Patent Application: US-201415101174-A

Abstract:
autoimmune diseases are thought to be initiated by exposures to foreign antigens that cross - react with endogenous molecules . analyses of peripheral blood lymphocytes and serum suggested that mutations in autoimmune antigen targets sparked cellular immunity and cross - reactive humoral immune responses . acquired immunity to autoimmune antigens can help control naturally occurring cancers .

Description:
the inventors have found somatic mutations in autoimmune protein targets in cancer cells in patients that have both an autoimmune disease and a cancer . patient sera contain t cells specific for the autoimmune protein targets and antibodies specific for the autoimmune protein targets . the autoantibodies do not distinguish between mutant and wild - type forms of the antigen and do not bind to peptides containing the mutant or wild - type residue . however , some cd4 + t cells are reactive with such mutant peptides . moreover , the cancer cells containing the somatic mutations appear to comprise only a subset of the cancer cells in the patient , i . e ., they are subclonal . these findings have implications for the pathogenesis of autoimmune diseases and for the immunological control of naturally occurring cancers . autoantigens are antigens to which humans can raise an autoimmune response . autoantigens are a family of proteins that have been found to be targets of an anti - self immune response associated with diseases . examples of such diseases are scleroderma , auoimmune rheumatic diseases , such as myositis , vasculitis , and sle , sjogren &# 39 ; s syndrome , and lupus . autoantigens known to be involved in such diseases include , but are not limited to the following : mutant peptides may display any change in amino acid sequence from the wild type . this can be conveniently determined with regard to the patient &# 39 ; s own normal tissues . alternatively , wild type found in reference data bases can be used to compare to potential mutant peptides . a mutant peptide may have one or more single nucleotide substitutions , deletions , or insertions . typically the peptide will have a single nucleotide substitution , deletion , or insertion . in general , it will have less than four , less than three , or less than two nucleotide substitutions , deletions , or insertions . other than the substitution , deletion , or insertion , the sequence of the peptide will be that of a contiguous stretch of amino acid residues of an autoimmune target antigen . the contiguous stretch will typically be less than 50 , less than 40 , less than 30 , less than 20 , less than 18 , less than 17 , or less than 16 amino acid residues . the contiguous stretch will typically be at least 8 , at least 10 , at least 12 , or at least 13 , or at least 14 amino acid residues . an isolated peptide may be made by any means that is practical , including by chemical synthesis , enzymatic synthesis , or in vivo biosysnthesis . isolated peptides are not in a cell or in a whole cell lysate . typically an isolated peptide is a predominant constituent in a composition , i . e ., greater than 10 , 20 , 30 , 40 , or 50 % of the active ingredients in a composition . a mutant peptide may also be in admixture with a full length protein of the autoimmune antigen . in such case the peptide and the protein may collectively be the predominant constituents in the composition . a peptide may have other moieties attached to the contiguous stretch of autoimmune target antigen sequence . such moieties may be , for example , radioactive , fluorescent , enzymatic , or adjuvant moieties . cancers which can be treated by use of the mutant peptides include any to which humans are subject . these include solid and hematological cancers , such as breast , lung , ovarian , colorectal , and b cell lymphoma . other cancers which can be treated include adenoid cystic carcinoma , adrenal gland tumor , amyloidosis , anal cancer , appendix cancer , astrocytoma - childhood , ataxia - telangiectasia , attenuated familial adenomatous polyposis , beckwith - wiedemann syndrome , bile duct cancer , birt - hogg - dubé syndrome , bladder cancer , bone cancer , brain stem glioma - childhood , brain tumor , breast cancer , breast cancer - inflammatory , breast cancer - male , breast cancer - metaplastic , carcinoid tumor , carney complex , central nervous system - childhood , cervical cancer , childhood cancer , colorectal cancer , cowden syndrome , craniopharyngioma - childhood , desmoplastic infantile ganglioglioma - childhood tumor , endocrine tumor , ependymoma - childhood , esophageal cancer , ewing family of tumors - childhood , eye cancer , eyelid cancer , fallopian tube cancer , familial adenomatous polyposis , familial malignant melanoma , familial non - vhl clear cell renal cell carcinoma , gallbladder cancer , gardner syndrome , gastrointestinal stromal tumor - gist , germ cell tumor - childhood , gestational trophoblastic tumor , head and neck cancer , hereditary breast and ovarian cancer , hereditary diffuse gastric cancer , hereditary leiomyomatosis and renal cell cancer , hereditary mixed polyposis syndrome , hereditary pancreatitis , hereditary papillary renal cell carcinoma , hiv and aids - related cancer , islet cell tumor , juvenile polyposis syndrome , kidney cancer , lacrimal gland tumor , laryngeal and hypopharyngeal cancer , leukemia - acute lymphoblastic - all - childhood , leukemia - acute lymphocytic - all , leukemia - acute myeloid - aml , leukemia - acute myeloid - aml - childhood , leukemia - b - cell prolymphocytic leukemia and hairy cell leukemia , leukemia - chronic lymphocytic - cll , leukemia - chronic myeloid - cml , leukemia - chronic t - cell lymphocytic , leukemia - eosinophilic , li - fraumeni syndrome , liver cancer , lung cancer , lymphoma - hodgkin , lymphoma - hodgkin - childhood , lymphoma - non - hodgkin , lymphoma - non - hodgkin - childhood , lynch syndrome , mastocytosis , medulloblastoma - childhood , melanoma , meningioma , mesothelioma , muir - tone syndrome , multiple endocrine neoplasia type 1 , multiple endocrine neoplasia type 2 , multiple