# Genetic Profiling Differentiates Second Primary Tumors from Metastases in Adult Metachronous Soft Tissue Sarcoma

Authors: Josefin Fernebro; Ana Carneiro; Anders Rydholm; Henryk A. Domanski; Anna Karlsson; Åke Borg; Mef Nilbert
Journal: Sarcoma
 (2008)

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## Abstract

Purpose. Patients with soft tissue sarcomas (STS) are at increased risk of second primary malignancies, including a second STS, but distinction between metastases and a second primary STS is difficult. Patients and Methods. Array-based comparative genomic hybridization (aCGH) was applied to 30 multiple STS of the extremities and the trunk wall from 13 patients. Different histotypes were present with malignant fibrous histiocytomas/undifferentiated pleomorphic sarcomas being the predominant subtype. Results. aCGH profiling revealed genetic complexity with multiple gains and losses in all tumors. In an unsupervised hierarchical cluster analysis, similar genomic profiles and close clustering between the first and subsequent STS were identified in 5 cases, suggesting metastatic disease, whereas the tumors from the remaining 8 patients did not cluster and showed only weak pairwise correlation, suggesting development of second primary STS. Discussion. The similarities and dissimilarities identified in the first and second STS suggest that genetic profiles can be used to distinguish soft tissue metastases from second primary STS. The demonstration of genetically different soft tissue sarcomas in the same patient  suggests independent tumor origin and serves as a reminder to consider development of second primary STS, which has prognostic and therapeutic implications.


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## Body

## 1. Introduction

The first observations of an
increased risk of secondary sarcoma development came from individuals treated
for childhood cancers such as retinoblastoma, leukemia, Wilms' tumor, Hodgkin's
lymphoma, and sarcoma [1–13]. However,
also adult soft tissue sarcoma (STS) patients have been shown to be at higher
risk of a second primary malignancy, with a particularly increased risk of a
second primary STS [14–17]. STS have
been associated with several hereditary syndromes, the most common being neurofibromatosis
and the Li-Fraumeni syndrome [18]. Treatment-induced sarcomas include sarcomas
associated with Stewart-Treeves syndrome and radiation-induced angiosarcomas,
which develop median 10 years after radiotherapy [19]. However, after exclusion
of STS in neurofibromatosis patients and STS that developed in irradiated
fields, an increased risk of a second primary STS remains [17].  Metachronous STS has been described in 1% of
sarcoma patients [16, 17, 20] and this observation constitutes the basis for
our study on similarities/differences in the genetic profiles of tumors from
patients with multiple STS. We applied array-based comparative genomic
hybridization (aCGH) that utilizes BAC clones with tiling coverage of the whole
genome and allows detailed copy-number analysis, to a series of 30 metachronous STS of different histopathological
subtypes from 13 patients.


