Research Article HIV-1 Evolutionary Patterns …downloads.hindawi.com/journals/sarcoma/2016/4510483.pdfResearch Article HIV-1 Evolutionary Patterns Associated with Metastatic Kaposi

Research ArticleHIV-1 Evolutionary Patterns Associated with MetastaticKaposirsquos Sarcoma during AIDS

Susanna L Lamers1 Rebecca Rose1 David J Nolan2 Gary B Fogel3 Andrew E Barbier1

Marco Salemi2 and Michael S McGrath4

1Bioinfoexperts LLC 718 Bayou Lane Thibodaux LA 70302 USA2Department of Pathology and Laboratory Medicine University of Florida 2055 Mowry Road Gainesville FL 32610 USA3Natural Selection Inc 5910 Pacific Center Blvd 6480 Weathers Place San Diego CA 92121 USA4The AIDS and Cancer Specimen Resource University of California at San Francisco and the Department of Laboratory MedicinePathology andMedicine University of California at San Francisco 1001 Poterero Ave Bldg 3 Rm 207 UCSF Box 1317 San FranciscoCA 94110 USA

Correspondence should be addressed to Michael S McGrath mikemcgrathucsfedu

Received 11 May 2016 Accepted 7 August 2016

Academic Editor C Verhoef

Copyright copy 2016 Susanna L Lamers et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Kaposirsquos sarcoma (KS) in HIV-infected individuals can have a wide range of clinical outcomes from indolent skin tumors to alife-threatening visceral cancer KS tumors contain endothelial-related cells and inflammatory cells that may be HIV-infected Inthis study we tested if HIV evolutionary patterns distinguish KS tumor relatedness and progression Multisite autopsies fromparticipants who died from HIV-AIDS with KS prior to the availability of antiretroviral therapy were identified at the AIDSand Cancer Specimen Resource (ACSR) Two patients (KS1 and KS2) died predominantly from non-KS-associated disease andKS3 died due to aggressive and metastatic KS within one month of diagnosis Skin and visceral tumor and nontumor autopsytissues were obtained (119899 = 12) Single genome sequencing was used to amplify HIV RNA and DNA which was present in alltumors Independent HIV tumor clades in phylogenies differentiated KS1 and KS2 from KS3 whose sequences were interrelatedby both phylogeny and selection HIV compartmentalization was confirmed in KS1 and KS2 tumors however in KS3 nocompartmentalizationwas observed among sampled tissuesWhile the sample size is small theHIV evolutionary patterns observedin all patients suggest an interplay between tumor cells and HIV-infected cells which provides a selective advantage and couldpromote KS progression

1 Introduction

Prior to the human immunodeficiency virus (HIV) epidemicKaposirsquos sarcoma (KS) was considered a very rare diseasefound primarily in Mediterranean countries and in organtransplant recipients [1] Although Human Herpes Virus 8(HHV8) is the etiological cause of KS [2] most HHV8+individuals do not develop KS Early in theHIV epidemic KSwas so frequently diagnosed that it was considered an AIDS-defining illness [3ndash5] Prior to the mid-1990s and the use ofcombined antiretroviral therapy (cART) HIV+ patients hada 40 lifetime risk of developing KS [6] Although combined

antiretroviral therapy (cART) has undeniably reduced KS-associated mortality among HIV-infected patients [7ndash9] thestatistical impact is unclear due to differing clinical defini-tions and cART adherence problems [10 11] FurthermoreKS is still one of the most common cancers among HIV+patients [12] despite cART-improved CD4+ T-cell countslow viral loads and reduced risk of opportunistic infections[13ndash16] KS is particularly problematic in resource-poorsettings where cART and chemotherapy are limited [6 17ndash19] however even in resource-rich settings full remission ofKS using cART and chemotherapy occurs in less than 50 ofHIV+ patients with disseminated disease [20]

Hindawi Publishing CorporationSarcomaVolume 2016 Article ID 4510483 10 pageshttpdxdoiorg10115520164510483

2 Sarcoma

KS lesions can be solitary localized or disseminatedbut the relationship among tumors in a single individualis largely unknown While many KS lesions are cutaneousand indolent tumors can occur in the oral cavity lymphnodes and viscera which can result in fatal complica-tions due to interference with normal body function Thegrowth of lesions can be very slow or explosively fastThe KS defining cell-type is a spindle cell (SC) [21 22]that expresses markers of both endothelial and macrophagelineages [23] KS tumors are polyclonal positive for thepresence of HHV8 virus which infects the tumor-associatedB-cells and SCs [23] and highly vascularized giving thema distinguishing purple color Inflammatory lymphocytesand monocytesmacrophages populate and enrich the KStumor environment for tumor growth through productionof a variety of cytokines [23] These immune cells are allpotential targets for HIV however to our knowledge onlyone study assessed KS tumors for the presence of HIV usingan early hybridization technique [24] SCs can be culturedfrom PBMCs of patients with KS suggesting that circulatingSCs may lead to the appearance of multiple KS lesions [23]

HIV has the potential to infect and evolve in manyanatomical tissues including tumors [25ndash27] Previouslywe showed that the viral populations in lymphoma tumorswere distinct from HIV in healthy tissues [25] Here ourgoal was to characterize HIV populations in KS tumors todetermine whether HIV evolution could be used to tracktumor relatedness and progression in native KS uncompli-cated by the use of cART We obtained tumor and nontumortissues sampled post mortem from the AIDS and CancerSpecimen Resource (ACSR) from three HIV+KS+ patientswho died from aggressive AIDS and we generated HIVenv-nef sequences using single genome sequencing (SGS) tocharacterize viral evolutionary patterns

2 Materials and Methods

21 Patients and Biomaterial The three male patientsincluded in this studywere diagnosedwithHIV infection anddied with advanced AIDS without receiving cART (Table 1)100mg of frozen autopsy tumor and nontumor tissues wasobtained through the ACSR (httpacsrucsfedu) Tissueswere classified as tumor or nontumor by histological exam-ination The ACSR is a National Cancer Institute-fundedtissue-banking program that obtains tissues from patientsafter appropriate consent and releases tissues to investigatorsfollowing approval of the proposed study and deidentificationof samples and clinical histories The ACSR is recognized bytheOffice of Biorepositories and BiospecimenResearch at theNational Institutes of Health as being HIPAA compliant andin accordance with the ethical standards of the Declarationof Helsinki Additionally all material was obtained underapproval from the University of California at San FranciscoCommittee on Human Research

22 RNADNA Extractions Total RNA and genomic DNAwere isolated separately from each tissue section (30ndash50 ng)using AllPrep DNARNAMini Kit (Qiagen 80204) Tissueswere homogenized just prior to extraction in Buffer RLT Plus

(lysis buffer) using a TissueRupter rotor-stator homogenizer(Qiagen 9001271) with a fresh sterile disposable probe(Qiagen 990890) for each sampleManufacturerrsquos guidelineswere followed with the exception of two 50 120583L final elutionsusing RNase-free water during the RNA isolationThe 100 120583Lfinal volume of RNA generated was concentrated usingRNeasyMinElute Cleanup Kit (Qiagen 74204) RNA andDNA extractions cDNA synthesis and first-round PCRsetup were conducted in a restricted-access amplicon-freeroom with separate air handling and laboratory equipmentwhere no amplified PCR products or recombinant clonedplasmids were allowed and work surfaces and equipmentwere thoroughly cleaned before and after use with Eliminase(Decon Labs Inc) cDNA was synthesized using Super-Script III First-Strand Synthesis System (Invitrogen LifeTechnologies 18080-051) using the provided Oligo(dT)

