Tumor Biopsy Principles and Techniques

The diagnosis of neoplastic and other pathologic conditions in animals depends on the procurement of an accurate biopsy specimen. Without an appropriate histologic diagnosis, it is impossible to plan appropriate therapy. Histopathologic results aid the clinician in providing an accurate prognosis and thereby guide the owner in the selection of various treatment options.

The ideal biopsy should procure enough tissue for specific pathologic diagnoses without jeopardizing the patient’s well being or the surgeon’s ability to achieve local tumor control. Many biopsy techniques can be used on any given mass. The procedure used is determined by 1) the clinician’s goals for the patient (i.e., diagnosis with no treatment versus diagnosis with treatment); 2) the skill and preference of the clinician; 3) the anatomic site of the mass; and 4) the general health status of the patient.1 Cytologic preparations obtained by fine needle aspirate are often helpful in guiding the selection of the optimal biopsy technique.

General Considerations

Biopsies can be obtained before the initiation of definitive therapy (pretreatment biopsy) or histologic specimens may be evaluated after the mass is removed in its entirety. In most situations, pretreatment biopsy is the optimum route of action because it provides a diagnosis before the institution of invasive or aggressive therapeutics.

Pretreatment biopsy is warranted when the type of treatment would be significantly altered by knowing the tumor type. For example, if an animal presents with a mediastinal mass, the distinction between a thymoma (responsive to surgery) and lymphoma (responsive to chemotherapy) would be important to make before instituting treatment.

If the extent of treatment would be altered by knowing the tumor type, pretreatment biopsy should be performed. Certain cancer types (e.g., mast cell tumors and soft tissue sarcomas) have high local recurrence rates and therefore require removal with wider margins than benign or lower grade malignant tumors. Many studies in both animals and human patients have shown that the best chance for surgical cure is to remove the lesion completely the first time. Clinicians who are tempted to “peel out” or “shell out” a lesion without knowing the histologic diagnosis are playing a dangerous game that may leave microscopic disease in the patient. If the lesion is malignant and incompletely excised, it will often grow back more quickly and invasively than the initial mass, thus potentially compromising further attempts at treatment.

Pretreatment biopsy should be considered when the tumor is in a difficult location for surgical reconstruction, such as a distal extremity, tail, or head and neck, or when the procedure could carry significant morbidity (e.g., maxillectomy or hemipelvectomy).

Finally, pretreatment biopsy is warranted when knowledge of the diagnosis would change the owner’s willingness to treat the disease. An owner may be more willing to allow the veterinary surgeon to perform a thoracic wall resection for a low grade soft tissue sarcoma (slow to metastasize) than for a high-grade osteosarcoma (metastasizes quickly).

In two situations, pretreatment biopsy is not indicated. The first is when knowledge of the tumor type would not change the surgical therapy. Examples of this are a splenectomy for a localized splenic mass or a lung lobectomy for a solitary lung mass. The second situation is when the biopsy procedure is as dangerous or as difficult as the definitive treatment (brain biopsy). In these cases, biopsy information is obtained after surgical removal of the lesion.

Soft Tissue Biopsy

Needle Core Biopsy

The most common use of the needle core biopsy is for externally palpable masses. This procedure can be done on an outpatient basis with local anesthesia and sedation. The method uses various types of needle core instruments (Tru-Cut [Tru-Cut biopsy needle, Travenol Laboratories, Inc., Deerfield, IL 60015] or A.B.C. Needles [A.B.C. Needles, Monoject, St. Louis, MO 63310]) to obtain a piece of tissue 1 to 2 mm in width and I to 1.5 cm long. The most commonly used size is a 14 gauge diameter needle; however, these needles are available in 16 and 18 gauge sizes as well. Any mass larger than 1 cm in diameter can be sampled using this instrument. These instruments can also be used for deep tissues, such as kidney, liver, and prostate, in a closed method or an open method at the time of surgery. Despite the small sample size, the pathologist is usually able to discern tissue architecture and tumor type. With experience, the clinician can usually tell whether representative samples have been obtained. Fibrous and necrotic tumors may not yield diagnostic tissue cores. If the clinician believes that representative samples have not been obtained, an incisional biopsy is indicated.

