Head and Neck Pathology: Practical Points to Ponder

Abstract

As anatomical pathologists seeking to serve clinicians and their patients, we aim to help clinicians to send us the specimens that allow us to do our job well. We have hints about taking, orientating and sending pathology specimens to the laboratory. We explain transport media and give guidance on fine needle aspiration, frozen sections, biopsies and resections. We discuss the ancillary techniques like special stains, immunohistochemistry and molecular studies that distinguish modern pathology from its historical roots. We explain the elements of pathological reports, hopefully demystifying tumour thickness, depth of invasion, pattern of invasion, perineural spread and the pathology of surgical margins.

FormalPara Core Messages

• Pathologists need information from clinicians and radiologists.

• Taking better specimens leads to better feedback to clinicians.

• Preparing the optimum specimen for your differential diagnosis.

• Being aware of all the testing modalities at the pathologists’ disposal.

• Forming a symbiotic relationship with your pathologist.

1 Introduction

Anatomical pathology is the branch of medicine that deals with the examination of cells, tissues and organs, mainly for diagnostic purposes. It is a self-standing medical discipline, and its practitioners require specialist knowledge and skills. It best serves the needs of patients if it is conducted not in isolation but in harmony with the clinicians who are in direct patient contact [1]. The aim of our chapter is not to review the pathology of malignancies of the head and neck but rather to give the pathologists’ perspectives of how best to achieve that harmony. We shall cover some practical information about what clinicians can do to enhance the subsequent contribution that pathologists can make to the team, and we shall cover how best to interpret the pathological report. Particularly in the field of head and neck pathology, providing appropriate clinical and radiological information, and the best possible specimens, massively assists what the pathologist can do to ensure the best outcome for the patient.

The specimens submitted by clinicians managing head and neck pathology most frequently are biopsy specimens. For the pathology laboratory to perform all the relevant testing and analysis required for the most accurate diagnosis, the most suitable specimen needs to be submitted, in the most appropriate transport medium. Pathologists have little leeway if the specimen is unavoidably small, and non-ideal handling of a small specimen before it reaches the laboratory can have irrecoverable consequences. So we will offer the pathologists’ perspectives on specimen collection techniques and transport media.

Once a specimen has been received and processed through the laboratory, samples are examined by pathology technicians and pathologists. The main tool of the anatomical pathologist remains the optical microscope, but today pathologists have access to a large range of special stains. Immunohistochemical stains and other molecular studies have vastly improved their diagnostic and prognostic capabilities, as well as their contribution to treatment planning. We shall offer a brief overview of some of these ancillary procedures.

If a cancer is identified in the specimen, the pathologist usually will submit a synoptic report that ensures that vital information about each cancer is recorded. Some of the important prognostic factors from these synoptic reports are tumour thickness and depth of invasion, pattern of invasion, perineural invasion and surgical margins, and we shall discuss how those factors are determined.

Not just at the report stage but throughout the process, we urge clinicians in direct contact with patients to interact with their pathologists. Discussions between pathologists and clinicians can help resolve a challenging diagnosis and encourage pathologists to review cases where initial pathological interpretations do not reconcile with the clinical picture. Also, when you really are in a bind and need something special, like rapid turnaround time, a pathologist, who you have established as an ally, can move mountains.

2 Obtaining a Specimen

We offer pathologists’ views on the procedures that clinicians need to undertake to obtain and submit the best specimens. Practices in the hands of clinicians determine the capacity of pathologists to derive information from the specimen and consequently the value of the report submitted back to the clinicians. The procedures that we shall discuss are fine needle aspiration (FNA), biopsy for frozen section and biopsy for routine histology.

3 Fine Needle Aspiration

Fine needle aspiration, which can be freehand or ultrasound guided, trumps other procedures with regard to rapid diagnostic capability. When used in the appropriate setting, a fine needle aspirate may provide material for a quick, sometimes immediate diagnosis as well as material for ancillary testing. The procedure is technically simple, usually has few side effects, and is a great addition to the diagnostic armoury of the surgeon/pathologist team. Cellular tumours, lymph nodes, metastatic skin lesions, salivary tumours and thyroid lumps are examples of lesions that are good candidates for cytology. Certain other lesions, for example, fibrotic lesions, small ill-defined lesions, exclusively cystic lesions and stromal tumours, may not be amenable to, or easily diagnosed by, FNA.

Here is how to get the most from an FNA.

3.1 Sample Procurement

Whilst fine needle aspiration is a simple technique, there is an art to taking a good sample and preparing a beautiful slide.