20

primer according to manufacturerrsquos recommendations RNAwas incubated at 65∘C for 5 minutes with deoxynucleosidetriphosphates (05mM) and 5 120583M Oligo(dT)

20and then

cooled quickly to 4∘C First-Strand cDNA Synthesis wasperformed in a 40 120583L reaction volume containing 1x reversetranscription buffer (10mM Tris-HCl [pH 84] 25mMKCl)5mMMgCl

2 10mM dithiothreitol 2 U120583L of RNase-OUT

(RNase inhibitor) and 10U120583L SuperScript III RT Thereaction was heated to 50∘C for 50 minutes followed by85∘C for 5 minutes The reaction was cooled to 37∘C and01 U120583L of E coli RNase H was added followed by a 20-minute incubation cDNA was stored at minus20∘C

23 Sequencing Protocol Amodified single genome sequenc-ing protocol was used to amplify linked HIV env andnef sequences [28] cDNA and genomic DNA were seri-ally diluted until an average of 30 or less of the nestedPCR reactions were positive During the first-round PCRdiluted cDNA or genomic DNA was amplified in 25120583Lreactions containing 1x Platinum Blue PCR SuperMix(Invitrogen Life Technologies) and 02 120583M of each primerEF2 51015840-ACAGTCTATTATGGGGTRCC-31015840 and NR1 51015840-AGCTCCCAGGCTCAGATCT-31015840 (6333ndash6352 bp and 9558ndash9576 bp of HIV HXB2 resp) with the following cyclingparameters initial denaturation 95∘C for 5 minutes followedby 35 cycles of denaturing at 94∘C for 1 minute annealing at58∘C for 1 minute and extension at 72∘C for 4minutes with afinal extension time of 10 minutes at 72∘C A second round ofgp120 PCR consisted of 1120583L of the first-round PCR added toa 24120583L second-round reaction consisting of 1x PlatinumBluePCR SuperMix (Invitrogen Life Technologies) and 02 120583Mof each primer EF3 51015840 CATAATGTTTGGGCCACACA-31015840 and ER2 51015840-CACCACTCTTCTYTTTGCC-31015840 (6420ndash6439 bp and 7724ndash7742 bp of HIV HXB2 resp) with thefollowing cycling parameters initial denaturation 95∘C for 5minutes followed by 35 cycles of denaturation at 94∘C for1 minute annealing at 58∘C for 1 minute and extension at72∘C for 2 minutes with a final extension time of 10 minutesat 72∘C This second-round PCR generated a 13 Kb productnearly covering the entire env gp120 gene Second-roundenv gp120 PCR products were visualized on 1 agarose gelsstained with ethidium bromide and reactions containing asingle 13 Kb product were considered positive and selected

Sarcoma 3

Table1Patie

ntclinicalh

istory

Patie

ntAge1

Major

comorbiditie

s2KS

locatio

nTimes

ince

KSdiagno

sis(m

onths)

Leng

thof

HIV

infection(years)

Anatomicallocatio

nof

biop

sy

Con

centratio

n(ng120583L)

Num

bero

fenv

sequ

ences

Num

bero

fnef

sequ

ences

RNA

DNA

RNA

DNA

RNA

DNA

KS1

34Lymph

omaMAC

PC

Pcytomegaloviru

s

Skinpleuralung

strachealymph

nodeliver

adrenalgland

sUnk

nown

7

KSskin

144

154

228

249

KSlymph

node

168

276

1321

1421

Lymph

node

120

121

3722

3526

Kidn

eygt200

818

50

90

KS2

31BC

BLSkinpleuralung

sesop

hagussm

allbow

elsim5

Unk

nown

KSskin

165

4414

1018

12KS

smallbow

el192

984

40

50

Liver

191

842

00

00

Spleen

190

139

231

312

KS3

28AggressiveK

S

Skinpleuralung

stracheaesop

hagussm

all

bowel

colon

rectum

iliaclym

phno

des

mesenteric

lymph

nodespara-aortic

lymph

nodesparatracheallymph

nodes

sim1

9

KSskin

4951

62

26

KSsm

allbow

el164

394

1625

1424

Liver

161

334

145

155

Stom

ach

782

402

248

208

1Age

inyears2MAC

Mycobacteriu

mavium

complexP

CPP

neum

ocystis

pneumoniaB

CBLbo

dycavitybasedlymph

oma

4 Sarcoma

for sequencing Subsequently the first-round reactions thatcorresponded to positive second-round gp120 PCRs werethen used to amplify the nef gene sequence Second-roundnef PCR consisted of 1 120583L of the first-round PCR added to a24120583L second-round reaction consisting of 1x Platinum BluePCR SuperMix (Invitrogen Life Technologies) and 02 120583Mof each primer NF1 51015840-TTAGGCAGGGATAYTCACC-31015840and NR2 51015840-ATCTGAGGGCTCGCCACT-31015840 (8347ndash8365 bpand 9488ndash9505 of HIV HXB2 resp) with the followingcycling parameters initial denaturation 95∘C for 5 min-utes followed by 35 cycles of denaturation at 94∘C for 1minute annealing at 58∘C for 1 minute and extension at72∘C for 2 minutes with a final extension at 72∘C for 10minutes Second-round nef PCRs were visualized on 1agarose gels stained with ethidium bromide and reactionscontaining single 11 Kb products were considered positiveand selected for sequencing The primers were designedusing Primer3 (httpbioinfouteeprimer3-040) and byobserving regions of conservation in alignments of publishedHIV subtype B sequences downloaded fromLosAlamosHIVSequence Database (httpwwwhivlanlgov) Sequencingwas performed on an Applied Biosystems 3730xl DNA Ana-lyzer (Life Technologies) at the University of Florida Inter-disciplinary Center for Biotechnology Research (UF ICBR)All sequences were assembled and aligned with the GeneiousR7 software package (Biomatters httpwwwgeneiouscom)followed by a manual optimization of regions containinginsertions and deletions Sequence data has been submittedto GenBank (Accession numbers KU709129ndashKU709831)

24 Sequence Analysis Env and nef alignments were gen-erated using ClustalW [29] in MEGA5 [30] with furtheroptimization performed by hand Due to a large numberof insertions and deletions that are typically problematic toalign regions with substantial insertions and deletions in envV1 V2 andV4 domains were removed An initial maximum-likelihood phylogeny was inferred using sequences from allpatients for both genes to ensure no cross-contaminationPairwise distance analysis within and between RNA andDNA tissue sequence populations over time was assessed inMEGA5 [30] using the Tamura-Neimolecularmodel (identi-fied usingModel-Selection) with standard error estimated by1000 bootstrap replicates The Kruskal-Wallis test was usedto determine significance between the mean pairwise dis-tances in different anatomical tissues Individual maximum-likelihood phylogenies were inferred for each patient under aGTR model of nucleotide evolution and gamma distributedrate variation among sites (+G) using PhyML [31] Statisticalsupport was assessed with 200 bootstrap replicates TheSlatkin-Maddison test [32] was used to determine com-partmentalization between sequences derived from differenttissues and significance was assessed using 1000 replicatesof tip randomization implemented in HYPHY v10 [33]Thefast unconstrained Bayesian approximations for inferringselection (FUBAR) model implemented in Data Monkey(httpdatamonkeyorg) were used to identify amino acidsites under selection in sequences derived from differenttissues for each individual Sites with posterior probabilitygt09 of an increased (diversifying) or decreased (purifying)

rate of nonsynonymous relative to synonymous substitutionswere considered to have experienced a significant level ofselective pressure Selected sites were numbered accordingto the molecular HIV clone HXB2 (GenBank AccessionAF358141)

