Paraneoplastic and oncologic profiles of patients seropositive for type 1 antineuronal nuclear autoantibodies

Seropositivity for type 1 antineuronal nuclear autoantibodies (ANNA-1, also known as "anti-Hu") is a valuable marker of small-cell lung carcinoma(SCLC) in adult patients who present with subacute neurologic or gastroenterologic symptoms and have a significant history of smoking.^1-7 The onset of neurologic or gastrointestinal symptoms typically precedes tumor diagnosis. Henson and Urich⁸ concluded from clinicopathologic correlations that the most common neurologic syndromes associated with SCLC are paraneoplastic sensory neuronopathy (PSN) and paraneoplastic encephalomyelitis (PEM). Investigators at Sloan-Kettering Cancer Center first described antineuronal nuclear autoantibodies in serum and CSF of patients with PSN.^1,2,9 Their finding was confirmed by Mayo Clinic investigators,⁴ and ANNA-1 was soon recognized in association with other SCLC-related neurologic syndromes.¹⁰ IgG antibodies of ANNA-1 specificity recognize proteins of relative molecular weight 35 to 40 kD,^1,11 expressed in neurons and SCLC cells,^1,11-14 and constituting a family of RNA-binding proteins (HuD, HuC, Hel-N1, and Hel-N2) that is characterized by an RNA recognition motif of ∼80 amino acids.^15-17 These proteins are thought to have a role in the development and maintenance of the nervous system because of their restricted expression in neurons and their homology to the Drosophila protein Elav.¹²

In tumor cells and in neurons, antigens accounting for the ANNA-1 specificity are highly concentrated in nuclei, less abundant in cytoplasm, and absent in the nucleolus.^{1,7,11,13,14,18} Altermatt et al.¹⁴ demonstrated immunohistochemically that autoantibodies of ANNA-1 specificity bind to neurons in both the central and peripheral nervous system, including sensory and autonomic ganglia, myenteric plexus, and adrenal medulla. These autoantibodies may not be pathogenic, but the widespread distribution of their antigens in the nervous system reflects the diversity of paraneoplastic symptoms and signs that have been reported in ANNA-1-seropositive patients.

This study of 162 patients was undertaken to determine the spectrum of symptoms and signs as well as the frequency of cancer in adult patients who were found to be seropositive for ANNA-1 in a comprehensive immunofluorescence screening assay for paraneoplastic autoantibodies. Most of the patients were not known to have cancer at the time of serologic testing, and in one-fifth of the cases ANNA-1 (anti-Hu) was an incidental finding, not having been the test the physician had requested. The conclusion drawn from our study is that appropriate serologic testing based on an appreciation of the great diversity of clinical signs and symptoms encountered with ANNA-1 seropositivity increases the likelihood of earlier detection and treatment of an otherwise occult SCLC.

Methods. We reviewed case records for 204 sequential adult patients who had symptoms or signs suggestive of a paraneoplastic neurologic process and whose serum was found to be positive for ANNA-1 by a comprehensive indirect immunofluorescence screening assay in Mayo Clinic's Neuroimmunology Laboratory.⁷ Forty-two patients were excluded from further consideration because of insufficient information or because of the possibility that neurologic or gastroenterologic symptoms may have been secondary to metastasis or identifiable metabolic, infectious, vascular, or iatrogenic causes. Data for the remaining 162 patients were analyzed. Seventy patients had been examined at the Mayo Clinic, and information for the other 92 was obtained by telephone interviews, form letters, and review of case records sent by outside physicians. Thus, two groups of patients, "Mayo" and"non-Mayo," were compared to assess referral bias.