myeloma , myelodysplastic syndromes - mds , myh - associated polyposis , nasal cavity and paranasal sinus cancer , nasopharyngeal cancer , neuroblastoma - childhood , neuroendocrine tumor , neurofibromatosis type 1 , neurofibromatosis type 2 , nevoid basal cell carcinoma syndrome , oral and oropharyngeal cancer , osteosarcoma - childhood , ovarian cancer , pancreatic cancer , parathyroid cancer , penile cancer , peutz - jeghers syndrome , pituitary gland tumor , pleuropulmonary blastoma - childhood , prostate cancer , retinoblastoma - childhood , rhabdomyosarcoma - childhood , salivary gland cancer , sarcoma , sarcoma - alveolar soft part and cardiac , sarcoma - kaposi , skin cancer ( non - melanoma ), small bowel cancer , stomach cancer , testicular cancer , thymoma , thyroid cancer , tuberous sclerosis syndrome , turcot syndrome , unknown primary , uterine cancer , vaginal cancer , von hippel - lindau syndrome , vulvar cancer , waldenström &# 39 ; s macroglobulinemia , werner syndrome , wilms tumor - childhood , and xeroderma pigmentosum . binding to human hla proteins and measurement of the binding can be performed according to any methods known in the art . one method which can be employed uses in silico tools such as immune epitope database ( iedb ) analysis resource consensus tools ( 7 - 9 ). high affinity binding is assessed when the ic 50 is & lt ; 50 nm . moderately high affinity binding is assessed when the ic 50 is & lt ; 125 nm . extremely high affinity binding is assessed when the ic 50 is & lt ; 10 nm . ascertainment of the type of hla proteins present in a human can be performed using any methods known in the art . exemplary methods include serotyping , cellular typing , gene sequencing , and phenotyping . a subset of patients with scleroderma and other autoimmune rheumatic diseases manifest cancer around the time of autoimmune disease diagnosis , suggesting that the two processes might be linked mechanistically ( 3 ), ( 4 ), ( 17 ). in scleroderma , this temporal clustering of scleroderma and cancer appears limited to the subgroup of patients with antibodies to rpc1 ( 3 ). in the current work , we demonstrated that the polr3a locus is genetically altered ( by somatic mutation or loh ) in six of eight cancers of patients with antibodies to rpc1 , but not in cancers from scleroderma patients with other autoantibody specificities . moreover , t cells reactive with the mutant forms of rpc1 could be identified in the peripheral blood of two of the three patients tested . these t cells did not simply cross - react with the wild - type form of the peptides , because t cells from subject scl - 4 were not stimulated by the wild type form , and the sequence of the tcrs conferring responsiveness to the wild type and mutant peptides in scl - 42 were largely unrelated . these genetic and immunologic findings suggest mutation in polr3a as the initiator of the immune response to rpc1 in an important subset of scleroderma patients . the only tenable alternative to this conclusion is that the onset of scleroderma and the cancer genomes of these patients were unrelated and that the missense mutations and t cell responses directed against the same mutations were coincidental . we believe this alternative is unlikely given the rarity of polr3a mutations in cancer in general ( 0 . 7 %, p & lt ; 10 − 20 , cosmic database , ( 18 ), and the absence of alterations at this locus in scleroderma patients without rpc antibodies ( p & lt ; 0 . 01 ). additionally , in patient scl - 42 , there were multiple different nucleotide sequences encoding tcrs with the identical amino acid sequence in t cells stimulated by the mutant peptide ( table 3 ). this provides strong support for the conclusion that the mutant polr3a gene product acted as an immunogen initiating the anti - rpc1 immune response in vivo . antibodies from all patients with polr3a mutations recognized wild type and mutant versions of rpc 1 similarly , and no antibodies directed specifically against the wild type or mutant peptides could be demonstrated . this suggests that the humoral response does not directly target the area of the mutation or discriminate between mutant and wild type versions of rpc 1 . the inability of autoantibodies to discriminate between the mutant and wt forms of the antigen is consistent with previous studies showing that a crossreactive humoral response is typical when a novel form of an antigen initially stimulates t cells that specifically recognize the modified antigen ( 19 , 20 ). the antibody cross - reactivity might contribute to b cell - mediated diversification of autoimmunity , spreading t cell responses to the wild type autoantigen ( 21 , 22 ). our data therefore suggest that the “ foreign ” antigen triggering the autoimmune response in scleroderma patients is actually a tumor antigen . this complements previous observations indicating cancers can elicit immune responses . it is known that some cases of paraneoplastic syndrome are caused by autoimmunity to proteins expressed in tumors ( 23 ); these responses are directed exclusively to the normal protein and there is no evidence that the gene ( s ) are mutated in the tumors . conversely , it has been shown that mutant genes in human tumors can elicit an immune response against the mutant gene product ( 24 - 26 ); these immune responses have not been shown to elicit a cross - reactive response to the normal gene product that could result in autoimmunity . finally , it has been shown that an in vitro - generated protein containing multiple ( but not single ) mutations , when injected into mice , can elicit a broad , cross - reactive immune response against the normal protein that results in autoimmunity ( 27 ). in these mice , tumor cells expressing only the wt protein can also be targeted by the subsequent immune response . our results show that an analogous situation appears to occur in humans when a single , strongly immunogenic epitope is created by somatic mutation in a patient with an appropriate mhc type . however , the generation of an autoreactive immune response alone may not be sufficient to generate the self - sustaining tissue injury seen in scleroderma , and additional factors ( genetic , environmental , or target tissue - specific ) may be required ( 28 ). our cohort included cancer patients without anti - rpc1 antibodies ( table 1 ). while the interval between scleroderma and cancer onset for patients in these patients was long ( median of 14 . 