• 1. Have all the necessary equipment, such as needles, clean syringes, labelled glass slides and transport medium, at hand before starting the procedure. This preparation reduces the time between aspiration of the material and its transfer to the slide. The longer the lag time, the greater the risk of creating a drying artefact (see Fig. 5) or allowing the material to clot in the needle, preventing expulsion.

• 2. Positioning the patient is equally important, especially if the fine needle aspirate is to be done freehand. Aim to achieve good access to the lesion and easy immobilisation of the lesion with your non-aspirating hand. Improving positioning can be as simple as tilting the chin or hyperextending the neck, the latter a useful technique for thyroid FNAs.

• 3. There is no universal agreement on the right needle gauge. As a general guideline, larger bore needles do not always yield better results. Avoid large needles such as 1.27 mm outer diameter (18 gauge) needles because they can promote bleeding, leading to blood clot obscuring target cells on the slide. Our preference is either 0.64 mm (23 gauge) or 0.52 mm (25 gauge) and a 10 mL syringe.

• 4. Tissue procurement can be done with or without suction. Both techniques have advantages and disadvantages. Suction may produce more tissue volume but at the risk of generating more bleeding. Our recommendation is to stop suction if there is bleeding at the site of sampling.

• 5. It is the fine backward and forward motion of the needle bevel that cuts loose target cells. The repeated forward motion forces the loose cells to move up the needle shaft, even without suction. FNA expert Susan Rollins suggests that the ideal needle oscillation is three per second with an intralesional dwell time of between 3 and 5 seconds [2].

3.2 Smear Preparation

If you are making the smear yourself, using a reliable technique will ensure that the pathologist can use the material to your mutual best advantage. Whilst there are many possible techniques, we shall describe one [3].

• 1. Immediately after aspiration has been completed, remove the needle from the syringe, and pull back on the plunger to provide sufficient force to express the material onto the slide (Fig. 1).

  Pitfall: Failure to disconnect the syringe barrel from the needle may pull all the aspirated material back into the syringe, making transfer of the specimen onto the slide difficult or impossible.

• 2. Re-attach the needle, place the needle onto the upper third of the glass slide, and depress the plunger of the syringe to express the material (Fig. 2).

  Pitfall: If your needle is held too far above the slide, the material will splatter. Worse than splatter is the dart-like needle projectile after excessive force is applied to express material clotted in the hub.

• 3. Invert a second slide over the first so that the material spreads from the weight of the slide only (Fig. 3).

  Pitfall: Additional pressure creates the risk of introducing crush artefact (see Fig. 6).

• 4. Pull the slides apart with a gentle horizontal motion, creating two slides for every needle pass, facilitating Pap and Giemsa stains (Fig. 4).

• 5. Air-dry one of the slides with the assistance of a fan or hair dryer if necessary, and wet fix the other slide immediately using 95% alcohol or spray fixative.

Fig. 1

Remove the needle containing the aspirated material from the syringe and then pull the plunger back

Fig. 2

Express the material onto the upper third of the slide

Fig. 3

Invert a second slide over the first, so that the material spreads from the weight of the slide only

Fig. 4

Gently pull the slides apart completely using a horizontal motion

3.3 Papanicolaou Stain vs Giemsa/Diff-Quik Stain

The stains usually employed for histopathological examination of FNA smears are the Papanicolaou stain, for nuclear detail, and the Romanowsky-type stains (Giemsa/Diff-Quik), for cytoplasmic detail (Table 1). Some pathologists may have a personal preference for one of these stains, but they highlight different cellular features [4]. The reason that we advised that one FNA smear be air-dried and the other wet-fixed is the Romanowsky-type stains require an air-dried smear and the Papanicolaou stain a wet-fixed smear. Providing both smears allows the pathologist to use both stains and the best opportunity to assess all aspects of the specimen.

Table 1 What pathologists can do with Romanowsky-type and Papanicolaou stains of FNA smears

3.4 Artefacts: What Can Go Wrong in FNA Smears and How to Avoid It

3.4.1 Drying Artefact

The thyroid follicle cells show artefactual degenerative changes (Fig. 5). The nuclei are pale and have lost the nuclear detail, making assessment for lesions like papillary carcinoma challenging for the pathologist.

Fig. 5

Thyroid aspirate (Giemsa stain 200× magnification)

Problem: The material has dried or has been fixed too slowly.

Solution: Dry with a fan or hair dryer or fix material immediately after smearing.

3.4.2 Crush Artefact

Problem: Too much force was used whilst smearing the slide (Fig. 6).

Fig. 6

Lymph node aspirate for metastatic Merkel cell carcinoma (Giemsa stain 200× magnification). In this slide the nuclei have been crushed leaving long blue strands of nuclear material (yellow arrow)

Solution: Use no more force than the weight of the top slide when preparing a smear.

