Introduction

Biopsies are essential procedures for any practitioner caring for patients with head and neck pathology. Before performing a biopsy, a comprehensive medical history must be obtained. The characteristics and timeline of the relevant symptoms, aggravating and alleviating factors, and presence of comorbidities help to establish a working diagnosis. Practical knowledge of oral pathology is paramount to determining which type of biopsy is best in any given circumstance.

Biopsies of the oral cavity sample two broad classes of tissue: soft tissue and hard tissue. Soft tissue biopsies are performed on the oral mucosa, gingiva, and tongue. Hard tissue biopsies collect samples from bony structures or access intraosseous areas to obtain tissue for histopathologic examination. Biopsies of deeper structures in the airway, such as the pharynx and hypopharynx, are beyond the scope of this activity.

A radiographic examination of any hard tissue lesions is recommended to evaluate the size of the lesion(s) and facilitate procedural planning. Generally, an initial office-based orthopantomogram or cone-beam computed tomography (CBCT) is sufficient.[1] Noncontrast medical-grade computed tomography plays a role in evaluating more extensive or invasive hard tissue lesions, except in cases of suspected infection such as osteomyelitis.

The 3 biopsy procedures commonly performed in clinical practice are fine needle aspiration, core biopsy, and brush biopsy.

Fine needle aspiration (FNA) is commonly performed to evaluate lesions within deeper tissues where access is otherwise surgical, and a definitive diagnosis is required to plan the final extirpation of the lesion. FNA is commonly used for lesions in the salivary glands and lymph nodes in the head and neck.[2] FNA is performed by passing a hollow needle in and out of the lesion several times and collecting the small tissue fragments within the needle for cytological examination. FNA can only reliably indicate if a lesion is benign or malignant; the obtained tissue is insufficient for a definitive diagnosis. However, the limited information obtained through FNA helps plan further treatments.

A core biopsy is similar to an FNA but obtains a tissue sample adequate for a definitive histological diagnosis of the lesion. At some institutions, the interventional radiology department offers this service.

Lastly, a brush biopsy involves sampling a soft tissue lesion with a specialized brush to harvest cells for examination. The technique could represent a predictable and less invasive biopsy method in the future; however, it is not yet recommended as the biopsy of choice due to a lack of supporting evidence.[3][4][5][6]

Anatomy and Physiology

Soft Tissues: Anatomical Considerations

The oral cavity soft tissues comprise the mucosae, gingivae, and tongue. The mucosae of the cheeks, vestibules, alveolar ridges, and lips inside the vermillion borders is a loose, nonkeratinized squamous epithelium supported by areolar tissues that allow for movement. This contrasts the keratinized epithelia of the gingivae and hard palate, which are firmly bound to the underlying periosteum.[7] The junction of these 2 mucosae is readily appreciated during clinical oral cavity examination.

The floor of the mouth is also covered in a loose nonkeratinized epithelial mucosa, and several salivary ducts emerge through the mucosa, draining from the submandibular and sublingual glands. The mucosa of the dorsal surface of the tongue differs from that of the ventral surface.

The oral cavity demonstrates a marked healing capacity as part of the gastrointestinal tract. Normal cellular turnover occurs approximately every 14 to 21 days; a biopsy of the oral mucosa will generally heal quickly and without prolonged discomfort.[8]

A soft tissue biopsy is a relatively simple procedure. Complications may arise if important anatomical structures near the biopsy site are injured. The lip mucosa is generally safe to biopsy. Common benign lip lesions, such as fibromas and mucoceles, are usually extirpated uneventfully, though they tend to bleed, given their high vascularity. Damage to the labial arteries can occur during deeper biopsies and may require cauterization. Care must be taken to avoid injuring the parotid (Stenson) duct when biopsying the cheek and vestibular mucosae. The opening of the parotid duct is the parotid papilla or puncta located along the buccal mucosa adjacent to the second molar.[9] Damage to the parotid papilla may inhibit drainage of the parotid gland.

