Identification of autoantibodies elicited in a patient with prostate cancer presenting as dermatomyositis

Authors


Douglas G. McNeel md phd, Section of Medical Oncology, K4/518 Clinical Science Center, 600 Highland Avenue, Madison, WI 53792, USA. Email: dm3@medicine.wisc.edu

Abstract

Objectives:  Dermatomyositis is an uncommon autoimmune disease distinguished by proximal muscle weakness and a characteristic skin rash. Dermatomyositis has also frequently been associated with malignancy, typically heralding the diagnosis of ovarian, lung, gastric, or colorectal cancer. We report an unusual case of prostate adenocarcinoma preceded by a diagnosis of dermatomyositis. We hypothesized that in this particular patient, proteins produced by the neoplastic prostatic tissue, which might be normally expressed in muscle tissue, were immunologically recognized as autoantigens.

Methods:  Serum from this patient was used to screen a cDNA lambda phage expression library from normal prostate tissue for prostate protein-specific IgG.

Results:  We identified several immunoreactive plaques encoding known autoantigens, and several encoding known muscle-related proteins, including aldolase C, eukaryotic translation elongation factor 1 α 1, transgelin, and acetyl-coenzyme A acyltransferase 1. IgG specific for these proteins were not specifically recognized in sera from other patients with prostate cancer compared with male control blood donors, and were not specifically recognized in a small panel of sera from patients with breast or ovarian cancer and dermatomyositis.

Conclusions:  Our results demonstrate that this patient with prostate cancer presenting as dermatomyositis had autoantibodies to specific proteins, possibly associated with his autoimmune myopathy. Moreover, given this patient's history and the multiple treatment options for prostate cancer, the identification of dermatomyositis in men should prompt an evaluation to exclude a concurrent diagnosis of prostate cancer.

Introduction

Prostate cancer continues to be a significant health problem worldwide. It is currently the most commonly diagnosed malignancy in the United States.1 The incidence of prostate cancer, and the mortality rate due to prostate cancer, have similarly been increasing over the last decade in several Asian countries, including Japan and Singapore.2 There has been much interest over the last several years in developing immunotherapeutic approaches to the treatment of prostate cancer, and at present many clinical vaccine trials are being conducted targeting known prostate-specific proteins such as prostate-specific antigen (PSA),3 prostatic acid phosphatase (PAP),4,5 and prostate-specific membrane antigen (PSMA).6 We have previously reported that some patients with prostate cancer have autoantibodies and T-cell responses to prostate cancer-associated proteins.7,8 The identification of immune responses to proteins produced by prostate cancer suggests that immunological tolerance to these proteins can be overcome in vivo with cancer development and/or progression. Moreover, the identification of immune responses to prostate cancer-associated proteins might suggest antigens that could be explored as tumor antigens in future vaccine trials.9

Dermatomyositis is an unusual disease characterized by proximal muscle weakness and a facial rash. It is believed to be an autoimmune disease, and autoantibodies to muscle antigens, notably a nuclear protein Mi-2, have been previously identified in patients with dermatomyositis.10,11 At this point, however, it is unknown whether autoantibodies detected in dermatomyositis are causally related to the myositis, or are a byproduct of this response.12 Dermatomyositis is commonly associated with malignancy, and a recent review suggests that lung cancer and ovarian cancer have the strongest associations with dermatomyositis.13 At present, to our knowledge there have only been nine case reports in the medical literature of dermatomyositis being associated with prostate cancer.14–22

In the current report, we identified a patient with dermatomyositis whose symptoms were associated with his clinical course of prostate cancer. We hypothesized that autoantibodies to antigens shared by the prostate cancer and muscle tissue might have been elicited in this patient. To test this, sera collected from this patient were used to screen a prostate tissue cDNA expression phage library using SEREX (serological analysis of recombinant cDNA expression library). We identified several muscle-associated proteins from the prostate tissue library, suggesting that the dermatomyositis observed was associated with his prostate cancer and possibly autoimmune mediated.