## 2. Patients and Methods

### 2.1. Patients

Adult patients (≥16 years of age)
who developed two or more STS at different anatomical sites before development
of any detectable pulmonary metastases were eligible for the study. Patients with
neurofibromatosis type I and the Li-Fraumeni syndrome were excluded. In the
southern Swedish cancer registry, 20 patients who fulfilled these criteria were
identified. The tumors had been operated either at the musculoskeletal tumor
center in Lund
(n=24) or at local hospitals in the southern Swedish health care region (n=6).
The clinicopathological reports were reviewed to confirm tumor location and to
rule out that the second primary STS represented a local recurrence and the histopathological
slides were reviewed by a sarcoma pathologist (H.D.) to confirm the diagnoses.Frozen tumor tissue was available
from 16 tumors and paraffin-embedded tissue was used from 28 tumors without systematic
differences related to tumor source. After DNA extraction, 7 individuals (14
tumors) were excluded because of poor DNA quality in at least one of the tumors
from the same patient. High-quality aCGH data were obtained from 30 tumors (in
15 of which DNA was extracted from frozen tissue) from 13 patients (Table1).
These patients contributed with two to four STS and were mean 73 (28–83) years at diagnosis
of the first STS. The second STS developed median 3 (1–7) years after
the first STS. No neoadjuvant chemotherapy was given, and only one patient
(case 5) had recieved postoperative chemotherapy after the first STS.
Radiotherapy had been administered to four patients (postoperatively in case 5,
6, and 8, and preoperatively in case 1), but none of the second STS developed
within the irradiated field.Table 1
Summary of clinical data from the 30 STS analyzed from 13 patients.
Case no.SexAgeSiteTypeYears after first tumorSiteTypeYears after first tumorSiteTypeFollow-up status/monthsTumor source frozen/paraffinFirst tumorSecond tumorThird tumorSimilar genomic profiles suggestive of metastatic disease1M80Trunk wallLMS3Left cheekLMSMET/50f/f2F82Right upper armMFH/UPS2Left buttockMFH/UPS3ScalpMFH/UPSMET/47p/f/p6F80Right
thighLS1Left shoulderLS2Right lower leg†LSTD/22f/p/p9F79Lower legMFH/UPS1Right thighMFH/UPSNED/36p/f12M76Right lower legMFH/UPS7Right thigh*MFH/UPSTD/164p/fDifferent genomic profiles suggestive of second primary tumors3F77Right shoulderMFH/UPS5Left thighMFH/UPS9Right lower armLMSNED/163f/f4F75Left lower armMFH/UPS4Right lower legMFH/UPSNED/54f/p5F60Right lower armMPNST4Right thighLMSNED/56f/p7M83Left thigh (laterally)LMS4Left thigh (medially)LMSTD/28p/f8M73Left kneeMFH/UPS1Left lower legMFH/UPSMET/15f/p10M28Right trunk wallMPNST1ScalpMPNSTTD/32f/p11F77Right thighMFH/UPS2BackMFH/UPSMET/51p/f13M76Right footMFH/UPS4Right thighMFH/UPSMET/51p/fMFH/UPS = malignant fibrous histiocytoma/undifferentiated pleomorphic sarcoma,
LMS = leiomyosarcoma, LS = liposarcoma, MPNST = malignant peripheral nerve sheath
tumor.
*Located 15 cm from the first tumor.
†The patient developed one additional LS, left thoracic wall.
MET: metastases, TD: tumor death, NED: no evidence of disease.Clinical data for the 13 cases are
presented in Table1. The lower extremity was the most common tumor site (16
tumors) and 28 tumors were high-grade (grades 3 and 4 on a 4-tiered scale). The
first STS included eight malignant fibrous histiocytomas/undifferentiated
pleomorphic sarcomas (MFH/UPS), two leiomyosarcomas, two malignant peripheral
nerve sheath tumors (MPNST), and one pleomorphic liposarcoma. The
histopathological diagnosis of the second STS differed from the first in two
patients; a leiomyosarcoma was diagnosed in a patient with two prior MFH/UPS
and a leiomyosarcoma was diagnosed in a patient previously operated on for an
MPNST. In the remaining patients, including the three cases from which three or four distinct tumors were analyzed, multiple STS of the same
histopathological type were diagnosed.In 9/13 patients the STS developed
at different anatomical locations, for example, different extremities or
extremity and trunk wall. Three patients developed metachronous STS in the same
extremity but at different locations, for example, lower leg and thigh (cases
8, 12, and 13) and one patient (no. 7) developed two STS in the same extremity;
a deep-seated leiomyosarcoma in the medial thigh and five years later a subcutaneous
leiomyosarcoma in the lateral part of the thigh (Table1). Clinical follow-up
was complete for a minimum of 8 years for the survivors. During follow-up, two
patients (cases 12 and 13) developed local recurrences, 1 and 10 years after
primary surgery. Lung metastases developed in 5/13 patients, median 50 (range
15–51) months after
diagnosis of the primary tumor. Apart from the metachronous STS, two patients
(cases 4 and 7) developed adenocarcinomas of the breast and the colon,
respectively. Ethical permission for the study was granted from the Lund
University ethics committee.