The area to undergo biopsy is clipped and prepared as for minor surgery. Sensation in overlying skin and muscle can be blocked using a local anesthetic along the area that the needle will penetrate. The mass is fixed in place with one hand, and a 1-mm stab incision is made in the overlying skin. The needle biopsy instrument is introduced through the stab incision, and several needle cores are removed from different sites in the tumor through the same skin hole (Figure 5-1). The tissue is then removed from the trough of the instrument with a hypodermic needle and is placed in formalin. Samples can be gently rolled on a glass slide for a cytologic preparation before fixation if desired. Skin sutures are usually not required. The biopsy tract, including the stab incision, should be removed at the time of definitive surgery.
 

Punch Biopsy

Another simple biopsy technique is the punch biopsy method (Figure 5-2). This technique uses Baker’s biopsy punch (Baker Cummons, Key Pharmaceuticals, Inc., Miami, FL 33169) instrument to obtain the specimen. The skin is prepared for minor surgery, and the overlying skin is anesthetized with a local anesthetic. Baker’s punch is applied to the mass in a manner that will yield a composite of normal and abnormal tissue. Pressure is applied as the instrument is twisted. The specimen is grasped and lifted with forceps while the operator uses scissors or a scalpel blade to cut the base. Care should be taken to not deform the tissue. Impression smears can be made for cytologic evaluation before placement in formalin. Multiple specimens may be taken from a single mass. A single skin suture per biopsy site is usually sufficient to close the defect and to control hemorrhage.

lncisional Biopsy

Incisional biopsy (Figure 5-3) is used when neither cytologic examination nor needle core biopsy yields a diagnosis. As mentioned, incisional biopsy is preferred for ulcerated or necrotic tissue because core biopsy rarely yields a diagnosis. Tumors are often poorly innervated, and as long as overlying skin is anesthetized, a wedge of tissue can often be removed without general anesthesia. Externally located tumors that are ulcerated may undergo biopsy without even the use of local anesthetics. The goal is to obtain a composite biopsy of abnormal tissue and adjacent normal tissue without compromising subsequent resection. The incisional biopsy tract always must be removed with a tumor at curative resection. Thus, the surgeon must not open uninvolved tissue planes that can become contaminated with tumor cells. In general, any normal tissue that the scalpel or surgical instruments have touched during an incisional biopsy is considered contaminated with tumor cells and is at risk for eventual tumor growth.

Endoscopic Biopsy

Endoscopic biopsy is used most commonly in the gastrointestinal, respiratory, and urogenital systems. It is convenient, safe, and cost effective; however, it has several limitations. Visualization may be inadequate, resulting in nonrepresentative biopsy samples. Full-thickness biopsy specimens are often impossible to acquire in these organs, and therefore, inflamed tissue or normal tissue overlying a tumor may undergo biopsy, not the tumor itself. A histopathologic diagnosis of inflammation in an animal suspected of having neoplasia should be interpreted with caution.

Laparoscopy and Thoracoscopic Biopsy

These techniques are best used when all staging and diagnostic procedures suggest inoperable and diffuse disease or when precise staging is indicated and an open procedure is not desired. Laparoscopic and thoracoscopic biopsy can yield important information regarding the extent of disease. Its disadvantages are that it can take as long as an exploratory laparotomy, it requires general anesthesia, and it does not give the clinician visualization as clear as that attained during open exploratory. In most cases, it cannot provide for excision. This procedure also carries some risk of hemorrhage and leakage of fluid from hollow organs and tumors. Animals staged by whatever means as having resectable disease are often best served by open exploratory laparotomy or thoracotomy, whereby resection with curative intent can be performed.¹

Image-Guided Biopsy

The use of fluoroscopy, computed tomography, and ultrasonography has greatly expanded the clinician’s ability to stage and diagnose neoplasia. Image guided biopsy may result in the avoidance of more invasive diagnostic procedures. A disadvantage of image-guided biopsy is that the technique requires specialized equipment and training. Biopsy in a closed space with limited visualization of the lesion carries some risk. As with laparoscopy and thoracoscopy, image guided biopsy is best done when the clinician is fairly certain that an excisional attempt would be unsuccessful or when pretreatment biopsy results would change the owners’ willingness to pursue more aggressive medical or surgical therapy.