Gingival and hard palate mucosal biopsies are usually straightforward without much risk to critical structures. However, the greater palatine neurovascular bundle emerging from the greater palatine foramina near the maxillary second molar and the nasopalatine neurovascular bundle arising from the incisive canal behind the maxillary central incisors must be avoided. The hard palate mucosa is so tightly adherent to the underlying periosteum that closure is nearly impossible and not recommended; biopsy sites are left to heal by secondary intention. Local tissue can sometimes be rearranged to cover the biopsy site.

The tongue is a large muscle with a remarkable regenerative capacity, which facilitates the collection of biopsy samples from its surface. The most common location of oral squamous cell carcinoma is the posterolateral borders of the tongue; therefore, providers should have a very low threshold for biopsying any suspicious lesion in this area.

The floor of the mouth is the most challenging area for biopsy in the oral cavity, as the mucosa here is very thin and fragile, and underneath lie various critical anatomical structures, including the salivary ducts of the submandibular and sublingual glands, the lingual nerve, and a diffuse network of arteries and veins. A less experienced provider could risk injuring these structures.

Soft Tissue: Lesions

A detailed description of oral pathology is beyond the scope of this activity, but a generalized framework helps understand commonly encountered lesions with malignant potential. Any lesion present for more than two weeks warrants consideration for biopsy.[5][10] Careful clinical observation past two weeks is a proper treatment if thoroughly discussed with the patient; a follow-up and surveillance plan should be initiated. Refusal of a biopsy should be documented in the medical record.

Benign lesions tend toward a longer clinical course of weeks to months, with little or no associated symptoms, though patients commonly report biting them, causing cyclic irritation and swelling.[11] Benign lesions are often pink or similarly colored to the surrounding mucosa, may be pedunculated or sessile, soft or firm, and nontender when palpated. Benign lesions are rarely ulcerated and often an idiopathic finding during a routine examination. Common benign soft tissue lesions of the oral cavity are fibromas, granulomas, and mucoceles.[12][13]

In addition to the major salivary glands, the oral cavity contains minor salivary glands spread ubiquitously throughout the oral mucosa. Salivary outflow tract obstruction of these small glands from trauma, such as direct injury or chewing, results in a mucocele, a large, soft, painless mass that fluctuates in size. Mucoceles commonly manifest on the lower lips and buccal mucosa. A lesion with these same characteristics but a pebbled appearance may represent a papilloma, an infection, a sexually transmitted disease, or a viral lesion.[14]

Ulceration is a common feature of malignant lesions; any ulcerated lesion warrants consideration of biopsy.[15][16] However, an ulcerated lesion may be traumatic ulcerative granulomas with stromal eosinophilia, a viral infection like primary herpetic gingival stomatitis or hand-foot-mouth disease that is past the vesicular phase, necrotizing sialometaplasia, and fungal infections in lesions >1 cm.[17][18][19]

Lesions on the alveolar ridge may be any of the abovementioned processes or may arise from the alveolar bone. Alveolar bony lesions are commonly caused by odontogenic infections that have formed at the apex of a tooth, and the localized infection has spread through the bone.

As previously stated, tongue lesions, particularly of the lateral-posterior border, should be strongly considered for biopsy. Biopsy should be strongly considered for posterolateral tongue lesions, particularly in the presence of concerning features, including pain, paresthesia, raised borders, ulceration, and associated systemic symptoms such as dysphagia, weight loss, and bleeding.[18][20] Red or purple lesions could indicate vascular lesions and may present with a palpable vibratory sensation (thrill) or audible sound if auscultated (bruit).[21][22] Vascular lesions are best managed with an electrosurgical unit or electrocautery in a setting where hemostatic agents are readily available. A contrast CT scan can help identify lesions of vascular origin.

The threshold for biopsying purple, dark blue, or black lesions must also be low due to the concern of oral melanoma.[23] However, in many cases, lesions with such features tend to be retained metallic fragments in the mucosa from amalgam dental restorations, commonly referred to as amalgam tattoos.

