Close temporal relationship between onset of cancer and scleroderma in patients with RNA polymerase I/III antibodies

Authors


Abstract

Objective

This study was undertaken to examine the temporal relationship between scleroderma development and malignancy, and to evaluate whether this differs by autoantibody status among affected patients.

Methods

Study participants had a diagnosis of scleroderma, a diagnosis of cancer, cancer, an available serum sample, and a cancer pathology specimen. Sera were tested for autoantibodies against topoisomerase I, centromere, and RNA polymerase I/III by immunoprecipitation and/or enzyme-linked immunosorbent assay. Clinical and demographic characteristics were compared across autoantibody categories. Expression of RNA polymerases I and III was evaluated by immunohistochemistry using cancerous tissue from patients with anti–RNA polymerase antibodies.

Results

Twenty-three patients were enrolled. Six patients tested positive for anti–RNA polymerase I/III, 5 for anti–topoisomerase I, and 8 for anticentromere, and 4 were not positive for any of these antigens. The median duration of scleroderma at cancer diagnosis differed significantly between groups (−1.2 years in the anti–RNA polymerase I/III group, +13.4 years in the anti–topoisomerase I group, +11.1 years in the anticentromere group, and +2.3 years in the group that was negative for all antigens tested) (P = 0.027). RNA polymerase III demonstrated a robust nucleolar staining pattern in 4 of 5 available tumors from patients with antibodies to RNA polymerase I/III. In contrast, nucleolar RNA polymerase III staining was not detected in any of 4 examined tumors from the RNA polymerase antibody–negative group (P = 0.048).

Conclusion

Our findings indicate that there is a close temporal relationship between the onset of cancer and scleroderma in patients with antibodies to RNA polymerase I/III, which is distinct from scleroderma patients with other autoantibody specificities. In this study, autoantibody response and tumor antigen expression are associated. We propose that malignancy may initiate the scleroderma-specific immune response and drive disease in a subset of scleroderma patients.

Patients with scleroderma may have an increased risk of malignancy compared with the general population (1–6). A wide array of cancers has been reported in scleroderma, although lung and breast cancers are thought to be the most common (3, 4, 6, 7). Although it is controversial whether malignancy risk is truly increased in scleroderma patients, reports detailing a close, at times concurrent, onset of scleroderma and malignancy raise the possibility of malignancy triggering an autoimmune disease process in a subset of scleroderma patients (8–10). Among scleroderma patients, this tight temporal association is most striking for breast cancer, with the majority of patients developing scleroderma within 18 months of cancer diagnosis (11–14). In 2 case series reviewing scleroderma patients with breast cancer, it has been estimated that up to 50% of breast cancer cases closely preceded or were diagnosed simultaneously with scleroderma (12, 14). Additionally, it has been reported that prompt treatment of a malignancy can abrogate the scleroderma disease process (8, 9, 15), suggesting that in these unique cases the biologic response to the malignancy or the malignant process itself may be driving the expression of scleroderma.

Despite this reported association between malignancy and scleroderma onset, few studies have evaluated scleroderma disease characteristics that associate with the presence or risk of malignancy, and little is known about potential mechanisms underlying this connection. We hypothesized that scleroderma-specific autoantibody production in a subset of patients with scleroderma is a manifestation of the immune response to tumor antigens that may be associated with or induce the scleroderma disease process. In this study, we evaluated whether clinical characteristics, including the temporal relationship between scleroderma and malignancy onset, differed by autoantibody status among patients with scleroderma and cancer. After demonstrating a temporal clustering between cancer onset and scleroderma in the RNA polymerase antibody–positive group, we investigated the expression of RNA polymerases I and III in cancerous tissue from these scleroderma patients compared with cancers from RNA polymerase antibody–negative patients, as well as noncancerous tissue from controls.

PATIENTS AND METHODS

Patients.

Participants were scleroderma patients who were followed up at the Johns Hopkins Scleroderma Center and had a new or past diagnosis of malignancy, an available serum sample, and an existing cancer pathology specimen available for histologic confirmation of cancer diagnosis. Patients with scleroderma who had a prior diagnosis of malignancy were identified via the Center's research database. Eligibility criteria included informed consent and either meeting the American College of Rheumatology (ACR; formerly, the American Rheumatism Association) criteria for scleroderma (16), having at least 3 of 5 features of the CREST syndrome (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, telangiectasias), or having definite Raynaud's phenomenon (RP), abnormal nailfold capillaries, and the presence of a scleroderma-specific autoantibody. For all patients, the closest available serum sample to cancer diagnosis was studied.