Tissue Procurement and Fixation Guidelines

The concept that performing a biopsy releases tumor cells and leads to early metastasis and decreased survival has proved false. Although biopsy procedures do release tumor cells into the circulation, neoplastic cells are constantly shed into vessels and lymphatics on a day to day basis.¹ No evidence in either human patients or animals indicates that a properly performed biopsy leads to a decrease in survival or early metastases. On the other hand, a poorly planned or improperly executed biopsy can result in significant alterations in the optimum treatment plan.

Biopsies should be planned so the tract may subsequently be removed with the entire mass. The ideal circumstance is when the biopsy is performed by the surgeon who will eventually perform the curative intent procedure. Biopsies performed within a body cavity (either open or closed) should be done so tumor cells are not “spilled” into the cavity. This precaution prevents seeding of peritoneal or pleural cavities. The sample size of the specimen affects the accuracy of the diagnosis. Because tumors are not homogenous and often contain areas of necrosis and inflammation, larger samples or multiple samples from different areas in a mass are more likely to yield a diagnosis. The smaller the sample, the less representative it is of the whole tumor. Thus, if needle core biopsy specimens are obtained, several samples should be submitted. Biopsies should not be obtained with electrocautery because this technique will disturb and deform the tissue architecture. Likewise, the clinician should take care not to deform the sample with forceps, suction, or other handling methods. Cautery can be used after blade removal of a specimen to control hemostasis if necessary.

The junction of normal and abnormal tissue is frequently the best area for sampling. This aids the histopathologist in comparing normal and abnormal tissue architecture. It is important to plan the incision so the normal tissue incised during the biopsy can easily be removed and is not necessary for reconstruction of the surgical defect. (The exception to the tissue junction rule is bone biopsies, discussed later in this chapter.) Biopsies performed on the legs or the tail should be done using an incision parallel to the long axis of the structure. This technique aids in resection of the biopsy scar if needed.

Excisional specimens submitted for biopsy should be evaluated for surgical margins. The surgeon should mark any areas of question or submit a margin from the patient in a separate container. It is good practice to mark all excisional margins routinely with ink. The pathologist samples tissue from several areas of the specimen. If tumor cells extend to the inked margin microscopically, the excision should be considered incomplete (“dirty”). Lateral and deep margins of an excised mass can be painted with India ink and allowed to dry before placement in formalin. Commercially available colored inks can be used to denote different sites on the tumor if desired (Davidson Marking System, Bloomington, MN).

Ultimately, the surgeon has the responsibility to communicate to the pathologist what is expected when evaluating margins on an excisional sample. Of course, incisional biopsies, needle core biopsies, and punch biopsies have incomplete margins by definition. Pathologists may not know whether the sample is intended to be excisional and do not always evaluate margins unless asked. Good communication between the pathologist and the clinician is vital to the care of the patient. Waiting until recurrence of the tumor to reoperate on a known malignancy that has been incompletely resected is a disservice to the client and the animal. Incomplete surgical resection of malignant disease is best dealt with early so further surgery or adjuvant therapy can be instituted immediately.

Tissues should be fixed in 10% neutral buffered formalin in a ratio of I part specimen to 10 parts fixative. Proper fixation is vital for accurate pathologic diagnosis. Tissue thicker than 1 cm does not fix deeply. Large masses can be sliced like a bread loaf, leaving one edge intact to allow for orientation. Alternatively, representative samples from the tumors can be sent while the larger portion of tumor is saved in formalin and further sections submitted if the pathologic diagnosis is in question. It is possible, especially in some large splenic masses, for only a small portion of the mass to be neoplastic and for the rest to consist of hematoma, necrosis, or fluid. This possibility emphasizes the need to submit several representative samples or, when possible, the entire mass. Tissue that is prefixed over 2 to 3 days in formalin can be mailed with a tissue - to - formalin ratio of 1:1.