3 Results

31 Clinical Characteristics of the Patient Cohort All patients(KS1ndashKS3) had CD4 counts lt50 cellsmm3 at death and werediagnosed with KS only months prior to death (Table 1)KS1 lived with HIV infection for seven years He was hospi-talized due to Mycobacterium avium complex Pneumocystispneumonia and wasting syndrome eleven months prior todeath Five months prior to death medical notes indicatemultipleAIDS-like symptoms however KSwas not specifiedAt autopsy numerous infections were identified throughouthis body including disseminated KS which either had notbeen previously reported or had originated closer to deathKS2rsquos clinical history is similar to that of KS1 in havingmultiple infections however this patient had an initial KSdiagnosis five months prior to death Also this patient wasdiagnosed with an aggressive body cavity based lymphoma(BCBL) four months prior to death that was treated withchemotherapy BCBL may be related to primary effusionlymphoma which is typically caused by HHV8 the Kaposisarcoma herpes virus KS3 had a nine-year history of HIVinfection with reoccurring upper respiratory tract infectionsHe was diagnosed with KS one month prior to death HisKS lesions became rapidly disseminated on the skin andinternally which lead directly to his death due to respiratorydistress

32 HIV RNA and DNA Was Successfully Sequenced fromAll KS Tumors Viral sequences were obtained from RNAin 1314 tissues and from DNA in 1214 tissues (Table 1)A preliminary maximum-likelihood (ML) phylogenetic treeof all sequences confirmed a distinct population of HIVsequences for each patient indicating no laboratory cross-contamination

33 KS Tumors Can Contain Distinct HIV Isolates ML phy-logenies were inferred for env and nef sequence populationsfor all three patients In the KS1 env phylogeny four distinctand well-supported clades (AndashD) were apparent that con-tained the majority of tumor-derived sequences (Figure 1)The relatively long internal branches leading to each cladeindicate considerable divergence from the rest of the viralpopulation The majority of lymph node tumor sequences(1934) were within two distinct clades (A D) The majorityof skin tumor sequences (2032) were also located in twodifferent distinct clades (B-C) One of these (B) containedboth DNA and RNA sequences while the other (C) wascomprised exclusively of RNA skin tumor sequences Mostof the terminal branches leading directly to skin tumor-derived sequences were very short while those leading tothe lymph node tumor-derived sequences were considerablylonger and showed more branching within the clade The

Sarcoma 5

env

A

B

C

D

E

F

001

Lymph node tumor DNA Lymph node tumor RNASkin tumor DNA Skin tumor RNA

Kidney RNA Lymph node DNALymph node RNAgt85 bootstrap

(a)

nef



A

B

C

D

E

001

(b)

Figure 1Maximum-likelihood phylogenies for KS1 Phylogenies were inferred for each patient using env (a) and nef (b) sequence alignmentsTips are labeled with symbols corresponding to the tissue of origin for each sequence Branches with red lines indicate bootstrap valuesgt85200 Branches are scaled in substitutionssite according to the scale under each tree Clades labeled AndashF are discussed in the text

remaining lymph node and skin tumor RNA and DNAsequences were located in highly mixed clades (E-F) The nefphylogeny for KS1 had similar branching patterns with evenmore pronounced diversity in one lymph node tumor clade(C) Similarly all skin tumor-derived sequences except oneclustered together in a distinct and well-supported clade inKS2 for both env and nef (A) (Figure 2) Small bowel tumor-derivedRNA sequenceswere interspersedwith the nontumorspleen-derived sequences in both genes however few bowel-derived sequences were obtained (119899 = 9) Branches in thenontumor clade showed considerable structure in both genesconsistent with a continuously diversifying virus populationIn the nef phylogeny two distinct clades were evident withinthemixed clade whichwas not present in the env tree In con-trast for KS3 all tumor- and nontumor-derived sequenceswere largely interspersed in both env and nef phylogenies(Figure 3) Three well-supported clades were present in bothgenes (AndashC) Interestingly this patient was diagnosed withadvanced and aggressive tumorigenesis less than one monthprior to death

34 KS-Associated Virus Is Compartmentalized in Tumorsin KS1 and KS2 but Not in KS3 When pairwise distanceswere calculated for sequences derived from each tumor andnontumor tissue only one significant differencewas observed

(119901 lt 0001) (KS1 lymph node tumor nef versus nontumornef sequences) Viral populations were significantly com-partmentalized within each of the two tumors and normaltissues for both env and nef in KS1 and KS2 (119901 lt 0001) Onthe other hand gene flow among the three sites was foundin both genes in KS3 (env 119901 = 0021 nef 119901 = 0171)Viral populations from combined tumor locations were againsignificantly compartmentalized compared with nontumorpopulations in env and nef in KS1 and KS2 as well as in neffor KS3 (119901 lt 0001)

35 Positively and Negatively Selected Codons Were Fewer andTissue-Specific in KS1 and KS2 in Contrast to KS3 (Tables 2and 3) The proportion of codons under selection in envdid not significantly vary between positively and negativelyselected sites overall or within any single subject Howeverthe number of positions under either selective pressure wassignificantly higher in KS3 than in KS1 and KS2 (119901 lt 001)In nef a significantly higher number of negatively selectedcodons over positively selected codons were observed in allpatients (119901 lt 001) Codons under selection were tissue-specific in both KS1 and KS2 with only one being sharedbetween two tissues in KS2 In contrast in KS3 env sequencepopulations all shared positively and negatively selectedcodons in tumor tissues were also identifiedwithin nontumor

6 Sarcoma

env

A

B

C

001

Small bowel tumor RNASkin tumor DNASkin tumor RNA

Spleen DNASpleen RNAgt85 bootstrap



nef

A

B

C

001

Figure 2 Maximum-likelihood phylogenies for KS2 See legend under Figure 1

env

A

B

C

001

Small bowel tumor DNASmall bowel tumor RNASkin tumor DNASkin tumor RNA

Liver DNALiver RNAStomach DNAStomach RNAgt85 bootstrap



nef

A

B

C

001


Sarcoma 7

Table 2 Number (and percent) of codons under positive or negative selection in tumor and nontumor sites in env sequence populations

Patient Tissue positivelyselected sites ()

Codon position of sharedpositively selected sites

negativelyselected sites

Codon position of sharednegatively selected sites

KS1 (359)

Skin tumor 7 (19) None 7 (19) naLymph node

tumor 1 (03) None 7 (19) 122

Nontumor None None 8 (22) 122

KS2 (394)

Skin tumor 8 (20) 464 5 (12) 150Small bowel

tumor None None None None

Nontumor 5 (12) 373 4 (10) 150

KS3 (373)

Skin tumor 3 (08) 464 6 (16) 207 247Small bowel

tumor 10 (26) 183 336 396 16 (42) 103 118 207 228 238 246254 276 374 408 415 445

Nontumor 11 (29) 143 167 183 336 454 21 (56) 103 118 207 228 238 246254 276 374 408 415 445