3.4.3 Too Thick

This aspirate from the lymph node is so thick that individual cell characteristics have been lost and it is difficult for the microscope to focus on the group of cells (Fig. 7).

Fig. 7

Lymph node aspirate (Papanicolaou stain 200× magnification)

Problem: Too much material in a localised area means individual cells cannot be seen.

Solution: Limit the amount of material and smear evenly. If you aspirate abundant material, it is better to prepare more slides than put a large drop on one slide.

3.5 Preparing FNA Specimens for Ancillary Studies

Harvesting as much of the sample as possible allows for smears to be supplemented with additional procedures (see “Ancillary histological studies”). The remaining material can be placed in liquid medium after smears have been prepared or primarily if a designated FNA pass is performed for this purpose. A small amount of the liquid should be drawn into the syringe and then expelled back into the medium container. This process may be repeated several times. In this way, a sample may be provided for flow cytometry, microbiology, a cell block, immunocytochemistry or molecular studies [5]. Whilst specific media are ideal, if they are not available, saline may be used, as long as the specimen is transported quickly to the laboratory. Because processing in the laboratory needs to be immediate if the specimen is in saline, let your pathologist know it is on its way. Try to avoid sending FNA specimens in saline late in the day, or before a weekend, because of the risk of deterioration of valuable cellular material.

4 Frozen Sections

Frozen sections are used when pathological analysis is required immediately, during surgery with the patient remaining anaesthetized. A specimen taken from the patient is frozen, and a thin section is cut on a cryostat machine (Fig. 8), which is a microtome with a freezing stage. The thin section is placed on a glass slide where it melts and is dried, stained and examined under the microscope. Pathologists can report on a histological examination by frozen section within 15–20 min of receiving the specimen.

Fig. 8

Leica™ CM1860UV cryostat machine, used for cutting frozen sections either in theatre or in the laboratory

Frozen section histological analyses usually are performed for one of three reasons [6]:

• 1. To guide intra-operative management, e.g. examination of margins of excisions.

• 2. For triaging of tissue for later special studies.

• 3. To confirm that lesional tissue is present in the specimen.

Specimens for frozen sections need to be provided fresh (not in formalin or other fixative) and should be accompanied by patient details including relevant clinical history and information about the tissue site and the reason for requesting frozen section. If the clinician is aware of, or suspects, an infection like tuberculosis, hepatitis B or HIV, this information also should be communicated to the pathologist so that the necessary protective and cleaning protocols can be instituted. Orientation of the specimen is vital, and a discussion with the pathologist about which area on the specimen should be sampled, and what information is being sought, will improve the information the pathologist will be able to provide.

Frozen section histological examination is not a rapid substitute for what the pathologist can do with a permanent section. Table 2 summarises the differences.

Table 2 What pathologists can do with frozen section and permanent section histology

Requesting a frozen section in theatre because it would be “nice to know” the diagnosis but when knowing the diagnosis immediately actually does not impact on patient management is a waste of resources. Frozen sections are labour intensive and time-consuming. They require committed and concurrent time (often including travel time) from a pathologist and from a skilled technician. There are some frozen sections that would be deemed inappropriate, and even detrimental to the patient, including small melanocytic lesions where freezing the entire lesion may remove the possibility of better histological examination by permanent section.

Frozen section histology has a sensitivity of 89% and a specificity of 99% for the evaluation of margin status for head and neck squamous carcinomas [7]. The specificity and sensitivity of frozen section histology for some other lesions, like follicular lesions of the thyroid, are much lower, and frozen section histology then is inappropriate. Even in ideal circumstances, a discordance rate between the frozen section histology and permanent section histology of 3% has been reported [7].

Discrepancies between frozen section histology and permanent section histology may be due to sampling error or to interpretation error. A sampling error may occur, for example, if tumour tissue is not in the section that was stained at the time of the frozen section but is present on the deeper levels that can be examined in permanent sections. An interpretation error may occur, for example, if a pathologist, faced with the poorer resolution of a frozen section, interprets tissue on the slide as being tumour when it is not or interprets tumour tissue as benign. Interpretation errors can be reduced if pathologists specialise in an organ system and develop expertise, just as surgeons do.

5 Biopsy/Excision or Incision

The majority of the work undertaken by pathologists is excisional or incisional biopsies. Routine management of biopsy samples usually at least entails overnight processing. It is sometimes possible to accelerate the management, for example, with small samples that do not require lengthy fixation. Accelerating the management is labour intensive and requires the technical staff to bypass usual processes, so is best reserved for the exceptional case where an urgent result is of the utmost importance to the patient. Accelerated management also requires communication with the pathologist so that every step of the process can be streamlined. Depending on the individual laboratory, it is possible to have slides prepared for the pathologist to examine within 3 1/2 h of a specimen arriving in the laboratory.