Control sera from 38 age-matched healthy subjects and 68 patients with SCLC without an evident neurologic disorder were screened for ANNA-1 antibodies. Seropositivity was defined by strict immunohistochemical criteria⁷: (1) a characteristic pattern of IgG binding to both cytoplasm and nucleus in central and peripheral neurons, (2) sparing of the nucleolus and non-neuronal cells, and (3) neuronal reactivity not absorbed by homogenized non-neural tissue.¹⁸

Results. All but 2 of the 162 patients seropositive for ANNA-1 were white, 1 was African American, and 1 was Oriental. Women predominated by a ratio of 2:1. The mean age was in the seventh decade (Mayo 63 years, range 39 to 83; non-Mayo 66 years, range 46 to 81). Smoking history was positive in 99% of the Mayo patients and in 97% of the non-Mayo patients for whom information was available (unavailable for 30%). Follow-up information was available for 59 Mayo patients (range, 2 to 108 months) and 69 non-Mayo patients (range, 2 to 53 months). Deaths were reported for 28 Mayo and 29 non-Mayo patients.

The median serum ANNA-1 titer was 1:3,840 (range, 1:60 to 1:256,660). All 38 age-matched healthy controls were seronegative; 6% of 68 patients with SCLC without an evident neurologic disorder were seropositive.

The frequency of SCLC and other malignant neoplasms in ANNA-1-positive patients. By the end of the follow-up period, a malignant neoplasm had been identified in a total of 142 patients (91% of Mayo patients and 85% of non-Mayo patients). In a minority of patients (4% Mayo, 8% non-Mayo), a primary malignant neoplasm was found without evidence of SCLC. These cases included four prostate carcinoma, three breast carcinoma, one prostate carcinoma and melanoma, one lymphoma, and one squamous-cell lung carcinoma.

SCLC was the most common neoplasm associated with ANNA-1 seropositivity. It was found in 87% of Mayo patients and 77% of non-Mayo patients; it was confined to the mediastinum in 93% of Mayo patients and in 85% of non-Mayo patients. Of note, SCLC was not found at initial workup in 62% of Mayo patients or in 51% of non-Mayo patients. However, more aggressive searching after documentation of seropositivity yielded evidence of tumor. MRI of the chest revealed a definite mass in 10 of 45 patients who had equivocal findings on chest roentgenograms or CTs. In seven patients who had no evidence of tumor by noninvasive investigation, SCLC was found by either bronchoscopy, mediastinoscopy, or thoracotomy. In 21 patients who had a nondiagnostic bronchoscopy, SCLC was found by a more invasive procedure, either mediastinoscopy or thoracotomy. Seven patients who lacked any evidence of SCLC in life were found to have SCLC at autopsy, whereas another three patients lacked evidence of SCLC even at autopsy.

An unrelated primary malignancy coexisted with SCLC in 13% of patients(table 1). In six patients renal-cell carcinoma was identified, and four had a second primary neoplasm of the lung.

Neurologic manifestations. The evolution of symptoms and signs in ANNA-1-positive patients was subacute (<6 months) in 78% of Mayo and 73% of non-Mayo patients. In fewer than 3% of patients, the symptoms evolved within 24 hours. A chronic presentation (>6 months) occurred in 20% of Mayo cases and 13% of non-Mayo cases. In 38 patients (63 of 64 Mayo patients[98%] and 75 of 78 non-Mayo patients [96%]), a paraneoplastic disorder preceded the diagnosis of cancer, either new or recurrent. These patients included 132 patients with SCLC and 10 in whom another cancer was found without evidence of SCLC. The mean latency between presentation and tumor diagnosis was 11 months in the Mayo group (range, 3 weeks to 8 years) and 8 months in the non-Mayo group (range, 2 weeks to 3 years). The four patients in whom the diagnosis of cancer preceded the paraneoplastic disorder all had SCLC. Twenty patients who were thought to have a paraneoplastic syndrome had no identifiable cancer. Symptoms in this group included 16 neurologic, 1 solely gastrointestinal, and 3 both neurologic and gastroenterologic.