Demographic data, scleroderma subtype (limited versus diffuse skin disease), disease duration, date of cancer diagnosis, smoking status (never, former, or current), most recent Medsger disease severity scores (17), peak modified Rodnan skin thickness score (MRSS) (18), medication use prior to cancer diagnosis, autoantibody status, results of pulmonary function tests, and echocardiogram data were obtained from the Center's database and, when necessary, medical chart review. Patients were classified as having limited cutaneous or diffuse cutaneous disease according to the criteria of LeRoy et al (19); patients were considered to have limited cutaneous disease if scleroderma skin changes were noted only on the face and/or distal to the knees and elbows, and diffuse cutaneous disease if the trunk and/or proximal extremities were involved. Disease duration was defined as the period of time from the first non-RP symptom to the date of cancer diagnosis. Cancer onset was defined by the date of cancer diagnosis. All forced vital capacity (FVC) and diffusing capacity for carbon monoxide results were standardized by age, sex, and height according to National Health and Nutrition Examination Survey criteria and the criteria of Knudson et al (20, 21). Interstitial lung disease (ILD) was defined as an FVC <70% predicted, and pulmonary hypertension (PH) was defined as right ventricular systolic pressure ≥45 mm Hg on resting echocardiography (22) or by right-sided heart catheterization evidence of pulmonary arterial hypertension.

All studies of human materials were performed using samples provided in compliance with Johns Hopkins Institutional Review Board (IRB) and HIPAA regulations. Surgical procedures were performed for the management of disease in patients; the research tissue used in our studies was in excess of the biopsied tissue required for routine diagnostic purposes. Serum samples were collected under an IRB-approved protocol. Each serum sample was tested for autoantibodies against topoisomerase I by enzyme-linked immunosorbent assay (ELISA) using commercially available kits (Inova Diagnostics). The presence of antibodies against RNA polymerase I/III in each patient serum sample was assessed using 2 different assays. Serum samples were tested by immunoprecipitation using radiolabeled HeLa cell extracts. The presence of anti–RNA polymerase I/III antibodies was determined based on comigration of immunoprecipitated bands with those detected using an RNA polymerase I/III scleroderma reference serum, which was included in each precipitation set (data not shown). The findings for RNA polymerase III were also validated using a commercially available RNA polymerase III ELISA kit (Inova Diagnostics); in all cases, both assays demonstrated the presence of antibodies against RNA polymerase in the identified sera. The presence of anticentromere antibodies was determined by immunoprecipitation using in vitro transcription–translated 35S-methionine–labeled centromere protein B as previously described (23).

Immunohistochemistry.

Paraffin sections from affected cancerous tissue in 6 patients with RNA polymerase antibodies were initially available for study. Since the patient tissues were obtained and paraffin-embedded at various pathology units at different times, we first assessed fixation variation (and subsequent loss of antigenicity) by staining sections of each tissue with a monoclonal antibody to CD31 (Dako). This evaluation confirmed excellent tissue preservation and comparable antigenicity in tissues from 4 different patients (patients 1 and 35 [who both had breast cancer], patient 2 [who had lung cancer], and patient 4 [who had ovarian cancer]). Since 2 of the 6 originally identified patients with RNA polymerase antibodies did not have adequate tissue preservation, 1 additional patient (patient 42) with anti–RNA polymerase antibodies and breast cancer was recruited, and a tissue sample was obtained from this patient for further validation of our preliminary findings. Normal breast and ovary paraffin sections were purchased from US BioMax. Cancerous and normal tissue sections were stained with a monoclonal antibody against RNA polymerase I (polypeptide C; Abnova) or a polyclonal antibody against RNA polymerase III (POLR3A; Santa Cruz Biotechnology). Staining was visualized with diaminobenzidine according to the recommendations of the manufacturer (Dako), and all sections were counterstained with Mayer's hematoxylin.

Statistical analysis.