Hard Tissue: Anatomical Considerations

Hard tissues of the oral cavity comprise bony structures and teeth. Teeth are frequently involved and sometimes the source of lesions, especially those that occur in localized infectious processes. However, teeth are rarely removed during the biopsy phase. Mandibular and maxillary bony lesions are common, but lesions within the temporomandibular joint are less so.

Benign processes within hard tissues tend to present asymptomatically, with a long history indicating slow growth.[24] These benign lesions are often discovered on panoramic radiographs during routine dental exams, after which a medical-grade CT or CBCT is recommended for a more thorough assessment.[24][25]

Hard tissue malignancies often present more abruptly, within weeks to months, with pain, paresthesia, and bony destruction characterized by a moth-eaten or mottled appearance of the bone.[26][27] Benign but locally aggressive lesions, such as ameloblastoma and myxoma, are not usually associated with metastasis but will warrant consideration for local resection. Intraosseous vascular lesions, like arteriovenous malformation, must also be considered when identifying a hard tissue lesion.

Hard Tissue: Lesions

Bony lesions of the maxilla and mandible are characterized by their radiographic appearance. Many of these lesions arise from cells of odontogenic origin or cell lineages associated with tooth development, as teeth are the main structures within the gnathic complex. On imaging, bony lesions can be radiopaque (hyperdense), radiolucent (hypodense), or present with a mixed picture. Radiopaque lesions are common and often represent a subset of conditions that do not require biopsy. Single areas of asymptomatic hyperdensity with no destruction or expansion are often found, and the differential diagnosis includes idiopathic osteosclerosis, cemento-osseous dysplasia, and condensing osteitis, usually from a tooth cavity that creates a low-grade inflammatory stimulus over time.

Mixed lesions with areas of hypodensity and hyperdensity tend to have a more concerning differential diagnosis, including adenomatoid odontogenic tumor, calcifying odontogenic epithelial tumor, calcifying cystic odontogenic tumor, cementoblastoma, and osteomyelitis. Hypodense or radiolucent lesions are further described by their appearance within the bone as unilocular or multilocular, occupying a single space or cavity or having multiple generally connected areas within the bone, respectively.[24][28][29]

Radiolucent benign lesions, like cysts, tend to be unilocular. Conversely, benign but locally aggressive lesions tend to have a "soap bubble" appearance characterized by single or many spaces fully or partially divided by bony septae. Malignancies tend to present with a destructive or moth-eaten appearance due to their faster spread and destructive characteristics.[24][30]

The growth pattern of bony lesions differs between benign and malignant processes. Benign lesions expand anterior-posteriorly and stay within the confines of the cortices; softer marrow bone is easier to expand than denser cortical bone. However, benign lesions can expand the cortices and reabsorb tooth roots as they enlarge. More aggressive lesions can erode through and spread into surrounding hard or soft tissues. Hence, evaluation of the pattern of lesion spread is essential and informs the choice of biopsy.

Some common benign etiologies of unilocular, generally asymptomatic lesions include a periapical cyst, hyperplastic follicle, dentigerous cyst, odontogenic keratocyst, and ameloblastoma.[31] Lesions that frequently present with a multilocular appearance and symptoms of pain, cortical expansion or destruction, loosening or loss of teeth include myxoma, ameloblastoma, central giant cell granuloma, odontogenic keratocyst, hemangioma, cherubism, and hyperparathyroidism.[32][33][34]

Indications

Biopsy planning can only occur after obtaining a comprehensive medical history, performing a thorough clinical and radiographic examination, and interpreting necessary laboratory testing. Informed consent must be obtained and should include a detailed discussion of possible diagnoses and the risks, benefits, alternatives, complications, and a description of the biopsy procedure.

Performing a biopsy of any hard or soft tissue lesions present for 2 weeks or more is reasonable, as self-limiting conditions will frequently resolve within two weeks. If the lesion is new, observation and close follow-up are reasonable.[35] Patients may request a biopsy of a presumably benign lesion to ease their minds, and any discussion of shared-decision making should be reflected in the documentation.