The most common initial neurologic manifestation in both groups was neuropathy (table 2). Cerebellar symptoms were reported as the presenting problem more frequently in non-Mayo patients than in Mayo patients (18% versus 3%). Table 3 summarizes the total spectrum of neurologic paraneoplastic manifestations encountered. In most patients, symptoms and signs were multifocal. In decreasing frequency, the neurologic disorders associated with ANNA-1 seropositivity were as follows: neuropathy (sensory > mixed somatic > autonomic > motor), cerebellar syndrome, limbic encephalitis, cranial neuropathy (particularly sensorineural hearing loss), Lambert-Eaton myasthenic syndrome, polyradiculopathy, myopathy, brachial plexopathy, myelopathy, motor neuronopathy, movement disorder, and aphasia. All patients who had a diagnosis of limbic encephalitis were reported to have cognitive decline; however, less than 60% experienced seizures.

Gastrointestinal dysmotilities. Most patients (87% Mayo, 89% non-Mayo) presented with neurologic symptoms; however, 12% of patients presented to a gastroenterologist with gut dysmotility. Overall, 38 patients(23%) developed gastrointestinal dysmotility in the course of their illness. Gastroparesis was most common, but the spectrum of dysmotilities included all levels of the gut (table 4). These manifestations were typically independent of cardiovascular or sympathetic dysfunction and were often without initial or striking evidence of somatic neuropathy.

CSF findings. CSF profiles were available for 41 ANNA-1-seropositive patients (table 5). An abnormality was found in 27 (66%); 7 of these patients had solely an elevation of protein. Of note, 18 patients with CSF abnormalities had neurologic signs restricted to the peripheral nervous system.

Treatment response. Immunosuppressant treatments used in 49 patients included adrenal corticosteroids, plasma exchange, intravenous immunoglobulin, or Cytoxan. No patient improved.

Discussion. Paraneoplastic disorders are notorious for posing a diagnostic dilemma for the clinician. The broad spectrum of neurologic and gastrointestinal symptoms and signs that we have documented in association with ANNA-1 seropositivity emphasizes the heterogeneity of clinical presentations in these patients, who generally have occult SCLC. Their clinical presentations may mimic other diseases. For example, a subacute sensory neuropathy may be seen with Sjögren's syndrome¹⁹ or excessive ingestion of pyridoxine,²⁰ and a disorder indistinguishable from PEM has been reported in patients without apparent cancer.²¹

Despite the diagnosis of SCLC being more frequent in men than in women(from 1.8:1 to 3:1),²² our study confirms earlier reports^2,3 that women predominate among ANNA-1-seropositive patients with SCLC. This observation is consistent with the greater frequency of autoimmune disorders in women and offers insight into the immunologic determinants of human immune responsiveness to SCLC.

In most of our patients, SCLC was not found at initial workup. More aggressive searching after the finding of ANNA-1 seropositivity, including MRI of the chest, led to a diagnosis of SCLC in most cases. Confirmation often required either mediastinoscopy or thoracotomy after a nondiagnostic bronchoscopy. These findings underscore the small size and limited spread of SCLC in patients with paraneoplastic autoimmunity. The finding of ANNA-1 seropositivity in a tobacco abuser should initiate an aggressive search for malignancy in the chest and mediastinum, but one must bear in mind that primary small-cell carcinoma in some cases may be in a remote extrapulmonary site.²³

A minority of ANNA-1-positive patients in our study had tumors other than SCLC. Prostate carcinoma and melanoma have both been reported to express a Hu antigen,²⁴ but lymphoma and breast carcinoma are not known to express a Hu antigen. An important finding was the 13% frequency of an unrelated primary malignancy coexisting with SCLC in our ANNA-1-positive patients. Renal-cell carcinoma, the most common second tumor, was previously noted by our laboratory to coexist with SCLC in two patients with Lambert-Eaton myasthenic syndrome,²⁵ neither of whom was ANNA-1 positive. Renal-cell carcinoma and SCLC both have an interstitial deletion in the short arm of chromosome 3.²⁶ The coexistence of primary lung adenocarcinoma and SCLC in three ANNA-1-positive patients accords with the unitarian theory for the origin of squamous carcinoma, adenocarcinoma, and SCLC. This theory, however, does not explain the finding of bronchioalveolar carcinoma with SCLC in a fourth ANNA-1-positive patient. If a primary neoplasm other than SCLC is found in an ANNA-1-positive patient, therapeutic implications may justify continuing the search for SCLC.

