Pathogenesis, diagnosis and management of pneumorrhachis

Pneumorrhachis (PR), the presence of intraspinal air, is an exceptional but eminent radiographic finding, accompanied by different aetiologies and possible pathways of air entry into the spinal canal. By reviewing the literature and analysing a personal case of traumatic cervical PR after head injury, we present current data regarding the pathoanatomy, clinical and radiological presentation, diagnosis and differential diagnosis and treatment modalities of patients with PR and associated pathologies to highlight this uncommon phenomenon and outline aetiology-based guidelines for the practical management of PR. Air within the spinal canal can be divided into primary and secondary PR, descriptively classified into extra- or intradural PR and aetiologically subsumed into iatrogenic, traumatic and nontraumatic PR. Intraspinal air is usually found isolated not only in the cervical, thoracic and, less frequently, the lumbosacral regions but can also be located in the entire spinal canal. PR is almost exceptional associated with further air distributions in the body. The pathogenesis and aetiologies of PR are multifold and can be a diagnostic challenge. The diagnostic procedure should include spinal CT, the imaging tool of choice. PR has to be differentiated from free intraspinal gas collections and the coexistence of air and gas within the spinal canal has to be considered differential diagnostically. PR usually represents an asymptomatic epiphenomenon but can also be symptomatic by itself as well as by its underlying pathology. The latter, although often severe, might be concealed and has to be examined carefully to enable adequate patient treatment. The management of PR has to be individualized and frequently requires a multidisciplinary regime.
Introduction
Pneumorrhachis (PR), the phenomenon of intraspinal air, is an exceptional imaging finding, caused by various, mainly traumatic and iatrogenic aetiologies. It is associated with different pathologies and possible pathways of air entry into the spinal canal. Although PR has been previously described, mainly in the radiological literature, spine specialists are less acquainted with this unusual pathological entity.
Therefore, based on a complete review of the medical literature and the analysis of a personal case description, we offer contemporary theories of the development of PR and current management strategies for this uncommon epiphenomenon of coincident underlying injuries and diseases to elucidate this rare phenomenon and to raise awareness to PR and possible coexisting pathologies.
Materials and methods
A comprehensive literature search of the U.S. National Library of Medicine’s Medline bibliographic database was conducted and the international medical literature up to December 2005 has been reviewed for the term “pneumo(r)rhachis” and related appropriate medical subject headings such as intraspinal air, intraspinal pneumoc(o)ele, spinal epidural and subarachnoid pneumatosis, spinal and epidural emphysema, aerorachia, pneumosaccus, air myelogram, pneumomyelogram or pneumomyelography.
Each bibliography and retrieved article were then obtained in complete form, analysed and searched for further citations. Pertinent references cited in the identified articles and other relevant literature were also scanned. Further references of each text were identified, and the text was obtained in complete form and analysed. A new case of cervical PR due to severe head injury was added.
Results
After application of the methods and criteria described above, only isolated case reports and no series of more than three cases of this condition could be found. In total, 71 articles were identified that reported on 86 cases. In addition, we describe a previously unreported case of traumatic cervical PR, which was diagnosed in our department.
Illustrative case
A 51-year-old woman was admitted comatose after an automobile accident and attempted unsuccessful endotracheal intubation with prolonged insufficient mask ventilation. Multiple skin lacerations were present as were blunt thoracic trauma with several rib fractures and severe head injury. The patient was decerebrated with bilateral fixed and dilated pupils. Cranial computerized tomography (CT) examination (Fig. 1) revealed a dislocated left occipital skull fracture radiating into the foramen magnum and petrosal bone and a fracture of the sphenoid sinus accompanied by a large acute right hemispheric subdural haematoma causing midline brain shift, traumatic subarachnoid haemorrhage, cerebral oedema and signs of hypoxemia. Internal pneumocephalus with diffuse air distribution in the basal, prepontine and perimesencephal cisterns and fourth ventricle could be detected. Additional spinal CT (Figs. 2, 3) demonstrated air lucency in the cervical vertebral column indicating extradural PR. Intraspinal air was caused by the transphenoid sinus and petrosal bone fractures thus allowing direct communication of pneumatized air containing cavities with the intracranial space. The penetrated air was then forced caudally due to the elevated intracranial pressure resulting from severe brain injury with diminution of capacity of the intracranial space. Horizontal and head-down position of the patient finally allowed the entrapped air to pass through the foramen magnum into the spinal canal.
Fig. 1Axial cranial CT scans (corresponding soft-tissue and bone window setting) revealing severe head injury accompanied by traumatic internal pneumocephalus with air distributed prepontine, perimesencephally and intraventricullary accompanied by skull fractures of the sphenoid, left occipital and petrosal bone leading to PRFig. 2Sagittal multiplanar reformatted spinal CT images (corresponding soft-tissue and bone window setting) demonstrating traumatic cervical PRFig. 3Axial CT scans of the cervical spine (corresponding soft-tissue and bone window setting) showing extradural air collection within the ventral spinal canal
The patient remained decerebrated, was transferred to the intensive care unit, considered brain death after adequate examinations and devoted to organ donation.
Discussion
Pathogenesis
Definition and classification
Free air surrounding the dura mater spinalis is an uncommon phenomenon that was primarily reported by Gordon et al. [30] in 1977 and described under various terms such as intraspinal pneumocele [28, 38, 50] or pneumocoele [78], spinal epidural and subarachnoid pneumatosis [4–6, 9, 18, 19, 32–34, 41, 47, 61, 74], spinal and epidural emphysema [4, 11, 23, 27, 28, 33, 59, 65, 69, 70, 72, 77], aerorachia [18, 22, 28, 34], pneumosaccus [9, 28, 62], air myelogram [2, 30, 76], pneumomyelogram [2, 3, 12, 17, 20, 28, 30, 76] or pneumomyelography [26]. The term PR itself was first coined 10 years later by Newbold and co-workers [54]. PR per se usually represents an asymptomatic, probably underdiagnosed epiphenomenon of coincident underlying injuries and diseases. As imaging techniques improve, this pathologic entity has become somewhat more often diagnosed.
PR can be classified descriptively into internal, intradural (intraspinal air within the subdural or subarachnoid space) and external, extradural (intraspinal, epidural air) PR. External PR by itself is usually innocuous, whereas internal traumatic PR frequently is associated with major trauma and believed to be a marker of severe injury [28].
Pathomechanism and aetiology
Apart from artificial, usually traumatic spinal leaks and penetrating spine injuries as a possible explanation for a direct route of intraspinal entry, air may entrap due to a one-way air valve mechanism and dissect indirectly between the paraspinal soft tissues into the epidural space of the spinal canal via the neural foramina and along the vascular and nerve root sheaths, and vice versa, thereby producing PR.
Various conditions may directly or indirectly produce PR including trauma [1–3, 9, 10, 12, 13, 17, 20–22, 26–28, 30, 32, 37–42, 45, 50, 51, 54, 60, 62, 64, 66–68, 73, 74, 76, 78], respiratory complications and conditions that produce high intrathoracic pressure and barotraumas [5, 6, 11, 14, 19, 22–24, 33, 40, 47, 55, 56, 58, 59, 61, 69, 70, 72, 77], recent iatrogenic manipulations during surgical, anaesthesiological and diagnostic interventions [7, 9, 15, 18, 31, 33, 35, 36, 44, 48, 52, 53, 57, 65, 71], malignancy and its associated therapy [29, 49, 63, 75] or it may occur spontaneously [4, 24]. In some cases, PR is an accidental discovery [2] and the conditions and lesions causing PR remain unclear or undetected [12]. The pathologies leading to PR can be generally subsumed and further classified into iatrogenic, traumatic and nontraumatic causes [28].
