriction enzyme analysis.
No mutations were found in the Gs gene in any MMFC (data
not shown).
LOH Analysis
We analyzed allelic losses of nine loci on six chromosomes in the
MTC and follicular components of all MMFCs. Three tumors exhibited LOH
in the MTC portion (one each at D1S188 and D7S490; and one tumor at
both D7S480 and D7S490), which was not present in the respective
microdissected follicular component of the MMFC (Table 4
, Figure 2
).
One additional MMFC had a LOH at D3S1110 in the follicular tumor, part
which was not detectable in the MTC component (data not shown).
Clonality Analysis
Four MMFCs were from female patients, and three of these were
informative for PCR-based clonality analysis by identification of
X-chromosomal inactivation. In all three MMFCs, a monoclonal
X-chromosomal pattern was identified in the medullary portion, but the
respective follicular components exhibited a polyclonal pattern in two
patients (Figure 2
; M9, M7) and a monoclonal pattern with inactivation
of the allele opposite that of the medullary component in the remaining
MMFC (Figure 2
; M8).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. We
report here the first molecular analysis of the two components in MMFC
of the thyroid. Our data strongly suggest that the follicular and
medullary components in MMFCs are not derived from a single stem cell,
because they consistently exhibited different patterns of
RET proto-oncogene mutation, LOH, and X-chromosomal
inactivation (clonality) in seven tumors that were suitable for clonal
analysis or had detectable molecular aberrations at the genes and loci
investigated (Table 4)
. Furthermore, we demonstrate that the follicular
structures in MMFCs are often polyclonal and more frequently exhibit
hyperplastic than neoplastic histological characteristics.
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. However, our findings clearly demonstrate
differences for both the clonal and the LOH/mutation pattern of the two
tumor components. Specifically, in three MMFCs of female patients the
MTC component exhibited a monoclonal pattern, whereas the follicle part
displayed a polyclonal pattern in two tumors and a monoclonal pattern
with inactivation of the opposite allele in one tumor. Furthermore,
whereas three MMFCs demonstrated a LOH at different loci in the
medullary part, a normal composition was detectable in the follicular
part of the same patients. In an additional tumor we found a LOH at the
locus D3S1110 in the follicular component but not in the MTC part. Our
mutation analysis revealed additional differences: three sporadic MMFCs
contained somatic RET mutations that could not be detected
in their follicular components or in one examined lymph node
metastasis. In summary, different molecular and clonal patterns were
found in the two components of all seven MMFCs amenable for molecular
analysis by three independent molecular approaches.
Therefore, the present results do not support the "stem cell"
theory of development of MMFC. Likewise, our findings seem to exclude
the "divergent differentiation" theory, assuming that some MTC
cells differentiate toward a follicular phenotype by the acquisition of
additional molecular defects (Table 5)
. Such tumors should exhibit a
common clonality pattern in the two tumor components, which was not
detectable in the MMFCs studied.
Another pathogenetic mechanism that has been put forward for MMFC is
the "collision theory" (Table 5)
. It assumes that two independently
arising tumors, either a MTC and a follicular carcinoma, or a MTC and a
papillary carcinoma, collide in the thyroid. In fact, several authors
have reported such neoplasms,40-43
and others have
pointed out that such tumors may imitate a primary
MMFC.8,24
Our series included one patient who had two
topographically separated primaries consisting of a MTC and a papillary
carcinoma and mixed metastases in regional lymph nodes. This indicates
that a MTC can entrap and disseminate foci of another thyroid
neoplasms. The same holds true for residual thyroid follicles, which
can become entrapped by MTCs. Thus MTC exhibits a tumor growth
different from that of most other human cancers, which usually destroy
the tissue in which they arise.
Our molecular analysis does not entirely rule out the "field
effect" hypothesis (Table 5)
, although we did not find a common LOH
or mutation in the components investigated. The field effect hypothesis
assumes a common oncogenic stimulus triggering neoplastic
transformation of both follicular and C-cells,24
which
leads to the simultaneous development of two different tumor
components.44
Yet another pathogenetic mechanism may exist in at least one subset of
MMFC. As already pointed out, MTCs may exhibit entrapped,
well-preserved residual follicles. Although it is common to see such
residual follicles at the periphery of classical MTCs, it is typical
for MMFCs that the follicular structures are found deep within the
tumor and that they exhibit cytological features different from that of
the surrounding nonneoplastic follicles. It is conceivable that some
unknown trophic factors keep the entrapped follicles alive and might
even stimulate them to proliferate, and conversely that some follicles
might secrete substances preventing them from being destroyed by the
MTC.45
Such a "symbiosis" of neoplastic C-cells and
follicular cells might lead to a combined growth of follicular and
C-cell structures and result in the mixed tumor pattern of the majority
of MMFCs. The microenvironment provided by the MTC might result in
hyperplastic and adenomatous follicular foci, which eventually may
become neoplastic through the acquisition of molecular defects (Figure 4)
. Such a scenario could explain the
spectrum of follicular patterns and the occasional neoplastic foci in
MMFCs as well as the clonal and molecular changes found in this study.
The question that arises from this "hostage theory" is whether such
follicular structures can metastasize together with the MTC component
(Figure 4)
. This aspect, however, cannot be answered at present and
will require additional detailed analysis.
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One might argue that our molecular results are more easily explained by the fact that we have only accidentally analyzed entrapped residual follicles. However, we have only microdissected follicular structures deep within the MMFC, which exhibited a histology different from that of the peritumoral nonneoplastic follicles (eg, enlarged or clear nuclei), or which occurred in clusters. Thus we believe that our results are obtained from follicular components typical for MMFCs. Other technical reasons, eg, resulting in false clonality patterns, can be excluded because appropriate controls have been included in each assay and methods have been evaluated and tested in previous studies.29,37
From all of the data of the literature and the results of our study, it appears that the group of so-called MMFCs is in fact a rather heterogeneous group with various morphological features and different pathogenetic mechanisms. It includes 1) neoplasms that have to be classified as collision tumors, ie, that are composed of two separately arising tumors with different morphologies and an intermingled growth; 2) "true" mixed carcinomas with features as defined by the WHO classification7 ; and 3) MTCs with Tg-expressing cells that might be derived from a common stem cell.26 However, the latter tumor type should be separated from MMFCs because they do not present histological evidence of follicles or papillae.
In summary, our present investigation provides molecular evidence that Tg-positive follicles or clusters of follicles and the medullary portion in MMFCs are not derived from a common stem cell. We therefore postulate that the pathogenetic mechanisms of MMFC might be different from those ones previously proposed. These findings provide new insights into the development of tumors with mixed morphology and raise the possibility that trophic factors secreted by neoplastic and host cells might have a significant role in the tumorigenesis of MMFC.
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Acknowledgements |
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Footnotes |
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Dedicated to the memory of the late Prof. Ch. Hedinger (December 5, 1917-January 12, 1999), former head of the Institute of Pathology at the University of Zürich, Zürich, Switzerland.
Supported in part by grants from the Italian Ministry of University and Research (M.V., M.P.).
Accepted for publication July 12, 1999.
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