2 years ), there were two , patients ( scl - 8 and scl - 32 ) who had relatively short intervals . we did not identify genetic alterations of topo1 or cenpb in these two patients . whether their cancers were adventitious , related to therapy , or due to mutations in genes encoding homologs of topo1 or cenpb or proteins that interact with them is unknown but are intriguing hypotheses for future study . similar factors could also explain the absence of genetic alterations of polr3a in two of the eight patients with antibodies to rpc1 . the relatively low fraction of neoplastic cells with genetic alterations in the cancers from some of these patients ( tables 1 and 2 ) suggests that immunoediting of the cancer had occurred , with cells containing these mutations selected against during tumor growth ( 29 ). the emergence of cancer in rpc1 - positive scleroderma patients may thereby represent escape of the tumor from immune pressure . we speculate that cancers harboring polr3a mutations had stimulated scleroderma in most patients with the rpc1 form of the disease . however , in the majority of these patients , the immune response had eradicated the cancer by the time scleroderma developed . patients with a short cancer - autoimmune disease interval have also been described for other autoimmune rheumatic disease phenotypes ( e . g . myositis , vasculitis , sle ) and similar mechanisms may be operative in these diseases ( 17 , 30 , 31 ). given the ubiquitous presence of somatic mutations in solid tumors ( 32 ), these new data add credence to the idea that immunoediting could play a major role in limiting the incidence of human cancer — an old hypothesis ( 33 , 34 ) that has recently garnered more attention ( 35 - 37 ). the data also suggest that this family of autoantigens might be used to generate biologically effective anti - tumor immunity . the above disclosure generally describes the present invention . all references disclosed herein are expressly incorporated by reference . a more complete understanding can be obtained by reference to the following specific examples which are provided herein for purposes of illustration only , and are not intended to limit the scope of the invention . we began by searching for missense mutations in the polr3a gene in tumors from scleroderma patients . we were able to collect tumor and normal tissue samples from eight scleroderma patients who had autoantibodies to rpc1 . we also evaluated eight scleroderma patients who had autoantibodies to top1 or to cenpb and developed cancers ( table 1 ). five of the patients with antibodies to rpc1 developed cancer prior to scleroderma ( median of 0 . 4 years before scleroderma onset ), while the remaining 3 developed cancer 0 . 3 - 2 . 5 years after the onset of scleroderma ( table 1 ). in contrast , patients with autoantibodies to cenpb or top1 who developed cancers only did so a median of 14 . 2 years after the onset of their scleroderma ( table 1 ). the characteristics of the 16 scleroderma patients , including tumor type , age of diagnosis of cancer , cancer - scleroderma interval , and autoantibody status , are listed in table 1 ; additional clinical information is provided in table s 1 and ( 5 ). formalin - fixed , paraffin - embedded tumors from each of the 16 patients were microdissected to enrich for neoplastic cell content , and dna was purified , blunt - ended , and ligated to adapters suitable for library preparation ( 5 ). libraries from peripheral blood cells of each patient were similarly prepared . following amplification of the 32 libraries ( 16 tumor , 16 matched normal ), the pcr products were captured using pcr - generated fragments containing all coding sequences of the polr3a , top1 , and cenpb genes ( 5 ). the captured fragments were evaluated by sequencing on an illumina instrument , achieving an average coverage of 516 reads per base of the 53 coding - exons of the three genes ( range 95 - to 2011 - fold ). this sequence revealed three somatic , missense variants in polr3a and none in top1 or cenpb ( table 1 ). all three variants were in the patients with autoantibodies to rpc1 . the three somatic mutations were each validated by massively parallel sequencing of pcr products generated from the regions surrounding the mutations ( 5 ). of note , both the capture approach and the direct - pcr sequencing approach showed that one of the three somatic mutations was decidedly subclonal , that is , was present in only a subset of the neoplastic cells : the fraction of mutant alleles in the lung cancer from patient scl - 2 was only 4 . 3 %, far less than the estimated fraction of neoplastic cells in the microdissected sample used for dna purification ( table 1 ) ( 5 ). in light of the subclonal nature of one of these mutations , we considered the possibility that cells containing these mutations were selected against during tumor growth , perhaps even disappearing as a result of an immune response . the most frequent way to lose a mutant allele in human cancers is through a gross chromosomal event that results in loss of the entire gene and the surrounding chromosomal region ( loss of heterozygosity , loh ) ( 6 ). to search for such losses , we designed 19 primer pairs that could each amplify a small fragment containing at least one common single nucleotide polymorphism ( snp ) within or surrounding the polr3a gene ( table s 2 ). these primer pairs were used in a multiplexed protocol to evaluate all 16 tumors ( 5 ). five of the eight tumors from scleroderma patients with autoantibodies to rpc1 exhibited loh ( table 2 ). these five tumors included three that did not contain a detectable somatic mutation of polr3a ( table 1 ). the fraction of neoplastic cells that had undergone loh could be estimated from the allelic ratios of the snp data , and in four of the five cases , was subclonal ( table 2 ). importantly , none of the tumors from patients with antibodies to top1 or cenpb exhibited loii of the region containing polr3a ( table 2 ). as an additional control , we evaluated 21 snps within or surrounding the top1 locus on chromosome 20 ( table s 2 ) and found that none of the 16 tumors from scleroderma patients , regardless of autoantibody status , had undergone loh of this region ( table s 3 ). in summary , six of eight tumors from scleroderma patients with autoantibodies to rpc 1 harbored genetic alterations affecting the polr3a locus compared to zero of eight tumors from scleroderma patients without anti - rpc1 antibodies ( p & lt ; 0 . 