Clinical and demographic characteristics were compared across autoantibody categories. Statistical significance testing included Kruskal-Wallis test for continuous variables and Fisher's exact test for binomial and categorical variables. Comparison of tissue nucleolar RNA polymerase III antigen expression by serum anti–RNA polymerase III antibody status was performed using Fisher's exact test. Statistical analyses were performed using Stata 10.0 (StataCorp). P values less than 0.05 (2-tailed) were considered significant.

RESULTS

Among 2,367 patients who were seen at the Johns Hopkins Scleroderma Center, 210 had a known history of malignancy. Thirty-seven of these patients were seen between February and August of 2008 and were screened for entry into our study. Both serum and pathology samples were available for 23 of these individuals. These 23 cases therefore comprised our study population. Their mean ± SD ages at scleroderma and cancer diagnosis were 50.1 ± 12.1 years and 57.3 ± 11.0 years, respectively. The mean ± SD duration of scleroderma at cancer diagnosis was 7.2 ± 10.4 years, and the mean ± SD duration of RP at cancer diagnosis was 10.1 ± 14.1 years. The majority of the patients were female (95.7%) and white (95.7%). Nineteen patients (82.6%) met the ACR criteria for diagnosis of scleroderma, and the remaining 4 met CREST syndrome criteria. Fourteen patients (60.9%) had limited cutaneous disease, and 9 patients (39.1%) had diffuse skin involvement. The mean ± SD MRSS was 15.0 ± 14.6. Five individuals had ILD, 2 had scleroderma renal crisis, and 3 had a myopathy suggestive of inflammatory myositis. The types of malignancies were varied, but the majority (91.3%) were epithelial cell tumors, with the breast being the primary site in 13 cases (Table 1).

Table 1. Cancer site and histologic findings in the 23 patients with scleroderma
Breast, no. (%)13 (56.5)
 Ductal carcinoma in situ, no.4
 Invasive ductal carcinoma, no.5
 High-grade adenocarcinoma, no.1
 Invasive lobular carcinoma, no.3
Lung, no. (%)2 (8.7)
 Small cell carcinoma, no.1
 Adenocarcinoma, no.1
Lymphomas, no. (%)2 (8.7)
Skin, squamous cell carcinoma, no. (%)1 (4.3)
Ovary, poorly differentiated carcinoma, no. (%)1 (4.3)
Tongue, squamous cell carcinoma, no. (%)1 (4.3)
Uterus, endometrial adenocarcinoma, no. (%)1 (4.3)
Anus, squamous cell carcinoma, no. (%)1 (4.3)
Vagina, squamous cell carcinoma, no. (%)1 (4.3)

Of these 23 individuals, 6 tested positive for anti-RNA polymerase I/III, 5 for anti–topoisomerase I, 8 for anticentromere, and 4 for none of these 3 antibodies (Table 2). No individual produced antibodies to >1 of the tested autoantigens. Age, sex, race, smoking status, disease severity indices, medication use prior to cancer diagnosis, and the frequency of ILD or evidence of PH did not differ statistically between groups. In contrast, the median duration of scleroderma at cancer diagnosis differed significantly between groups. The median duration was −1.2 years in the anti-RNA polymerase I/III group (range −2 to 1.3 years), 13.4 years in the anti–topoisomerase I group (range 0.25 to 29 years), 11.1 years in the anticentromere group (range −2.0 to 36.9 years), and 2.3 years in the group of patients who were negative for all of these antibodies (hereafter referred to as the “antibody-negative group”) (range −1.2 to 5.0 years) (P = 0.027) (Figure 1).

Table 2. Characteristics of the study participants by autoantibody status*
 Pol I/III (n = 6)Topo (n = 5)CENP (n = 8)Negative (n = 4)P
  • *

    Pol I/III = anti–RNA polymerase I/III; topo = anti–topoisomerase I; CENP = anticentromere; ACR = American College of Rheumatology; MRSS = modified Rodnan skin thickness score.

  • The remaining patients met CREST syndrome (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, telangiectasias) criteria.

  • Defined as Medsger Raynaud's phenomenon (RP) severity score ≥2.

  • §

    Defined as Medsger general severity score ≥1; based on the presence of anemia or weight loss.

  • Defined as forced vital capacity <70% predicted.

  • #

    Defined as right ventricular systolic pressure ≥45 mm Hg or right-sided heart catheterization evidence of pulmonary arterial hypertension.