Methods

Patient case history

A 72 year-old man presented to his primary physician with a 1-month history of weakness and dull aches in his shoulders and thighs bilaterally, exacerbated by physical activity. Additionally, over the preceding 2 weeks he had developed an erythematous, flat, non-scaling, non-painful, and occasionally pruritic rash over the front and back of his scalp. Blood counts, creatinine and serum chemistries were normal with the exception of an elevated creatine kinase (CK) of 3102 U/L, elevated lactate dehydrogenase (LDH) of 419 U/L, and elevated erythrocyte sedimentation rate (ESR) of 62 mm/h. An electromyogram (EMG) demonstrated changes consistent with an inflammatory myopathy, and he was diagnosed with dermatomyositis. The patient was treated with prednisone 60 mg daily followed by a long steroid taper over a treatment period of 9 months, with complete resolution of symptoms within 3 months of therapy.

Four months prior to the initial presentation of dermatomyositis, the patient was incidentally found to have a 5 mm left upper lobe lung nodule by chest X-ray. A computed tomography (CT) scan was performed which demonstrated at least two suspicious lung nodular densities in the right lung. A follow-up chest CT scan performed 6 months later, and 3 months after the diagnosis of dermatomyositis, revealed multiple new bilateral pulmonary nodules, the largest 4–5 mm in size. A CT scan 4 months later, and now 7 months following his diagnosis of dermatomyositis, revealed a new small soft tissue mass in the retrocrural region consistent with a metastatic lymph node. An abdominal CT was then performed and revealed extensive retroperitoneal lymphadenopathy extending from the upper abdominal aorta to the bifurcation of the common iliac arteries, with at least seven lymph nodes measuring 2–3 cm in size. A fine needle aspirate was performed of one of the retroperitoneal lymph nodes and demonstrated adenocarcinoma of unclear etiology. Immunohistochemical analysis was negative for prostate specific antigen (PSA) staining. However, digital rectal exam revealed a 1-cm left-sided prostate nodule somewhat fixed to the pelvic sidewall, and the patient's serum PSA level was found to be elevated at 79.4 ng/mL. Of note, the most recent PSA measurement prior to this was 6.4 ng/mL and had been 15 months earlier, 5 months preceding the diagnosis of dermatomyositis.

A prostate biopsy was performed revealing an adenocarcinoma, Gleason grade (4 + 3 = 7), involving 30% of the biopsy volume in the left lobe, the right lobe biopsies being negative for malignancy. A follow-up bone scan was found to be negative for evidence of bone metastases. He was started on androgen-deprivation therapy with bicalutamide followed 2 weeks later by a depot injection of leuprolide acetate. The patient denied any genitourinary symptoms, abdominal pain or bone pain. However, coincident with the diagnosis of prostate cancer, and now 1 month after completing the initial steroid taper, he developed a return of proximal muscle weakness with a rise in his CK (to 276 U/L from 150 U/L the month prior). Given his other medical issues, steroid treatment was not reinitiated. His serum PSA declined from 79.4 to 1.4 ng/mL over 6 months, and curiously, his dermatomyositis symptoms also resolved during this time period. Repeat staging CT scans demonstrated a marked decrease in the size of the retroperitoneal adenopathy and lung nodules.

The patient did well for approximately 1 year before his serum PSA again began to rise. Specifically, 2.5 years after his initial presentation of dermatomyositis, and 1.5 years after his diagnosis of prostatic adenocarcinoma, the patient's PSA was found to have slowly increased from 1.9 to 2.3 ng/mL over 2 months, which prompted the addition again of bicalutamide. While on bicalutamide and leuprolide acetate therapy, his PSA continued to increase, to 11.5 ng/mL 2 months later, at which point the bicalutimide was discontinued. In addition, within 1 month after beginning bicalutamide, he had recurrence of a non-painful violaceous macular rash on his frontal scalp, with slight tenderness of his quadriceps and biceps muscle groups. Laboratory evaluation revealed a CK of 985 U/L, LDH of 384 U/L, and ESR of 55 mm/h. A prednisone burst was begun, 60 mg daily, followed by a rapid taper. His PSA subsequently decreased to 6.1 ng/mL the following month, coincident also with discontinuing bicalutamide, and with resolution of his dermatomyositis symptoms. At the time of this writing, the patient remains in follow up, nearly 4 years after the initial diagnosis.