Thirteen cases are found in the literature describing PR caused by violent coughing due to bronchial asthma or acute bronchitis [11, 14, 23, 24, 40, 55, 56, 58, 59, 70, 72], single cases of PR after cardiopulmonary resuscitation [58], airway obstruction due to foreign body aspiration [69] and two cases each of PR after physical exertion [19, 77] and inhalational drug abuse of 3,4-methylenedioxymethamphetamine (“Ecstasy”) [6] or marijuana [34]. Furthermore, three cases of PR because of prolonged and forceful emesis with diabetic ketoacidosis were documented [5, 47, 61]. Other authors describe PR secondary to traumatic causes including isolated head [3, 10, 17, 30, 38, 39, 62, 68, 50, 76, 78], cervical [2], thoracic [21, 22, 40, 60, 64, 66], abdominal [42] and pelvic injuries [13] or combinations of different injury patterns including spinal trauma [1, 9, 12, 20, 26, 27, 32, 37, 41, 45, 51, 54, 67, 73, 74].
Invasive tumour progression and postradiation changes [29, 49, 63, 75], surgical interventions [33, 36, 65, 71], nasotracheal intubation [18] and peri- or epidural anaesthesia involving a lumbar puncture [7, 9, 15, 31, 35, 44, 48, 52, 53, 57] have also been reported as a cause of PR. Introduction of air as a negative contrast medium into the lumbar subarachnoid space for diagnostic pneumoencephalography or pneumomyelogram, first performed and described by Dandy in 1919, is now only of historical interest [16].
Localization and distribution
Two documented cases with air in the entire spinal canal [12, 55, 56], single cases of air bubbles distributed in the cervical, thoracic and lumbar region [35], and in the cervical and lumbar spine [10], six cases of air in the thoracic and lumbar region [7, 9, 15, 26, 57] and eight cases with both cervical and thoracic PR were previously described [1, 11, 26, 32, 47, 52, 63, 77]. Most cases demonstrate isolated PR of the cervical [2, 3, 5, 10, 17, 20, 30, 38, 39, 45, 50, 51, 54, 60, 66, 68, 69, 74, 76, 78], thoracic [4, 6, 14, 18, 19, 21–24, 27, 29, 33, 34, 37, 40–42, 49, 58, 59, 61, 64, 65, 67, 70–75] or lumbosacral region [9, 10, 13, 31, 36, 44, 48, 53, 57, 62, 70, 72, 74]. Nevertheless, this might be due to the fact that in some cases of PR not a complete examination of the whole spine was carried out. Furthermore, location and distribution of air within the spinal canal is probably depending on the site of air dissection, rate and volume of intraspinal air with large volumes spreading widely, capacity of intraspinal space and positioning of the patient. In cases of external PR, because of the lower resistance from the loose connective tissue, as compared with the rich vascular network that is present anteriorly, the epidural air usually collects in the posterior epidural space [6].
PR may be primary or secondary depending on whether intraspinal air is the cause and origin or effect of associated further occurrence of air in the body.
Almost exceptionally, PR is found in combination with associated air distribution in other compartments and cavities of the body: particularly, in conjunction with pneumocephalus, pneumothorax, pneumomediastinum, peumopericardium or subcutaneous emphysema [3–6, 9–11, 13, 14, 17–24, 26, 27, 29, 30, 32–34, 36–39, 42, 47, 49–51, 54–56, 58–66, 68–70, 72–74, 76–78]. Thus, diagnosis of PR implicates the possibility of the coincidence of associated and hidden further air distributions in the body. Especially, patients with traumatic cervical and intradural PR will almost certainly have injuries of the anterior cranial fossa usually causing subdural PR and skull base or middle and posterior cranial fossae fractures with intracranial subarachnoid air and pneumocephalus, but also penetrating spinal injuries may cause isolated subarachnoid PR without accompanying head injury [2, 17, 76].
Diagnosis
Diagnostic procedures
Since intraspinal air is usually asymptomatic and clinically unspecific, PR is primarily a radiographic and not a clinical diagnosis. The diagnostic work-up of patients with PR should include plain roentgenograms and CT scanning of the spine.
X-ray may be helpful as an initial examination for the early detection of possible associated injuries and to detect larger amounts of intraspinal air [28, 56]. A linear lucency along the spinal canal on a lateral chest radiograph was described as a useful sign in the detection of PR [6]. Moreover, PR itself to some extent acts as a negative contrast agent to delineate canal morphology [78].
The diagnostic tool of choice for a reliable and prompt detection of PR is CT [9, 11, 49, 56]. Nevertheless, on CT, intra- and extradural PR may be difficult to differentiate [28, 60, 67]. Since PR is often combined with air distribution in other parts of the body and traumatic PR is a marker of severe injury, these observations suggest a wider indication for the application of CT, and if necessary, even a systematic total body CT scan or magnetic resonance imaging (MRI). After the initial CT examination follow-up of patients with PR should primarily rely on clinical observations.
Other, not only more sensitive but also more extensive diagnostic techniques such as MRI or myelography are indicated for determining coexisting aetiologies of PR and differential diagnostically examinations [56].
Differential diagnosis
Intraspinal air has to be clearly differentiated from free intraspinal gas collections due to degenerative [25], malignant [43], inflammatory [8] and infectious diseases by gas-forming organisms [46]. It has been demonstrated gas-chromatographically that in cases associated with the so-called vacuum disc phenomenon, the gas collection contains up to 92% nitrogen combined with oxygen, carbon dioxide and other trace gases, in contrast to atmospheric air with a lower nitrogen (78%) and a higher oxygen composition (21%) [25].
Intraspinal gas, which usually has the same low density as air collections on CT, cannot be clearly differentiated by CT examination. It can simulate PR and therefore has to be included in the differential diagnosis of PR. Furthermore, the coincidence of PR and intraspinal gas has to be considered.
Management
Symptomatology
PR in itself usually is asymptomatic, does not tend to migrate and reabsorbs spontaneously and completely with the air being passed directly into the blood in several days without recurrence [3, 4, 6, 10–14, 16, 19, 22–24, 27, 34, 36, 69, 70, 74]. Therefore, patients with PR usually are managed conservatively. As the entrapped air occupies parts of the cerebrospinal compartment thus may cause syndromes of both intracranial and intraspinal hypertension as well as hypotension secondary to either an increase or decrease of intracranial and intraspinal pressure.
Rarely, PR per se is symptomatic and associated with discomfort and pain or even neurological deficits [29, 31, 35, 41, 44, 48, 52, 53, 57, 71, 78]. One case of traumatic PR leading to sensory symptoms has been reported. It was associated with pneumocephalus resulting from closed head injury and treated conservatively by the administration of supplemental oxygen [78]. A single case of a patient with progressive motor deficit of the lower limbs as a result of entrapped intraspinal air, which compressed the spinal cord due to presumptive introduction of air into the intraspinal arachnoid space after repeated lumbar puncture, was demonstrated by Uemura et al. [71]. Altered intrathecal pressure caused by the adhesion of the arachnoid following spinal surgery for lipoma of the thoracic spine may have caused PR in this case. The patient was reoperated with removal of the spinal compression and closure of the dural defect. Gonzales et al. [29] reported a case of reversible spinal cord and lower cervical root dysfunction caused by air dissection through a bronchopleural-epidural-cutaneous fistula likely due to tumour erosion and postradiation changes. The patient underwent surgical exploration of the chest with improvement of symptoms postoperatively. Identification of the epidural space through loss of resistance to air is a widely applied anaesthetic procedure during placement of an epidural catheter. In contrast, intraspinal air may cause undesirable inadequate analgesia during epidural anaesthesia [7, 15]. But injected air also can act as a space-occupying lesion and exert pressure on nervous structures within the spinal canal [7, 15, 31, 48, 52, 53, 57]. Eight cases with postanaesthetic neurological symptoms and pain thought to be complications associated with the application of intraspinal air were reported in the literature [31, 35, 44, 48, 52, 53, 57] including two case descriptions of accidentally injected air into the epidural space via a permanent epidural catheter for continuous epidural anaesthesia [35, 44]. The treatment consisted primarily of removal of the spinal catheter, bed rest and medication for pain relief, without permanent sequelae in most cases [31, 35, 48, 57]. Other treatment modalities included intravenous dexamethasone [53], decompression of the epidural space by percutaneous insertion of a Tuohy needle with air aspiration [44], administration of high concentrations of inspired oxygen with the idea of promoting reabsorption of air from the subdural space or even trials of hyperbaric oxygen therapy [34, 57].
