In the context of a cutaneous lesion, incisional or excisional biopsies may be performed. Shave biopsies are generally not recommended as an accurate assessment of tumor thickness cannot be made using this technique. This is of particular importance with pigmented lesions since staging and subsequent therapy of malignant melanoma is dependent upon the depth of invasion (1). In addition, the dermatopathologist cannot differentiate between carcinoma in situ (CIS) and invasive carcinoma without evaluating the basement membrane—a feature that cannot be assessed on scrape biopsies.

The simplest and most efficient means of obtaining a skin sample is through the performance of a punch biopsy. A variety of skin punches are commercially available and typically range in size from 2 to 4 mm in diameter. These instruments are typically trephines attached to the end of a long plastic handle. The blade is inserted perpendicular to the skin and gently rotated through the epidermis and the dermis as to provide a full-thickness sample of the lesion in question. For all but the smallest lesions, punch biopsies are incisional rather than excisional. Thus a more definitive procedure will often be required once the diagnosis is confirmed. Alternatively, skin lesions may undergo incisional biopsy or, if small enough, be excised en bloc with a scalpel in the outpatient clinic. Care must be taken to orient
excisions within the relaxed skin tension lines within the face and neck.

When obtaining tissue from mucosal sites in the upper aerodigestive tract, through-cutting or cupped forceps are often employed. Using this method, a representative portion is grasped with the instrument and sharply divided or avulsed from the lesion in question. Though widely used, this forceps biopsy does have its drawbacks. The urologic literature suggests that this technique may result in inaccurate diagnosis due to limited sample size, distorted architecture, and crush artifact (2). In addition, the samples tend not to be full thickness. Consequently, the pathologist may not be able to differentiate in situ from invasive carcinoma. Conversely, incisional/excisional scalpel biopsies and punch biopsies allow for a more complete assessment of a mass, its interface with the surrounding tissue, and, in the context of malignancy, the depth of tumor invasion—a feature that has significant treatment implications for early-stage oral cavity tumors. Moreover, it has been demonstrated that when compared to other methods, oral biopsy samples obtained using a punch are significantly less prone to artifacts such as crushing, splitting, hemorrhage, and fracture—all features that might impede the pathologist’s ability to make a complete and accurate assessment (3). Given all of this information, it is the authors’ contention that punch biopsy be the first choice for mucosal biopsy in the upper aerodigestive tract and that the cup forceps should only be used when punch or scalpel biopsy is not technically feasible (i.e., due to inaccessibility because of the anatomic locale, trismus, or other factors).

Neck masses are among the most common reasons for referral to otolaryngologist-head and neck surgeons. An understanding of percutaneous biopsy techniques is essential to their management. The most common method for obtaining tissue diagnosis in the context of a neck mass remains the fine needle aspiration (FNA) biopsy. FNA has long been central to the diagnostic algorithm for thyroid nodules. It has also assumed a prominent role in the evaluation of enlarged cervical lymph nodes, salivary masses, and other subcutaneous tissues. FNA is relatively simple to perform in the outpatient setting, it is safe and well tolerated by most patients without the use of local anesthesia, and is highly accurate when used appropriately. Indeed, a recent meta-analysis by Tandon et al. (4) revealed that the aggregate sensitivity, specificity, positive, and negative predictive values across all head and neck subsites were 89.6%, 96.5%, 96.2%, and 90.3%, respectively. Despite its benefits and widespread use, there is no consensus as to the optimal technique for specimen acquisition and preparation. In addition, most otolaryngology programs do not formally train residents to perform FNA—oftentimes leaving them unaware of the potential pitfalls in performing the procedure. This has likely contributed to the high rates of specimen inadequacy that have been observed in some contexts (4,5,6,7).