5.1 Core Needle Biopsy

Core needle biopsy usually is performed with a larger-gauge needle, ranging from outer diameter of 2.1 mm (14 gauge) to 0.91 mm (20 gauge), than is FNA [8]. The larger diameter of the core biopsy in theory translates to a larger sample of target cells acquired and often with intact tumour architecture. We cannot over-emphasise the value of preserved tumour architecture in the slides used for cancer diagnosis. Indeed, tumour architecture preservation is the main advantage that core needle biopsy (or excisional biopsy for the same matter) has over fine needle aspiration. That said, core needle biopsy (and excisional biopsy) is not without flaws. The advantages and disadvantages of core needle biopsy and FNA are outlined in Table 3.

Table 3 Advantages and disadvantages, for the pathologist, of core needle biopsies and FNAs

5.2 The Complex Resection Specimen

There is no doubt that extreme facial surgery results in large complex histology specimens. Whilst we have general guidelines for the management of such samples when they get to the laboratory, pathologists benefit, and the outcomes for clinicians and patients are better, from attention to multiple steps before that, some of which begin before surgery has been completed.

5.2.1 Submitting Pathology Specimens

The requirement for correct patient identification, identification of the requesting surgeon/ clinician and date of the procedure goes without saying, and these details invariably are provided. Adequate clinical history, sometimes provided less reliably, is of equal importance and has a direct impact on the ability of the pathologist to interpret the pathological features meaningfully. Whilst the vote of confidence is flattering, pathologists cannot accurately derive that clinical information from information on glass slides alone [9].

Clinical information beneficial to pathologists includes relevant prior diagnoses and, if histology has been carried out previously, a copy of the original report, or at least an indication as to which laboratory performed that earlier histology. Tell your pathologist the type of specimen and what you are trying to achieve by the procedure. For instance, does the specimen represent a debulking procedure or an incisional biopsy, or is it an attempt at a complete excision. The former requires little attention from the pathologist with respect to margin status, but establishing margin status is imperative in the latter. The locations and nature of some lesions that are clearly evident in vivo may become less obvious after excision and cessation of blood flow (e.g. vascular lesions and cystic lesions). Small lesions may be difficult for the pathologist to pinpoint, and specimens from lesions treated with neo-adjuvant therapy may no longer be grossly identifiable [7].

If there is a specific request, or a specific question that needs to be answered, include that request/question on the form that accompanies the specimen. Pathologists will be able to respond better if they know about special needs before the specimen is processed as they may alter markedly the way in which a specimen is processed. For example, if the question is about clearance of a specific margin, the specimen needs to be sectioned to display that margin well. If the question is whether an infection is present, a sample of non-fixed tissue may be submitted for culture and sensitivity. If it is whether a lymphoma is present, fresh tissue can be sent for flow cytometry.

5.2.2 Orientation of Pathology Specimens

Preserving the three-dimensional orientation of tissue after resection can be difficult. When complex anatomy and formalin fixation compound the issue, the orientation of the specimen that arrives in the laboratory may be even more obscure. To assist the pathologist, please consider the following:

• 1. Mark the designated borders/points of orientation with sutures, clips or ink during or immediately after resection [6]. Sutures of variable composition, length or number may be used. Sutures and clips also are useful to mark the resection margins of pertinent nerves or to identify a sentinel point, for example, 12 o’clock using a clock face template [10] for orientating the specimen (Fig. 9).

• 2. Pinning the specimen to a cork board (Fig. 10) has multiple benefits. The tissue is fixed in a non-rotated plane which is easier to section. Tissue shrinkage and tissue curling at the edges are reduced by pinning. Pins of different colours (e.g. indicating different anatomic locations) may be used to attach the tissue to the board and can be referenced in a specimen diagram (see Fig. 12).

• 3. Alternatively, the clinician may wish to define the orientation of the specimen by writing or drawing directly on the cork board. If so, the specific pen and ink need to withstand exposure to formalin. Some ink washes away almost entirely in formalin or smudges so badly that the writing/drawing becomes illegible. The images in Fig. 11 show the consequences for cork board drawings with different inks of being submerged in formalin overnight. This test is an easy one for clinicians to replicate when selecting a pen for this purpose.

  Once the specimen is attached, the cork board should be inverted in the specimen container to ensure that the tissue is submerged in formalin.