### 2.2. DNA Extraction and Array-Based Comparative Genomic Hybridization

Genomic DNA from frozen (n=15) and
paraffin-embedded (n=15) tumors was extracted using the Wizard Genomic DNA
Purification kit (Promega, Madison, WI) and overnight proteinase-K digestion
treatment followed by phenol-chloroform purification. When paraffin-embedded
tissue was used, a fresh4-μm section was obtained, stained with hematoxylin
& eosin and a representative tumor area was chosen. Thereafter, 1-mm tissue
cores were obtained and used for DNA extraction. The tissue cores were
pretreated in xylene before proteinase-K treatment and phenol-chloroform
purification. DNA quality was checked using a Ready-To-Go RAPD analysis kit
(Amersham Biosciences, Buckinghamshire, UK), and the concentration was measured
using a Nano drop (NanoDrop Technologies, Wilmington, Del, USA). Commercial genomic
male DNA, derived from a pool of healthy individuals, was used as a reference
(Promega, Madison, Wiss, USA). CyDye coupling/labeling was carried out
using a random labeling kit (Invitrogen Life Technologies, Carlsbad, Calif, USA)
according to the manufacturer's recommendations. In short, 2-μg genomic tumor
DNA and reference DNA were differentially labeled with fluorescent dyes, Cy3
for tumor tissue, and Cy5 for reference DNA. After a purification step, these
were pooled, mixed with COT-1 DNA to block repetitive DNA sequences, dehydrated
and resuspended in a formamide-based buffer (Invitrogen). The labeled DNA was
then applied to arrays pretreated in washing solutions (Pronto! Microarray
Reagent System, Corning Labsystems, Corning, NY, USA) and hybridization was
performed for 48–72 hours at 37°C.
The incubation was performed under cover slips for
the DNA isolated from the frozen tumor material whereas the MAUI hybridization
System (BioMicro systems Inc., Salt
Lake City, Utah, USA) was
used for the DNA derived from paraffin-embedded tumors. Dye-swaps (i.e.,
complementary hybridization in which Cy5 was used for tumor tissue and Cy3 for reference DNA) were used in three cases and allowed subtraction of dye-related noise. The slides were treated in post-hybridization
washing solutions and finally scanned using an Agilent Microarray scanner
(Agilent Technologies, Palo Alto, Calif, USA).


### 2.3. BAC Array Platform

The BAC array slides used were
produced at the Swegene DNA Microarray Resource Center,
Department of Oncology, Lund University. These have an
average resolution of 80 kb and contain a total of 32 433 BAC clones from
the 32 k human genome high-resolution BAC rearrayed clone set, version 1.0 from
the BACPAC Resource Center at Children's Hospital Oakland Research Institute
(Oakland, Calif, USA) (http://bacpac.chori.org/).
The clones provide >99% coverage of the fingerprint map and current sequence
assembly with a resolution of 100 kb.


