Histology: The gold standard for diagnosis?

Histopathology is an important diagnostic tool used during a workup for various problems, including cancer. It is generally recognized as the gold standard for the diagnosis of neoplasia. As both clinicians and pathologists play important roles in making a diagnosis, open and good communication are vital. Through this column, we hope to raise awareness among clinicians about the importance of the proper and critical interpretation of histopathology reports. Although the discussion here may apply to all histopathological reports, this column focuses specifically on the histopathology of neoplasia.

A common reason to consider obtaining a biopsy sample is to determine whether an animal has cancer. Many features are assessed on a biopsy in order to make this determination, including architectural features (e.g., type/origin of neoplasia, presence of vascular or stromal invasion, and biopsy margins) and potential criteria of malignancy (abnormal cell morphologies such as anisocytosis, anisokaryosis, multinucleation, abnormal nuclei; increased mitotic rate; and presence of necrosis) (1). Collectively, these features provide information on the type of cancer, the histologic grade (if applicable), and whether the biopsy margins are clear of neoplastic cells (and if so, to what extent).

Histopathological assessment requires in-depth knowledge of both clinical and pathological aspects of diseases, but it also relies on skill and experience; such assessments can thus be variable and subjective. Indeed, studies have documented interobserver variability in the histopathological assessments of certain diagnoses, such as mast cell tumors and digital masses (2,3), and anecdotal examples of such variability exist for many biopsy types. In these cases, a re-evaluation or a second review of the tissue slides by an additional pathologist is ideally requested — but what does this mean in practice?

A series of retrospective (4,5) and prospective (6) studies assessed the utility of second-opinion histopathology requested by a veterinary oncologist at 2 referral institutes. Second opinions resulted in a partial disagreement, defined as a change to the grade or subtype, or some other minor disagreement, in 20 to 34% of cases; whereas a complete disagreement, defined as a change between benign and malignant or 2 different diagnoses, was observed in 10 to 19% of cases. Overall, such disagreements led to a significant change in either the prognosis or the treatment in 17 to 39% of the cases. Although the high likelihood of a second review being inconsistent with the original diagnosis is probably due to selection bias, it is still an important consideration for clinical diagnosis. The studies also noted that the disease progression tended to align better with the revised histopathological diagnosis. This may be because clinicians requesting a second review of histopathology findings tend to provide additional clinical information to the reviewing pathologists. It is probable that the inclusion of additional clinical history and findings would allow the reviewing pathologist to consider a diagnosis that better matches the animal’s clinical picture. This emphasizes the importance of providing a detailed clinical history when submitting histopathology samples. Furthermore, if a discrepancy between the clinical suspicion and the histopathological diagnosis of the submitted tissue is found, the authors would encourage the clinician to discuss the discrepancy directly with the pathologist and request a second review of the sample. The reader is encouraged to remain critical of the second review, keeping in mind the fact that a diagnosis may constantly evolve with the disease progression, and a third or even a fourth review might be warranted.

Another important aspect of a histopathology report is the interpretation of the histological margins. This can only be done when the tissue is properly processed. Inappropriate trimming before tissue submission, or aggressive use of electrocautery at the surgical margins can lead to tissue architecture damage at the margins and preclude margin assessment. Margin assessment for a surgically excised tumor involves sectioning the tissue at an area where the tumor was grossly closest to the “deep” and “lateral” edges of the tissue and measuring the width of nonneoplastic tissues, microscopically, from the cancer cells to the tissue edge. This is then reported as a measurement in millimeters in the histopathology report, along with a description of “complete,” “complete but narrow,” or “incomplete” margins. However, a clinical challenge is that local tumor recurrence can still occur even with histologically “complete” margins. This phenomenon can be explained by multiple factors relating to both the histological assessment and the biological behavior of a tumor. Histological factors leading to a discrepancy between histopathological margins and local tumor recurrence include i) tissue shrinkage related to formalin fixation and processing, ii) the identification of relevant surgical planes, and iii) the impracticality of assessing every single tissue edge. Biologically, tumor recurrence may still occur regardless of local tumor control, via metastases to lymph nodes or distant organs (7,8).