Subject populations

Blood was obtained from the patient described above at three different time points between March 2003 and July 2004 under a University of Wisconsin IRB-approved protocol. Specifically, sera were collected at time points of 6 months after the diagnosis of prostate cancer (without symptoms of dermatomyositis; serum PSA 1.4 ng/mL), approximately 18 months after the diagnosis of prostate cancer on casodex (with symptoms of dermatomyositis; serum PSA 7.6 ng/mL), and 1 month later after stopping casodex (symptomatic dermatomyositis, hormone-refractory disease, serum PSA 11.5 ng/mL). Sera were stored in aliquots at −80°C until use. Sera were also obtained from 25 other consenting patients with prostate cancer (six of whom had early stage prostate cancer without primary therapy and 19 of whom had androgen-independent disease), eight female patients with breast or ovarian cancer and a history of dermatomyositis, four female volunteer blood donors without cancer or dermatomyositis, and 25 male volunteer blood donors without cancer or dermatomyositis, from the University of Wisconsin, Madison, and the University of Washington, Seattle, all under IRB-approved protocols. These sera were also stored in aliquots at −80°C until use.

Serological analysis of recombinant cDNA expression library

The sera obtained were first precleared of antibodies to Escherichia coli and lambda phage by ‘panning’ sera with nitrocellulose membranes that had been coated with lambda phage-transduced E. coli. Serological analysis of recombinant cDNA expression library (SEREX) analysis was then performed similar to that previously described by us and others.23–25 Specifically, a lambda phage cDNA expression library derived from normal human prostate tissue (Clontech, Palo Alto, CA, USA) was used to transduce the XL1-Blue E. coli strain. The transduced bacteria were then plated in a lawn on multiple 150-mm culture dishes, such that over 700 000 PFU were ultimately screened with the patient's sera. After the plaques were allowed to develop, nitrocellulose filters, impregnated with 10 mmol/L isopropyl β-D-thiogalactopyranoside (IPTG), were overlaid to permit induction of protein expression and transfer to the nitrocellulose membrane. Membranes were then removed, washed, and blocked with 10 mmol/L Tris 8.0, 150 mmol/L NaCL, 0.5% Tween-20 (TBST) + 1% bovine serum albumin (BSA). The precleared sera were then diluted 1:100 and incubated with the nitrocellulose membranes overnight at 4°C. The membranes were then washed, and bound human IgG was detected with an alkaline phosphatase-conjugated monoclonal antihuman IgG antiserum diluted in TBST + 1% BSA. The membranes were washed again and then developed with 0.3 mg/mL nitroblue tetrazolium chloride (NBT) and 0.15 mg/mL 5-bromo 4-chloro 3-indoylphosphate (BCIP) in 100 mmol/L Tris 9.5, 100 mmol/L NaCl, and 5 mmol/L MgCl2. Immunoreactive plaques, identified as purple-colored spots on the membranes, were isolated as cores from the corresponding agar plates, and the phage were eluted overnight in 100 mmol/L NaCl, 10 mmol/L MgSO4, 35 µmol/L Tris 7.5, and 0.01% gelatin. A secondary screen was then carried out with the eluted phage to identify and remove false positives. Specifically, positive eluted phage from the primary screen were plated at lower plaque density and rescreened with the original sera in identical fashion as described. Plaques were reviewed at this point by multiple blinded observers to reduce the subjectivity in identifying immunoreactive plaques. Plaques that were above background intensity but were qualitatively paler in color than an IgG positive control phage were labeled ‘weakly reactive’, and those that were as dark or darker than an IgG positive control phage were labeled ‘strongly reactive.’ Purified phage plaques were converted to phagemids by transduction of the recombinase-positive E. coli strain BM25.8 (Clontech). pBluescript-derived plasmids encoding the gene product of interest were selected in ampicillin-containing media, and sequenced using plasmid specific primers and the ABI BigDye terminator sequencing reaction by the University of Wisconsin Biotechnology Center. Gene products encoded by the recombinant phage were identified by GenBank database searches.26