Contraindications

Absolute contraindications to biopsies of structures within the oral cavity are limited. Most contraindications are relative and depend on the type of biopsy (incisional versus excisional) and the practitioner's experience. If the practitioner does not have experience operating at the site of the lesion, a referral is recommended.

However, several clinical situations warrant further consideration.

Vascular malformations may manifest as hard or soft tissue lesions. Soft tissue lesions can exhibit a thrill or bruit, blanching with pressure, and be arterial, venous, or both. Bony vascular lesions commonly present with the triad of pain, bleeding, and mobile teeth.[36] Surgical excision of vascular malformations may result in profound blood loss; CT angiography and consultation with interventional radiology for potential embolization before excision should be considered.[37][38] Excision in an operating room setting is strongly advised.

Vascular lesions of the bone are potentially more problematic than those of the soft tissues; intraoperative bleeding is harder to control. When entering a bony lesion for a biopsy, the first step is to aspirate the lesion; if blood is aspirated, a vascular lesion should be suspected, and the procedure should be terminated pending further workup.

Potentially malignant lesions also warrant special consideration. The clinical findings of malignancy include pain, paresthesia or altered nerve sensation, ulcers with rolled borders, and destruction with a moth-eaten appearance in the case of bony lesions. Practitioners should be familiar with malignancy workup guidelines in their respective countries; in the United States, the National Comprehensive Cancer Network has procedural guidelines available online for no cost.[39] When biopsying a potentially malignant lesion, a description of the lesion, the location and depth of the biopsy, and proper marking of the specimen are critical. Photography of the lesions in situ and tissue resected is helpful to future providers.

Lastly, osteomyelitis, osteoradionecrosis, and medication-related osteonecrosis of the jaws (MRONJ) warrant special consideration due to the extent of the necessary evaluation, which is beyond the scope of this activity.[40][41][42][43][44]

Equipment

Soft Tissue Lesions

Most soft tissue biopsies can be performed safely in an outpatient clinical setting under local anesthesia and are performed as an excisional biopsy with complete removal of the lesion for subsequent histopathologic examination. An incisional biopsy is warranted if a more destructive or malignant lesion is suspected.

Biopsy of soft tissue lesions can usually be performed with the following equipment.

• Local anesthetic with vasoconstriction agent[45]
• 15-blade or similar incisional device
• Toothed forceps
• Small dissecting scissors
• Suction with a small tip
• Gauze
• Suture material for wound closure and specimen marking
• Electrocautery device should be readily available

Hard Tissue Lesions

Bony lesions often require extensive preoperative planning, and the decision between an incisional or excisional biopsy is typically made during the procedure.

When a bony lesion is opened, the presence of yellow or clear fluid suggests a cystic pathology. In such cases, an excisional biopsy is performed. After removing the lesion, the possibility of bone grafting and, in some instances, stabilizing the mandible or maxilla with hardware is considered, depending on the remaining bone structure.

If no fluid aspirate is obtained from the lesion and solid tissue is observed upon exposure, an incisional biopsy may be performed to remove a sample of the lesion.

In addition to the instrumentation required for a soft tissue biopsy, burrs for entering the bone, a needle and syringe to aspirate the bony lesion and hemostatic agents are required when biopsying a nard tissue lesion.

Specimen Transport

Specimens requiring routine formal histopathological evaluation should be placed in formalin.[46] If an underlying immunological issue is suspected, such as lichen planus, pemphigus vulgaris, mucous membrane pemphigoid, or lupus, a separate tissue sample should be obtained for direct immunofluorescence and placed in Michel solution.[47] Biopsies requiring immunological evaluation should be discussed with the pathologist before initiating the procedure. If the biopsy is performed in a facility with an onsite pathologist, evaluation of a frozen section may be possible to rule out malignancy intraoperatively.

Technique or Treatment

Soft Tissue Lesion

Field anesthetic infiltration or regional anesthetic blocks are superior to localized anesthetic infiltration, which can distort the lesion.