Failure to obtain adequate tissue for cytologic analysis remains a vexing problem in clinical practice. Several studies have addressed this issue and the means by which the rates can be improved. The process begins with using the appropriate equipment. It is recommended that either a 23 or 25-gauge needle of adequate length be employed for FNA (8). In the authors’ experience, the 25-gauge needle is of sufficient caliber to obtain a specimen while at the same time minimizing patient discomfort. The authors use a modification of the technique described by Dusenberry (8): After decontaminating the skin with an alcohol wipe, the tip of the needle is passed into the center of the mass. A “series of short, staccato strokes” are performed while holding continuous suction on the plunger (among head and neck masses, active suction during aspiration appears to yield significantly higher rates of specimen adequacy when compared to needle strokes without suction (9)). The procedure is continued until material is observed within the hub of the needle. At this point, the aspiration should stop and the needle should be withdrawn from the skin. If a cystic lesion is encountered, it should be completely evacuated in order to reduce the risk of a falsenegative aspiration—particularly when nodal metastasis is in the differential diagnosis. Once the sample is collected, a smear is made of the aspirate and it is prepared for the pathologist. If lymphoma is suspected, additional material should be placed into a specialized transport material and submitted for flow cytometry.

If sufficient material is available, a cell block may also be prepared. A cell block is a means of preparing an FNA specimen that allows it to be processed, sectioned, and stained as if it were a histology section. Cell blocks are prepared from small tissue fragments, blood clots, and mucus within the FNA needle. This material is then compacted into a “block” for further processing. Cell blocks can be made manually, with albumin or agar, and with or without the aid of centrifugation. The cell block is a valuable tool in that it can supplement the information gathered through cytologic smears. Additionally, special stains (including immunohistochemistry [IHC]) can be performed on cell blocks (10).

After aspiration and smearing, slides are allowed to air dry or are placed into 95% ethanol for Papanicolaou staining. Air-dried slides may be evaluated on-site using Diff-Quik staining. The Diff-Quik stain may be performed within minutes of specimen procurement and allows for immediate examination by the cytopathologist. On-site evaluation is not simply for establishment of an “on the spot” diagnosis. Rather its value is in the determination of specimen adequacy. The importance of this practice cannot be understated. In essence, it provides the physician with immediate confirmation that an appropriate amount of the desired material was collected—thereby decreasing
the likelihood that the patient will be subject to the discomfort, distress, and expense of rebiopsy. A 2010 report by Moberly et al. supports this notion. They found that both the rates of specimen adequacy and the ability to obtain a definitive diagnosis by FNA were improved by immediate cytologic analysis (7). Given these findings, it is recommended that whenever feasible, a cytotechnologist or cytopathologist be present during performance of an FNA so that they may assess the specimen for adequacy.

The addition of image guidance also appears to have a significant effect upon the success of FNA. Early experience with CT and ultrasound-guided biopsy yielded favorable results—with these techniques being utilized to improve needle placement and diagnostic yield during FNA, even in the presence of palpable disease. Due to the reduced cost, lack of radiation exposure, improved sensitivity, and widespread availability of ultrasound, it has to a great extent supplanted CT for use in image-guided biopsy. Indeed, office-based ultrasound is now employed by a growing number of otolaryngologists and is particularly useful for assisting with needle placement. When compared to the traditional palpation method of needle placement, ultrasound-guided FNA has been shown to reduce the number of false-negative biopsies (11) and improve the diagnostic accuracy and adequacy rates by 23% and 84%, respectively (12). Surgeon-performed ultrasound-guided FNA appears to yield similar results to those achieved by interventional radiologists and cytopathologists. A randomized controlled trial conducted by Robitschek et al. (6) found that the number of inadequate specimens decreased by nearly 30% when ultrasound was utilized to perform FNA in the otolaryngology clinic.

While FNA yields cellular material suitable for cytologic characterization, the technique does not preserve tissue architecture—a feature that may reveal additional clues as to the definitive diagnosis. Core biopsies, on the other hand, provide a more substantial and intact piece of tissue that may be fixed and stained for review by a surgical pathologist. Core biopsies are generally performed using larger bore needles (18 or 20 gauge) than those used for FNA. There are a number of commercially available devices for core needle biopsy. These typically consist of a springloaded mechanism that deploys the needle once a trigger is depressed. The needle enters the specimen and is rapidly withdrawn, taking with it a core of tissue that may subsequently be fixed and processed by the histology laboratory. Alternatively, core needle biopsy may be performed using a standard needle and syringe, making a single swift pass into and out of the lesion in question. Given the size of the needle, local anesthetic is often injected into the overlaying skin in order to minimize discomfort. A small skin incision may also be made to facilitate needle entry without contamination by the overlaying epidermis.