Table 3 Number (and percent) of codons under positive or negative selection in tumor and nontumor sites in nef sequence populations

Patient Tissue positivelyselected sites




KS1 (207)

Skin tumor 0 None 3 (15) 188Lymph node

tumor 4 (19) None 18 (87) 8 37 188

Nontumor 1 (05) None 4 (19) 8 37 188

KS2 (205)

Skin tumor 1 (05) 190 0 NoneSmall bowel

tumor 1 (05) 190 4 (20) 81

Nontumor 2 (10) 190 5 (24) 81

KS3 (207)

Skin tumor 1 (05) None 7 (34) 30 65 147 151 191 195Small bowel

tumor 1 (05) None 14 (68) 4 30 63 65 111 129 151164 191 195

Nontumor 1 (05) None 21 (101) 4 63 111 129 147 151 164191 195

sequences similarly in KS3 nef sequence populations allshared selected codons from tumors were identified innontumor tissues except codon positions 31 and 67

4 Discussion

In this study we examined if HIV evolutionary patterns coulddifferentiate metastatic KS in patients untreated with cARTWe analyzed HIV env and nef sequences from tumor andnontumor anatomical sites from three subjects who died withKSMedical records indicated that subjects KS1 andKS2 had aslower KS progression than subject KS3 however all patientshad visceral KS indicating an advanced cancer at deathfrom AIDS HIV RNA andor DNA was amplified from alltumors in the study demonstrating that similar to a previousstudy [25] cancer tissues contain substantial amounts ofHIVFurthermore HIV sequence populations from most tissueshad similar genetic distances indicating no constraints toHIV evolution in any of the tissue environments examined

HIV from tumor sites in KS1 and KS2 was compart-mentalized relative to the nontumor virus with limited geneflow between compartments however subpopulations ofvirus were also present within tumors Additionally codonselection analysis demonstrated that at the protein level KS1and KS2 had sequence populations evolving under indepen-dent selective pressures in different tumornontumor sitesIn contrast the patient who died from highly aggressive KS(KS3) showed remarkably different patterns Here the virusin tumor and nontumor tissue was completely interspersedwith a non-tissue-specific viral population structure Thesefindings could result from a number of scenarios includingthe following (1) subpopulations of virus in tumor couldresult from recent viral migration from another unsampledanatomical location (2) the viral population in the tumors isevolving independently due to a physical barrier restrictinggene flow (3) the viral populations in the tumors are evolvingin response to local selective pressures (4) viral sequencesin one or more sites could represent archival virus that was

8 Sarcoma

reactivated because of the cancer growth The first scenariois untestable here however we are conducting additionalstudies withmore tumor and nontumor tissues fromotherKSpatients including KS derived from patients on cART withno detectable plasma viral load While the second scenariois possible unlike the brain in which viral flow is impededby the blood-brain barrier KS tumors are highly vascular-ized which would in theory provide ample opportunity formigration Furthermore a small number of tumor viruseswere found in nontumor tissues also suggesting a lack ofphysical barrier In considering the third and fourth scenariowe did find that codon positions were under varied selectivepressures in the tumors fromKS1 and KS2 when compared tonontumor tissues (as expected from the phylogeny) For KS3most but not all selected codons were shared among tissueshowever this patient had widely metastatic KS at the time ofdeathTherefore it is possible that tumor-compartmentalizedHIV populations could have existed in this patient priorto the rapid outgrowth and spread of the cancer perhapsrepresented by one of the distinct subpopulations

Reactivation and clonal expansion of viral populationscould be consistent with the large clades of identical or nearlyidentical tumor-associated virus in KS1 and KS2 phylogeniesand with the separation of DNA and RNA derived sequencesinto different clades within tumors of KS1 This hypothesiscould be investigated further by identifying the infectedcells types within each tumor Furthermore identifying theinsertion sites within the host genome in these tumors couldhelp understand if HIV-infected cells in tumors are physicallymigrating to other tumor sites A recent HIV integration sitestudy showed that one mechanism of HIV persistence withinT-cells is related to clonal expansion [34] Interestingly theauthors also found that insertion sites were enriched for genesinvolved in cell growth [34] suggesting that the insertionsites themselves may promote tumorigenesis Studies withincancer and AIDS dementia tissue macrophages performed inour lab showed that major HIV integration sites in tissue-associated macrophages (TAMs) were related to cellular acti-vation including those just upstream of the c-fes oncogenewhich could contribute to clonal expansion [35ndash38] This isconsistent with a sequential pathogenesis model in whichthe insertion site of the virus promotes persistent cellularactivation clonal expansion andmigration of HIV+ from thetumor to nontumor sites [36]

The patients studied here were all cART-naıve howeverKS remains a substantial comorbidity even for patientsundergoing cART For some patients rapid progression of KSoccurs under cART even with an undetectable viral load [39]These cases are frequently classified as related to immunereconstitution inflammatory syndrome (IRIS) [19 39 40]a well-studied phenomenon of resurgence of underlyingpathologies during cART [41ndash46] IRIS-related worseningof coinfections typically occurs within the first 6 monthsof therapy is associated with localized tissue inflammationand low CD4 counts and is most often observed in limited-resource settings [41 47] Although T-cell restoration andactivation have been suggested as causing KS progression inthese patients no direct link has been demonstrated [47]Innate immune dysfunction (eg macrophage-activation)

has also been offered as an explanation for progressive KSduring cART whereby the interaction of restored T-cellpopulations with existing activated macrophages leads toincreased IRIS [19]

5 Conclusions

Kaposirsquos sarcoma (KS) is a major cancer that still occurs inHIV positive patients despite immune-restorative combinedantiretroviral therapy (cART) In this study we isolated HIVfrom tumor and nontumor tissue sites frompatients who diedwith KS which had metastasized to the viscera (KS1ndashKS3)KS1 andKS2 died fromotherAIDS-associated complicationswhereas KS3 died directly due to a highly aggressive KS thatmetastasized to lungs and caused death within 1 month ofinitial KS diagnosisWe compared the HIV sequences amongpatientrsquos tissue sites and found that KS1 and KS2 had uniqueHIV in tumors compared to nontumor sites whereas HIVwas freelymigrating among all tissues inKS3While the studyis based on a very small sample size the results suggest thatan HIV-associated mechanism may promote the metastaticprocess in aggressive KS It is also important to note that itis unclear how the other AIDS-associated cancers may haveimpacted the study (eg lymphoma in KS1 and BCBL inKS2) Continued studies of the evolutionary patterns withinand among KS tumors could help in understanding themechanisms of metastasis as well as describing an additionalHIV reservoir during cART Evolutionary studies of HIV intumor tissues are sorely lacking and further investigationcould provide additional clarity into themechanisms of HIV-associated cancers

Disclosure

The funders had no role in study design data collectionand interpretation or the decision to submit the work forpublication

Competing Interests

The authors declare that they have no competing interests

Authorsrsquo Contributions

Susanna L Lamers and Rebecca Rose contributed equally tothis work

Acknowledgments

This work was supported by the US National Institutes ofHealth under Grants NIMH R01-MH100984 and NCI ACSRUM1-CA181255-03 to Michael S McGrath and Grants P50-GM103297 and R01-NS063897 to Marco Salemi Additionalsupport was provided by Navidea Biopharmaceuticals Inc

References

[1] P Cook-Mozaffari R Newton V Beral and D P BurkittldquoThe geographical distribution of Kaposirsquos sarcoma and of