Treatment
Because of the rareness and the different pathogenesis and aetiologies, no empiric guidelines for the treatment of PR and standards of care exist. PR is thought to be associated with an increased morbidity and mortality [14]. Therefore, the whole extent of the conditions causing PR has to be evaluated and the contributing causes leading to PR have to be appropriately treated. PR associated with decreased intraspinal pressure secondary to cerebrospinal fluid leakage usually has a more benign character, whereas entrapped intraspinal air under pressure entering the cranio-spinal compartment usually in combination with a one-way air valve mechanism might cause tension PR and pneumocephalus with nervous tissue compression requiring intervention. Therefore, the authors suggest recognizing the importance of differential diagnosis of the phenomenon of altered intraspinal pressure with either hypo- or hypertension of the cerebrospinal compartment to allow adequate management.
Because of the higher risk of possible meningitis, demonstrable cerebrospinal fluid leaks, if significant or persistent, may have to be repaired neurosurgically [50, 55] or treated by a temporary lumbar spinal catheter [38]. Furthermore, in some cases the underlying pathologies causing PR such as fistulous tracts between intrathoracic structures and the subarachnoid space (thoracoarachnoid fistulas) [29, 63], traumatic lung injury [66, 73] or lung herniation into the spinal canal [37] may require surgical intervention. Although operative repair usually may be uncomplicated, diagnosis and identification of the fistula remains challenging. Other treatment modalities include bronchoscopy for extraction of aspirated foreign bodies causing airway obstruction [69] and transient high concentration oxygen therapy to achieve nitrogen washout [17, 34, 78]. The use of application of prophylactic antibiotic treatment to prevent meningitis and its potential benefit is not instituted and discussed controversial [47, 78]. In principle, apart from antibiotic treatment of underlying infectious diseases such as acute bronchitis [11], even in traumatic PR, we do not recommend prophylactic management with antibiotics in cases of extradural PR and in patients with intradural PR without signs and symptoms of meningitis. The management of patients with PR has to be decided on an individual basis and frequently requires an inter- and multidisciplinary regime.
If intraspinal air is present, especially if caused by trauma, the spine physician has to consider PR as an initial sign of potentially associated, hidden and severe diseases or injuries [55, 56].
If general anaesthesia is required in a patient with PR, because of the presumptive diagnosis of a perforation of the dura mater and additional pneumocephalus, the involved anaesthetist should not use inhalational nitrous oxide, because it may cause expansion of intracavitary air and result in an increase in CSF pressure, as nitrous oxide diffuses into the air-filled space. In addition, pressurisation of the oro- and nasopharynx should be avoided, and alternative anaesthetic techniques such as intermitted positive pressure ventilation with transient high-concentration oxygen should be used, thus preventing an increase in the volume of any intraspinal and intracranial air and promote faster reabsorption of air [17, 34, 57].
The radiologist necessarily has to search for other pathological conditions during the evaluation of a patient with PR and should further investigate the underlying disease or whole extent of injuries. For further treatment of underlying pathologies, other disciplines such as internal medicine, thoracic surgery or ORL may have to be consulted.
The subjects of patients with severe and life-threatening causes leading to PR should be carefully monitored, followed-up and considered for admission to an intensive care unit.
Conclusions
Pneumorrhachis can be caused by a multitude of sources and the evaluation of aetiologies of PR could be a diagnostic challenge. Although PR per se usually is self-limiting and without further therapeutic consequences, prompt recognition of the underlying cause is essential. The attending spine specialist has to carefully evaluate the associated pathologies leading to PR to enable adequate therapy.

pneumorrhachis
intraspinal air
spinal canal


Human Coenzyme Q10 Deficiency

Ubiquinone (coenzyme Q10 or CoQ10) is a lipid-soluble component of virtually all cell membranes and has multiple metabolic functions. Deficiency of CoQ10 (MIM 607426) has been associated with five different clinical presentations that suggest genetic heterogeneity, which may be related to the multiple steps in CoQ10 biosynthesis. Patients with all forms of CoQ10 deficiency have shown clinical improvements after initiating oral CoQ10 supplementation. Thus, early diagnosis is of critical importance in the management of these patients. This year, the first molecular defect causing the infantile form of primary human CoQ10 deficiency has been reported. The availability of genetic testing will allow for a better understanding of the pathogenesis of this disease and early initiation of therapy (even presymptomatically in siblings of patients) in this otherwise life-threatening infantile encephalomyopathy.
Introduction
Coenzyme Q10 (CoQ10) is the predominant human form of endogenous ubiquinone. Synthesized in the mitochondrial inner membrane, CoQ10 is comprised of a ubiquinone head group attached to a tail of 10 five-carbon isoprenoid units, that anchors the molecule to the membranes [1]. In additions to its central role in the mitochondrial respiratory chain as electrons carrier from complex I and II to complex III [2], CoQ10 is now thought to be involved in a number of cellular functions (Table 1) [1]. Intracellular synthesis is the major source of CoQ10, although a small proportion is acquired through diet. The complexity of the biosynthesis (Fig. 1) suggests that defects in different biosynthetic enzymes or regulatory proteins may cause different clinical syndromes.
Table 1Functions of coenzyme Q (modified from [1])Electron carrier in mitochondrial respiratory chain [3]Extra-mitochondrial electron transport [4]Antioxidant [5]Regulation of mitochondrial permeability transition pore [6]Regulation of physiochemical properties of membranes [7]Modulation of the amount of β-integrins on the surface of blood monocytes [8]Modulator of endothelial function [9]Oxidation of sulfide in yeast [10]Introduction of disulfide bonds in bacteria [11]Fig. 1CoQ10 biosynthetic pathway with eight known biosynthetic enzymes denoted as COQ1-8. CoQ10 is composed of a benzoquinone and a decaprenyl side chain. While the quinone ring is derived from amino acids tyrosine or phenylalanine, the isoprenoid side chain is produced by addition of isopentenyl pyrophosphate molecules to geranylgeranyl pyrophosphate (derived from mevalonate pathway) by decaprenyl diphosphate synthase. After para-hydroxybenzoate and decaprenyl pyrophosphate are produced, at least seven enzymes (encoded by COQ2-8) catalyze condensation, methylation, decarboxylation, and hydroxylation reactions to synthesize CoQ10
Primary CoQ10 deficiency is an autosomal recessive condition with a clinical spectrum that encompasses at least five major phenotypes: (1) encephalomyopathy characterized by the triad of recurrent myoglobinuria, brain involvement and ragged red fibers; (2) severe infantile multisystemic disease; (3) cerebellar ataxia; (4) Leigh syndrome with growth retardation, ataxia and deafness; and (5) isolated myopathy [12–26].
Generalized weakness, exercise intolerance, and recurrent myoglobinuria are the distinctive features in the first four patients reported with the encephalomyopathic form, [12–15]; however, myoglobinuria was absent in another patient [16]. All the patients described so far have proximal muscle weakness [12–16] The brain involvement is variable; seizures are a common finding [12, 13], in association with cognitive impairment [12] and cerebellar symptoms [12, 15, 16].