01 , fisher exact probability test , two - tailed ). we began the immunological analysis of these patients by addressing whether rpc1 autoantibodies recognized the mutated protein differently from the wild type ( wt ) form of the protein . each of the three abnormal forms of the protein found in scleroderma patients was synthesized by in vitro transcription - translation ( ivtt ) ( 5 ). wild type and patient - matched mutant rpc1 were then subjected to immunoprecipitation analysis with sera from patients or from normal individuals ( control sera ). in each case , mutant and wt proteins were precipitated similarly by patient serum , but not precipitated by control sera ( fig . s1 ), demonstrating that the autoantibodies do not discriminate between wild type and mutant versions of the antigen . we next constructed a custom peptide microarray to comprehensively identify linear antigenic regions of the rpc1 protein . we synthesized 276 overlapping peptides of 15 amino acids in length , each offset by five amino acids from the previous peptide and covering the entire length of rpc1 ( table s 4 ). peptides that contained each of the three somatic mutations described above were also synthesized ( three peptides for each mutant ; table s 4 ). these peptides were printed on microarrays and used to assess serum from the three patients with antibodies to rpc1 ( scl - 02 , scl - 04 , and scl - 42 ) whose cancers harbored polr3a mutations , and four control patients ( scl - 200 , scl - 201 , scl - 202 , scl - 203 ) who had scleroderma and antibodies to rpc1 but who did not have cancers . each of the seven serum samples displayed reactivity with at least two of the peptides on the array ( fig . s2 ). notably , there was no reactivity to the mutant peptides or their wild type counterparts ( i . e ., wt amino acids in place of mutant amino acids ) in sera from the patients whose cancers harbored these mutations ( or in the other patients ). having shown that there was no demonstrable humoral immune response specific to the mutant rpc1 proteins , we sought to determine if there was a cellular immune response directed against the mutants . we first performed high - resolution class i and ii hla typing on the three scleroderma patients in whom somatically mutated polr3a genes were identified ( table s 5 ). iedb analysis resource consensus tools ( 7 - 9 ) were then used to determine whether peptides containing the specific mutations in individual patients were likely to bind with high affinity to that patient &# 39 ; s hla alleles . in patient scl - 42 , both wild type and mutant epitopes were predicted to bind with high affinity to both alleles of the patient &# 39 ; s class ii dr hla ( table s 6 ). this was particularly dramatic for hla - dr * 0701 , where the predicted ic 50 was & lt ; 1 nm for both the mutant ( fhvgyfravigtlqmi ; seq id no : 95 ) and wild type peptides ( fhvgyfravigilqmi ; seq id no : 96 ; table s 6 ). high affinity binding of the wild type and mutant peptides to this patient &# 39 ; s other dr allele ( hla - dr * 1001 ) was also predicted ( table s 6 ). in patient scl - 4 , the mutant peptide was predicted to bind to this patient &# 39 ; s hla - dr * 0101 allele with an affinity of 4 nm , 18 - fold higher than the affinity of the wt peptide ( table s6 ). the wild type peptide in this region was also predicted to bind , albeit less strongly , to this patient &# 39 ; s second allele ( 26 nm to hla - dr * 1101 ). neither wild type nor mutant peptides were predicted to bind with high affinity to the class ii molecules of patient scl - 2 ( table s 6 ). the algorithms also predicted binding of patient - matched wild type and mutant peptides to a single hla class i allele in each patient , though the binding affinities were only moderate ( 27 to 78 nm , table s 6 ). cd4 cells are known to recognize peptides presented by mhc class ii alleles and play central roles in both tumor immunity and autoimmunity ( 10 , 11 ). in light of this knowledge and our finding that the predicted affinities for class ii peptides were much higher than for class i peptides , we searched for cd4 t cells recognizing the predicted peptides in pbmcs from patients whose tumors contained polr3a mutations . cd154 expression at 18 hours after peptide stimulation was used to identify peptide - activated cd4 + t cells ( 12 , 13 ). in patient scl - 4 , cd4 t cell activation was observed in response to the patient - matched mutant peptide but not to the wt peptide ( fig1 a , c ). moreover , no cd4 t cell responses to these peptides were observed in t cells from a healthy control matched with scl - 4 at hla - dr * 1101 ( fig1 a ). thus , the experimental data confirmed the in silico predictions . the experimental data also confirmed the predicted reactivity of t cells from patient scl - 42 , with a 2 - fold increase in the number of cd4 + cd154 + t cells in response to both the wt and mutant scl - 42 peptides over control conditions . the frequency of responding cells was about a log lower in scl - 42 compared to scl - 4 , with ˜ 1 : 5 , 000 cd4 t cells responding ( fig1 b , c ). the cd4 t cell responses to wt and mutant scl - 42 peptides were abolished by treatment with anti - hla - dr antibodies but not by an isotype control ( fig . s3 ). as in patient scl - 4 , no response to rpc1 peptides was observed in t cells from a healthy control matched with scl - 42 at hla - dr * 0701 ( fig1 b ). as predicted by the in silico binding algorithms ( table s 6 ), patient scl - 2 did not respond to either wild type or mutant peptides , but did express cd154 in response to the positive control stimulus , demonstrating that her cells were immune competent ( fig . s5 ). these data document the existence of cd4 t cells reactive with peptides containing the rpc1 mutations in two of the three patients studied . the reactivity was patient , peptide , and hla - type specific . the frequencies of mutant peptide - reactive cd4 t cells observed in these scleroderma patients (˜ 1 : 600 to ˜ 1 : 5000 , fig1 ) were in the range observed for antigen - specific cd4 + t cells observed in other autoimmune processes ( 14 ). scl - 4 responded only to the mutant peptide , while patient scl - 42 responded to the mutant as well as to the wt peptides ( fig1 c ). it was possible that the cd4 t cells that were activated in response to the mutant peptide in scl - 42 were the same as those responding to the wt peptide . to evaluate this issue , we performed tcr spectratyping of t cells stimulated by either wt or mutant peptides . out of the 22 vβ families analyzed , 12 displayed a similar distribution of their cdr3 lengths in response to wt and mutant peptides including vβ8 , vβ17 , and vβ20 ( fig2 a - c ). in contrast , significant differences in the distribution of cdr3 lengths were observed for several other vβs ( vβ3 , vβ5 , vβ7 , vβ12 , vβ16 and vβ24 ) ( fig2 d - f ). for some vβs , marked skewing in cdr3 lengths was observed , with & gt ; 25 % of tcrs from cells - treated with either the mutant or the wt form of the peptide represented by a single cdr3 length . these data suggested that the t cells responding to the mutant peptides were not , in general , those responding to the wt peptides . to characterize the tcrs in more detail , we determined the sequence of the cdr3 regions in the vβ7 , vβ12 and vβ24 pcr products ( 5 ). two striking findings were revealed by massively parallel sequencing of these regions . first , the sequences of the dominant tcrs generated from tcells stimulated with the wt peptide were completely distinct from those stimulated by the mutant peptide ( table 3 ). in 5 of 6 dominant tcr &# 39 ; s identified by sequencing , the wt - and mutant - specific cdr3 sequences were precisely the lengths predicted by the spectratype analysis ( table 3 ). the sequencing results therefore strongly supported the conclusion from spectratyping that the mutant and wt peptides had stimulated many distinct t cell clones . second , there was a high degree of redundancy among the amino acid sequences — but not the nucleotide sequences — of the tcrs identified in this experiment . for example , we identified 17 different nucleotide sequences ( represented by 2066 clusters on the sequencing instrument ) that encoded the identical cdr3 amino acid sequence in t cells stimulated by the mutant peptide ( table 3 ). as t cells , unlike b - cells , do not undergo continued evolution once a successful vdj rearrangement has occurred ( 15 , 16 ), these data document the existence of multiple , independent t cell clones responding , and presumably binding , to the same mutant peptide . finally , we developed cdr3 - specific taqman assays to verify that distinct populations of wt and mutant - specific t cells were present in the peripheral blood of scl - 42 prior to the short - term cultures used in the experiments described above . the vβ24 tcrs were chosen for this experiment because their cdr3 sequences were the most abundant in the sequencing analysis and were each encoded by multiple distinct nucleotide sequences ( table 3 ). the tcrs expected to bind the mutant and wild type peptides were detected in uncultured scl - 42 pbmcs ( fig . s4 ). neither tcr was detectable in the pmbcs of patient scl - 4 , used as a control . consenting scleroderma patients with confirmed cancer diagnoses were recruited from the johns hopkins scleroderma center . scleroderma patients met the american college of rheumatology criteria for scleroderma ( 38 ). existing cancer pathology specimens were obtained from prior surgical procedures performed as part of routine clinical care . the closest serum sample to cancer diagnosis was studied in all patients , and dna and pbmc samples were obtained in consenting participants . the johns hopkins institutional review board approved the acquisition of clinical data and all biological samples for this study . demographic and clinical data were abstracted from the johns hopkins scleroderma center database and careful medical record review . cancer diagnosis dates and histology were determined by review of the initial diagnostic pathology report . the clinical onset of scleroderma was defined by the first scleroderma symptom , either raynaud &# 39 ; s or non - raynaud &# 39 ; s . the interval between scleroderma onset and cancer diagnosis was calculated for each subject ( cancer date — scleroderma onset date ). the scleroderma cutaneous subtype and modified rodnan skin score were defined by established criteria ( 39 , 40 ). all sera were tested for autoantibodies against rpc1 , top1 , and cenpb as previously described ( 3 ). demographic and clinical data were compared across autoantibody groups , and differences in continuous and dichotomous / categorical variables were assessed by the kruskal - wallis and fisher &# 39 ; s exact tests , respectively . the clinical features and cancer types of the 16 patients evaluated in this study are listed in table 1 . eight patients were positive for anti - rpc1 antibodies , five for anti - top1 antibodies and three for anti - cenpb antibodies . enhanced nucleolar staining with the anti - rpc1 antibodies ( 3 ) was observed in the tumors of all eight patients . no subject was positive for more than one autoantibody . clinical phenotypic characteristics were representative of those expected in each autoantibody group ( e . g . severe diffuse disease in rpc1 - positive patients ), and patients with rpc1 autoantibodies had a shorter interval between scleroderma onset and cancer diagnosis ( median of − 0 . 1 years vs . 13 . 4 years for patients with top1 autoantibodies and 34 . 0 years for patients with cenpb autoantibodies ; p = 0 . 