• 4. Drawing a diagram (Fig. 12) is an excellent way of orientating a specimen. It gives the pathologist a clear indication of the surgeon’s view of the sample and provides a platform for specific landmarks and margins to be identified. Diagrams also are convenient for identifying specific issues regarding the specimen for the pathologist’s attention, for example, identifying a margin of concern.

• 5. Inking of the specimen to delineate margins or other landmarks usually takes place in the pathology laboratory. However, it also can be a valuable tool for the surgeon [11]. The surgeon may apply ink to orientate the specimen, to indicate a specific margin of concern or, conversely, to identify an area that does not represent a true surgical margin (Fig. 13). The ink can be applied with a cotton swab, patted dry and then dabbed with acetic acid (vinegar) as a mordant that combines with the ink to fix it to the material.

Fig. 9

Orientation of a specimen in situ using the clock face template. Orientation of a specimen is important especially if there is a positive margin on histology and the lesion requires re-excision

Fig. 10

A neck dissection specimen, including a level III cystic mass, pinned on a cork board and orientated with coloured pins

Fig. 11

(a) Drawings made with four different marking pens on cork boards (BIC vivid™, Premier™ ballpoint, Artline 210™ medium, Sharpie finepoint™ from left to right). The cork boards were then submerged in 10% neutral buffered formalin for 24 h. (b) The same drawings after 24 h. Some pens resist formalin but are more difficult to use. Use a convenient pen in theatre but test its formalin resistance in advance

Fig. 12

Example of a drawing for which your pathologist would be very grateful

Fig. 13

(a) A range of ink colours (Aero Color Professional finest acrylic ink™) is available and can be used to orientate specimens. The ink can be applied with cotton swabs. Be sure to annotate which ink colour corresponds to which margin or other anatomical point of interest. (b and c) A lipoma inked with and without a mordant. (b) The lipoma has been inked with blue ink without a mordant. The inked margin denoted by the yellow arrow is fuzzy, and the ink has smudged and run into all the adjacent cracks. This problem may result in a pathology report with a false-positive margin. (c) The same lipoma has been inked with blue ink which has then been set with acetic acid (vinegar). The margin denoted by the red arrow is much sharper

6 Transporting a Specimen

Tissue undergoes cytolytic degeneration very quickly once extracted from the body. The smaller the volume of tissue, the more rapidly this degeneration occurs. Therefore it is crucial to prepare slides and to place any remaining material into an appropriate transport medium swiftly, for immediate preservation. The volume of transport medium should be at least ten times the volume of the specimen. Neutral buffered formalin is perhaps the best-known medium. There are situations, however, where alternative mediums are preferred. Table 4 below gives advice about which transport medium to use in different situations.

• 1. 10% neutral buffered formalin

  Formalin is formaldehyde gas dissolved in water. Formaldehyde stabilises (“fixes”) tissue by creating crosslinks in macromolecules like proteins. Raw formalin is an acid solution, which reacts with haemoglobin to give a black precipitate, hampering subsequent histological assessment. To avoid this problem, a buffer such as sodium phosphate is added to the solution, to create “neutral buffered formalin”.

  As long as formalin can penetrate all parts of the specimen, tissue preservation usually is consistent and reliable. For most core biopsies, punch or small open biopsies where the working diagnosis does not include lymphoma or require identification of microorganisms by culture, formalin remains the default option as the transport medium. Formalin-fixed paraffin-embedded tissue can be processed for routine haematoxylin and eosin staining and for immunohistochemistry and other molecular studies.

• 2. Alcohol-based transport medium

  Alcohol stabilises tissue by coagulation. Alcohol-based preserving transport medium usually is supplied commercially in individual vials. Two products used commonly in Australasia are ThinPrep Cytolyt™ (by Hologic) and CytoRich™ (by BD). They are similar in the way that they render cells as monolayer films on the slides. Alcohol-based preservatives have become more popular over the years due to the consistency they produce, the ease of use and good level of cell preservation. Cells preserved in alcohol-based medium will be suitable for routine cytomorphology (either in addition to a direct smear or without), haematoxylin and eosin histology, immunohistochemistry and molecular studies [12].

• 3. Roswell Park Memorial Institute Medium (RPMI)

  RPMI (also known as RPMI 1640) is a nutrient-rich medium containing 19 different amino acids, 11 vitamins, sodium bicarbonate and various salts. Iscove’s Modified Dulbecco’s Medium (IMDM) is a similar medium but with a higher calcium concentration. RPMI and IMDM are not fixatives. They do not preserve cells but maintain viability of adherent cells. A nutrient-rich medium is the preferred medium for preserving and transporting cells destined for flow cytometry and mutational studies, including karyotyping. These nutrient-packed media need to be stored in a dark chilled environment (recommended storage temperature is 2–8 °C). They contain a pH-sensitive indicator to monitor possible degradation. Too long elapsed time and failure to store the medium in an appropriate manner will lead to degradation which is recognised by a change in colour from salmon orange to dark pink (Fig. 14).