Excisional biopsies are most commonly performed via an elliptical excision with the lesion in the middle of the excised tissue. Elliptical incisions allow for wound closure with less tissue tension. The longest dimension of the ellipse should be 3 times the longest dimension of the lesion to achieve a cosmetic closure.[48]

Lesions on the lips should be excised with the ellipse oriented vertically to achieve an aesthetic closure; horizontal orientation of the ellipse may result in a misaligned closure due to scar contracture. Biopsies performed on facial skin should be oriented within the resting tension lines to achieve an aesthetically pleasing scar[49].

Incision of the mucosa is performed with a 15-blade or similar scalpel. Sharp dissection facilitates tissue undermining and complete removal of the lesion. Best practices dictate limiting electrocautery to avoid distortion destruction of the resected tissue, which may limit histopathological analysis. The presumptive etiology of the lesion dictates the depth of the biopsy; the depth of invasion of the tumor frequently dictates the staging and subsequent treatment of benign and malignant neoplasms.[5]

The resulting wound should be thoroughly examined, irrigated, and hemostasis must be achieved before closure. In the oral cavity, any minor salivary glands present should be removed to prevent mucocele formation. If the excision is extended down to the bone, the local periosteum can be elevated to achieve soft tissue closure. Oral soft tissues rarely require undermining to achieve closure. Soft tissue biopsy sites can also be allowed to heal by secondary intention, as can sites on the gingivae or hard palate.

An incisional biopsy is recommended when a malignant diagnosis is suspected, as further surgical intervention is usually required. For example, if an excisional biopsy of a lesion suspicious for oral squamous cell carcinoma removes the lesion in its entirety, the consulting oncologist will not know exactly where the lesion was, which can adversely affect medical decision-making. However, incisional biopsy of highly suspicious malignancies is controversial and should be discussed with the pathologist and local cancer team. A wedge-shaped biopsy to capture some normal and abnormal tissue may be preferred to facilitate histopathological examination.[50][51]

When a more aggressive lesion is suspected, photographs of the lesion in situ and the specimens should be taken. Orienting a specimen is essential and should be done along two axes; different lengths or colors of sutures are commonly used for specimen orientation. The depth achieved during an incisional biopsy is critical and will vary with the location and suspected diagnosis of the lesion.[52] Incisional biopsies can be irrigated, hemostasis achieved, and closed with sutures in a fashion similar to an excisional biopsy.

Bony Lesion

Before the procedure, the practitioner should plan out the biopsy, the design of the flap used to access the bone, where the bone will be entered, critical structures during both the approach and dissection, and any tooth extractions. Patients undergoing bony biopsies often consented to an incisional and excisional biopsy as the decision is frequently made based on intraoperative findings. It is paramount to have the patient consent to the multiple eventualities that give the provider the necessary intraoperative flexibility to perform an incisional or excisional biopsy, possible extraction of teeth, and possible bone grafting or plating.

After administration of local anesthesia, an incision is made to expose the bony area of the lesion. If the bone is relatively thin, needle aspiration of the lesion should be performed. If the lesion is vascular, this pinpoint bleeding can be controlled, hemostasis achieved, and the site closed. Angiography, other diagnostic studies, or consultation for possible preoperative embolization can be scheduled to prevent potentially life-threatening bleeding.[53] The lesion is likely cystic if aspiration yields a clear, turbid, yellow fluid. If aspiration yields a caseous substance, the lesion is highly suspicious for an odontogenic keratocyst. If nothing is aspirated, this indicates a more solid tumor or tissue. An incisional biopsy is preferred for solid lesions; the differential diagnosis for solid lesions includes more entities that require margins of up to 1 cm and further treatment planning before definitive treatment. If the bone is too thick to enter with a needle directly, a burr can be used to thin the bone until a needle can be introduced. After aspiration, the lesion is entered with a burr under irrigation.

If an incisional biopsy is decided upon, a representative piece or pieces of tissue should be excised, with a size adequate to ensure proper histopathological examination. However, this incision compromises the margin of the tumor. At the subsequent surgery for definitive treatment, if a lesion requires more than simple enucleation and curettage, the periosteum overlying the incisional biopsy hole should be considered compromised and removed.