05 ). seven of the 16 patients had a short interval (+/− 2 years ) between scleroderma onset and cancer diagnosis , and 6 of these 7 patients ( 85 . 7 %) were positive for anti - rna polymerase iii antibodies . genomic dna libraries were prepared following illumina &# 39 ; s ( illumina , san diego , calif .) suggested protocol with the following modifications . ( 1 ) 50 to 75 microliters ( μl ) of genomic dna from tumor or normal cells in a total volume of 100 μl te was fragmented in a covaris sonicator ( covaris , woburn , mass .) to a size of 100 to 500 bp . dna was purified with a nucleospin extract ii kit ( cat # 740609 , macherey - nagel , germany ) and eluted in 50 μl of elution buffer included in the kit . ( 2 ) 45 μl of purified , fragmented dna was mixed with 40 μμl of h 2 o , 10 μμl end repair reaction buffer and 5 μl of end repair enzyme . all reagents used for this step and those described below were from new england biolabs ( neb cat # e6040 , ipswich , mass .) unless otherwise specified . the 100 μl end - repair mixture was incubated at 20 ° c . for 30 min , purified by a pcr purification kit ( cat # 28104 , qiagen ) and eluted with 42 μl of elution buffer ( eb ). ( 3 ) to a - tail , all 42 μl of end - repaired dna was mixed with 5 μl of 10 × da - tailing reaction buffer and 3 μl of klenow fragment ( 3 ′ to 5 ′ exo -). the 50 μl mixture was incubated at 37 ° c . for 30 min before dna was purified with a minelute pcr purification kit ( cat # 28004 , qiagen ). purified dna was eluted with 27 μl of 70 ° c . eb . ( 4 ) for adaptor ligation , 25 μl of a - tailed dna was mixed with 10 μl of pe - adaptor ( illumina ), 10 μl of 5x ligation buffer and 5 μl of quick t4 ligase . the ligation mixture was incubated at room temperature ( rt ) or 20 ° c . for 15 min . ( 5 ) to purify adaptor - ligated dna , 50 μl of ligation mixture from step ( 4 ) was mixed with 200 μl of nt buffer from nucleospin extract ii kit ( cat # 636972 , clontech , mountain view , calif .) and loaded into a nucleospin column . the column was centrifuged at 14000 g in a desktop centrifuge for 1 min , washed once with 600 μl of wash buffer ( nt3 from clontech ), and centrifuged again for 2 min to dry completely . dna was eluted in 50 μl elution buffer included in the kit . ( 6 ) to obtain an amplified library , ten or twenty pcrs of 50 μl each were set up , each including 30 μl of h 2 o , 2 . 5 μl dimethyl sulfoxide ( dmso ), 10 μl of 5x phusion hf buffer , 1 . 0 μl of a dntp mix containing 10 mm of each dntp , 0 . 5 μl of illumina pe primer # 1 , 0 . 5 μl of illumina pe primer # 2 , 0 . 5 μl of hot start phusion polymerase , and 2 . 5 or 5 μl of the dna from step ( 5 ). the pcr program used was : 98 ° c . 1 minute ; 10 to 16 cycles of 98 ° c . for 20 seconds , 65 ° c . for 30 seconds , 72 ° c . for 30 seconds ; and 72 ° c . for 5 min . to purify the pcr product , 250 μl pcr mixture ( from the ten pcr reactions ) was mixed with 500 μl nt buffer from a nucleospin extract ii kit and purified as described in step ( 5 ). library dna was eluted with 70 ° c . elution buffer and the dna concentration was estimated by absorption at 260 nm . the targeted regions included all 53 exons of cenpb , polr3a , top1 . capture probes were designed ( 41 ) to capture both the plus and the minus strand of the dna and had a 33 - base overlap and were custom - synthesized by agilent technologies en masse on a solid phase and used for capture , essentially as described ( 42 ). approximately 3 μg of library dna was used per capture . after washing , the captured libraries were ethanol - precipitated and redissolved in 20 μl of tris - edta ( te ) buffer . the dna was then amplified in a pcr mix containing 51 μl of distilled water ( dh 2 o ), 20 μl of 5x phusion buffer , 5 μl of dimethyl sulfoxide ( dmso ), 2 μl of 10 mm dntps , 50 pmol of illumina forward and reverse primers , and 1 μl of hotstart phusion enzyme ( new england biolabs ) with the following cycling program : 98 ° c . for 30 s ; 15 cycles of 98 ° c . for 25 s , 65 ° c . for 30 s , 72 ° c . for 30 s ; and 72 ° c . for 5 min . the amplified pcr product was purified with a nucleospin column ( macherey nagel inc .) according to the manufacturer &# 39 ; s suggested protocol , except that the nt buffer was not diluted and the dna bound to the column was eluted in 45 μl of elution buffer . the captured libraries were quantified using an agilent bioanalyzer . captured dna libraries were sequenced with the illumina gaiix genome analyzer . sequencing reads were analyzed and aligned to human genome hg18 with the eland algorithm in casava 1 . 6 software ( illumina ). a mismatched base was identified as a mutation only when ( i ) it was identified by ten or more distinct pairs ; ( ii ) the number of distinct tags containing a particular mismatched base was at least 2 . 5 % of the total distinct tags ; and ( iii ) it was not present in & gt ; 0 . 5 % of the tags in the matched normal sample . mutations were confirmed by amplification of the relevant region with a single primer pair and evaluated as described in ( 43 ). loh analysis was performed in a similar way , using the primer pairs described in table s 3 . a patient was considered “ informative ’ for the snp if dna from the normal tissue of that patient was heterozygous for the snp . a tumor was determined to have undergone loh if & gt ; 75 % of the informative primer pairs in that patient had an allelic ratio less than the mean minus 2 standard deviations of those measured in control individuals without scleroderma . note that this analysis can only assess allelic imbalance , i . e ., a gain in one allele or a loss in the other allele , though it is often ( including in the current study ) interpreted as loh , i . e ., loss of an allele . all experiments with the peptide microarray were performed at proimmune inc . ( oxford , uk ). peptides were synthesized as 15 - mers with 10 overlapping amino acids from the previous peptide , spanning the entire rpc1 protein . peptides were printed on glass slides with multiple arrays per slide separated with gaskets , allowing for multiple donor sera to be tested