We found a broader spectrum of neurologic symptoms associated with ANNA-1 seropositivity than suggested by early reports.^1-4 Neuropathy, the most common presentation, was more frequent in the Mayo group of patients than in non-Mayo patients. This may reflect a referral bias to Mayo Clinic's large peripheral nerve practice and a possible misclassification of some patients' signs as "cerebellar" when there was a sensory ataxia due to a large-fiber neuropathy.

The finding that subacute hearing loss was the most commonly encountered cranial neuropathy associated with ANNA-1 seropositivity is a novel observation. Auditory symptomatology or pathology has seldom been reported in patients with paraneoplastic disorders.^27-29 For otolaryngologists, ANNA-1 seropositivity provides a valuable marker for underlying SCLC in tobacco abusers presenting with acute or subacute sensorineural hearing loss. If positive, this simple test will unambiguously distinguish paraneoplastic hearing loss from other causes of sudden sensorineural hearing loss.

ANNA-1 is proving to be an extremely valuable marker of occult SCLC in patients who present with a gastrointestinal dysmotility disorder, particularly when evidence of more widespread autonomic failure is lacking. Chronic intestinal pseudo-obstruction has long been recognized as a paraneoplastic accompaniment of SCLC,^30-34 but the association of ANNA-1 with pseudo-obstruction⁵ and a broader spectrum of dysmotility disorders⁶ was recognized for the first time in 1991. The association of ANNA-1 and pseudo-obstruction was confirmed in 1993.³⁵ The present study reports the association of ANNA-1 with focal dysmotilities involving any level of the gut. The paraneoplastic basis of these disorders can be recognized early by appropriate serologic testing. For the clinical gastroenterologist, paraneoplastic autoimmune serologic testing offers a valuable diagnostic aid for determining the likelihood of an underlying SCLC in patients presenting with varied gastrointestinal dysmotility disorders. Additional serological markers include N-type calcium channel antibodies³⁹ and neuronal acetylcholine receptor antibodies.⁴⁰

No patient whom we reviewed benefited from immunosuppressant therapy with steroids, Cytoxan, plasma exchange, or IV immunoglobulin. The report of improvement in a patient with paraneoplastic cerebellar degeneration after IV therapy with high doses of normal donor IgG³⁶ was confounded by the patient's having also been treated with 5-fluorouracil, an agent known to cause a reversible cerebellar ataxia. Another ANNA-1-positive patient with a subacute paraneoplastic cerebellar syndrome and Lambert-Eaton syndrome associated with SCLC was reported to improve after treatment with IV immunoglobulin.³⁷ However, we have observed at our own institution the spontaneous improvement of cerebellar function in a patient with an almost identical presentation whose only treatment was chemotherapy for the SCLC. Graus et al.³⁸ reported seven ANNA-1-positive patients with SCLC and PEM who did not improve neurologically after plasmapheresis and/or prednisone, cyclophosphamide, or chemotherapy. The generally poor response to immunosuppressant therapy does not refute an auto-immune basis for these disorders, but rather is consistent with rapid and irreversible damage to neurons.

In conclusion, the spectrum of paraneoplastic symptoms associated with ANNA-1 seropositivity is broader than generally recognized. Seropositivity in an adult patient with a history of tobacco abuse mandates an aggressive search for malignancy directed to the chest and mediastinum. MRI imaging may be justifiable given the high likelihood of detecting limited SCLC. The search for SCLC should not end with the discovery of another primary neoplasm. ANNA-1 seropositivity in a smoker with any subacute gastrointestinal dysmotility should alert the gastroenterologist to underlying SCLC and spare unnecessary surgeries.