Immunoblot analysis

IgG responses to the antigens identified above were identified in other patients with prostate cancer, other subjects with dermatomyositis, and volunteer control blood donors using similar methods to those above and to what we have previously reported.23 Briefly, the purified phage encoding the identified antigens were directly spotted onto lawns of E. coli in multiple replicates. IPTG-impregnated membranes were placed over the transduced bacteria as above, blocked, and probed individually with different sera obtained from individual subjects diluted 1:100 in TBST buffer. Membranes were washed, and human IgG detected as above. Immunoreactivity to individual plaques was scored as positive or negative by visual inspection compared with positive control (encoding human IgG) and negative control (not encoding an antigen) phage plaques. Plaques were scored positive if they were more immunoreactive than the phage encoding the negative control.

Results

Prostate protein-specific IgG were identified in the sera of the patient with dermatomyositis

In order to identify possible autoreactive immune responses, sera from the patient with dermatomyositis were used to probe a prostate cDNA expression library. Specifically, sera had been previously obtained from this patient at three different time points following the diagnosis of prostate cancer, once during a period of disease quiescence, and twice during periods of dermatomyositis flare. Because it was not known a priori if there were an optimal time to evaluate for immunological responses, sera were used from each time point, and the results pooled, to identify a panel of immunologically recognized proteins. Specifically, sera from each of these time points were used to screen over 200 000 PFU of a lambda phage cDNA expression library prepared from normal prostate tissue. From this analysis 84 possibly immunoreactive plaques were identified, as illustrated in Figure 1. Immunoreactive phage plaques were then removed from the corresponding agar plates, and eluted phage were replated at lower plaque density and rescreened as above with patient sera to isolate specific, purified phage. An example is shown in Figure 2. From this analysis, false positives were eliminated from the primary screen, and 11 phage were ultimately identified as weakly reactive (Fig. 2a) or strongly reactive (Fig. 2b). Phage were then sequenced and identified (Table 1). As indicated, two of these strongly reactive proteins were identified twice from different phage isolates.

Figure 1.

Prostate protein-specific IgG were identified in the sera from this patient with dermatomyositis. Shown is a representative serological analysis of recombinant cDNA expression library nitrocellulose blot with approximately 10 000 PFU per blot screened with patient sera. The insets show magnifications of the screen, with arrows pointing to immunoreactive plaques.

Figure 2.

Purification of immunoreactive phage plaques. Immunoreactive phage were isolated and purified by rescreening at lower phage density. Shown are representative blots for phage with ‘weak’ immunoreactivity (a) or ‘strong’ immunoreactivity (b).

Table 1.  Immunoreactive proteins identified
Phage cloneNumber of isolatesGenBank accessionDescription
  1. Shown are the identities, and GenBank accession numbers, for the nine purified immunoreactive phage.