### 2.4. Data Analysis

Image analysis and data extraction
were carried out using GenePix Pro 4.1.1.4 version (Axon Instruments Inc.,
Foster City, CA, USA) and the quantified data matrix was then uploaded into the
web-based BioArray Software Environment (BASE;http://gothmog.thep.lu.se/int/index) [21], where all data management and analysis were carried out. The
background correction and intensities of Cy3 and Cy5 were calculated using the
median feature and median-local background intensities of the uploaded files,
and the intensity ratios were calculated using the background corrected spot
intensities by calculating the log2 ratios of tumor to reference
intensity. In BASE a preliminary filter, based on the flagging in the image
analysis, was applied, and spots with a diameter <55 μm and a signal to
noise (SNR) ratio ≤3 in the tumor or reference channel were flagged as “bad”
and filtered away from further analysis. The intensity-dependent LOWESS
algorithm [22] was used to normalize the data within individual arrays. To
correct for spatial bias, the data were normalized within groups of 8 print-tip
blocks. A moving average smoothing algorithm with a 250 kbp sliding window was
then applied, and a BASE-adapted CGH-plotter software was used to identify
regions of gains and losses [23]. In the CGH-plotter, each clone was assigned a
calculated level log2 ratio value, corresponding to the level that
the clone belongs to, in order to reduce the noise. Hereafter an unsupervised
hierarchical cluster analysis, using the Pearson correlation distance metric
and the average linkage method, was applied to the data derived from the
CGH-plotter (the TMeV application from the TM4 microarray software suite was used; http://www.tm4.org/mev.html). The CGH-plotter was also used to generate a ternary scale, where all clones were designated gained, lost or unchanged. These values were used to calculate the percentage
of altered clones in each assay and the mean number of altered clones in tumor
subgroups. Pearson correlation was used to determine the correlation between
tumors within the same individual, based on the number of altered clones. Gains
and losses were defined as a log2 ratio ±0.2. Amplifications were
defined as clones with a log2 ratio ≥0.5, whereas high-level
amplifications were defined as a log2 ratio ≥1.5. Homozygous
deletions were suspected when the log2 ratio was ≤1.5.


## 2.1. Patients

Adult patients (≥16 years of age)
who developed two or more STS at different anatomical sites before development
of any detectable pulmonary metastases were eligible for the study. Patients with
neurofibromatosis type I and the Li-Fraumeni syndrome were excluded. In the
southern Swedish cancer registry, 20 patients who fulfilled these criteria were
identified. The tumors had been operated either at the musculoskeletal tumor
center in Lund
(n=24) or at local hospitals in the southern Swedish health care region (n=6).
The clinicopathological reports were reviewed to confirm tumor location and to
rule out that the second primary STS represented a local recurrence and the histopathological
slides were reviewed by a sarcoma pathologist (H.D.) to confirm the diagnoses.Frozen tumor tissue was available
from 16 tumors and paraffin-embedded tissue was used from 28 tumors without systematic
differences related to tumor source. After DNA extraction, 7 individuals (14
tumors) were excluded because of poor DNA quality in at least one of the tumors
from the same patient. High-quality aCGH data were obtained from 30 tumors (in
15 of which DNA was extracted from frozen tissue) from 13 patients (Table1).
These patients contributed with two to four STS and were mean 73 (28–83) years at diagnosis
of the first STS. The second STS developed median 3 (1–7) years after
the first STS. No neoadjuvant chemotherapy was given, and only one patient
(case 5) had recieved postoperative chemotherapy after the first STS.
Radiotherapy had been administered to four patients (postoperatively in case 5,
6, and 8, and preoperatively in case 1), but none of the second STS developed
within the irradiated field.Table 1
Summary of clinical data from the 30 STS analyzed from 13 patients.
Case no.SexAgeSiteTypeYears after first tumorSiteTypeYears after first tumorSiteTypeFollow-up status/monthsTumor source frozen/paraffinFirst tumorSecond tumorThird tumorSimilar genomic profiles suggestive of metastatic disease1M80Trunk wallLMS3Left cheekLMSMET/50f/f2F82Right upper armMFH/UPS2Left buttockMFH/UPS3ScalpMFH/UPSMET/47p/f/p6F80Right
thighLS1Left shoulderLS2Right lower leg†LSTD/22f/p/p9F79Lower legMFH/UPS1Right thighMFH/UPSNED/36p/f12M76Right lower legMFH/UPS7Right thigh*MFH/UPSTD/164p/fDifferent genomic profiles suggestive of second primary tumors3F77Right shoulderMFH/UPS5Left thighMFH/UPS9Right lower armLMSNED/163f/f4F75Left lower armMFH/UPS4Right lower legMFH/UPSNED/54f/p5F60Right lower armMPNST4Right thighLMSNED/56f/p7M83Left thigh (laterally)LMS4Left thigh (medially)LMSTD/28p/f8M73Left kneeMFH/UPS1Left lower legMFH/UPSMET/15f/p10M28Right trunk wallMPNST1ScalpMPNSTTD/32f/p11F77Right thighMFH/UPS2BackMFH/UPSMET/51p/f13M76Right footMFH/UPS4Right thighMFH/UPSMET/51p/fMFH/UPS = malignant fibrous histiocytoma/undifferentiated pleomorphic sarcoma,
LMS = leiomyosarcoma, LS = liposarcoma, MPNST = malignant peripheral nerve sheath
tumor.
*Located 15 cm from the first tumor.
†The patient developed one additional LS, left thoracic wall.
MET: metastases, TD: tumor death, NED: no evidence of disease.Clinical data for the 13 cases are
presented in Table1. The lower extremity was the most common tumor site (16
tumors) and 28 tumors were high-grade (grades 3 and 4 on a 4-tiered scale). The
first STS included eight malignant fibrous histiocytomas/undifferentiated
pleomorphic sarcomas (MFH/UPS), two leiomyosarcomas, two malignant peripheral
nerve sheath tumors (MPNST), and one pleomorphic liposarcoma. The
histopathological diagnosis of the second STS differed from the first in two
patients; a leiomyosarcoma was diagnosed in a patient with two prior MFH/UPS
and a leiomyosarcoma was diagnosed in a patient previously operated on for an
MPNST. In the remaining patients, including the three cases from which three or four distinct tumors were analyzed, multiple STS of the same
histopathological type were diagnosed.In 9/13 patients the STS developed
at different anatomical locations, for example, different extremities or
extremity and trunk wall. Three patients developed metachronous STS in the same
extremity but at different locations, for example, lower leg and thigh (cases
8, 12, and 13) and one patient (no. 7) developed two STS in the same extremity;
a deep-seated leiomyosarcoma in the medial thigh and five years later a subcutaneous
leiomyosarcoma in the lateral part of the thigh (Table1). Clinical follow-up
was complete for a minimum of 8 years for the survivors. During follow-up, two
patients (cases 12 and 13) developed local recurrences, 1 and 10 years after
primary surgery. Lung metastases developed in 5/13 patients, median 50 (range
15–51) months after
diagnosis of the primary tumor. Apart from the metachronous STS, two patients
(cases 4 and 7) developed adenocarcinomas of the breast and the colon,
respectively. Ethical permission for the study was granted from the Lund
University ethics committee.