Tissue shrinkage is an inevitable by-product of the routine tissue processing methodology. Studies in canine and feline cadavers showed a 10 to 36% decrease in mass size between pre-surgical and histological measurements (9,10). Antemortem studies similarly reported a 7 to 43% decrease in mass size between post-surgical measurements and histological measurements (11,12). This implies that a measurement of 1 mm on histology does not directly relate to 1 mm in vivo. This discrepancy may be critical, especially for tumors such as high-grade mast cell tumors or canine oral squamous cell carcinomas, for which measurement of the tumor-free margins dictates the necessity of adjuvant therapy (7).

Surgical planning involves using fascial planes as a natural barrier against tumor invasion. However, these planes may not be obvious upon a histological assessment. Proper identification of relevant surgical planes through appropriate tagging of surgical margins and proper communication between pathologists and clinicians will help pathologists identify which planes to evaluate (13). In the authors’ experience, not all commercially available dyes for margin inking are recommended. It is advised to check with the histopathology laboratory for preferred methods of labelling. Margin assessments of clinically relevant planes will increase the likelihood that histological margins will be accurately measured.

Routine sectioning cannot practically evaluate all edges of the excised tissue. Although errors can be minimized with the use of different trimming methodologies, such as the tangential method (14), it is impossible to be definitive. Reported margins can differ widely depending on how the trimming is carried out (15). Clinicians should consider this limitation when making recommendations about tumors for which an incomplete excision will change the prognosis. Clinicians can discuss the trimming methods and the confidence in a clear excision with the pathologist.

Even in cases where a tumor is excised completely with wide histological margins, it may recur locally due to other factors. Literature in the veterinary field is limited, but “self-seeding” by circulating tumor cells has been shown in preclinical breast and colon cancers, melanomas (16), and osteosarcoma models (17). Local residual tissue may also be predisposed to local tumor recurrence due to the field effect (18); and histologically “normal” tissue may still be predisposed to neoplastic transformation, as was demonstrated in human head and neck cancers (19).

Due to these concerns about margin evaluation, a significant risk of local recurrence is always present in certain tumors, such as canine high-grade mast cell tumors, even when margins are histologically tumor-free (20). Clinicians should use their judgment when recommending an appropriate adjuvant therapy as needed.

Histopathology remains the gold standard for the diagnosis of canine and feline solid cancers. However, the histopathology report must be interpreted carefully in light of the clinical picture. To help pathologists create a clinically meaningful assessment and diagnosis, clinicians should provide a thorough clinical history and physical examination findings when submitting a sample. If any discrepancy between the clinical and pathological findings arises, a second histopathological review may be requested after discussion with the pathologist. Finally, although histological margins should be used as a guideline, clinicians should always remain vigilant for tumor recurrence by carrying out recheck examinations and monitoring based on the suspected clinical prognosis.

Questions

• 1. An animal is presented to you with a mass that you believe to be clinically benign. The animal appears otherwise completely healthy. You surgically excise the mass with narrow margins and submit it for histopathology. However, the histopathology report concludes the mass is an extremely malignant neoplasia. Which is the most appropriate next step?

  1. Assume that the histopathology report is erroneous and proceed with treatment for a benign mass.

  2. Trust the histopathology report completely and proceed with aggressive recommendations (therapy or referral).

  3. Request a second opinion for the histopathology and trust that second report over the primary opinion.

  4. Contact the pathology laboratory to explain your clinical suspicions and the reasons why you believe there might be a disagreement between the clinical picture and the histopathological report. Request a second, third, or even fourth opinion on the histopathology if needed, providing updates to the pathologists on the animal’s clinical picture throughout the process.

Answer: D

• 2. Which of the following best explains why local recurrence of neoplasia might occur after a wide surgical excision, even if the margins appear to be clear on histopathological examination?

  1. The tissue was not sectioned at an edge with dirty margins, causing the appearance of a false clean excision.

  2. The tumor was excised completely, but the “healthy” tissues in the area that appeared normal on histopathology have already undergone some changes that predispose them to new tumor formation.

  3. The tumor was excised completely but circulating tumor cells in the body “self-seeded” the site of the surgical excision and caused a new growth.

  4. All the above.

Answer: D