Though core biopsy is common in non-head and neck sites, it has yet to gain widespread popularity for our purposes. This may be due to concern regarding bleeding and trauma to structures such as the facial nerve and great vessels as these complications have been reported (13). However, larger studies specifically addressing head and neck biopsies have demonstrated complication rates similar to those reported for FNA (14,15,16,17). Another concern regarding head and neck core biopsy is that it may have the potential to seed tumor cells along the needle tract— though this has not been shown to be the case (18).

Moreover, Saha et al. (19) demonstrated that ultrasound-guided core needle biopsy was significantly more accurate than palpation-guided FNA at detecting cervical metastases (97% vs. 85%) and that seeding was not an issue. Among their series of 49 core biopsies and 50 FNAs, the nondiagnostic rates were 2% and 42%, respectively. The data is somewhat misleading given the fact that FNA was performed without ultrasound guidance. However, this and other similar studies do suggest that in skilled hands and with image guidance, core needle biopsy is a safe and useful tool in the evaluation of head and neck masses. It has been promoted as a less traumatic, less time consuming, and more easily performed alternative to open biopsy in the context of a neck mass. As such, core biopsy may be best suited as in an intermediate step between FNA and surgical exploration in patients with nondiagnostic or indeterminate cytology (14,15).

In head and neck cancers (and in some benign diseases such as pleomorphic adenoma) margin status has long been known to affect subsequent treatment and ultimately prognosis. Both locoregional control and overall survival may be compromised in the context of grossly or microscopically positive margins. Using relatively nonstringent criteria for margin positivity (defined as frank carcinoma, CIS, or premalignant change at the margin), Loree and Strong (20) found that the incidence of
local recurrence increased twofold and that the survival rate was reduced by 13% in patients with positive surgical margins. The absolute distance from a margin is also of importance in head and neck cancers. Nason et al. (21) evaluated patients with margins that were 5 mm or more, 3 to 4 mm, 2 mm or less, and clearly involved. In their series of 277 patients with oral cavity carcinoma, there was a highly significant difference in overall and recurrence-free survival that varied with the size of the margin. After matching for stage and other confounding variables, patients with positive surgical margins had a 2.5-fold increase in risk of death at 5 years when compared to patients with negative but close (3-mm) margins. Patients with 3-mm margins had a 1.5-fold increase in risk of death compared to those with clear (greater than or equal to 5 mm).

Despite the findings of these and similar studies, there currently is no consensus in the literature as to what constitutes an adequate margin for head and neck squamous cell carcinomas (SCCs). Meier et al. surveyed members of the American Head and Neck Society in 2005. Of the 476 responses, the most common definition of a clear margin was greater than 5 mm. Though this was the most frequently used distance, it accounted for less than 50% of the total responses in the study—reflecting the significant diversity of definitions used throughout the literature and applied in clinical practice. This is further confounded by the fact that 14% of the participants considered CIS to be a negative margin while nearly 26% considered dysplasia to be a positive margin (22). Given the inconsistencies in the literature, it is imperative that individual cases be discussed at multidisciplinary tumor boards with consistent application of local standards and frequent re-evaluation of outcomes and practice patterns.

Regardless of what standard is chosen, an understanding of how the pathologist evaluates margins will ultimately assist in clinical decision making. When a specimen is received, it is initially fixed in buffered formalin. The tissue is then examined grossly and measured. It is then marked at critical points and presumed margins with one of a variety of commercially available inks or dyes. These inks withstand the fixation and slide preparation process and are visible to the pathologist on light microscopy. Under the microscope, the distance between disease and ink is measured by the pathologist and reported. This essentially is how margins are assessed. However, procedures for specimen inking vary from institution to institution and from pathologist to pathologist. When coupled with the complexity of head and neck anatomy and the fact that excisions often do not obey true anatomic boundaries, the subjective nature of the inking process can confound margin interpretation. This makes communication between the surgeon and the pathology team paramount.