The second variant, described in three families, manifests as an infantile encephalopathy with renal involvement. Rötig and colleagues initially described two siblings with defects of multiple quinone-dependent enzymes ascribed to a deficiency of CoQ10 in various tissues [17]. The patients presented with retinitis pigmentosa, optic nerve atrophy, bilateral sensorineuronal deafness, nephrotic syndrome, progressive ataxia, and cardiomyopathy. In Rahman’s patient, hypothermia, lactic acidosis, cerebral and cerebellar atrophy, and developmental delay were associated with renal tubulopathy and ventricular hypertrophy [18]. Last year, we described two siblings who presented with nephrotic syndrome due to glomerulosclerosis, and the older sibling developed hypotonia, psychomotor delay, seizures, stroke-like episodes, and cerebellar and cerebral atrophy [19]. Interestingly, in these infantile-onset patients, deficiency of CoQ10 is present not only in skeletal muscle, but also in other tissues and cells including skin fibroblasts. In addition, two adult sisters with encephalopathy, growth retardation, infantilism, ataxia, deafness and lactic acidosis have been described by Van Maldergam [23].
Cerebellar ataxia is the most common phenotype associated with CoQ10 deficiency with 21 reported patients [20–22, 26]. Cerebellar ataxia and cerebellar atrophy are present in all the patients. Epilepsy is the most common associated feature, but pyramidal signs, mental retardation, myopathic weakness, and delayed motor milestones are other variably associated symptoms and signs. Except for two adult brothers with cerebellar ataxia and hypogonadism [22], the condition begins in childhood or adolescence. Muscle morphology does not show ragged-red fibers and lipid storage myopathy and CoQ10 is moderately reduced in skin fibroblasts.
The clinical presentation of the variant isolated myopathy, recently described in four patients, is subacute onset of exercise intolerance and proximal limb weakness at variable ages. All patients have lipid storage and ragged-red fibers in muscle, as well as increased serum lactate and creatine kinase (CK) [24, 25]. In all the patients with different phenotypes, respiratory chain enzymes analyses show reduced activities of complex I + III and II + III with normal activities of isolated complex I and III.
In most of the reported patients, the exact site and nature of the defects in the biosynthesis of CoQ10 have not yet been identified. Because ubiquinone biosynthesis is complex and not fully defined (Fig. 1), identification of the molecular genetic defect is not straightforward.
Undetectable CoQ10 and low levels of decaprenylpyrophosphate, an intermediate compound in the synthesis of the lateral chain of CoQ10 were found in the fibroblasts of the patient reported by Rötig and colleagues suggesting a defect in the synthesis of the decaprenyl side-chain, but they did not find any mutation in the cDNA of COQ1, the gene encoding transprenyltransferase, the enzyme that synthesizes decaprenylpyrophosphate [17].
This year, in two siblings with the infantile form of CoQ10 deficiency, we reported a homozygous missense mutation (Y297C) in the COQ2 gene, which encodes para-hydroxybenzoate (PHB) polyprenyl transferase. PHB-polyprenyl transferase mediates the conjugation of the benzoquinone ring with the decaprenyl side chain. Biochemical assays measuring incorporation of radiolabeled PHB or decaprenyl-PP into CoQ10 in skin fibroblasts from the proband showed 23–25% of control fibroblasts activity [27] confirming the defect of CoQ10 biosynthesis.
The finding of a homozygous stop codon mutation in the APTX gene which is known to cause ataxia-oculomotor-aprataxia 1 (AOA1), in three siblings with cerebellar ataxia and CoQ10 deficiency, supports the hypothesis that the ataxic form is a genetically heterogeneous disease in which CoQ10 deficiency can be secondary [28]. Aprataxin is a member of the histidine triad superfamily and may be involved in nuclear DNA single strand break repair [29–31]. Despite the lack of an obvious link between aprataxin and regulation of CoQ10 synthesis or catabolism, our findings, coupled with the clinical improvement of patients after CoQ10 supplementation, suggest that CoQ10 deficiency plays a pathogenic role in AOA1. Intriguingly, both CoQ10 and cholesterol share a common biosynthetic pathway, therefore, in AOA1, altered levels of these molecules could be due to aberrant biosynthesis.
Patients with all forms of CoQ10 deficiency have shown clinical improvement with oral CoQ10 supplementation. The beneficial effects of exogenous CoQ10 require high doses and long-term administration [32, 33]; its oral bioavailability is poor due to its extreme hydrophobicity; therefore, only a small fraction of orally administered CoQ10 reaches the circulatory system, and augmentation of mitochondrial CoQ10 content is even less effective. Beneficial effects of CoQ10 supplementation are supported by in vitro correction of biochemical and histological abnormalities [12–14, 17]. However, while muscle abnormalities improve clinically and biochemically, in general, cerebral symptoms are only partially ameliorated [16]. This difference could be explained by the presence of irreversible structural brain damage before treatment or the poor penetration of CoQ10 across the blood-brain barrier. Increases in plasma concentrations of CoQ10 after oral supplementation in both humans and animals has been well-documented, but early work questioned whether CoQ10 accumulates in tissues [34] and studies of 45-day-old rodents showed little or no change in rodent brain CoQ10 concentrations after oral administration perhaps because levels of CoQ10 are tightly regulated in young animals or may be saturated in membranes [34, 35]. In contrast, administration of very high doses of CoQ10 (200 mg/kg daily) to 12–24 month-old rats produced significant increases in brain CoQ10 levels [36], but a more recent study of the regional distribution of CoQ in 16 week-old rat brain before and after 4 weeks of moderately high exogenous dietary CoQ10 (20 mg/kg daily) supplementation showed that the concentration of CoQ was essentially unchanged in the cortex and in the striatum [37]. That study also demonstrated that the cerebellum, of the 7 brain regions dissected, contains the lowest levels of CoQ. If human brain has the same regional distribution of CoQ10 as rat brain, the cerebellum may have the narrowest safety margin and therefore would be the first tissue to suffer from a pathological shortage of CoQ10. Moreover, to function as an antioxidant, CoQ10 must be in the reduced form but normally only 20% of the lipid is reduced in the brain. The high proportion of oxidized CoQ10 in the brain could be a reflection of the high oxygen consumption in this tissue, causing an increased demand of anti-oxidants [38]. Therefore, CoQ10 deficiency could be another form of inherited ataxia due to oxidative damage, as vitamin E deficiency.
Oxidative damage and mitochondrial dysfunction have also been implicated in neurodegenerative diseases such Parkinson disease (PD), Alzheimer disease (AD), and Friedreich’s ataxia (FRDA) [39–41]. Preclinical studies have indicated that CoQ10 can protect the nigrostriatal dopaminergic system and high doses (1200 mg daily) of CoQ10 for 16 months appeared to slow progression PD [40, 42]. In 10 FRDA patients treated for 47 months high doses of CoQ10 (400 mg daily) with vitamin E (2100 IU daily), improved bioenergetics in heart and skeletal muscle, improved cardiac function by echocardiography, and, possibly less clinical decline compared to historical controls [43]. Larger studies are necessary to better define the short- and long-term effects of CoQ10 as primary or adjunctive therapy in neurodegenerative diseases.
Deficiency of CoQ10 has been associated with a variety of neurological disorders that respond to oral supplementation. We have demonstrated that CoQ10 deficiency can be primary due to a defect of biosynthesis or secondary as in patients with APTX mutations. The response of both groups of patients to oral supplementation suggests that exogenous CoQ10 is taken up by affected tissues and can correct biochemical derangements. Further studies to define CoQ10 deficiency syndrome will reveal new causes, enhance our understanding of its pathogenesis, and may provide novel insights that may be relevant to other neurodegenerative diseases.