For the pathologist to provide the most accurate diagnosis, each sample must be accompanied by a complete history. Whenever the histopathologic diagnosis does not concur with the history, clinical signs, or clinician’s impression, a call to the pathologist is warranted. In some cases, a small but vital piece of information left out of the patient’s history can drastically change the pathologist’s impressions. Pathology is a combination of art and science, and diagnoses are only as accurate as the information provided by the clinician.

A veterinary trained pathologist is always preferable to a pathologist trained in human disease. Although similarities exist across species lines, there are enough histologic differences to result in interpretive errors.

Frozen Sections

Frozen sections are becoming more common in the perioperative setting in veterinary medicine. This process provides a rapid means to a diagnosis at the time of surgery, as well as information on adequacy of tumor resection and the presence or absence of metastases. Although the use of this technique in veterinary medicine is limited to those institutions with specialized personnel and equipment, it is of potentially great value to the surgeon. Accuracy rates are high (93%) when results are compared with those from traditional paraffin embedded tissues.²

Bone Biopsy

Bone biopsy is essential in the diagnosis of proliferative and lytic bone lesions. Results of a bone biopsy often determine the course of treatment and may drastically change proposed operative intervention. As with all biopsies, the clinician must plan the biopsy with the intended curative treatment in mind. The most common instruments used for bone biopsies are the Michelle trephine (Michelle trephine, Kirschner Co., Timonium, MD) and the Jamshidi type bone marrow biopsy needle (Jamshidi bone marrow/aspirate needle, American Pharmaseal, Valencia, CA 91335; Bone marrow biopsy needle, Sherwood Medical, St. Louis, MO 63130). When used properly, both instruments provide a suitable sample with minimal complications. The small size of the Jamshidi biopsy needle cannula is advantageous in that it requires a smaller skin approach (1-mm stab incision) and leaves a small diameter bone defect, making biopsy related fractures less likely than with a trephine. Trauma to soft tissue structures and hemorrhage are minimal with the Jamshidi method.

Jamshidi needles are available in single use and reusable models.3 The reusable model is “self sharpening” and steam sterilizable. In our experience, the single use model may be reused 10 to 15 times after gas sterilization. Jamshidi type needles are available in various sizes, but the 8 and 11 gauge needles (4 inches long), are most commonly used. A Jamshidi-type needle features a pointed stylet that facilitates passage through the soft tissues (Figure 5-4). The stylet is secured by a screw on cap. The tip of the cannula is tapered, allowing the specimen to be locked into the cannula. This tapering eliminates the rocking motion necessary to break off and retrieve a tissue specimen when using a trephine. A small probe is also provided to assist in removing the specimen from the needle. The specimen must be pushed out the handle because damage and compression distortion of the specimen will occur if it is pushed out the tapered cannula tip.
 

Indications and Preoperative Considerations

Bone biopsies are most often performed to confirm the presence of a neoplasm suspected on radiographic and clinical evaluation. Primary malignant tumors of bone in dogs include osteosarcoma, chondrosarcoma, fibrosarcoma, and hemangiosarcoma. Plasma cells, myeloma, and other round cell tumors can also originate from bone. Metastatic spread to bone from other primary tumors must also be considered. Metastasis to bone can occur with almost any type of tumor. The clinical and radiographic signs of primary and metastatic bone tumors can be similar; they include lameness of the affected limb, a warm swelling that is sensitive when palpated, and lytic and proliferative changes, which are apparent on radiographs. Other conditions that can mimic bone tumors include fungal and bacterial osteomyelitis. Dogs with fungal infection have generally traveled in fungus-endemic areas. Dogs with bacterial osteomyelitis usually have intermittent drainage from the lesion and a history of penetrating trauma or previous surgery.