Along these lines, it is vital that the surgeon provide an adequate clinical history and description of the procedure. The pathologist may not have a detailed understanding of otolaryngologic procedures. For example, surgeries such as partial laryngectomy and tongue base resection can be quite foreign to nonsurgeons and involve threedimensionally complex anatomy. The resulting specimens are often small and amorphous without any identifiable orienting landmarks or directionality. Large specimens such as composite resections or laryngopharyngectomies present another problem as it is not feasible to histopathologically examine the entire surface. In the absence of input from the surgeon, the pathologist must essentially make an educated guess as to what or where the true resection margin is in these cases. Hand delivering a specimen to the pathologist and orienting it for their team can help to minimize ambiguity in margin determination.

It may also be helpful for the surgeon to ink specimens himself or herself. This will ensure that the precise area of concern is being evaluated by the pathologist. Methylene blue is the author’s preferred ink for this purpose as it is readily available in most operating rooms and can be easily applied using a cotton-tipped applicator. It should be noted that methylene blue is water soluble—hence the site must be reinked once it reaches the histology laboratory to avoid being washed away during processing. Though little has been written about surgeon-initiated inking in otolaryngology, the breast surgery literature suggests that it is indeed a valuable tool. Numerous studies have implicated surgeon inking as a means of reducing discrepancies and ambiguity in the identification of tumor margins. They have also detailed the shortfalls of suture orientation and the benefits of well-orchestrated specimen handoffs to the pathology team in this regard (23,24,25).

The inadequacies of contemporary margin procurement and analysis for head and neck surgery have been elegantly discussed by Black et al. (26). Framing this issue in the larger context of quality improvement in medicine, the authors applied a business model—governed by what are commonly known as the “Toyota” principles—to the process. Under this model, processes and production are subdivided into multiple steps with the ultimate goal of reducing redundancy, waste, and error. Each step is carefully regulated and a strong emphasis is placed upon the quality of “handoffs”—the points at which a product is transferred to another team member for performance of the next step. Black’s paper highlights the fact that inconsistencies and discrepancies with respect to tumor margins can in large part be attributed to poor communication during handoffs. They contend that interdisciplinary communication must occur earlier in the overall process of margin determination—with the greatest benefit coming at the specimen orientation step. If the process fails at this step, then all subsequent steps and, eventually, the final pathology report, are jeopardized. With this in mind, it is again strongly advocated that the surgeon play an active role in specimen handoff and orientation.

Yet even when all of these measures are taken, we may still be left with a positive margin. However, surgeondirected orientation—either by intraoperative inking or through a face-to-face handoff to the pathology team— should make the task of re-excision or targeted radiotherapy less daunting.

Typically a head and neck resection is outlined on the tissue surface with a wide margin. However, it is not uncommon to see an assumedly appropriate cuff of normal tissue wither down to no more than a few millimeters once the specimen is excised. This margin may be even smaller by the time the pathologist reviews the slides and renders his or her report. While there may be multiple reasons for discrepancies between gross margins and those observed histologically, specimen shrinkage appears to play a major role. Most shrinkage appears to occur immediately after resection. It is thought to be the result of unopposed contractility in the submucosal musculature and release from surrounding supporting structures (27). In a 1996 study of canine oral mucosa, Johnson et al. demonstrated that specimen margins were reduced by 30% to 47% between the time of excision and the time of histologic review. The majority of shrinkage occurred after resection and before fixation (27). Subsequent to this, Mistry (28) presented a series of patients with SCC. In this report, 1 cm in situ margins were measured by the surgeon. Tumors were removed along this margin using monopolar cautery. The margin was subsequently remeasured on the specimen 30 minutes after resection. The overall mean margin shrinkage was 22.7%, with T1 and T2 tumors experiencing significantly greater rates of shrinkage than T3 and T4 tumors (25.6% vs. 9.2%). Similarly, Cheng et al. (29) demonstrated significantly increased shrinkage rates among T1 and T2 tumors as compared to T3 and T4 disease. This might have significant implications for adjuvant therapy as it is commonplace to use margin adequacy to guide adjuvant therapy—particularly in early-stage disease. Tumor location may also play a role in the degree of shrinkage, as the effects appear to be most marked in the buccal mucosa, mandibular alveolar ridge, and retromolar trigone (29). Knowing that significant shrinkage may be anticipated when resecting small tumors in these areas, the surgeon should—being mindful of the functional consequences—plan accordingly and design wider (i.e., greater than 1 to 2 cm) margins.