Median age at scleroderma diagnosis, years51.845.148.954.60.511
Median age at cancer diagnosis, years51.064.960.956.90.337
Sex, no. (%) female6 (100)5 (100)7 (87.5)4 (100)1
Race, no. (%)    0.391
 White6 (100)4 (80)8 (100)4 (100) 
 African American0 (0)1 (20)0 (0)0 (0) 
No. (%) of patients meeting ACR criteria6 (100)5 (100)5 (62.5)3 (75)0.176
Scleroderma classification, no. (%)    <0.001
 Limited0 (0)3 (60)8 (100)3 (75) 
 Diffuse6 (100)2 (40)0 (0)1 (25) 
Median duration of scleroderma at cancer diagnosis, years−1.213.411.12.30.027
Median duration of RP at cancer diagnosis, years0.2513.223.84.00.113
Smoking status, no. (%)    0.832
 Never4 (66.7)2 (40)5 (62.5)3 (75) 
 Former2 (33.3)3 (60)3 (37.5)1 (25) 
Disease severity, no. (%)     
 Severe RP1 (16.7)3 (60)5 (62.5)0 (0)0.100
 Abnormal general severity score§4 (66.7)2 (40)3 (37.5)1 (25)0.681
 History of renal crisis2 (33.3)0 (0)0 (0)0 (0)0.123
 History of myopathy2 (33.3)0 (0)0 (0)1 (25)0.206
Median MRSS369460.012
Interstitial lung disease, no. (%)2 (33.3)2 (40)0 (0)1 (25)0.231
Pulmonary hypertension, no. (%)#1 (16.7)1 (20)2 (25)1 (25)1
Medications prior to cancer diagnosis, no. (%)     
 Hormone replacement therapy1 (16.7)1 (20)2 (25)0 (0)0.892
 Prednisone1 (16.7)1 (20)1 (12.5)0 (0)1
 Methotrexate1 (16.7)0 (0)1 (12.5)0 (0)1
 Azathioprine0 (0)1 (20)0 (0)0 (0)0.391
 Cyclophosphamide0 (0)2 (40)0 (0)0 (0)0.063
 Mycophenolate mofetil0 (0)1 (20)0 (0)1 (25)0.202
Figure 1.

Distribution of scleroderma duration at cancer diagnosis by autoantibody status. There was a tight temporal relationship between malignancy diagnosis and scleroderma onset in patients producing anti–RNA polymerase I/III antibodies (anti–RNA Pol I/III) compared with patients with anti–topoisomerase I antibodies (antitopo) and patients with antibodies to centromere (anticentromere). The antibody-negative group (negative) showed a similar close relationship between scleroderma and cancer onset. Data are presented as box plots, where the boxes represent the 25th to 75th percentiles, the lines within the boxes represent the median, and the lines outside the boxes represent the 10th and 90th percentiles. Circles indicate outliers.

The median duration of RP at cancer diagnosis followed a similar trend, with a duration of 0.25 years in the anti–RNA polymerase I/III group (range −2.4 to 1 years), 13.2 years in the anti–topoisomerase I group (range 0.25 to 34 years), 23.8 years in the anticentromere group (range −5.0 to 36.9 years), and 4.0 years in the antibody-negative group (range −1.2 to 7.9 years) (P = 0.113). Patients in the anti–RNA polymerase I/III group had diffuse disease exclusively, whereas 40% of the patients in the anti–topoisomerase I group, none of the patients in the anticentromere group, and 25% of the patients in the antibody-negative group had diffuse cutaneous disease (P < 0.001). Correspondingly, the median MRSS was significantly higher in the anti–RNA polymerase I/III group (median score 36) than in the anti–topoisomerase I group (median score 9), anticentromere group (median score 4), or antibody-negative group (median score 6) (P = 0.012).

Characteristics of the 6 initially identified patients with anti–RNA polymerase I/III and a tight temporal clustering of scleroderma onset and malignancy diagnosis are reported in Table 3. Five of the malignancies were epithelial cell tumors, 3 of which originated in the breast. Cancer diagnosis closely preceded scleroderma onset in 4 individuals, and the range of scleroderma duration at cancer diagnosis was −2 to 1.3 years. Similarly, cancer preceded RP onset in 2 individuals and was diagnosed concurrently in 1 patient. These patients had aggressive skin disease with MRSS ranging from 14 to 48. Scleroderma renal crisis developed in 2 patients.