• 4. Normal saline

  Normal saline is the simplest transport medium. Without any cell fixing property or nutrient, normal saline is not a good choice if the sample is expected to be in anything but urgent transit. However, if a specimen in saline can be sent to the laboratory sufficiently quickly, the specimen can be used for routine histology (cell block); flow cytometry; biochemical studies, e.g. thyroglobulin level for metastatic thyroid carcinoma; microorganism culture; and potentially molecular studies.

  If a wide differential diagnosis has to be considered, more material than usual is required, which may require multiple specimens. Preparing multiple samples can be a complex exercise. If a pathologist or a trained lab technician can attend for rapid on-site evaluation, specimen triaging can be done immediately, and the differential diagnosis may be narrowed, reducing the number of specimens required.

Table 4 Recommended transport mediums for different clinical scenarios and tests potentially to be done

Fig. 14

The bottle on the left shows the normal colour of IMDM. The bottle on the right shows a degraded IMDM which is unsuitable for specimen preservation

7 Ancillary Studies

Only decades ago, a simple distinction between benign and malignant tumours was enough for cancer management. Up until the 1990s, classifying lung cancer as small cell carcinoma versus non-small cell carcinoma was all that was required for treatment. Tumour classification, including for head and neck tumours, has become much more complex and beyond the capacity of simple light microscopy alone. So-called ancillary studies have become essential components of diagnostic anatomical pathology. Special stains, immunohistochemistry, cell markers and molecular studies are amongst the techniques that a modern pathologist uses.

8 Special Stains

Use of special stains probably was the first step away from simple light microscopy and haematoxylin and eosin. The advent of more sophisticated ways of marking tissue has resulted in a decline in the use of special stains in diagnostic pathology. The few stains that still are used fairly commonly today include the mucin stains (e.g. mucicarmine) used to identify glandular differentiation, the Perl’s stain (Perl’s Prussian blue) to aid in the distinction between haemosiderin and melanin, silver stains (e.g. Grocott-Gömöri methenamine silver stain) to identify fungal elements and Congo red stain for amyloid.

9 Immunohistochemistry

Immunohistochemistry has revolutionised diagnostic surgical pathology. There is a growing catalogue of immunohistochemical markers for histology. Whilst the interpretation of immunohistochemical markers can be challenging, the basic mechanism of action of immunohistochemical marking is quite simple. Immunohistochemical markers contain antibodies that bind to specific antigens and a coloured histochemical tag that makes the antigen-antibody binding visible. Most pathology laboratories tend to use a brown tag (chromogen) like 3,3′-diaminobenzidine tetrachloride [13].

Every tumour type has a unique antigen profile. Pathologists rely on those profiles to aid in a diagnosis. The catalogue of immunohistochemical markers already is extensive. In Table 5 we list some that one may encounter in pathologists’ reports concerning the histological diagnosis of head and neck cancer.

Table 5 Immunohistochemical markers used commonly in histological diagnosis of head and neck cancers

10 Flow Cytometry

Flow cytometry is a technique for detecting and measuring properties of cells from their diffraction patterns as they transit through a laser beam [14]. Flow cytometry is a highly efficient way of identifying a cell population of interest in a large volume of tissue. In head and neck cancer, it is particularly helpful in determining if there is clonality of lymphoid cells, indicating likely lymphoma. Flow cytometry is hardly ever used in the diagnosis of carcinoma. Flow cytometry requires fresh tissue so specimens should be submitted in RPMI/IMDM (preferred) or normal saline (see “transporting a specimen”). The specimen cannot be submitted in formalin or alcohol-based fixative.

11 Molecular Studies

Fluorescence in situ hybridization (FISH) has become an integral part of diagnostic pathology. In head and neck pathology, it plays an important role in the diagnosis of salivary gland tumours and sarcomas (Table 6). FISH is a cytogenetic technology based on the complementary nature of DNA and RNA double strands. FISH probes are single-strand fragments of DNA or RNA that bind to complementary nucleotide sequences in target cells, including tumour cells [16]. The probes are tagged with fluorescent nucleotides.