Although history, clinical signs, and radiographic changes can aid in making a presumptive diagnosis, the definitive diagnosis of bone lesions can be obtained only through histologic evaluation of a tissue specimen. Radiographic evaluation before biopsy should include two different views (craniocaudal and lateral) of the lesion. As previously mentioned, biopsies are traditionally obtained at the junction of tumor and normal tissue. However, in bone, the center of neoplastic lesions is most likely to yield diagnostic material.⁴ Bones surrounding almost any insult, including trauma, infection, and tumor, can become reactive. Although biopsy specimens obtained at the center of bone tumors often contain considerable necrotic tissue, tumor identification is not impeded.⁴ Inadequate sampling may result in a report of reactive bone. In these cases, the clinician should consider rebiopsy, especially if the diagnosis of reactive bone does not fit the clinical picture. The center of the lesion can be measured on the radiograph with reference to a nearby landmark, generally the adjacent joint. The radiograph should be in view and a sterile ruler available at the time of biopsy.

The skin incision and route of the biopsy needle should be made with subsequent surgical procedures in mind (i.e., limb sparing operations). Questions of preferred location of biopsy are best directed to the referral institution that would perform the definitive surgery. In any case, a joint should never be entered and dissection through the planes or neurovascular bundles should be avoided. If evidence points toward primary bone tumor and if the clients are interested in pursuing limb sparing surgery, referral for biopsy may be the best alternative. General anesthesia is usually necessary for bone biopsy. Selection of the anesthetic regimen depends on the general condition of the animal, on personal preference, and on experience. Because many of these patients are geriatric, complete blood count, serum biochemistry, and urinalysis are indicated. In some cases, particularly in animals with a lytic lesion, heavy sedation and local anesthesia may suffice.

Surgical Technique

The surgical site should be aseptically prepared and routinely draped. Adhesive drapes covering the biopsy site offer excellent protection allowing palpation and manipulation of the limb. A 1 - to - 2 mm stab incision in the skin is made at the desired location. The Jamshidi cannula, with the stylet locked in place, is gently pushed through the soft tissue structures. When bone is reached, the location of the cannuta should be evaluated using the radiographs as reference (Figure 5-5). The cannula can be shifted to a different location if desired. The stylet is removed. With a gentle twisting motion and the application of firm pressure, the cortex is penetrated. The cannula is advanced through the medullary cavity, taking care to avoid penetrating the opposite cortex (Figure 5-6). After the instrument is removed, the specimen is pushed from the tip out through the base of the cannula with the probe, not with the stylet (Figure 5-7). The procedure is repeated, following the soft tissue tract previously established. The instrument can be angled in different positions after reaching the bone. Two or three specimens should be obtained. If the center of the lesion is so soft that a core of tissue cannot be obtained, the cannula should be directed toward the peripheral aspect of the lesion. Hemostasis is generally not a problem with this technique; however, if bleeding occurs, direct pressure is sufficient to control it. The Jamshidi instrument bends if excessive pressure is applied. 

Damage to the cannula and stylet can occur during biopsy of normal cortical bone or of an extremely proliferative and organized bony lesion. If the cannula cannot be inserted, its position should be reevaluated to ensure that the cannula is not on adjacent normal bone. If the position appears correct, a trephine may be indicated to obtain an adequate sample. A skin suture may be placed after the procedure. For biopsies of the lower extremities, a soft wrap may be applied.

Biopsy specimens should be placed in a 10% neutral buffered formalin solution as soon as possible to prevent desiccation. Specimens can also be placed in culture medium if desired. Samples should be sent to a pathologist and laboratory experi- enced in evaluating and processing bone specimens.
 