Sarcoma 9

lymphomas inAfrica before theAIDS epidemicrdquoBritish Journalof Cancer vol 78 no 11 pp 1521ndash1528 1998

[2] Y Chang E Cesarman M S Pessin et al ldquoIdentification ofherpesvirus-like DNA sequences in AIDS-associated Kaposirsquossarcomardquo Science vol 266 no 5192 pp 1865ndash1869 1994

[3] httpwwwcdcgovmmwrpreviewmmwrhtmlrr5710a2htm[4] A Stewart S Chan Carusone K To N Schaefer-Mcdaniel M

Halman and R Grimes ldquoCauses of death in HIV patients andthe evolution of an AIDS hospice 1988ndash2008rdquo AIDS Researchand Treatment vol 2012 Article ID 390406 7 pages 2012

[5] R Rothenberg M Woelfel R Stoneburner J Milberg RParker and B Truman ldquoSurvival with the acquired immunod-eficiency syndrome Experience with 5833 cases in New YorkCityrdquoThe New England Journal of Medicine vol 317 no 21 pp1297ndash1302 1987

[6] J H Campbell A C Hearps G E Martin K C Williams andS M Crowe ldquoThe importance of monocytes and macrophagesin HIV pathogenesistreatmentand curerdquo AIDS vol 28 no 15pp 2175ndash2187 2014

[7] M A Eltom A Jemal S M Mbulaiteye S S Devesa andR J Biggar ldquoTrends in Kaposirsquos sarcoma and non-Hodgkinrsquoslymphoma incidence in the United States from 1973 through1998rdquo Journal of the National Cancer Institute vol 94 no 16pp 1204ndash1210 2002

[8] A M Cattelan M L Calabro P Gasperini et al ldquoAcquiredimmunodeficiency syndrome-related Kaposirsquos sarcoma regres-sion after highly active antiretroviral therapy biologic correlatesof clinical outcomerdquo Journal of the National Cancer InstituteMonographs no 28 pp 44ndash49 2001

[9] G Di Lorenzo P A Konstantinopoulos L Pantanowitz R DiTrolio S De Placido and B J Dezube ldquoManagement of AIDS-related Kaposirsquos sarcomardquoThe Lancet Oncology vol 8 no 2 pp167ndash176 2007

[10] A S Semeere N Busakhala and J N Martin ldquoImpact ofantiretroviral therapy on the incidence of Kaposirsquos sarcoma inresource-rich and resource-limited settingsrdquo Current Opinionin Oncology vol 24 no 5 pp 522ndash530 2012

[11] S E Krown ldquoHighly active antiretroviral therapy in AIDS-associated Kaposirsquos sarcoma implications for the design of ther-apeutic trials in patients with advanced symptomatic Kaposirsquossarcomardquo Journal of Clinical Oncology vol 22 no 3 pp 399ndash402 2004

[12] E L Yanik K Tamburro J J Eron B Damania S Napravnikand D P Dittmer ldquoRecent cancer incidence trends in anobservational clinical cohort ofHIV-infected patients in theUS2000 to 2011rdquo Infectious Agents and Cancer vol 8 no 1 articleno 18 2013

[13] D Serraino A De Paoli A Zucchetto et al ldquoThe impact ofKaposi sarcoma and non-Hodgkin lymphoma on mortality ofpeople with AIDS in the highly active antiretroviral therapieserardquo Cancer Epidemiology vol 34 no 3 pp 257ndash261 2010

[14] LMartın-Carbonero R Palacios E Valencia et al ldquoLong-termprognosis ofHIV-infected patients withKaposi sarcoma treatedwith pegylated liposomal doxorubicinrdquo Clinical Infectious Dis-eases vol 47 no 3 pp 410ndash417 2008

[15] M Bower J Weir N Francis et al ldquoThe effect of HAART in254 consecutive patients with AIDS-related Kaposirsquos sarcomardquoAIDS vol 23 no 13 pp 1701ndash1706 2009

[16] S E Krown J Y Lee and D P Dittmer ldquoMore on HIV-associated Kaposirsquos sarcomardquo The New England Journal ofMedicine vol 358 no 5 pp 535ndash536 2008

[17] A Jemal F Bray M M Center J Ferlay E Ward and DForman ldquoGlobal cancer statisticsrdquo CAmdashA Cancer Journal forClinicians vol 61 no 2 pp 69ndash90 2011

[18] E A Mesri E Cesarman and C Boshoff ldquoKaposirsquos sarcomaand its associated herpesvirusrdquo Nature Reviews Cancer vol 10no 10 pp 707ndash719 2010

[19] E Letang J J Lewis M Bower et al ldquoImmune reconstitu-tion inflammatory syndrome associated with Kaposi sarcomahigher incidence andmortality in Africa than in the UKrdquoAIDSvol 27 no 10 pp 1603ndash1613 2013

[20] H Q Nguyen A S Magaret M M Kitahata S E VanRompaey A Wald and C Casper ldquoPersistent Kaposi sarcomain the era of highly active antiretroviral therapy characterizingthe predictors of clinical responserdquoAIDS vol 22 no 8 pp 937ndash945 2008

[21] S Uccini S Scarpino F Ballarini et al ldquoIn situ study ofchemokine and chemokine-receptor expression in Kaposi sar-comardquo The American Journal of Dermatopathology vol 25 no5 pp 377ndash383 2003

[22] N Dupin and P A Grange ldquoLooking for the target cellof Kaposirsquos sarcoma-associated herpesvirusrdquo The Journal ofInvestigative Dermatology vol 126 no 3 pp 545ndash547 2006

[23] B Ensoli C Sgadari G Barillari M C Sirianni M Sturzl andP Monini ldquoBiology of Kaposirsquos sarcomardquo European Journal ofCancer vol 37 no 10 pp 1251ndash1269 2001

[24] P Delli Bovi E Donti D M Knowles II et al ldquoPresenceof chromosomal abnormalities and lack of AIDS retrovirusDNA sequences in AIDS-associated Kaposirsquos sarcomardquo CancerResearch vol 46 no 12 part 1 pp 6333ndash6338 1986

[25] M Salemi S L Lamers L C Huysentruyt et al ldquoDistinctpatterns of HIV-1 evolution withinmetastatic tissues in patientswith non-hodgkins Lymphomardquo PLoS ONE vol 4 no 12Article ID e8153 2009

[26] S L Lamers M Salemi D C Galligan et al ldquoHumanimmunodeficiency virus-1 evolutionary patterns associatedwith pathogenic processes in the brainrdquo Journal of NeuroVirol-ogy vol 16 no 3 pp 230ndash241 2010

[27] S L Lamers M Salemi D C Galligan et al ldquoExtensive HIV-1intra-host recombination is common in tissues with abnormalhistopathologyrdquo PLoS ONE vol 4 no 3 Article ID e5065 2009

[28] S Palmer M Kearney F Maldarelli et al ldquoMultiple linkedhuman immunodeficiency virus type 1 drug resistance muta-tions in treatment-experienced patients are missed by standardgenotype analysisrdquo Journal of Clinical Microbiology vol 43 no1 pp 406ndash413 2005

[29] J D Thompson T J Gibson F Plewniak F Jeanmougin andD G Higgins ldquoThe CLUSTAL X windows interface flexiblestrategies for multiple sequence alignment aided by qualityanalysis toolsrdquoNucleic Acids Research vol 25 no 24 pp 4876ndash4882 1997