When discussing margin shrinkage, it is important to recall that prognostic statistics pertaining to margin status are based upon histopathologic data and not in situ or gross margins. Thus it is a fallacy to believe that because of shrinkage a close or positive margin may be disregarded as artifact in the context of a wide clinical margin.

The choice of surgical instruments may also influence the pathologist’s interpretation of margins. While “cold steel” has for generations been the standard for surgical incisions and excisions, a number of new technologies and instruments—such as the monopolar cautery pencil, the CO₂ laser, and the harmonic scalpel—have been introduced for this purpose. These have been variably touted for their improved hemostasis, reduced operative time, and positive effects on wound healing and postoperative pain (30,31). However, what is often lost in these discussions is the fact that each technique can impart artifactual changes to the tissues. As these artifacts can interfere with the interpretation of margins, it is helpful to inform the pathologist about which technique was used for the resection. Monopolar cautery can induce significant thermal injury and has been shown to induce a wider area of tissue damage when compared to CO₂ laser excision (31,32). Both monopolar and laser techniques result in vacuolar degeneration and nuclear elongation. The nuclei may appear pyknotic and distorted—features that can be confused with dysplasia or even overt carcinoma. If the laser is used, the thermal effects may be reduced using the pulsewave mode. Likewise, the harmonic scalpel results in fewer artifacts than monopolar cautery, but significantly more than cold steel technique. In comparing these methods, Kakarala et al. showed that harmonic scalpel resulted in significantly less tissue fragmentation and nuclear change than the monopolar. However, the scalpel was superior to both with respect to margin distortion (30). Though the histologic benefits must be weighed against other factors such as bleeding, it is recommended that any surface being evaluated as a resection margin be incised using cold sharp technique.

Cervical lymph node excision is one of the most common procedures performed by otolaryngologists. Whether this is in the context of a diagnostic biopsy or a neck dissection, the quality and quantity of information in the pathology report can be significantly affected by specimen handling in the operating room. This is perhaps no more evident than in the context of a suspected lymphoma. If a lymphoproliferative disorder is in the differential diagnosis, the lymph node (or Waldeyer ring tissue) should not be placed into a fixative solution. Instead, it should immediately be sent to the surgical pathology laboratory “fresh”—with the concern for lymphoma clearly indicated on the requisition. The fresh tissue should be hand delivered to the laboratory immediately as fixation, time, and heat will result in protein denaturation. If target antigens denature, the accuracy of flow cytometry may be limited. Furthermore, flow cytometry requires that specimens be separated into individual cells. This allows them to freely flow through the analyzer during characterization. The fixation process makes it difficult to separate the cells from one another— thus making flow cytometry an impossibility. It is important to remember that most flow laboratories only operate during business hours (i.e., Monday to Friday day shift). Specimens that are allowed to sit all weekend or even overnight may significantly degenerate and produce less than optimal results. Hence the surgeon must make the pathology team aware if such a biopsy is being performed at night or on weekends. Many hospitals have established “lymphoma protocols” whereby the specimen cup is explicitly labeled as a possible lymphoma. This initiates the abovementioned cascade of procedures in an attempt to systematically reduce misadventures in processing.

Neck dissection presents different challenges with regard to specimen handling. In the context of head and neck cancers, a great deal of prognostic information may be derived from histologic evaluation of the cervical lymph nodes. Data such as the number of involved nodes, their location (i.e., level), and the presence or absence of extracapsular extension are used to determine whether or not a patient would benefit from adjuvant therapy and, in some cases, what that therapy might consist of (35,36

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