per slide . donor serum was diluted 1 : 100 , 1 : 500 and 1 : 1000 and incubated on the array . a fluorescent anti - human igg antibody was used as a secondary antibody , results were detected using a ccd camera and analysis was done using ms excel . peptides were determined to be potential binders if the normalized average signal intensity was greater than 4 × the respective background negative control . binding was considered positive if the signal intensity was 4 × background for all three serum dilutions . rpc1 antibodies were assayed by elisa using a commercially available kit ( inova diagnostics ). cenp and top1 autoanitibody assays were performed as described in ( 3 ). to define whether patient antibodies recognized patient - specific mutated forms of rpc1 , full - length wild type human polr3a cdna was purchased from origene , and site - directed mutagenesis was performed to generate the three different rpc1 mutants , each with a single point mutation : e1072q , k1365n and 1104t , corresponding to the tumor mutations detected in patients scl - 2 , scl - 4 and scl - 42 , respectively . all were sequence verified before use . 35 s - methionine - labeled products were generated from the wild type and mutant dnas by ivtt reactions ( promega kit ). prior to use in immunoprecipitations , the radiolabeled proteins were electrophoresed on sds - page gels and visualized by fluorography . the radiolabeled signal generated by each of these products was similar ( 1 μl e1072q equivalent to : 1 . 1 , 1 . 2 and 1 . 7 μl of i104t , wt and k1365n , respectively ). equivalent radioactive amounts 35 s - methionine - labeled wt and mutated rpc1 proteins were used in immunoprecipitations performed as described in ( 44 ) with sera from three cancer scleroderma patients . one μl of each serum was used to immunoprecipitate the wild - type form of rpc1 as well as the specific rpc1 mutation found in the tumor from that patient . pbmcs were freshly isolated from whole blood by density - gradient centrifugation ( ficoll - paque plus , ge healthcare ), and were used fresh ( scl - 42 ) or frozen ( scl4 and scl2 ). for each patient , pbmcs from a donor expressing one matching hla - drb1 allele was selected and used as a control . cells were resuspended to a concentration of 1 . 5 × 10 6 cells / 150 μl in rpmi medium supplemented with serum , 2 mm l - glutamine , 100 u / ml penicillin , and 100 μg / ml streptomycin , and were plated onto 96 - well flat bottom plates . 1 μg / ml anti - human cd40 blocking antibody ( g28 . 5 , biolegend ) was added , and after 30 minutes , cells were stimulated for 18 hours as indicated : 4 μg / ml wild - type or mutant patient - matched rpc1 peptide , 4 μg / ml of peptidyl arginine deiminase 4 ( pad4 ) peptide as a negative control , or 4 μg / ml of a pool of class - ii peptides from infectious agents antigens ( ceft ) ( axxora ) as a positive control . peptide storage buffer was used for the unstimulated control . hla restriction was assessed by stimulating cells in the presence of 1 μg / ml anti - hla - dr blocking antibody ( l243 , biolegend ) or 1 μg / ml igg - 2a κ isotype control ( mopc - 173 , biolegend ). cells were washed with pbs after stimulation , stained with live / dead fixable blue dead cell stain ( molecular probes ), and then stained with bv510 - conjugated cd3 antibody ( okt3 , biolegend ), pacific blue - conjugated anti - cd4 antibody ( rpa - t4 , bd pharmingen ), apc - h7 - conjugated anti - cd8 ( sk1 , bd ), and pe - conjugated anti - cd154 antibody ( trap1 , bd pharmingen ). facs analysis was performed on facsaria flow cytometer - cell - sorter using facsdiva ( becton dickinson ) and flowjo software ( tree star inc . ashland , oreg ., usa ). the diversity of cdr3 regions for 22 tcr vβ regions was assessed using the tcrexpress quantitative analysis kit ( biomed immunotech , tampa fla .). briefly , rna was isolated from scl - 42 pbmcs after culture for 6 days with wt or mutant peptides ( invitrogen , carlsbad calif . ), and cdna was generated using random hexamers ( invitrogen ) and you prime first strand beads ( ge , buckinghamshire uk ) following the manufacturers protocols . cdr3 regions were amplified from cdna using two rounds of pcr with vβ - family specific pcr primers as per the manufacturer &# 39 ; s instructions . fragment length analysis was performed by the johns hopkins dna analysis facility . the distribution of cdr3 lengths for each vβ - family was determined and expressed as “ proportion of tcr ”. peaks were considered to be antigen - driven when the observed proportion of a given fragment size differed by more than 10 % between wt and mutant - stimulated cells . tcr libraries were prepared for sequencing using a truseq sample preparation kit following the manufacturer &# 39 ; s suggestions with the following modifications . input dna was prepared from the pcr products obtained from spectratyping analysis . product from wells using primers specific to the cdr3 regions of vβ3 , vβ5 , vβ7 , vβ12 , vβ16 and vβ24 were purified using a qiagen pcr purification kit . dna was eluted in 30 ul of 65 ° c . elution buffer . after a - tailing and igation to adaptors , the library was amplified in six reactions with a pcr mix containing 10 ul h20 , 1 . 5 ul dmso , 6 ul 5x phusion buffer , 6 ul dntps , 3 ul each of forward and reverse primers , 3 ul phusion polymerase ( 2 u / ul ) and 2 ul ligation reactions , and cycled using the following program , 98 ° c . for 30 s ; 14 cycles of 98 ° c . for 10 s , 65 ° c . for 30 s , 72 ° c . for 30 s ; and 72 ° c . for 5 min . the resulting product was purified using ampure beads , quantified with an agilent bioanalyzer , and sequenced using an illumina instrument . custom taqman assays for specific tcrs were developed using primer express v2 . 0 software and synthesized by applied biosystems . for detection of the vβ24 jβ1 . 1 wt tcr , a fam - labeled probe ( actgaagctttctttggac ; seq id no : 1 ), forward primer ( gcaccgggacagtgatgaa ; seq id no : 2 ), and reverse primer ( ggtcctctacaactgtgagtttggt ; seq id no : 3 ) were synthesized . for detection of the vβ24 jβ1 . 