## 2.2. DNA Extraction and Array-Based Comparative Genomic Hybridization

Genomic DNA from frozen (n=15) and
paraffin-embedded (n=15) tumors was extracted using the Wizard Genomic DNA
Purification kit (Promega, Madison, WI) and overnight proteinase-K digestion
treatment followed by phenol-chloroform purification. When paraffin-embedded
tissue was used, a fresh4-μm section was obtained, stained with hematoxylin
& eosin and a representative tumor area was chosen. Thereafter, 1-mm tissue
cores were obtained and used for DNA extraction. The tissue cores were
pretreated in xylene before proteinase-K treatment and phenol-chloroform
purification. DNA quality was checked using a Ready-To-Go RAPD analysis kit
(Amersham Biosciences, Buckinghamshire, UK), and the concentration was measured
using a Nano drop (NanoDrop Technologies, Wilmington, Del, USA). Commercial genomic
male DNA, derived from a pool of healthy individuals, was used as a reference
(Promega, Madison, Wiss, USA). CyDye coupling/labeling was carried out
using a random labeling kit (Invitrogen Life Technologies, Carlsbad, Calif, USA)
according to the manufacturer's recommendations. In short, 2-μg genomic tumor
DNA and reference DNA were differentially labeled with fluorescent dyes, Cy3
for tumor tissue, and Cy5 for reference DNA. After a purification step, these
were pooled, mixed with COT-1 DNA to block repetitive DNA sequences, dehydrated
and resuspended in a formamide-based buffer (Invitrogen). The labeled DNA was
then applied to arrays pretreated in washing solutions (Pronto! Microarray
Reagent System, Corning Labsystems, Corning, NY, USA) and hybridization was
performed for 48–72 hours at 37°C.
The incubation was performed under cover slips for
the DNA isolated from the frozen tumor material whereas the MAUI hybridization
System (BioMicro systems Inc., Salt
Lake City, Utah, USA) was
used for the DNA derived from paraffin-embedded tumors. Dye-swaps (i.e.,
complementary hybridization in which Cy5 was used for tumor tissue and Cy3 for reference DNA) were used in three cases and allowed subtraction of dye-related noise. The slides were treated in post-hybridization
washing solutions and finally scanned using an Agilent Microarray scanner
(Agilent Technologies, Palo Alto, Calif, USA).