Weak reactivity
 11NM_001648Prostate-specific antigen
 21BC003613Aldolase C
 31NM_000969Ribosomal protein L5 (RPL5)
 41BC029343Eukaryotic translation elongation factor 1 alpha 1
 51BC024296Transgelin
 61BX936369.5RP11–430J6 DNA on chromosome 16
 71AL390074.17RP11–294H11 DNA on chromosome 6
Strong reactivity
 82NM_015358Zinc-finger protein, CW type with coiled-coil domain 3 (ZCWCC3)
 92NM_001607Acetyl-coenzyme A acyltransferase 1 (peroxisomal 3-oxoacyl-coenzyme A thiolase; ACAA1)

Sera from other patients with dermatomyositis or prostate cancer were evaluated for IgG responses to identified antigens

The purified phage encoding identified antigens were used to transduce bacteria for immunoblot screening with sera from eight patients with breast or ovarian cancer and with a history of dermatomyositis, four volunteer female blood donors without a history of cancer or dermatomyositis, 25 patients with prostate cancer, and 25 volunteer male blood donors without a history of cancer. An example of this type of analysis is shown in Figure 3, and the results from all of these studies is shown in Table 2. No significant differences were found in the immunoreactivity to individual antigens among these different populations.

Figure 3.

Immunoblot analysis with purified phage encoding known proteins. Shown are examples of the immunoblot analysis performed with sera from multiple patients with prostate cancer, dermatomyositis, or normal volunteer blood donors. (a) Immunoblot with sera from index patient. (b) Immunoblot from separate patient with prostate cancer. *ZFCCW3-encoding phage; ^acetyl-CoA acyltransferase 1-encoding phage. Positive control phage encodes human IgG.

Table 2.  Summary of immunoblot analysis
Antigen-encoding phageProstate cancerMale controlsDermatomyositisFemale controls
  1. Shown is the number of positive immunoreactive plaques found for each purified phage per subject population.

Prostate-specific antigen 3/252/250/80/4
Aldolase C 0/251/250/80/4
Ribosomal protein L5 (RPL5) 1/250/250/80/4
Eukaryotic translation elongation factor 1 alpha 1 0/250/250/80/4
Transgelin 0/250/250/80/4
RP11–430J6 DNA on chromosome 16 1/250/250/80/4
RP11–294H11 DNA on chromosome 6 7/255/250/80/4
Zinc-finger protein, CW type with coiled-coil domain 3 (ZCWCC3) 4/254/250/80/4
Acetyl-coenzyme A acyltransferase 1 (peroxisomal 3-oxoacyl-coenzyme A thiolase; ACAA1)12/259/251/81/4

Discussion

In this report we describe an unusual case of prostate cancer in a patient who presented with dermatomyositis. Dermatomyositis has a strong association with malignancy; however, it has most commonly been associated with ovarian cancer and lung cancer.13,27 To our knowledge, there have only been nine previous case reports of dermatomyositis being associated with prostate cancer.14–22 Most of these reports describe an initial presentation of dermatomyositis followed by a diagnosis of prostate cancer. The first reported case by Rapoport and Omenn in 1968,15 as well as a similar case by Ansai and Koseki in 1996,14 describe patients who presented with dermatomyositis, had rapidly progressive and fatal illnesses, and were found on autopsy to have widespread metastatic prostate cancer. In the reports of Joseph,18 Masuda,19 and Subramonian22 the authors described an improvement in the dermatomyositis symptoms following prostatectomy or androgen deprivation therapy. In an attempt to understand the association between dermatomyositis and prostate cancer, Otnes and colleagues reported a case in 1978 in which a patient presented with dermatomyositis and prostate cancer and in whom prostate and skin biopsies were performed. These authors described large numbers of plasma cells within the prostate tissue, and IgG staining within prostate tissue as well as skin biopsies. They could not detect circulating antibodies using techniques available at that time, but suggested that an autoimmune reaction might be involved in the disease process.16 There has otherwise been no investigation, to our knowledge, prior to our report of an autoimmune association between dermatomyositis and prostate cancer.