coq10
myopathy
cerebellar ataxia
encephalopathy
mitochondria


Diagnostic Accuracy of Fine Needle Biopsy for Metastatic Melanoma and Its Implications for Patient Management

Background The use of fine needle biopsy (FNB) for the diagnosis of metastatic melanoma can lead to the early removal and treatment of metastases, reduce the frequency of unnecessary surgery, and facilitate the staging of patients enrolled in clinical trials of adjuvant therapies. In this study, the accuracy of FNB for the diagnosis of metastatic melanoma was investigated.
Fine needle biopsy (FNB) is frequently used in the diagnostic workup of clinically or radiologically detected mass lesions that are suspicious for metastatic melanoma. By determining whether they represent metastatic melanoma, the use of FNB in melanoma patients can expedite detection of metastases, leading to earlier removal and treatment; facilitate the staging of patients enrolled in clinical trials of adjuvant therapies (particularly in deep-seated lesions); reduce the frequency of unnecessary surgery; and assist in the planning of the most appropriate surgery.
FNB is a swift, minimally invasive and cost-effective technique employed in the diagnostic workup of mass lesions occurring in a wide variety of organs.1,2 The technique has been extensively evaluated in assessing the nature of lesions located in the tissues of the breast, thyroid, lung, liver, pancreas, lymph nodes, salivary glands, and kidneys, among other locations.3–6
There have been several previous case studies assessing the use of FNB in patients with melanoma. The majority of these studies lacked sufficient case numbers to precisely determine the diagnostic accuracy of FNB.7–10 However, in 1986, Perry and colleagues11 analyzed almost 300 FNBs from melanoma patients and found the procedure to be accurate, with a sensitivity of 86.5% and a specificity of 96.1%. Since this study, there have been important changes in the procedure, including improvements in immunochemical characterization and radiological guidance. In 2000, Voit and colleagues12 published a study of 739 FNBs from melanoma patients with palpable suspicious lymph nodes or small lesions that were only detectable by ultrasound B-scan examination. Although the authors reported a sensitivity of 97.8% and a specificity of 100.0%, the sensitivity depended on lesion size.
The objective of this retrospective cohort study was to evaluate the diagnostic accuracy of the FNB procedure in the detection of metastatic melanoma. To accomplish this, a very large consecutive sample was collected of FNBs performed on melanoma patients who attended the Sydney Melanoma Unit (SMU), Sydney, Australia. This large sample also allowed evaluation of the effect of several clinicopathologic features and factors related to the procedure on the diagnostic accuracy of FNB.
MATERIALS AND METHODS
Patients
For all patients with melanoma who attended the SMU and gave informed consent, clinical and histologic details of their disease were recorded on the SMU database, and follow-up information was entered prospectively. Patients with suspicious clinically palpable or radiologically identified mass lesions, detected by a variety of imaging modalities, were further investigated by FNB. All SMU patients who underwent an FNB that had been reported by cytopathologists from the Department of Anatomical Pathology at the Royal Prince Alfred Hospital in Sydney, Australia, between January 1992 and December 2002 were identified from the SMU database, and their clinical records and FNB reports were reviewed. Details of the patients are provided in Table 1.
TABLE 1.Patient characteristicsCharacteristicn%Sex  Male88862.7  Female52837.3Age at melanoma diagnosis (y)  10–301107.8  31–401359.5  41–5025618.1  51–6030321.4  61–7030221.3  70–8021915.5  81+624.4  Unknown292.0Cancer diagnosis  Other cancer diagnoses15911.2  More than one other cancer diagnoses151.1No. of primary melanoma lesions  1123687.3  214810.4  ≥3322.3No. of FNBs  195367.3  228720.3  3986.9  >3785.5Total no. of patients1416100.0FNB, fine needle aspiration biopsy.
FNB Procedure
For palpable lesions, the FNBs were performed by the reporting cytopathologist or a trainee pathologist under their supervision. Following localization and stabilization of the lesion with one hand, a hollow bore needle (22G, 23G, or 25G) was inserted directly into the mass and the needle was moved swiftly in and out for approximately 10 seconds. Aspiration with a syringe was not used in the vast majority of cases, hence our preference for the term ‘fine needle biopsy’ and ‘fine needle aspiration biopsy’. In our experience, the use of a needle without an attached syringe allows better control of the movement of the needle. Furthermore, aspiration often yields blood, promotes clotting and hampers optimal interpretation of cytologic detail. The procured material was ejected from the needle onto glass slides by pushing air from a syringe through the needle. The material was spread evenly across the slide using another glass slide. One slide was air-dried and stained immediately with Diff-Quik (Lab Aids, Narrabeen, NSW, Australia) and another fixed in alcohol and later stained by the Papanicolaou method.2 Residual material was washed into Hanks balanced salt solution for later preparation of cell blocks using the serum-prothrombin method2 or by cytocentrifuge preparations, to be used for immunochemistry. The air-dried slides were examined by the cytopathologist at the time of the procedure. Further passes were performed if necessary, depending on the amount and type of cellular material obtained.
Review of Clinical Material and Follow-up
The accuracy of the FNB procedure in diagnosing metastatic melanoma was evaluated by two reference standards: a) histopathologic evaluation of the excised lesion (1120 cases) or b) follow-up in those cases for which histologic material was not available (462 cases). The duration of follow-up was 6 months or greater (mean 50.2 months, median 45.7 months, range 6.1–144.4 months) in 456 cases. In six cases, the length of follow-up was less than 6 months (mean 4.2 months, median 4.0 months, range 3.0–5.7 months). The mass was considered benign if it was stable in size or resolved after clinical follow-up.
Cytodiagnosis
Cytodiagnoses were categorized as positive, suspicious, or negative for metastatic melanoma (Table 2). Cases were considered positive for metastatic melanoma if the specimen included sufficient numbers of well-preserved malignant cells with typical cytological features, pigment, and/or confirmatory immunochemistry for a confident diagnosis of melanoma to be made. On verification, these samples were classified as truly positive (n = 800, 50.5%) or falsely positive (n = 5, 0.3%).
TABLE 2.Categories of cytodiagnosesCharacteristicTotal n%ConfirmedNot confirmedPositive for Metastatic Melanoma  Malignant cells—melanoma108949.4805284Suspicious for Metastatic Melanoma  Malignant cells—suspicious for melanoma442.0395  Malignant cells—unknown cancer331.52211  Suspicious for malignancy401.8355Negative for Metastatic Melanoma  Malignant cells—other cancer924.24745  No malignant cells—other cells present41618.9308108  No malignant cells—scant other cells29613.420492  No malignant cells—no other cells1757.912253  No malignant cells—procedure not performed190.9019Total22041001582622
Samples that contained cells from unclassified/unspecified malignancies or cases categorized as suspicious for melanoma (those with small numbers of atypical cells, poorly preserved cells, and cells that lacked specific features of melanoma, such as cytoplasmic pigment, and where insufficient material was present for immunochemistry) were classified as suspicious for metastatic melanoma. These were determined to be true suspicious (n = 78, 4.9%) or falsely suspicious (n = 18, 1.1%) results after verification.
FNBs classified as negative for metastatic melanoma contained no material that could be diagnosed as metastatic melanoma. They included cases containing malignant cells diagnosed as another neoplasm or b) various amounts of cellular material from the tissue of the site that was sampled. After verification, these procedures were found to be either true negative (n = 612, 38.7%) or false negative (n = 69, 4.4%).
Statistical Analysis
All analyses were performed using the S-PLUS software package (Insightful Corporation, Seattle, WA) and Microsoft Excel, version 2000 (Microsoft, Redmond, WA). Diagnostic accuracy of FNB for metastatic melanoma was measured by the sensitivity and specificity of the test.