## 2.3. BAC Array Platform

The BAC array slides used were
produced at the Swegene DNA Microarray Resource Center,
Department of Oncology, Lund University. These have an
average resolution of 80 kb and contain a total of 32 433 BAC clones from
the 32 k human genome high-resolution BAC rearrayed clone set, version 1.0 from
the BACPAC Resource Center at Children's Hospital Oakland Research Institute
(Oakland, Calif, USA) (http://bacpac.chori.org/).
The clones provide >99% coverage of the fingerprint map and current sequence
assembly with a resolution of 100 kb.


## 2.4. Data Analysis

Image analysis and data extraction
were carried out using GenePix Pro 4.1.1.4 version (Axon Instruments Inc.,
Foster City, CA, USA) and the quantified data matrix was then uploaded into the
web-based BioArray Software Environment (BASE;http://gothmog.thep.lu.se/int/index) [21], where all data management and analysis were carried out. The
background correction and intensities of Cy3 and Cy5 were calculated using the
median feature and median-local background intensities of the uploaded files,
and the intensity ratios were calculated using the background corrected spot
intensities by calculating the log2 ratios of tumor to reference
intensity. In BASE a preliminary filter, based on the flagging in the image
analysis, was applied, and spots with a diameter <55 μm and a signal to
noise (SNR) ratio ≤3 in the tumor or reference channel were flagged as “bad”
and filtered away from further analysis. The intensity-dependent LOWESS
algorithm [22] was used to normalize the data within individual arrays. To
correct for spatial bias, the data were normalized within groups of 8 print-tip
blocks. A moving average smoothing algorithm with a 250 kbp sliding window was
then applied, and a BASE-adapted CGH-plotter software was used to identify
regions of gains and losses [23]. In the CGH-plotter, each clone was assigned a
calculated level log2 ratio value, corresponding to the level that
the clone belongs to, in order to reduce the noise. Hereafter an unsupervised
hierarchical cluster analysis, using the Pearson correlation distance metric
and the average linkage method, was applied to the data derived from the
CGH-plotter (the TMeV application from the TM4 microarray software suite was used; http://www.tm4.org/mev.html). The CGH-plotter was also used to generate a ternary scale, where all clones were designated gained, lost or unchanged. These values were used to calculate the percentage
of altered clones in each assay and the mean number of altered clones in tumor
subgroups. Pearson correlation was used to determine the correlation between
tumors within the same individual, based on the number of altered clones. Gains
and losses were defined as a log2 ratio ±0.2. Amplifications were
defined as clones with a log2 ratio ≥0.5, whereas high-level
amplifications were defined as a log2 ratio ≥1.5. Homozygous
deletions were suspected when the log2 ratio was ≤1.5.