In the current report, similar to most other reported cases, our patient's symptoms of dermatomyositis preceded the diagnosis of metastatic prostate cancer. The time frame of less than 2 years suggested a relationship between the two diagnoses, as has been observed with other malignancies and dermatomyositis.27 In addition, the response of the dermatomyositis symptoms to the treatment of prostate cancer (androgen deprivation), further suggests a link between these two diagnoses, as has been reported by Joseph and Masuda.18,19

There is a growing appreciation of the relationship between various autoimmune diseases and malignancy. With tumor ‘immune surveillance’ it is generally assumed that an immune response is elicited to antigens presented by the tumor that are normally expressed in other tissues, producing an autoimmune disease. Whether autoantibodies detected in autoimmune diseases are a byproduct of this response, are causally related, or are completely unrelated, remains unknown and elusive to prove or disprove.12 Autoantibodies to muscle antigens, however, have previously been identified in patients with dermatomyositis,28 and a dominant nuclear protein autoantigen, Mi-2, has been previously identified in other patients with dermatomyositis.10,11 In the current report, we hypothesized that if our patient had an autoimmune disease associated with his prostate cancer, autoantibodies to prostate-derived proteins might be detectable. We report the identification of several proteins using this patient's sera to screen a normal prostate cDNA expression library, including PSA, aldolase C, eukaryotic translation elongation factor 1 α 1, transgelin, acetyl-coenzyme A acyltransferase 1, ribosomal protein L5, a putative zinc-finger protein, and two uncharacterized antigens. Immune responses to these proteins were not more common in a small cohort of other patients with prostate cancer versus a male control population, and immune responses to these proteins were not detected in sera from a small cohort of female cancer patients with dermatomyositis, suggesting that these responses were, for the most part, specific to this patient and not commonly associated with prostate cancer or dermatomyositis. However, many patients with advanced androgen-independent prostate cancer exhibit non-neurological weakness. While the etiology of this weakness may be multifactorial, it would be interesting in the future to evaluate immune responses to the proteins identified in a larger population of patients with prostate cancer with such symptoms to determine if immune responses to muscle-related proteins are more common in this specific population.

Of the proteins identified, several are known muscle-related proteins, including aldolase, a protein known to be highly expressed in muscle tissue.29 There are also multiple reports of elevated serum levels of aldolase in patients with prostate cancer.30,31 Transgelin, similarly, is a poorly characterized protein with predominant expression in smooth muscle.32 Curiously, however, this protein has recently been identified as a protein overexpressed in prostate stromal tissue.33 Finally, translation elongation factor 1α has predominant expression in brain, heart, and muscle tissue.34 Certainly, while the identification of proteins with muscle expression is not proof of a causal relationship between dermatomyositis and prostate cancer in this patient, it is nonetheless interesting that these proteins were identified from a normal prostate cDNA expression library. Of note, these three proteins were not significantly immunologically recognized antigens in sera from other patients with prostate cancer or the volunteer blood donors. Some of the other proteins identified in this study may be commonly recognized autoantigens. We have previously reported the detection of autoantibodies to PSA.7,23 Autoantibodies to other ribosomal proteins, including L1435 and elongation factor 2 kinase,36 have been detected in other patients with autoimmune connective tissue disorders. The finding of antibodies to acetyl-coenzyme A acyltransferase 1 and the putative zinc finger protein in multiple other patients and controls suggests that these may be common autoantigens without specificity for dermatomyositis or prostate cancer.

In summary, we report an unusual case of prostate cancer presenting as dermatomyositis. The timing of these diagnoses, and the response of the dermatomyositis to treatment of the prostate cancer, suggests a link between these diseases. The presence of antigen-specific IgG in this patient, several of which were specific for antigens known to be expressed in muscle tissue, suggests a possible autoimmune component to the disease. Furthermore, given that therapy for prostate cancer can be curative for localized disease, and that even with metastatic disease early treatment can prolong survival, the diagnosis of dermatomyositis in men should prompt an evaluation for prostate cancer.

Acknowledgments

This work is supported for D.G.M. by NIH (K23 RR16489), the Howard Hughes Medical Institute, and the Prostate Cancer Foundation (CaPCURE).

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