Table 6 Some of the common nucleotide abnormalities that can be revealed by FISH in head and neck pathology [22]

Two common types of FISH probes used in diagnostic pathology are break-apart and fusion probes. The probes are used to detect translocations, the most frequent genetic defects in cancer cells. Break-apart probes reveal nucleotide sequence separations that occur typically in a cancer cell. Two tags of different fluorescent colours are placed at opposite ends of a probe with a nucleotide sequence complementary to a targeted gene breakpoint. In normal cells, there is no break in the gene, so under fluorescence microscopy one observes the two tags (usually one red and one green) joined as a pair. When there is a translocation at that gene location, there is a break between the two tags, so they separate into individual colours. Fusion probes work in the opposite way and are used to reveal a specific rearrangement of nucleotides. Markers of different colours are placed on two different gene locations. In normal cells, the red and green signals remain separate, but when there is an abnormal fusion of those gene locations, the two colours emerge as a pair. FISH cytogenetics can be done on formalin-fixed paraffin-embedded tissue.

12 Making Sense of the Pathologist’s Report

In the past pathology reports were entombed in long-winded text, but often important information was omitted. In recent times pathologists submitting reports on most malignancies rather use synoptic reports constructed on a prescribed format that ensures that all the relevant information is covered and succinctly. They frequently employ the TNM staging system of the American Joint Commission on Cancer (AJCC), which assigns T codes for the extent of the tumour, N codes for lymph node involvement and M codes reporting whether metastases are present or not. The benefits of synoptic reports include completeness of pathologists’ reports for surgeons or other clinicians, easier readability and digestibility of information and providing a useful framework for research. Currently the synoptic reports tend to be paper-based, but in the future they undoubtedly will move to web-based systems, which, amongst other benefits, will allow for greater accuracy through the body of the report [17].

To help clinicians make sense of pathologists’ reports, we comment on concepts that appear routinely in synoptic reports, in the context of squamous carcinomas, melanomas, basal cell carcinomas and salivary gland carcinomas, the most frequent tumours of extreme facial malignancies.

12.1 Tumour Thickness and Depth of Invasion

Tumour thickness and depth of invasion are not synonymous terms (Fig. 15). Tumour thickness refers to the largest vertical dimension of the tumour, whilst depth of invasion gives an indication of how far a tumour has invaded into normal tissue. Tumour thickness/depth of a primary tumour is associated with adverse biological outcomes like risk of recurrence, metastasis or death [18]. The thicknesses of melanomas and depth of invasion of squamous cell carcinomas are measured differently. Melanomas are measured from the granular layer of the epidermis to the deepest level of invasion in the area of the tumour, whereas squamous carcinomas are measured from the granular layer of the nearest adjacent normal epidermis to the deepest part of the tumour. This method for squamous carcinomas adjusts for exophytic lesions. Ulcerated melanomas may have their thickness underestimated. Oral cavity carcinomas are measured differently.

Fig. 15

Illustration of tumour thickness versus depth of invasion. (a) Depth of invasion is measured from the top of the granular layer of adjacent uninvolved skin to the base of the tumour (AJCC eighth edition). (b) Tumour thickness is measured from the granular at the apex of the tumour to its deepest base

12.2 Pattern of Invasion at the Tumour Front

Table 7 shows the three main patterns of tumour growth at the infiltrative front.

Table 7 The three main patterns of tumour growth at the infiltrative front

12.3 Perineural Invasion

Perineural invasion (Fig. 16) occurs when a tumour has invaded into the nerve sheath [19]. Squamous carcinomas of the head and neck often affect nerves and those that do tend to have poor outcomes, as the tumour may track the cranial nerves back to the brain. AJCC eighth Edition recognises the importance of perineural invasion in the staging for primary cutaneous carcinoma; involvement of a named nerve, an unnamed nerve of at least 0.1 mm diameter or an unnamed nerve deeper than dermis will upstage a tumour from a pT2 to pT3. For some of the salivary gland tumours, the presence of perineural invasion aids with the diagnosis of malignancy. This feature is particularly prominent in adenoid cystic carcinomas. Perineural invasion far away from the invasive front portends a poor prognosis.

Fig. 16

Perineural invasion by moderately differentiated keratinising squamous carcinoma (haematoxylin and eosin 100× magnification). The nerve is surrounded by squamous carcinoma that is depicted by the yellow arrow

12.4 Margin Status

Pathologists pay a lot of attention to the margins of surgical specimens, especially resections for malignancy, because it is the condition of the margins that determines the completeness of the resection (Fig. 17). Sections can be taken either perpendicularly to the margin or en face at the margin. Perpendicular sections allow the pathologist to visualise the distance between the tumour and the surface of the specimen (the surgical margin) as both tumour and margin appear in the same section and so to advise the surgeon, if the margin is negative, how close to the resection surface the malignant tissue was. If the tumour is close to the margin, the pathologist can measure and report its separation from the margin using the graticule within the microscope’s eyepiece or the scale on the microscope’s stage. An en face section allows the pathologist to decide only whether the specimen is positive or negative for malignancy, so whether the margin from which it was taken was healthy or diseased.