[30] K Tamura D Peterson N Peterson G Stecher M Nei andS Kumar ldquoMEGA5 molecular evolutionary genetics analysisusing maximum likelihood evolutionary distance and max-imum parsimony methodsrdquo Molecular Biology and Evolutionvol 28 no 10 pp 2731ndash2739 2011

[31] S Guindon F Delsuc J-F Dufayard and O Gascuel ldquoEstimat-ing maximum likelihood phylogenies with PhyMLrdquoMethods inMolecular Biology vol 537 pp 113ndash137 2009

[32] M Slatkin andWPMaddison ldquoA cladisticmeasure of gene flowinferred from the phylogenies of allelesrdquo Genetics vol 123 no3 pp 603ndash613 1989

10 Sarcoma

[33] D W Huang B T Sherman and R A Lempicki ldquoBioin-formatics enrichment tools paths toward the comprehensivefunctional analysis of large gene listsrdquo Nucleic Acids Researchvol 37 no 1 pp 1ndash13 2009

[34] F Maldarelli X Wu L Su et al ldquoSpecific HIV integration sitesare linked to clonal expansion and persistence of infected cellsrdquoScience vol 345 no 6193 pp 179ndash183 2014

[35] B Shiramizu B Herndier H Abby and M S McGrathldquoClonal HIV is present in a wide variety of HIV-associatedmalignancies and cell separation studies map the clone totumor-associated macrophagesrdquo Journal of Acquired ImmuneDeficiency Syndromes amp Human Retrovirology vol 14 no 4article A50 1997

[36] M SMcGrath B T Shiramizu and B G Herndier ldquoIdentifica-tion of a clonal form of HIV in early Kaposirsquos sarcoma evidencefor a novel model of ontogenesis lsquosequential neoplasiarsquordquo Journalof Acquired Immune Deficiency Syndromes and Human Retrovi-rology vol 8 no 4 pp 379ndash385 1995

[37] B Shiramizu B G Herndier and M S McGrath ldquoIdentifi-cation of a common clonal human immunodeficiency virusintegration site in human immunodeficiency virus-associatedlymphomasrdquo Cancer Research vol 54 no 8 pp 2069ndash20721994

[38] K D Mack X Jin S Yu et al ldquoHIV insertions within andproximal to host cell genes are a common finding in tissuescontaining high levels ofHIVDNAandmacrophage-associatedp24 antigen expressionrdquo Journal of Acquired Immune DeficiencySyndromes vol 33 no 3 pp 308ndash320 2003

[39] R S Leidner and D M Aboulafia ldquoRecrudescent Kaposirsquossarcoma after initiation of HAART a manifestation of immunereconstitution syndromerdquo AIDS Patient Care and STDs vol 19no 10 pp 635ndash644 2005

[40] E Letang J M Almeida J M Miro et al ldquoPredictorsof immune reconstitution inflammatory syndrome-associatedwith kaposi sarcoma in mozambique a prospective studyrdquoJournal of Acquired Immune Deficiency Syndromes vol 53 no5 pp 589ndash597 2010

[41] M Muller S Wandel R Colebunders S Attia H FurrerandM Egger ldquoImmune reconstitution inflammatory syndromein patients starting antiretroviral therapy for HIV infectiona systematic review and meta-analysisrdquo The Lancet InfectiousDiseases vol 10 no 4 pp 251ndash261 2010

[42] S A Shelburne and R J Hamill ldquoThe immune reconstitutioninflammatory syndromerdquoAIDS Reviews vol 5 no 2 pp 67ndash792003

[43] S A Shelburne III R J HamillMC Rodriguez-Barradas et alldquoImmune reconstitution inflammatory syndrome emergenceof a unique syndrome during highly active antiretroviral ther-apyrdquoMedicine vol 81 no 3 pp 213ndash227 2002

[44] M A French P Price and S F Stone ldquoImmune restorationdisease after antiretroviral therapyrdquo AIDS vol 18 no 12 pp1615ndash1627 2004

[45] S F Stone P Price and M A French ldquoImmune restorationdisease a consequence of dysregulated immune responses afterHAARTrdquoCurrent HIV Research vol 2 no 3 pp 235ndash242 2004

[46] P Price N Mathiot R Krueger S Stone N M Keane and MA French ldquoImmune dysfunction and immune restoration dis-ease in HIV patients given highly active antiretroviral therapyrdquoJournal of Clinical Virology vol 22 no 3 pp 279ndash287 2001

[47] N F Walker N F Walker N F Walker et al ldquoImmune recon-stitution inflammatory syndrome in HIV-infected patientsrdquoHIVAIDS - Research and Palliative Care vol 7 pp 49ndash64 2015

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

2 Sarcoma

KS lesions can be solitary localized or disseminatedbut the relationship among tumors in a single individualis largely unknown While many KS lesions are cutaneousand indolent tumors can occur in the oral cavity lymphnodes and viscera which can result in fatal complica-tions due to interference with normal body function Thegrowth of lesions can be very slow or explosively fastThe KS defining cell-type is a spindle cell (SC) [21 22]that expresses markers of both endothelial and macrophagelineages [23] KS tumors are polyclonal positive for thepresence of HHV8 virus which infects the tumor-associatedB-cells and SCs [23] and highly vascularized giving thema distinguishing purple color Inflammatory lymphocytesand monocytesmacrophages populate and enrich the KStumor environment for tumor growth through productionof a variety of cytokines [23] These immune cells are allpotential targets for HIV however to our knowledge onlyone study assessed KS tumors for the presence of HIV usingan early hybridization technique [24] SCs can be culturedfrom PBMCs of patients with KS suggesting that circulatingSCs may lead to the appearance of multiple KS lesions [23]

HIV has the potential to infect and evolve in manyanatomical tissues including tumors [25ndash27] Previouslywe showed that the viral populations in lymphoma tumorswere distinct from HIV in healthy tissues [25] Here ourgoal was to characterize HIV populations in KS tumors todetermine whether HIV evolution could be used to tracktumor relatedness and progression in native KS uncompli-cated by the use of cART We obtained tumor and nontumortissues sampled post mortem from the AIDS and CancerSpecimen Resource (ACSR) from three HIV+KS+ patientswho died from aggressive AIDS and we generated HIVenv-nef sequences using single genome sequencing (SGS) tocharacterize viral evolutionary patterns

2 Materials and Methods

21 Patients and Biomaterial The three male patientsincluded in this studywere diagnosedwithHIV infection anddied with advanced AIDS without receiving cART (Table 1)100mg of frozen autopsy tumor and nontumor tissues wasobtained through the ACSR (httpacsrucsfedu) Tissueswere classified as tumor or nontumor by histological exam-ination The ACSR is a National Cancer Institute-fundedtissue-banking program that obtains tissues from patientsafter appropriate consent and releases tissues to investigatorsfollowing approval of the proposed study and deidentificationof samples and clinical histories The ACSR is recognized bytheOffice of Biorepositories and BiospecimenResearch at theNational Institutes of Health as being HIPAA compliant andin accordance with the ethical standards of the Declarationof Helsinki Additionally all material was obtained underapproval from the University of California at San FranciscoCommittee on Human Research

22 RNADNA Extractions Total RNA and genomic DNAwere isolated separately from each tissue section (30ndash50 ng)using AllPrep DNARNAMini Kit (Qiagen 80204) Tissueswere homogenized just prior to extraction in Buffer RLT Plus