## 3. Results

Genomic profiles from the 30
metachronous STS of five different histopathological subtypes revealed multiple
gains and losses and identified several high-level amplifications (HLA) and homozygous
deletions (Table2). The alterations affected mean 39 (9–70)% of the whole
genome with 19% amplifications and 20% deletions. When the first STS (n=13)
were compared to the subsequent STS (n=17) a small difference in the total
number of alterations was found with 35 (16–54)% and, 42 (9–70)% of the
genome altered, respectively. Several recurrent aberrations were identified
with the most frequent changes (present in >60% of the tumors) being
deletions of 10q24.3–25.2, 13q12.1–12.2, 13q21.1–21.2, 16q13–23.2, 18q12.2–12.3, and
amplifications of 1q21.3–23.1 and 19p13.3.Table 2
Recurrent high-level amplifications and homozygous deletions.
ChromosomeNo of tumorsCytobandStart positionSize (Mbp)HLA/Hz del*Cancer related genes121p32.1585714821.2HLAJUN131q442415581901.0Hz del323q231410824840.51Hz del333p12.1–3787802187.0HLA424q12540163341.5HLAPDGFRA, KIT, CHIC2636p 12.3–21.23736064011.4HLA969p21.3216474332.4Hz delCDKN2A, CDKN2B, MTAP10210q23.31895384201.5Hz delPTEN11211q13.4–5717300975.3HLA131313q14.2479011600.65Hz delRB1*HLA = high level amplification, Hz del = homozygous deletion.Unsupervised hierarchical cluster
analysis, based on the ~27 000 clones that survived the filters in BASE,
revealed close clustering of the tumors from five individuals without
significant differences between the first and subsequent STS (43% and 41% of
the genome altered) (Figure1 and Table 1, cases 1, 2, 6, 9, and 12). These
tumor pairs showed strong similarities between the genomic plots (Figure 2(a)) with
a mean correlation of 0.7 (0.5–0.9).  The many shared alterations outnumbered the
few differences in all five cases and deletions identified in the first tumor
were always present in the second STS. The median time interval between the
first and second STS in these five patients was 1 (1–7) year, and two
of these patients subsequently developed lung metastases. In the remaining eight
cases STS from the same individual did not cluster together and showed a
significantly weaker correlation, mean 0.1 (0–0.4). These
tumors had pronounced intertumor variability (30% of the genome being altered
in the first tumor compared to 42% in the subsequent tumors), which was
comparable to the interpatient variability, which had a mean correlation of 0.1
(0.04–0.2). In four of
these cases, deletions present in the first tumor were not present in the
subsequent tumor, which supports independent tumor origin. The second STS in
these eight cases developed median 4 (1–5) years after
the first STS and three of the patients later developed pulmonary metastases.Figure 1
Unsupervised hierarchical cluster analysis of all 30 soft tissue sarcomas from
13 patients. The analysis was based on the ~17 000 clones that survived
the preprocessing filters, and close clustering of the metachronous STS was demonstrated
in 5 patients (case 1, 2, 6, 9, and 12 marked with different colours), whereas
the STS from the remaining 8 patients were scatter in the analysis. Clustering
was done using the TMeV application from theTM4 microarray software suit.Superposition
of whole genome plots from two different tumors from the same patient showing (a)
similarities in gene copy number changes in two liposarcomas (case 6). (b) Differences
in gene copy number changes in two malignant fibrous histiocytomas (case 3).
(a)(b)