If an excisional biopsy is chosen, the lesion should be removed in its entirety via curettage or a similar procedure. Residual cells left in situ may promote recurrence. Specific treatments vary among etiologies. A round burr can often be used to gently go over the bone cavity to ensure complete lesion removal.[54]

One of the most commonly encountered situations in the mandible is a lesion abutting or surrounding the inferior alveolar nerve. Benign pathological entities are well-encapsulated, pushing surrounding tissue away rather than invading it. Care should be taken when dissecting lesions away from the nerve or performing curettage to avoid direct injury to the neurovascular bundle.[55][56]

The remaining bony structure should be examined for strength and integrity; plating can be performed to prevent a fracture during the healing period, and bone grafting may be considered.[57] The advantages of bone grafting are facilitating the regenerative process and easier radiographic identification of the lesion should it recur. The site should be closed in a standard fashion as the initial flap design dictates.

Marsupialization and Drain Placement

Decompression with marsupialization or drain placement is an alternative approach to oral cavity lesions.[58][59][60] By facilitating decompression, the lesion's expansion mechanism is removed, and the lesion may shrink over months. 

Marsupialization is more commonly performed when treating soft tissue lesions; the lesion is incised, and the edges sutured to the surrounding tissues allowing a cyst to drain freely, decompress, and shrink with time. Marsupialization can be considered for large ranulas and other pathology of the floor of the mouth. Marsupialization also induces a minor inflammatory response, thickening the tissues and making eventual removal more manageable and predictable.

Drains are an excellent procedural adjunct that facilitates decompression in bony cystic lesions and other entities like the odontogenic keratocyst. After representative tissue is taken during the biopsy, a drain can be placed into the lesion and sutured to the outer mucosa or wired to nearby teeth. When combined with daily postoperative irrigation, a drain can shrink large lesions dramatically, facilitating enucleation and curettage for a lesion that would otherwise have required extensive resection. Excellent materials for drains include pediatric nasopharyngeal airways or various intravenous tubing trimmed to the appropriate size. Drains may be left in place if the lesion continues to shrink objectively on radiography and the patient can tolerate the foreign body. Drains should be placed to not interfere with occlusion and minimize damage to surrounding tissues.

Complications

Complications of biopsying lesions within the oral cavity can arise and should be thoroughly discussed and documented before the procedure. These complications include bleeding, infection, pain, swelling, damage to adjacent structures like teeth, temporary or permanent damage to sensory and motor nerves, and the potential need for hospitalization or revision surgery. If an incisional biopsy is planned, future procedures may be required. If a graft is performed, there is a risk of infection or graft failure.

Clinical Significance

Pathology represents a sizeable portion of the head and neck surgical care workload. Working knowledge of soft and hard tissue biopsies, techniques, and considerations allows thoughtful and detailed planning to ensure predictably safe and optimal treatment.

Head and neck lesions can range from benign processes requiring observation and follow-up to malignant tumors requiring significant medical and surgical intervention. The initial evaluation and biopsy represent the starting point for treating any lesion. The practitioner must understand the rationale and thought processes behind a biopsy, fully considering the medical history, risk factors, patient age, presenting signs and symptoms, and radiographic features to guide the location and type of biopsy. The practitioner must also possess the skill set required to perform various biopsies, identify and manage potential complications, and either manage the disease process or refer the patient for continued care.

Enhancing Healthcare Team Outcomes

Successfully performing a biopsy of a lesion within the oral cavity can require a large amount of interdisciplinary care.[61] Ensuring optimal patient outcomes requires coordination within the clinical team, including office-based clinical staff, operating room staff, general or oral pathologists, and pharmacy staff. The use of specialized equipment or the need for a frozen section must be communicated clearly and in advance. 

Patients with final diagnoses that require further or more complicated treatment are best served by coordinating care between teams. Malignancies frequently require interdisciplinary care based on input from the institutional tumor board. Large lesions, even if benign, may require dental rehabilitation, grafting, implants, or prosthetics, and the restoring dentist or prosthodontist should be included early in treatment planning to collaborate on the final restorative plan.