The effect of the type of tissue involved by melanoma, anatomic location, and 15 clinicopathologic and procedural factors were analyzed. These factors included features of the primary melanoma such as tumor thickness, dermal mitotic rate, presence of ulceration, predominant cell type and histopathologic subtype of melanoma; patient attributes such as sex, American Joint Committee on Cancer (AJCC)/International Union Against Cancer (UICC) stage,13 age at FNB and location of the FNB (local or distant to the primary lesion); aspects of the FNB procedure such as number of needle passes, needle size, the experience (based on caseload) of the reporting cytopathologist; year of procedure, use of immunostains; and presence of necrosis. Statistical significance was determined by the 95% confidence intervals (95% CIs) of these parameters. When comparing different samples, a two-sample test for binomial proportions was used. All equations used have been described elsewhere.14,15
RESULTS
In the 11-year study period, 2204 consecutive FNBs were performed in 1416 patients. Nineteen FNBs (0.9% of all FNB cases) were excluded from the analysis because the procedure could not be performed, i.e., no aspirate was obtained (Table 3). In half of these cases, the lesion could not be located (inappropriate referrals), but patient pain tolerance and difficulties with the procedure also contributed.
TABLE 3.Fine needle biopsy procedures that could not be performedLocationExplanation for failureConfirmationHistologyLeft neckUnable to locate lesionNegative—follow-up–Left face (subcutis)PainNot confirmed–Right faceUnable to locate lesionNot confirmed–Left neck (LN)Unable to locate lesionNegative—surgeryNo evidence of malignancyRight breastAdjacent to prothesisNegative—follow-up–Right neckPainNot confirmed–Right axilla (LN)Unable to locate lesionNegative—follow-up–Right neckPainNegative—surgeryNo evidence of malignancyLeft neck (LN)PainNegative—follow-up–Left neck (LN)Unable to locate lesionNegative—follow-up–Right groinPainNegative—surgeryNo evidence of malignancyLeft axilla (LN)Unable to locate lesionPositive—surgeryMelanomaRight axilla (LN)Unable to locate lesionNegative—follow-up–Right neck (LN)PainNot confirmed–Left axilla (LN)Unable to locate lesionNot confirmed–Left neckUnable to locate lesionNegative—follow-up–ThyroidUnable to locate lesionNegative—follow-up–Right axilla (LN)Unable to locate lesionNegative—follow-up–Right sternum (LN)Unable to locate lesionNot confirmed–LN, lymph node.
Verification of the remaining 2185 FNBs was partial; in 1582 (71.8%), the true disease status was confirmable by either histopathologic evaluation of the excised lesion or by clinical follow-up (Fig. 1). A total of 1120 FNBs were verified by histopathologic evaluation of the excised lesion; all confirmed positive FNBs and FNBs with false-positive or negative results were verified histologically. In those cases (mostly FNBs with negative results) where histopathologic material was not available (462 cases), verification was made by clinical follow-up. The mass was considered benign if it was stable in size or if it resolved after clinical follow-up. The duration of follow-up was >6 months in 456 cases and 3 to 6 months in 6 cases. Of the verified FNBs, 1435 (90.7%) were palpation-guided FNBs performed by trainee cytopathologists or cytopathologists, while 147 (9.3%) were image-guided FNBs performed by radiologists or other physicians.
FIG. 1.Flow diagram showing fine needle biopsy (FNB) result distribution.
A total of 603 FNBs (27.4%) could not be verified; this was due to loss to follow-up, particularly by death in patients with advanced metastatic melanoma, uncertainty regarding sites (where FNBs from more than one site were obtained at the same visit to the clinic), and where there were multiple metastases, which led to uncertainty in lesion location and the correlation of FNB and histopathologic results. Unconfirmed FNBs differed from confirmed procedures in several ways. They were more likely to be from lesions located in visceral organs or to be in patients with AJCC/UICC stage IV disease. FNBs with inconclusive cytodiagnoses were more likely to be followed up with clinical observation or further biopsy.
Confirmation of FNBs was unaffected by the year of procedure, the number of needle passes during sampling, the use of immunostains, the reporting cytopathologist, or the age or sex of the patient.
Approximately 12% of the patients were diagnosed with additional cancers (most commonly breast cancer, colorectal cancer, and chronic lymphocytic leukaemia), with some suffering from multiple types of other cancers. Multiple primary melanomas occurred in an eighth of this patient population. Almost a third of the patients underwent multiple FNBs. These procedures were performed both concurrently and sequentially.
There were 1582 FNB procedures for metastatic melanoma with histologic verification or clinical follow-up. The overall sensitivity was 92.1% (95% CI, 93.7–90.0) and the specificity was 99.2% (95% CI, 99.7–98.1). Five cases were determined to be false positive, resulting in a false-positive rate of 0.6% (Table 4). The false-negative rate was 10.2%, with no metastatic melanoma identified in 69 FNB cases in which metastatic melanoma was identified by later histologic evaluation (Fig. 2).
TABLE 4.False-positive fine needle biopsy findings for metastatic melanomaCytodiagnosisHistologyLocationCommentMelanomaMetastatic adenocarcinomaRight axillary (LN)–MelanomaMetastatic papillary carcinomaLeft supraclavicular fossa (LN)–MelanomaHematomaLeft axilla–MelanomaChronic osteomyelitisLeft skull (bone)S100 positiveaMelanomaMetastatic adenocarcinomaRight axilla–LN, lymph node.aS100-positive histiocytes were identified in the excision specimen, which probably caused the misdiagnosis.FIG. 2.Distribution of false-negative fine needle biopsy (FNB) cytodiagnoses.
The large numbers of confirmed cases permitted detailed analysis of this procedure for metastatic melanoma. The effect of FNB site was studied (Table 5). Lymph node tissue was the most common site for FNB evaluation, with 926 procedures, 753 of them confirmed. The sensitivity and specificity of FNB for metastatic melanoma in lymph nodes were not significantly different compared with that for all sites. However, the sensitivity for FNBs performed on lymph nodes located in the axilla was approximately 9% less compared with FNBs performed on lymph nodes in other locations (z = −3.9, P = 0.0001). Increased FNB sensitivity (by 4%) was found when the procedure was performed on lesions located in the skin and subcutis compared with other sites. However, this was only of borderline significance (z = 1.9, P = 0.05). Too few confirmed FNBs were conducted on visceral organs (n = 79) to allow conclusions to be drawn regarding the success of the procedure in these locations.
TABLE 5.Diagnostic accuracy of FNB: Effect of tissue type and anatomic locationLocationnConfirmed%TPFNTSFSFPTNSn(95% CI)Sp(95% CI)All FNB2204158271.880069781856120.92(0.90–0.94)0.99(0.98–1.00)Lymph nodes92675381.34134341732460.91(0.88–0.93)0.99(0.97–1.00)  Neck23518578.710431541580.97(0.92–0.99)0.98(0.91–1.00)  Axilla38331381.71552912221130.84a(0.78–0.89)0.98(0.94–1.00)  Groin27423385.0144111100670.93(0.88–0.96)1.00–  Other342264.710031081.00–1.00–Skin and subcutis71150470.92701726401870.94b(0.91–0.96)1.00–  Head and neck1319874.8545700320.92(0.82–0.96)1.00–  Trunk28819668.11014620830.96(0.91–0.99)1.00–  Limbs29221071.911581320720.93(0.88–0.97)1.00–Visceral organs1767944.9303520390.91(0.76–0.97)1.00–  Liver562239.371100130.88(0.53–0.98)1.00–  Lung944345.7182410180.90(0.70–0.97)1.00–Other39124662.98766521400.94(0.87–0.97)0.99(0.95–1.00) TP, true positive; FN, false negative; TS, true suspicious; FN, false suspicious; FP, false positive; TN, true negative; SN, sensitivity; SP, specificity; 95% CI, 95% confidence interval.aAxilla lymph nodes had significantly reduced sensitivity compared with other sites (z = -3.9, P = .0001).bSkin and subcutis FNBs had significantly increased sensitivity compared with other sites (z = 1.9, P = .05).