5 mutant tcr , a fam - labeled probe ( acagtaaatcagccccagc ; seq id no : 4 ), forward primer ( tgtgtgccaccagcagaga ; seq id no : 5 ), and reverse primer ( agtcgagtcccatcaccaaaa ; seq id no : 6 ) were synthesized . cdna from day 0 scl - 42 pbmcs was prepared as described and was amplified by one round of pcr using vβ24 - family specific pcr primers ( biomed immunotech , tampa fla .). expression of the specific vβ24 tcrs was determined in triplicate using standard abi chemistry and reagents . as detailed below , the anti - prc1 positive scleroderma patients with cancer shared many features , including a short interval between the first clinical signs of scleroderma and cancer diagnosis , aggressive cutaneous disease , and a high risk of scleroderma renal crisis . patient 1 palpated a breast mass in the summer of 2005 and was diagnosed with a breast invasive ductal carcinoma on 7 / 26 / 05 . around the time of her diagnosis , she developed hypertension , thrombocytopenia , seizures , and renal failure that progressed despite blood pressure control . she ultimately initiated peritoneal dialysis . her breast cancer was treated with lumpectomy , radiation therapy , and doxorubicin . in october of 2005 , she began to notice raynaud &# 39 ; s phenomenon , and in october of 2007 , she developed skin thickening . when first seen in our center on 2 / 4 / 08 , she was noted to have extensive and severe scleroderma skin thickening with a modified rodnan skin score ( mrss ) of 41 ( range is 0 - 51 with 51 representing most severe disease possible ). patient 2 was noted to have a mass on a chest radiograph , leading to a diagnosis of a small cell carcinoma of the lung on 3 / 22 / 06 . she clearly had raynaud &# 39 ; s phenomenon by april 2006 . she was treated with chemotherapy ( completed 7 / 06 ) and radiation therapy with prophylactic brain irradiation ( completed 9 / 06 ). by april of 2007 , she began to notice worsening of her raynaud &# 39 ; s phenomenon and swelling of her hands followed by the onset of rapid , diffuse skin thickening . she was initially treated for her cutaneous disease with mycophenolate mofetil and was noted to have a mrss of 47 on her visit to our center on 9 / 6 / 07 . she later required therapy with cyclophosphamide due to concern for interstitial lung disease , and by 12 / 13 / 11 , her mrss had decreased significantly to 5 . patient 4 had a known brca1 mutation and underwent a prophylactic oophorectomy based on her genetic risk . during this procedure , she was found to have stage iii ovarian adenocarcinoma with papillary serous features ( 4 / 5 / 06 ). she completed 6 cycles of paclitaxel and cisplatin in 8 / 2006 , and around this time developed raynaud &# 39 ; s phenomenon . her chemotherapy course was complicated by the development of pericarditis with tamponade physiology requiring drainage and a pericardial window , and there was no evidence of an infected or malignant effusion . when first seen here on 1 / 10 / 08 , she was noted to have significant skin disease ( mrss 21 ), numerous tendon friction rubs , a myopathy , and scleroderma renal crisis with a cr of 1 . 9 . in 2008 - 2009 , she was treated with a number of immunosuppressive agents targeting her cutaneous , muscle , and joint disease including mycophenolate , methotrexate , azathioprine and hydroxychloroquine , and her skin disease was significantly improved by 8 / 08 . in october 2011 , she developed a small bowel obstruction with imaging findings consistent with serosal implants in the context of a rising ca 125 level . she began weekly carboplatin , and her ca 125 level had normalized by april 2012 . patient 13 noticed bilateral hand and ankle swelling in may 2005 and raynaud &# 39 ; s phenomenon in august 2005 . she was diagnosed with invasive ductal carcinoma of the breast on 8 / 24 / 05 . she was treated with a mastectomy followed by doxorubicin and cyclophosphamide from 10 - 12 / 05 . in 01 / 06 , she developed worsening skin thickening in her hands , arthralgias and myalgias and began therapy with d - penicillamine and later methotrexate . she also initiated paclitaxel and trastuzumab for her cancer in 01 / 06 . she was seen at our center apr . 16 , 2007 and was noted to have progressive skin disease ( mrss 30 ); she was transitioned to mycophenolate for her cutaneous disease and gradually had an improvement in her skin disease ( mrss in 2012 was 2 ). patient 35 was diagnosed with breast ductal carcinoma in situ on aug . 16 2004 , treated with a mastectomy . in april 2006 , she developed symptoms consistent with carpal tunnel syndrome , and by 8 / 06 , she had lower extremity skin thickening and tendon friction rubs . raynaud &# 39 ; s phenomenon developed in 1 / 07 . when first seen at our center in 6 / 07 , her mrss was 48 , and she was on letrozole for her malignancy . after therapy with cyclophosphamide and mycophenolate , her cutaneous disease significantly improved ( mrss 3 by 3 / 11 ). patient 42 developed arthralgias and skin thickening in her fingers in 3 / 2007 that rapidly progressed to diffuse skin thickening . in 4 / 2007 she developed raynaud &# 39 ; s phenomenon also . by 10 / 07 , she developed hypertension requiring ace - inhibitor therapy , and her mrss was 18 . despite therapy with mycophenolate , her cutaneous disease progressed , and by 3 / 08 , her mrss had increased to 37 . in 4 / 08 , methotrexate was added to her regimen with some improvement in flexibility and new hair growth ; however her mrss remained at 35 in 7 / 08 . in 9 / 08 , in the setting of increased cutaneous activity , she was diagnosed with a stage ii , triple negative , invasive ductal cancer of the breast . she was treated with mastectomy ( 10 / 08 ) and chemotherapy with cyclophosphamide and docetaxel ( 12 / 08 - 2 / 09 ). her cutaneous symptoms gradually improved in the setting of ivig therapy , and by 12 / 11 her mrss was 8 . patient 81 was diagnosed with an adenocarcinoma of the colon with positive lymph nodes in 5 / 05 requiring bowel resection and 5 - fu and platinum chemotherapy . in 7 / 09 , he developed diffuse cutaneous skin disease and by 11 / 09 had raynaud &# 39 ; 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