Fig. 17

Atypical fibroxanthoma extends to the deep margin (yellow arrow) (haematoxylin and eosin 100×). The differential diagnosis of an atypical fibroxanthoma includes a pleomorphic dermal sarcoma. One of the distinguishing features is extension into subcutaneous tissue. In this biopsy where the tumour is extending to the deep margin and the subcutaneous tissue is not visualised, a pleomorphic dermal sarcoma cannot be entirely excluded

What do pathologists report as a positive margin? If a specimen has been inked and tumour extends to the inked surgical margin, that margin is reported as positive. If tumour extends to a cautery artefact, that too is reported as positive. In some situations, for instance, in the case of a basal cell carcinoma, if the characteristic stroma of the tumour extends to the margin, even though the actual epithelial tumour islands are not at the margin, the margin also is reported as positive.

An adequate excision depends on the type of tumour but also on the anatomical site of the tumour. If a tumour has been excised inadequately, does the anatomy allow room for additional surgery? If not, is the tumour type amenable to treatment with modalities other than surgery? Again communication between the surgeon and the pathologist is beneficial to arrive at the decision best for the patient, given the evaluation of the status of the margins, especially if the specimen is fragmented.

12.5 Nodal Status and Pathological Staging

To deliver a good service, pathologists would like a selective neck dissection to yield at least 10 lymph nodes and a comprehensive neck dissection to yield 15 or more lymph nodes. We consider the proportion of nodes that are positive, the maximum dimension of the largest node and that of the largest metastatic deposit as important elements in the pathological assessment, though the UICC and AJCC staging systems base their pN categories on the size of the involved node, rather than the size of the tumour deposit. In some cases there may be a considerable difference in the dimensions of the largest metastatic deposit and the largest involved node, and, when both sizes are recorded, the oncologist will have extra information upon which to plan treatment.

Another element that we consider, and report, is extranodal extension, which is the spread of tumour outside the capsule of the lymph node into the perinodal soft tissue (Fig. 18). It is measured perpendicular to the external aspect of the node capsule and may be categorised as microscopic (≤2 mm in extent) or macroscopic (>2 mm in extent). Extranodal extension is a risk factor for a poor prognosis in cervical node positive carcinoma, except in HPV-mediated oropharyngeal cancer, where its significance has yet to be established. The presence of extranodal extension in head and neck cancers correlates with a risk of regional local recurrence and distant metastasis and may be an indication for adjuvant combined chemotherapy and radiotherapy. The risk of regional recurrence and distant metastasis is higher with macroscopic rather than microscopic extranodal extension. Tumours with extranodal extension should be excised with a clear margin. If a margin is positive, the risk of local recurrence is increased and is an indication for radiotherapy to that site [20].

Fig. 18

Extranodal extension (Pan cytokeratin immunohistochemical stain 40× magnification). The black arrow perpendicular to the node capsule (indicated by dashed blue line) shows how extranodal extension is measured. The extranodal extension is clear of the surgical margin, inked black

We draw attention to changes relevant to interactions between clinicians and pathologists that have been introduced in the eighth edition of the AJCC [21] and also in the International Union for Cancer Control (UICC) cancer staging manuals. These changes have resulted in changes to the synoptic reports and staging of tumours. The changes include:

• 1. Separating p16-positive HPV-related carcinomas from p16-negative carcinomas.

• 2. Including extranodal extension in the pN categorisation for p16 negative oropharyngeal, hypopharyngeal, oral cavity, laryngeal, skin, major salivary gland, nasal cavity, paranasal sinus and unknown primary cancers.

• 3. Introducing a separate category for occult primary tumours of the head and neck with p16 and Epstein-Barr virus testing recommended.

• 4. Introducing a separate chapter for cutaneous squamous cell and other carcinomas (with the exception of Merkel cell carcinoma).

13 A Take-Home Message from Pathologists

We have belaboured the theme of cultivating the clinician-pathologist relationship and amalgamating the diverse wealth of information to benefit patient care. A small consideration by clinicians about what they are going to biopsy and what they want to achieve with that biopsy, before taking the specimen, can make all the difference in the value to the clinician of what the pathologist can do subsequently. We cannot stress enough the importance of providing pathologists with all the relevant clinical and radiological details; our own experience is that important information is omitted regrettably often. Recognising, and making sense of, those features in a pathology report that may influence treatment will impact your patient’s prognosis. We also submit that cultivating the clinician-pathologist relationship is personally stimulating, educational and fulfilling. Take time to ponder. We look forward to hearing from you soon.