(lysis buffer) using a TissueRupter rotor-stator homogenizer(Qiagen 9001271) with a fresh sterile disposable probe(Qiagen 990890) for each sampleManufacturerrsquos guidelineswere followed with the exception of two 50 120583L final elutionsusing RNase-free water during the RNA isolationThe 100 120583Lfinal volume of RNA generated was concentrated usingRNeasyMinElute Cleanup Kit (Qiagen 74204) RNA andDNA extractions cDNA synthesis and first-round PCRsetup were conducted in a restricted-access amplicon-freeroom with separate air handling and laboratory equipmentwhere no amplified PCR products or recombinant clonedplasmids were allowed and work surfaces and equipmentwere thoroughly cleaned before and after use with Eliminase(Decon Labs Inc) cDNA was synthesized using Super-Script III First-Strand Synthesis System (Invitrogen LifeTechnologies 18080-051) using the provided Oligo(dT)

20

primer according to manufacturerrsquos recommendations RNAwas incubated at 65∘C for 5 minutes with deoxynucleosidetriphosphates (05mM) and 5 120583M Oligo(dT)

20and then

cooled quickly to 4∘C First-Strand cDNA Synthesis wasperformed in a 40 120583L reaction volume containing 1x reversetranscription buffer (10mM Tris-HCl [pH 84] 25mMKCl)5mMMgCl

2 10mM dithiothreitol 2 U120583L of RNase-OUT

(RNase inhibitor) and 10U120583L SuperScript III RT Thereaction was heated to 50∘C for 50 minutes followed by85∘C for 5 minutes The reaction was cooled to 37∘C and01 U120583L of E coli RNase H was added followed by a 20-minute incubation cDNA was stored at minus20∘C

23 Sequencing Protocol Amodified single genome sequenc-ing protocol was used to amplify linked HIV env andnef sequences [28] cDNA and genomic DNA were seri-ally diluted until an average of 30 or less of the nestedPCR reactions were positive During the first-round PCRdiluted cDNA or genomic DNA was amplified in 25120583Lreactions containing 1x Platinum Blue PCR SuperMix(Invitrogen Life Technologies) and 02 120583M of each primerEF2 51015840-ACAGTCTATTATGGGGTRCC-31015840 and NR1 51015840-AGCTCCCAGGCTCAGATCT-31015840 (6333ndash6352 bp and 9558ndash9576 bp of HIV HXB2 resp) with the following cyclingparameters initial denaturation 95∘C for 5 minutes followedby 35 cycles of denaturing at 94∘C for 1 minute annealing at58∘C for 1 minute and extension at 72∘C for 4minutes with afinal extension time of 10 minutes at 72∘C A second round ofgp120 PCR consisted of 1120583L of the first-round PCR added toa 24120583L second-round reaction consisting of 1x PlatinumBluePCR SuperMix (Invitrogen Life Technologies) and 02 120583Mof each primer EF3 51015840 CATAATGTTTGGGCCACACA-31015840 and ER2 51015840-CACCACTCTTCTYTTTGCC-31015840 (6420ndash6439 bp and 7724ndash7742 bp of HIV HXB2 resp) with thefollowing cycling parameters initial denaturation 95∘C for 5minutes followed by 35 cycles of denaturation at 94∘C for1 minute annealing at 58∘C for 1 minute and extension at72∘C for 2 minutes with a final extension time of 10 minutesat 72∘C This second-round PCR generated a 13 Kb productnearly covering the entire env gp120 gene Second-roundenv gp120 PCR products were visualized on 1 agarose gelsstained with ethidium bromide and reactions containing asingle 13 Kb product were considered positive and selected

Sarcoma 3

Table1Patie

ntclinicalh

istory

Patie

ntAge1

Major

comorbiditie

s2KS

locatio

nTimes

ince

KSdiagno

sis(m

onths)

Leng

thof

HIV

infection(years)

Anatomicallocatio

nof

biop

sy

Con

centratio

n(ng120583L)

Num

bero

fenv

sequ

ences

Num

bero

fnef

sequ

ences

RNA

DNA

RNA

DNA

RNA

DNA

KS1

34Lymph

omaMAC

PC

Pcytomegaloviru

s

Skinpleuralung

strachealymph

nodeliver

adrenalgland

sUnk

nown

7

KSskin

144

154

228

249

KSlymph

node

168

276

1321

1421

Lymph

node

120

121

3722

3526

Kidn

eygt200

818

50

90

KS2

31BC

BLSkinpleuralung

sesop

hagussm

allbow

elsim5

Unk

nown

KSskin

165

4414

1018

12KS

smallbow

el192

984

40

50

Liver

191

842

00

00

Spleen

190

139

231

312

KS3

28AggressiveK

S

Skinpleuralung

stracheaesop

hagussm

all

bowel

colon

rectum

iliaclym

phno

des

mesenteric

lymph

nodespara-aortic

lymph


nodes

sim1

9

KSskin

4951

62

26

KSsm

allbow

el164

394

1625

1424

Liver

161

334

145

155

Stom

ach

782

402

248

208

1Age

inyears2MAC

Mycobacteriu

mavium

complexP

CPP

neum

ocystis

pneumoniaB

CBLbo

dycavitybasedlymph

oma

4 Sarcoma




3 Results




Sarcoma 5

env

A

B

C

D

E

F

001



(a)

nef



A

B

C

D

E

001

(b)






6 Sarcoma

env

A

B

C

001





nef

A

B

C

001


env

A

B

C

001





nef

A

B

C

001


Sarcoma 7






KS1 (359)


tumor 1 (03) None 7 (19) 122


KS2 (394)



Nontumor 5 (12) 373 4 (10) 150

KS3 (373)


tumor 10 (26) 183 336 396 16 (42) 103 118 207 228 238 246254 276 374 408 415 445

Nontumor 11 (29) 143 167 183 336 454 21 (56) 103 118 207 228 238 246254 276 374 408 415 445






KS1 (207)


tumor 4 (19) None 18 (87) 8 37 188

Nontumor 1 (05) None 4 (19) 8 37 188

KS2 (205)


tumor 1 (05) 190 4 (20) 81

Nontumor 2 (10) 190 5 (24) 81

KS3 (207)


tumor 1 (05) None 14 (68) 4 30 63 65 111 129 151164 191 195

Nontumor 1 (05) None 21 (101) 4 63 111 129 147 151 164191 195


4 Discussion



8 Sarcoma





5 Conclusions


Disclosure


Competing Interests




Acknowledgments


References


Sarcoma 9

































10 Sarcoma





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Sarcoma 3

Table1Patie

ntclinicalh

istory

Patie

ntAge1

Major

comorbiditie

s2KS

locatio

nTimes

ince

KSdiagno

sis(m

onths)

Leng

thof

HIV

infection(years)

Anatomicallocatio

nof

biop

sy

Con

centratio

n(ng120583L)