Fifteen clinicopathologic and procedural factors were analyzed to assess their effect on the diagnostic accuracy of FNB for metastatic melanoma (Table 6). The FNB confirmation rate was similar for all clinicopathologic factors, with the exception of AJCC/UICC stage and location (distant or regional to the primary lesion), as described above.
TABLE 6.Diagnostic accuracy of fine needle biopsy effect of clinicopathologic factorsFactorNConfirmed%TPFNTSFSFPTNSN(95% CI)SP(95% CI)All FNB2204158271.880069781856120.92(0.90–0.94)0.99(0.98–1.00)AJCC/UICC stage at FNB  Stage I40032380.8128922631550.93(0.88–0.97)0.98(0.95–0.99)  Stage II70556980.72833124612240.90(0.86–0.93)1.00(0.98–1.00)  Stage III83656167.13102427511940.93(0.90–0.95)0.99(0.97–1.00)  Stage IV26312949.0795510390.94(0.87–0.97)1.00(0.91–1.00)Location  Regional1340107780.46175858643340.91(0.89–0.93)0.99(0.97–1.00)  Distant86450558.418311201212780.94(0.90–0.97)1.00(0.98–1.00)Use of immunochemistry  Yes58340669.631662681490.98a(0.96–0.99)0.98(0.90–1.00)  No1621117672.548463521045630.88(0.86–0.91)0.99(0.98–1.00)Year  1992–199439228572.71441515511050.91(0.85–0.94)0.99(0.95–1.00)  1995–199755541474.62161513611630.94(0.90–0.96)0.99(0.97–1.00)  1998–200069350072.22412126322070.92(0.88–0.95)0.99(0.97–1.00)  2001–200256438367.91991824411370.92(0.87–0.95)0.99(0.96–1.00)Sex  Male137099472.650048451013900.91(0.89–0.93)1.00(0.99–1.00)  Female83458870.53002133842220.93(0.90–0.96)0.98(0.96–0.99)Age at FNB  ≤50 y57241472.41981222121790.94(0.90–0.97)0.99(0.96–1.00)  >50 y1632116871.660257561734330.91(0.89–0.93)0.99(0.98–1.00)No. of FNB attempts  142630972.52324320680.98b(0.96–0.99)1.00(0.95–1.00)  251837171.61851321301490.93(0.89–0.96)1.00(0.97–1.00)  331521969.5791312601090.86(0.77–0.92)1.00(0.97–1.00)  ≥419213670.833172111630.66(0.52–0.78)0.98(0.92–1.00)  Unknown75354772.62712221642230.92(0.89–0.95)0.98(0.96–0.99)Needle sizec  22G1336045.1222410310.92(0.74–0.98)1.00(0.89–1.00)  23G17313879.8667820550.90(0.81–0.95)1.00(0.93–1.00)  25G91066473.03512439712420.94(0.91–0.96)1.00(0.98–1.00)Necrosis present  Yes674161.224490040.86(0.69–0.94)1.00(0.51–1.00)  No2137154172.177665691856080.92(0.90–0.94)0.99(0.98–1.00)Pathologist caseload  <100 cases16410463.4517540370.88(0.77–0.94)1.00(0.91–1.00)  100–500 cases65147472.82272927321860.89(0.84–0.92)0.99(0.96–1.00)  >500 cases1389100472.352233461133890.94d(0.92–0.96)0.99(0.08–1.00)First primary Breslow thickness   ≤2 mm95070574.23322334843040.94(0.90–0.96)0.99(0.97–0.99)  >2 mm91966272.03673833602180.91(0.87–0.93)1.00(0.98–1.00)  Unknown33521564.210181141900.93(0.86–0.96)0.99(0.94–1.00)First primary ulceration  Yes57140771.32471618101250.94e(0.90–0.96)1.00(0.97–1.00)  No100975775.036040351043080.90(0.87–0.93)0.99(0.97–1.00)  Unknown62441867.01931325711790.94(0.90–0.96)0.99(0.97–1.00)First primary lesion mitotic rate (/mm2)  ≤141430072.51361012421360.93(0.88–0.96)0.99(0.95–1.00)  1 to <448135273.21701920211400.90(0.85–0.93)0.99(0.96–1.00)  4 to <833223771.41417810800.95(0.91–0.98)1.00(0.95–1.00)  ≥839229675.5175221440810.89(0.84–0.93)1.00(0.95–1.00)  Unknown58539767.91781124721750.94(0.90–0.97)0.99(0.96–1.00)First primary lesion histologic subtype  Desmoplastic1309774.6426630400.88(0.75–0.94)1.00(0.91–1.00)  SSM51237072.31681022411650.94(0.90–0.97)0.99(0.97–1.00)  NM46134374.41982014411060.91(0.86–0.94)0.99(0.95–1.00)  Other75454972.82772622412190.91(0.88–0.94)1.00(0.97–1.00)  Unknown34722364.311571432820.94(0.89–0.97)0.98(0.92–0.99)First primary lesion predominant cell type  Epithelioid44332974.31851713401100.92(0.87–0.95)1.00(0.97–1.00)  Spindle1378965.0476900270.89(0.77–0.95)1.00(0.88–1.00)  Mixed17311164.2586830360.91(0.81–0.96)1.00(0.90–1.00)  Unknown1451105372.651040481154390.93(0.90–0.95)0.99(0.97–1.00)TP, true positive; FN, false negative; TS, true suspicious; FN, false negative; FP, false positive; TN, true negative; SN, sensitivity; SP, specificity; 95% CI, 95% confidence interval; SSM, superficial spreading melanoma; NM, nodular melanoma.aFNBs that used immunostains had significantly increased sensitivity compared with those that did not (z = 4.8, P < .001).bFNBs obtained in one pass had significantly increased sensitivity compared with FNBs which required more than one pass (z = 4.8, P < .001).cThe size of the needle used for the FNB procedure was not known in 988 cases.dFNBs reviewed by a cytopathologist who reported >500 cases had significantly increased sensitivity compared with FNBs reported by cytopathologists who had reported <500 cases (z = 2.4, P = .02).eFNBs obtained from patients with an ulcerated first primary lesion had increased sensitivity compared with FNBs from patients without ulcerated first primary lesions (z = 1.9, P = .05).
Patients with an ulcerated primary lesion experienced slightly better FNB test sensitivity compared to those with no ulceration (z = 1.9, P = 0.05). AJCC/UICC stage, anatomic location, year of procedure, needle size, sex, age at FNB, primary lesion thickness, dermal mitotic rate, predominant cell type, histologic subtype of melanoma, and presence of necrosis did not affect the diagnostic accuracy of the test (Table 6).
Three factors related to the conduct of the procedure affected FNB test accuracy. These were the number of needle passes needed to collect the sample, the number of FNBs for metastatic melanoma the cytopathologist had reported during the study period (caseload), and the use of immunostains. Samples that required only one attempt at FNB had a sensitivity increase by >10% compared to samples with more than one attempt (z = 4.8, P < 0.001). Sensitivity fell in a linear manner with each subsequent attempt (Fig. 3). Cytopathologists who had caseloads of >500 FNBs (2 cytopathologists) had greater FNB test sensitivity by about 5% compared to those who had reported <500 FNBs (16 cytopathologists) (z = 2.4, P = 0.02). Finally, FNB tests in which immunostains were used had better sensitivity (z = 4.8, P <0.001) (Table 6).
FIG. 3.Sensitivity of fine needle biopsy (FNB) in the diagnosis of metastatic melanoma. Sensitivity is reduced in a linear manner as the number of FNB attempts needed to obtain the sample increases. Bars, 95% confidence intervals.
DISCUSSION
Metastatic melanoma is an aggressive tumor with a high mortality rate. Patients with primary melanomas that are at high risk of metastasizing who attend the SMU are followed closely for evidence of metastatic disease with radiological investigations and regular clinical follow-up. Early surgery for metastatic disease may lead to longer disease-free periods and may ultimately improve survival in some of these patients. This underscores the importance of early detection of metastatic disease.