Table 3. Characteristics of the 6 initially identified anti–RNA polymerase I/III–positive patients*
Patient no.SexMalignancyScleroderma duration at cancer diagnosis, yearsRP duration at cancer diagnosis, yearsMRSSScleroderma complications
  • *

    RP = Raynaud's phenomenon.

  • Maximum modified Rodnan skin thickness score (MRSS) during disease course.

  • Complications evaluated included renal crisis, myopathy, interstitial lung disease (ILD), and pulmonary arterial hypertension (PAH).

1FemaleBreast, invasive ductal carcinoma−2.00.742Renal crisis, ILD
2FemaleLung, small cell carcinoma−1.0047ILD
4FemaleOvary, poorly differentiated metastatic carcinoma−1.3−0.321Renal crisis, myopathy
9FemaleNon-Hodgkin's lymphoma0.80.514Myopathy, PAH
13FemaleBreast, invasive ductal carcinoma1.31.030None
35FemaleBreast, ductal carcinoma in situ−2.0−2.448None

Since adequate tissue preservation allowed further study of tissue samples from only 4 of these 6 patients, one additional patient with scleroderma, cancer, an available cancer pathology specimen, and anti–RNA polymerase I/III autoantibodies was recruited (patient 42). Patient 42 had a breast ductal carcinoma that was detected 1.5 years after scleroderma onset.

To evaluate levels of RNA polymerase I and RNA polymerase III expression in vivo, paraffin sections from cancerous breast tissue (n = 3 [patients 1, 35, and 42]), cancerous lung tissue (n = 1 [patient 2]), and cancerous ovarian tissue (n = 1 [patient 4]) were analyzed by immunohistochemistry. (Selection of these tissues is described in Patients and Methods.) Tumors from 4 RNA polymerase antibody–negative patients were also evaluated (cancerous breast tissue [n = 3] and cancerous lung tissue [n = 1]). Three of these 4 patients had anti–topoisomerase I antibodies. Breast, ovarian, and lung paraffin sections were also obtained from normal individuals for purposes of comparison. Robust and extensive nuclear staining was detected in all of the cancerous ovarian tissue (Figures 2A and B), cancerous breast tissue (Figures 2E and F), and cancerous lung tissue (results not shown) sections stained with anti–RNA polymerase I antibody, irrespective of the patient's RNA polymerase antibody status. When staining was performed under identical conditions using an isotype-matched IgG1 antibody instead of the RNA polymerase I monoclonal antibody, no staining was detected (results not shown). In contrast to the prominent staining noted in cancerous tissue sections, normal breast (Figures 2G and H), ovarian (Figures 2C and D), and lung paraffin sections (results not shown) showed minimal/limited RNA polymerase I staining. Of note, in normal breast sections, RNA polymerase I staining was restricted to ductal cells (Figures 2G and H).

Figure 2.

RNA polymerase I staining is prominent in cancerous ovary and breast tissue from scleroderma patients as compared with normal ovary and breast tissue. A–D, Paraffin sections from cancerous ovary tissue from a scleroderma patient (patient 4) (A and B) and from normal ovary tissue (C and D) stained with antibodies against RNA polymerase I (see Patients and Methods). E–H, Paraffin sections from cancerous breast tissue from a scleroderma patient (patient 42) (E and F) and from normal breast tissue (G and H) stained with antibodies against RNA polymerase I. Brown shows RNA polymerase I staining; blue shows nuclei (Mayer's hematoxylin counterstain). In each set, the bottom panel is a magnification of part of the field shown in the top panel. (Original magnification × 10 in A, C, E, and G; × 40 in B, D, F, and H).

The pattern of staining of RNA polymerase III in cancers was strikingly different. An exclusively nucleolar staining pattern with the anti–RNA polymerase III antibody was detected in 4 of the 5 cancerous tissues from RNA polymerase antibody–positive patients (Figures 3A, B, E, and F and results not shown). In contrast, this nucleolar pattern was absent in all 4 of the tumors from RNA polymerase antibody–negative patients (P = 0.048). Additionally, it was not detected in the normal tissue sections (Figures 3C, D, G, and H) or in the cancerous sections when staining was performed under identical conditions but using normal goat serum instead of anti–RNA polymerase III goat polyclonal antibody (results not shown). Therefore, tumor RNA polymerase III staining is strikingly associated with the scleroderma patient's RNA polymerase antibody status, but tumor RNA polymerase I staining is not.