Num

bero

fenv

sequ

ences

Num

bero

fnef

sequ

ences

RNA

DNA

RNA

DNA

RNA

DNA

KS1

34Lymph

omaMAC

PC

Pcytomegaloviru

s

Skinpleuralung

strachealymph

nodeliver

adrenalgland

sUnk

nown

7

KSskin

144

154

228

249

KSlymph

node

168

276

1321

1421

Lymph

node

120

121

3722

3526

Kidn

eygt200

818

50

90

KS2

31BC

BLSkinpleuralung

sesop

hagussm

allbow

elsim5

Unk

nown

KSskin

165

4414

1018

12KS

smallbow

el192

984

40

50

Liver

191

842

00

00

Spleen

190

139

231

312

KS3

28AggressiveK

S

Skinpleuralung

stracheaesop

hagussm

all

bowel

colon

rectum

iliaclym

phno

des

mesenteric

lymph

nodespara-aortic

lymph


nodes

sim1

9

KSskin

4951

62

26

KSsm

allbow

el164

394

1625

1424

Liver

161

334

145

155

Stom

ach

782

402

248

208

1Age

inyears2MAC

Mycobacteriu

mavium

complexP

CPP

neum

ocystis

pneumoniaB

CBLbo

dycavitybasedlymph

oma

4 Sarcoma




3 Results




Sarcoma 5

env

A

B

C

D

E

F

001



(a)

nef



A

B

C

D

E

001

(b)






6 Sarcoma

env

A

B

C

001





nef

A

B

C

001


env

A

B

C

001





nef

A

B

C

001


Sarcoma 7






KS1 (359)


tumor 1 (03) None 7 (19) 122


KS2 (394)



Nontumor 5 (12) 373 4 (10) 150

KS3 (373)


tumor 10 (26) 183 336 396 16 (42) 103 118 207 228 238 246254 276 374 408 415 445

Nontumor 11 (29) 143 167 183 336 454 21 (56) 103 118 207 228 238 246254 276 374 408 415 445






KS1 (207)


tumor 4 (19) None 18 (87) 8 37 188

Nontumor 1 (05) None 4 (19) 8 37 188

KS2 (205)


tumor 1 (05) 190 4 (20) 81

Nontumor 2 (10) 190 5 (24) 81

KS3 (207)


tumor 1 (05) None 14 (68) 4 30 63 65 111 129 151164 191 195

Nontumor 1 (05) None 21 (101) 4 63 111 129 147 151 164191 195


4 Discussion



8 Sarcoma





5 Conclusions


Disclosure


Competing Interests




Acknowledgments


References


Sarcoma 9

































10 Sarcoma





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















4 Sarcoma




3 Results




Sarcoma 5

env

A

B

C

D

E

F

001



(a)

nef



A

B

C

D

E

001

(b)






6 Sarcoma

env

A

B

C

001





nef

A

B

C

001


env

A

B

C

001





nef

A

B

C

001


Sarcoma 7






KS1 (359)


tumor 1 (03) None 7 (19) 122


KS2 (394)



Nontumor 5 (12) 373 4 (10) 150

KS3 (373)


tumor 10 (26) 183 336 396 16 (42) 103 118 207 228 238 246254 276 374 408 415 445

Nontumor 11 (29) 143 167 183 336 454 21 (56) 103 118 207 228 238 246254 276 374 408 415 445






KS1 (207)


tumor 4 (19) None 18 (87) 8 37 188

Nontumor 1 (05) None 4 (19) 8 37 188

KS2 (205)


tumor 1 (05) 190 4 (20) 81

Nontumor 2 (10) 190 5 (24) 81

KS3 (207)


tumor 1 (05) None 14 (68) 4 30 63 65 111 129 151164 191 195

Nontumor 1 (05) None 21 (101) 4 63 111 129 147 151 164191 195


4 Discussion



8 Sarcoma





5 Conclusions


Disclosure


Competing Interests




Acknowledgments


References


Sarcoma 9

































10 Sarcoma





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Sarcoma 5

env

A

B

C

D

E

F

001



(a)

nef



A

B

C

D

E

001

(b)






6 Sarcoma

env

A

B

C

001





nef

A

B

C

001


env

A

B

C

001





nef

A

B

C

001


Sarcoma 7






KS1 (359)


tumor 1 (03) None 7 (19) 122


KS2 (394)



Nontumor 5 (12) 373 4 (10) 150

KS3 (373)


tumor 10 (26) 183 336 396 16 (42) 103 118 207 228 238 246254 276 374 408 415 445

Nontumor 11 (29) 143 167 183 336 454 21 (56) 103 118 207 228 238 246254 276 374 408 415 445






KS1 (207)


tumor 4 (19) None 18 (87) 8 37 188

Nontumor 1 (05) None 4 (19) 8 37 188

KS2 (205)


tumor 1 (05) 190 4 (20) 81

Nontumor 2 (10) 190 5 (24) 81

KS3 (207)


tumor 1 (05) None 14 (68) 4 30 63 65 111 129 151164 191 195

Nontumor 1 (05) None 21 (101) 4 63 111 129 147 151 164191 195


4 Discussion



8 Sarcoma





5 Conclusions


Disclosure


Competing Interests




Acknowledgments


References


Sarcoma 9

































10 Sarcoma





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















6 Sarcoma

env

A

B

C

001





nef

A

B

C

001


env

A

B

C

001





nef

A

B

C

001


Sarcoma 7






KS1 (359)


tumor 1 (03) None 7 (19) 122


KS2 (394)



Nontumor 5 (12) 373 4 (10) 150

KS3 (373)


tumor 10 (26) 183 336 396 16 (42) 103 118 207 228 238 246254 276 374 408 415 445

Nontumor 11 (29) 143 167 183 336 454 21 (56) 103 118 207 228 238 246254 276 374 408 415 445






KS1 (207)


tumor 4 (19) None 18 (87) 8 37 188

Nontumor 1 (05) None 4 (19) 8 37 188

KS2 (205)


tumor 1 (05) 190 4 (20) 81

Nontumor 2 (10) 190 5 (24) 81

KS3 (207)


tumor 1 (05) None 14 (68) 4 30 63 65 111 129 151164 191 195

Nontumor 1 (05) None 21 (101) 4 63 111 129 147 151 164191 195


4 Discussion



8 Sarcoma





5 Conclusions


Disclosure


Competing Interests




Acknowledgments


References


Sarcoma 9

































10 Sarcoma





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Sarcoma 7






KS1 (359)


tumor 1 (03) None 7 (19) 122


KS2 (394)



Nontumor 5 (12) 373 4 (10) 150

KS3 (373)


tumor 10 (26) 183 336 396 16 (42) 103 118 207 228 238 246254 276 374 408 415 445

Nontumor 11 (29) 143 167 183 336 454 21 (56) 103 118 207 228 238 246254 276 374 408 415 445






KS1 (207)


tumor 4 (19) None 18 (87) 8 37 188

Nontumor 1 (05) None 4 (19) 8 37 188

KS2 (205)


tumor 1 (05) 190 4 (20) 81

Nontumor 2 (10) 190 5 (24) 81

KS3 (207)


tumor 1 (05) None 14 (68) 4 30 63 65 111 129 151164 191 195

Nontumor 1 (05) None 21 (101) 4 63 111 129 147 151 164191 195


4 Discussion



8 Sarcoma





5 Conclusions


Disclosure


Competing Interests




Acknowledgments


References


Sarcoma 9

































10 Sarcoma





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















8 Sarcoma





5 Conclusions


Disclosure


Competing Interests




Acknowledgments


References


Sarcoma 9

































10 Sarcoma





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Sarcoma 9

































10 Sarcoma





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















10 Sarcoma





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Documents

Research Article HIV-1 Evolutionary Patterns …downloads.hindawi.com/journals/sarcoma/2016/4510483.pdfResearch Article HIV-1 Evolutionary Patterns Associated with Metastatic Kaposi