Nasal Biopsy

A nasal biopsy requires that the animal be anesthetized, with an endotracheal tube inserted. The cuff of the endotracheal tube should be inflated and checked periodically to prevent aspiration of blood during the procedure. Several procedures have been used to procure nasal biopsies. In our experience, the easiest and most successful procedure in dogs is the use of a rigid plastic tube, such as the outer sleeve of a Sovereign catheter (Sovereign indwelling catheter, Monoject, Division of Sherwood Medical, St. Louis, MO) or spinal needle.⁵ The actual catheter portion is discarded, and the metal stylet is cut off at the hub using bandage scissors. The catheter sleeve is slid over the remaining hub, and a 12-mL syringe is attached. The location of the tumor is visualized on radiographs, and the plastic sleeve is measured from the medial canthus of the eye to the tip of the nose. The sleeve can be marked or cut off so the clinician does not introduce the biopsy device further than this distance. This technique prevents disruption of the cribriform plate and invasion of the brain. The tube is introduced past the wing of the nostril using gentle pressure. It is then reamed in and out of the tumor repeatedly while suction is applied to the syringe. Hemorrhage is common but usually self-limiting and should not deter the clinician from being aggressive. The device is withdrawn from the nose, and the syringe is removed and filled with air. The specimen is then forced out by flushing the air through the tube using the syringe. Samples should be placed on a gauze sponge to allow blood to drain away. Tumor tissue is usually white to tan, although it may be hemorrhagic and mucoid. All tissues are placed in 10% buffered formalin for evaluation. Smaller pieces can be placed on filter paper before placement in formalin to preserve architecture.

In cats, smaller dogs, and brachiocephalic breeds, a curette can be used followed by flushing the nose with saline. Care is taken to properly inflate the endotracheal tube cuff to prevent aspiration. The instrument should not be introduced further than the distance from the tip of the nose to the medial canthus. It is helpful to mark the instrument with a piece of tape at this distance. Sponges should be placed above the soft palate and at the external nares to catch bits of tissue. The curette is then introduced into the nasal cavity and a scooping action is used to dislodge tumor fragments. Cool saline is used to flush out specimen pieces using a pulsing action. All tissue is submitted for histopathologic evaluation.

Mild hemorrhage is noted for several hours after the biopsy. Sneezing after the biopsy can aggravate this hemorrhage. Patients should undergo recovery in a quiet area with supervision and should be kept for several hours or overnight after anesthetic recovery. These techniques are safe, they have minimal morbidity when compared to open biopsies, and they yield excellent specimens.⁵

Interpretation of Results

The biopsy should be reviewed with respect to other data concerning the patient, such as clinical signs, history, and physical examination. A clinician should expect to receive the following information in a biopsy report: a determination of neoplasia versus no neoplasia; a diagnosis of benign versus malignant; a histologic type; grade of tumor if applicable; and margins if excisional. Interpretive errors can occur at any level of diagnosis. An estimated 10% of biopsy results may have some clinically significant inaccuracy. If the biopsy result is inconclusive or is inconsistent with the clinical findings, one of several actions should be taken. At the very least, the pathologist should be called and the concern expressed. This exchange should be looked on as welcome and helpful for both parties, not as an affront to the pathologist’s expertise. In many cases, added information may lead to resectioning of the available paraffin tissue block, use of special stains for certain tumors, or a second opinion. Rebiopsy is also a possibility if the tumor is still present in the patient.

A properly performed biopsy and interpretation are the most important steps in the management of the cancer patient. The decision to submit a tissue specimen for histopathologic examination should not be left to the owner. If necessary, the charge for submission and interpretation of the biopsy should be included in the surgery fee. Mass excision without interpretation is no longer considered the standard of care. Because of increasing legal concerns, much more is at stake than the satisfaction of medical curiosity.

References

1. Withrow SJ, MacEwen EC. Small animal clinical oncology. 2nd ed. Philadelphia: WB Saunders, 1996.
2. Whitebait JG, Griffey SM, Olander HJ, et al. The accuracy of intraoperative diagnoses based on examination of frozen sections: a prospective comparison with paraffin embedded sections. Vet Surg 1993;22:255 259.
3. Jamshidi K, Swain WR. Bone marrow biopsy with unaltered architecture: a new biopsy device. J Lab Clin Med 1971;77:335.
4. Wykes PM, Withrow SJ, Powers BE, et al. Closed biopsy for diagnoses of long bone tumors: accuracy and results. J Am Anim Hosp Assoc 1985;21:489.
5. Withrow SJ, Susaneck SJ, Macy DW, et al. Aspiration and punch biopsy techniques for nasal tumors. J Am Anim Hosp Assoc 1985;21:55 1.