Figure 3.

RNA polymerase III staining is prominent in cancerous ovary and breast tissue from scleroderma patients as compared with normal ovary and breast tissue. A–D, Paraffin sections from cancerous ovary tissue from a scleroderma patient (patient 4) (A and B) and from normal ovary tissue (C and D) stained with antibodies against RNA polymerase III (see Patients and Methods). E–H, Paraffin sections from cancerous breast tissue from a scleroderma patient (patient 42) (E and F) and from normal breast tissue (G and H) stained with antibodies against RNA polymerase III. Brown shows RNA polymerase III staining; blue shows nuclei (Mayer's hematoxylin counterstain). In each set, the bottom panel is a magnification of part of the field shown in the top panel. (Original magnification × 10 in A, C, E, and G; × 40 in B, D, F, and H).

DISCUSSION

In this pilot study, we evaluated whether clinical characteristics, including the temporal relationship between scleroderma and malignancy onset, differed by autoantibody status among patients with scleroderma and cancer. After identifying all patients with a history of cancer seen in our center, we evaluated the first 23 patients in whom histologic confirmation of cancer diagnosis was possible. Within this group, we found that RNA polymerase I/III autoantibodies were strongly associated with malignancy that occurred contemporaneously with scleroderma onset. In all patients who produced anti–RNA polymerase I/III antibodies, scleroderma developed within 2 years of cancer diagnosis. Because of this association, we evaluated the expression of RNA polymerases I and III in tumors from patients with anti–RNA polymerase I/III antibodies compared with tumors from patients without these antibodies. Interestingly, we found that nucleolar RNA polymerase III expression was enhanced exclusively in patients with RNA polymerase antibodies. Although RNA polymerase I was expressed at high levels in tumors, this was not restricted to tumors from patients with the anti–RNA polymerase immune response. The association of tumor RNA polymerase III expression with the presence of anti–RNA polymerase autoantibodies suggests that RNA polymerase III could be driving the immune response in these individuals. These preliminary findings require confirmation in a larger patient sample with an expanded control population. It is of interest that another subset of patients, the autoantibody-negative group, also had a similar close temporal relationship between scleroderma and cancer onset. This group may elaborate unique autoantibodies and express novel tumor antigens that remain to be identified.

Our study suggests that cases of paraneoplastic scleroderma may demonstrate hallmark scleroderma-specific reactivity. Prior studies have not investigated or detected this association between the contemporaneous onset of scleroderma and malignancy and anti–RNA polymerase I/III antibodies in scleroderma patients with cancer. This relationship may have been missed because RNA polymerase I/III antibody testing was not commercially available until recently, and prior investigations have focused on whether the relationship between scleroderma and cancer differed by scleroderma subtype or tumor origin and histology. By seeking to determine whether the relationship between scleroderma and malignancy onset differed by autoantibody status among patients with scleroderma and cancer, we were able to detect this association even in a relatively small group of patients.

This striking temporal relationship between scleroderma and malignancy onset among anti–RNA polymerase I/III–positive patients with cancer is similar to that observed in dermatomyositis (24–26) and systemic lupus erythematosus (27) and suggests that cancer and autoimmunity onset might be mechanistically related. There is strong evidence that anti-cancer immunity and autoimmunity are related. For example, effective initiation of anticancer immunity during immunotherapy is often accompanied by autoimmunity (28–33). Multiple immune effector pathways are likely involved in causing tissue damage, with prominent involvement of cytotoxic killing pathways. We therefore hypothesize that tumors expressing high concentrations of RNA polymerase III initiate an immune response to these autoantigens. In the appropriate setting, possibly involving enhanced expression of the same autoantigens in damaged or perturbed blood vessels, this antitumor immune response may also be directed against specific host tissues, with consequent tissue damage that generates the ongoing rheumatic phenotype. Direct visualization of specific autoantigen expression in tissues targeted in scleroderma is an important future priority.