In some melanoma treatment centers, FNB has been used for many years to verify clinically suspicious lesions and radiological abnormalities in patients with melanoma, prior to traumatic or costly surgical or adjuvant treatment. FNB is a cost-effective, rapid procedure that is well tolerated by patients when performed by proficient operators.2 However, the ability of the test to accurately diagnose suspicious lesions as metastatic melanoma has been infrequently studied.
Sensitivity and specificity are measures that are used extensively in the FNB literature. They were chosen as the best measures of diagnostic accuracy of FNBs in patients with melanoma because of the binary quality of the data.16 In this study, a large number of consecutive FNB procedures (2204) for metastatic melanoma were analyzed, with 1582 procedures confirmed by either histopathology after surgical resection or by clinical follow-up. This cohort represents more FNB procedures in melanoma patients than all previous reported series combined.7–12 The overall sensitivity of FNB for metastatic melanoma was 92.1%, and the specificity was 99.2%. These results are superior to those obtained by Perry and colleagues11 in their study of 298 (261 confirmed) cases of metastatic melanoma FNB more than 20 years ago (sensitivity of 86.5% and specificity of 96.1%). Other similar case series did not have the power to determine the true diagnostic accuracy of FNB because of their low numbers of FNBs (between 56 and 108 confirmed cases).7–10 Voit and colleagues12 examined 739 FNBs for metastatic melanoma and found the technique to be highly sensitive (97.9%) at their institution. However, regional node basins in their patient population were routinely evaluated by ultrasound B-scan, and a third of FNB cases were performed under ultrasound guidance.
Studies of FNB are frequently retrospective investigations and thus verification bias may be a hazard in these studies. Not only are there different reference tests, which are determined by the results of the FNB procedure, namely histopathology after surgical resection for all positive FNB tests and clinical follow-up for most negative procedures, but verification is also partial. Not every consecutive FNB can be verified. Differential verification can lead to overestimation of the measures of diagnostic accuracy.17,18 This bias is related to the quality of the reference tests. Confirmation of melanoma metastases by histopathologic examination of tissue removed after surgery is better at identifying true disease status than clinical follow-up of patients with negative FNB results.19 Melanoma metastases may regress spontaneously, resulting in failure to identify all false-negative results by clinical follow-up, or they may cause truly positive findings to be incorrectly classified as false positives if the lesion resolves before it is excised or during follow-up.20,21
In this study, there were differences in those FNBs that were verified by the reference tests compared with those that were not. Unconfirmed FNBs were more often performed on nonpalpable lesions in visceral sites, and in patients with advanced disease. Verification rates of negative, positive, and suspicious FNBs were 68.2%, 73.9%, and 82.1%, respectively.
Despite the high specificity rate, as a result of the large number of confirmed biopsy samples, five false-positive findings were detected (Table 4). The cytologic features of these cases have been reviewed in more detail elsewhere.22 In all cases, misinterpretation of the cellular material had occurred, three of which were found to be adenocarcinoma. Two of these represented metastatic breast adenocarcinoma in axillary nodes, and one was a metastatic papillary adenocarcinoma of renal origin. Two other false-positive cases were caused by the misinterpretation of large histiocytes or reactive fibroblasts as metastatic melanoma cells.
In 69 FNBs, no metastatic melanoma cells were identified cytologically; however, the presence of metastatic melanoma was subsequently identified by histopathologic examination (false negatives) (Fig. 2). Astute clinical and/or radiologic surveillance results in the detection of small suspicious lymph nodes. Difficulties in performing FNBs on such small suspicious lymph nodes contribute to some of the false-negative diagnoses (particulary small, mobile axillary nodes). In fact, approximately a third (n = 21) of these biopsy samples (which were reported as “no malignant cells identified”) did not contain any cellular material, suggesting that an absence of sampling of the malignant cells was the reason for the false-negative result. Further investigation is usually necessary to identify the cause of the mass lesion. Surgical examination is usually the next step when the index of clinical suspicion is high.
There were several sources of difficulty in performing the FNB procedure for metastatic melanoma. Often it occurred when there was a failure to locate the suspicious lesion because of its small size or its location was not communicated with adequate precision. Failure to identify metastatic melanoma occurred when cellular material showed too few typical morphologic characteristics of metastatic melanoma, or these characteristics were destroyed or masked by necrosis.
The large number of confirmed FNB in this study permitted the analysis of subgroups. Most FNB for metastatic melanoma were performed in lymph nodes as well as skin and subcutaneous tissues. No difference in sensitivity was noted among these tissue groups. However, FNBs of lymph nodes of the axilla were significantly less sensitive compared with those performed at other sites and with FNBs performed in lymph nodes of the groin and neck. A number of factors are likely to contribute to the low sensitivity of FNBs performed in the axilla, including the greater difficulty in gaining access to and locating lymph nodes, particularly those high in the axilla, and the presence of large amounts of fatty tissue in axillary lymph nodes (“horseshoe” nodes).
Fifteen clinicopathologic factors were examined for their effect on the accuracy of FNB for metastatic melanoma, most of which had no effect on the diagnostic accuracy of the test. However, the following four variables did influence the sensitivity of the FNB procedure. (1) Ulceration of the primary lesion led to a small increase in the sensitivity of the test (P = 0.05). (2) Additional needle passes were performed if the initial sampling failed to obtain sufficient cellular material; thus, the number of needle passes is an indicator of the difficulty in obtaining a sample from a specific lesion. FNBs that were performed in only one needle pass were found to have superior sensitivity than those which required additional passes to obtain an adequate sample. Furthermore, the sensitivity of the test seemed to decrease in a linear manner with each subsequent pass. (3) Training and experience have been shown to significantly influence the interpretation of FNB of the breast,23 and in this study, we found that the caseload (i.e., level of experience) of the cytopathologist who reviewed the slides influenced the sensitivity of the test. Those cytopathologists who reviewed >500 metastatic melanoma FNB samples in the study period performed better. Those cytopathologists who performed <100 reviews seemed to do as well as those who had a caseload of 100 to 500 cases. (4) The use of immunostains was associated with improved sensitivity of the test. However, this may reflect the fact that immunochemistry was only performed in those cases in which a sufficiently high cellular yield was obtained, in which case it may have been the high cellular yield that lead to an improvement in sensitivity, rather than the use of immunostains per se. The diverse cytological presentation of metastatic melanoma may also be a factor in the association of the use of immunochemistry with increased sensitivity.24,25 Nasiell et al.26 found that immunochemical characterization was necessary to conclusively diagnose >50% of metastatic melanomas that presented with an equivocal cytological picture. However, immunological characterization cannot be considered definitive when the FNB lacks typical cytologic features expected of metastatic melanoma. For example, one of the false-positive cases exhibited S100 positivity, whereas two of the false-negative FNBs contained melanoma cells that seemed to be S100 negative. This suggests that cytopathologists should be cautious when reporting the results of immunostains on limited samples.
Our study shows that FNB for metastatic melanoma is a procedure with very high specificity and good sensitivity. Several clinicopathologic factors were found to influence the diagnostic accuracy of the test for metastatic melanoma. These included factors relating to the original primary melanoma lesion, location of the sampled lesion, and factors relating to the performance of the test. The SMU employs an on-demand FNB service with assessment of the cytologic material and delivery of a provisional result to the clinician at the time of the patient’s visit. This helps guide subsequent diagnostic and therapeutic measures, and it reduces costs (e.g., by decreasing the need for additional patient visits to the clinic). A multidisciplinary approach involving clinicians, pathologists and radiologists enables an efficient and cost-effective management strategy in melanoma patients.

diagnostic accuracy
fine needle biopsy
melanoma
diagnosis
pathology
cytology