## 4. Discussion

Despite multidisciplinary and
multimodality treatment, distant metastases develop in about 30% of STS
patients. Hematogenous, pulmonary metastases predominate, whereas lymphatic
spread occurs in <5% of the patients [19, 24]. Soft tissue metastases are
rare and have mainly been reported in liposarcomas [16, 25]. Development of synchronous
or metachronous STS has been described in several case studies, but it remains
a rare clinical presentation and the interpretations hereof have varied [25–29].
Epidemiological data support an increased risk of a secondary primary sarcoma
among adult STS patients [14, 17]. In order to reduce bias from inclusion of
familial sarcoma syndromes, we excluded multiple sarcoma patients diagnosed with
neurofibromatosis or the Li-Fraumeni syndrome. The only patient who developed
more than one MPNST was carefully examined without any sign of neurofibromatosis
(until death 3 years later). Since
only one patient had received adjuvant chemotherapy after the first STS and
none of the second STS developed in irradiated fields, the second STS studied
are unlikely to represent treatment-induced secondary tumors.Application of CGH in STS has mainly
involved leiomyosarcoma and MFH/UPS and has in these highly malignant and
pleomorphic STS subtypes demonstrated extensive genomic complexity with
recurrent copy number changes, including losses of 2p, 2q, 10q, 11q, and 13q and
gains of 1q, 5p, 8q, and 17p [30–36]. Several of
these recurrent changes were also identified among the 30 STS in this study
with the most frequent being deletions of 10q24.3–25.2, 13q12.1–12.2, 13q21.1–21.2, 16q13–23.2, 18q12.2–12.3, and
amplifications of 1q21.3–23.1 and 19p13.3.
In order to obtain as many tumor pairs as possible, frozen as well as
paraffin-embedded tumor tissue was used. Among the tumors from which
high-quality DNA was obtained, no differences were identified related to tumor
source (Table 1). When the genomic profiles from the different tumor pairs were
compared, five pairs showed highly correlated genomic profiles suggestive of
metastatic disease, whereas eight cases showed different profiles suggestive of
distinct primary STS. In the latter STS, the differences by far outnumbered the
similarities, which resulted in a weak correlation, which was comparable to the
interpatient variability. Importantly, there was no systematic difference in
sample preparation methodology between the two groups suggestive of metastatic
disease and distinct primary STS, respectively. The similarities and
differences were evident in an unsupervised hierarchical cluster analysis (Figure 1) in which the five former tumor pairs clustered closely, whereas the latter eight
did not.Previous reports of multiple STS
have predominantly involved liposarcomas. In a mixed series of nine STS
patients who developed synchronous and metachronous STS, Grobemyer et al. identified liposarcomas (n=5)
and gastrointestinal stromal tumors (n=5) as the most frequent subtypes whereas
Blair et al. reported 16
patients with multiple STS of whom nine had liposarcomas [20, 37]. Our study only
included successfully analyzed liposarcomas from one patient (case no. 6) who
developed four liposarcomas in four years without signs of lung metastases and died
from locally advanced tumor masses 22 months later. Antonescu et al. applied Southern blot analysis
to tumors from six patients with multifocal myxoid liposarcomas and hereby
verified monoclonality, thus demonstrating that multiple myxoid liposarcomas in
the same individual most likely represent recurrent disease [16]. Similar
genetic profiles were in our series present in metachronous STS of the same
histopathological types; three MFH/UPS, one liposarcoma, and one leiomyosarcoma,
which developed with median 1 year interval. Subsequent development of lung
metastases occurred in two of these five patients (Table 1). Previous studies
that have examined genetic differences between primary and recurrent STS have
been carried out using conventional CGH and have demonstrated increasing genetic
complexity from primary STS to a local
recurrence [38–40]. However, the issue of development of second primary STS has to our
knowledge not been addressed using genetic profiling. We demonstrate strikingly
similar genetic profiles in the five STS likely representing soft tissue
metastases with mean 43% and 41% of the genome altered and several shared
deletions identified. This stands in contrast to the eight STS patients from
which the metachronous STS showed different genetic profiles. Among these,
multiple histologic subtypes (i.e., MFH/UPS, MPNST, and leiomyosarcomas) were
present and four of the deletions identified in the primary tumors were not
found in the second STS. In summary, the clinical presentation, histopathology,
and the genetic profile support independent sarcoma origin in 8 of the 13
patients (Table 1). Although development of metachronous STS is rare, our
demonstration of different genetic profiles in the majority of these cases
serves as a reminder to consider independent tumor origin, which has implications
for the choice of therapy, for example, use of adjuvant chemo- and/or
radiotherapy after surgery for a second STS that should not per se be perceived to represent metastatic
disease.



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*Source: 431019-2009-02-02.xml*