Although in the groups of patients with cancer and autoantibodies to centromere and topoisomerase I there was a prolonged interval between scleroderma onset and cancer diagnosis, there were outliers in each group (2 in the anticentromere group and 1 in the anti–topoisomerase I group), in whom cancer and scleroderma onset occurred closely together in time. It is of interest that increased topoisomerase I expression has been detected in a variety of cancers (34–36), and in 2 reported patients with preexisting scleroderma, anti–topoisomerase I titers were markedly increased, recognizing distinct epitopes, at the time of lung cancer diagnosis (37). The data suggest that, in some cases, anti–topoisomerase I antibody production might also be driven by malignancy (37). In our series of patients, this group appears to be a minority.

A variety of other mechanisms could explain the relationship between malignancy and scleroderma. Immunosuppressive therapy for autoimmune disease could account for the increase in malignancy risk in a subset of patients. Additionally, treatment of malignancy could result in the development of scleroderma. For example, multiple chemotherapeutic agents have been implicated as potential causes of scleroderma, scleroderma-like disease, or severe RP (38–42). Radiation therapy may also result in severe skin thickening in patients with scleroderma (43) or cause localized scleroderma in patients without a prior history of connective tissue disease (44). However, if cancer therapy were the inciting agent that triggered the development of scleroderma, we would not expect our findings to segregate by autoantibody status. Chronic inflammation and repair due to the scleroderma disease process may predispose cells to malignant transformation; this may especially be true of late lung cancers and esophageal adenocarcinomas in the setting of pulmonary fibrosis and longstanding gastroesophageal reflux disease, respectively. Other possible explanations for the relationship between cancer and scleroderma include genetic susceptibility to both malignancy and the development of autoimmune disease, or a common inciting exposure.

We propose that in scleroderma patients who produce anti–RNA polymerase antibodies but have not been diagnosed as having cancer, the full expression of an underlying malignancy was aborted by the now scleroderma-specific (originally antitumor) immune response. In the paraneoplastic neurologic diseases, available data suggest that a patient's immune response recognizes antigens expressed in the tumors and the target tissue, and that often patients have very small or undetectable tumors at disease diagnosis (45–47). Further investigation is needed to determine whether anti–RNA polymerase I/III antibodies are a marker for increased malignancy risk in scleroderma and whether more aggressive cancer screening should be performed in this patient population.

It is important to note that our small sample size limits the generalizability of these conclusions, and these results need validation in a larger patient sample with nonscleroderma cancer controls. The association of tumor RNA polymerase III expression, RNA polymerase III autoantibodies, and the interval between cancer and scleroderma diagnosis observed in scleroderma patients does not in any way predict that such antigen expression patterns are restricted to scleroderma-associated cancers. Indeed, it is likely that similar RNA polymerase III expression patterns occur in tumors from patients who do not have scleroderma, and that additional pathways and events are required to generate both the scleroderma-specific immune response and the clinical phenotype. We acknowledge that we cannot establish a causal relationship between cancer and scleroderma with our retrospective study design that focused on scleroderma patients with a history of malignancy. Another limitation of our study was that, in some cases, we lacked serum samples that were obtained concurrently with malignancy onset. To address this, we evaluated the closest available serum sample to cancer diagnosis; the median duration between cancer diagnosis and serum sample studied was 2.3 years. There are many issues that can only be addressed in a prospective study, including changes in autoantibody profiles in response to cancer therapy (range of autoantibodies targeted and titers).

We have demonstrated a tight temporal relationship between scleroderma onset and malignancy diagnosis in scleroderma patients with cancer who produce anti–RNA polymerase autoantibodies. Expression of RNA polymerase III was enhanced exclusively in the tumors from patients with RNA polymerase antibodies, demonstrating that tumor antigen expression and scleroderma autoantibodies are strongly associated, and highlighting RNA polymerase III as the tumor-associated antigen target. These findings provide evidence for a mechanistic relationship between malignancy, the immune response, and the development of scleroderma, and raise the possibility that RNA polymerase I/III autoantibodies are markers of malignancy in patients in whom scleroderma is newly diagnosed. These findings may have important diagnostic and therapeutic implications.

AUTHOR CONTRIBUTIONS

All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Shah had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Study conception and design. Shah, Rosen, Hummers, Wigley, Casciola-Rosen.

Acquisition of data. Shah, Casciola-Rosen.

Analysis and interpretation of data. Shah, Rosen, Hummers, Wigley, Casciola-Rosen.

Acknowledgements

We thank the Rheumatic Diseases Research Core Center (funding by NIH grant P30-AR-053503) for performing the ELISAs and Tonie Hines for excellent technical assistance.

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