Getting the most from the histopathology (Proceedings)

Biopsy and histopathology remains the gold standard diagnostic test for many conditions and for nearly all tumors and cancers. When performing this test, it is important to know what answers to look for, in order to obtain as much critical information as possible that may eventually impact the prognosis and the treatment planning. A good histopathology report will contain all the relevant findings observed in the submitted sample and, depending on the condition present, will describe details that may bear prognostic significance. Despite an excellent work by most anatomic pathologists, reports that do not contain all crucial or essential information are regularly encountered. The clinician's duty is then to correspond with the laboratory or pathologist directly, in order to obtain such information. Only then may the clinician discuss the findings with the owners and offer precise information that will lead to additional diagnostic tests, when deemed necessary, or prepare a specific therapeutic plan for the patient.

FIRST Step: the submitting clinician's responsibilities

It is often said that the information obtained from the histopathologic analysis will only be high-quality if all the steps preceding the sample submission were properly performed. The results of a recipe are only as good as the ingredients used in the first place.

The sample(s) submitted must be appropriate in order to obtain correct information through its analysis. If an incisional biopsy, then it must be large and deep enough. It may be difficult to obtain large biopsies in certain anatomical sites or organs. Ideally, at least 2 (or 3 or 4) samples should be obtained and submitted. It is occasionally possible to tell, just by the appearance of a biopsy sample, if it will be of diagnostic quality or not. Samples that are too small (less than 1 mm) often will not be diagnostic or will show marked distortion. Similarly one may obtain inadequate results if the sample seems to contain mucus, necrotic material, or a blood clot. A sample that floats on the surface of the formalin may contain mostly fat and, unless a lipoma was biopsied, may not be diagnostic. Soft-tissue lesions should be biopsied at the periphery, in order to avoid a necrotic center, and to permit the observation of the junction between normal and diseased tissue. When biopsying a bone lesion, we typically aim at the center of the lesion, in order to avoid the peripherally located reactive bone. Remember that performing a biopsy on a tumor will lead to an increased risk of dissemination, and will not change its biologic behavior, with a few exceptions such as the occasional abdominal carcinomas that may seed in the peritoneal cavity or biopsy tract, when biopsied in a suboptimal manner (ex: transcutaneous biopsies of a bladder transitional cell carcinoma located in the trigone, etc.).

If the sample is the result of an excisional biopsy, when such a procedure applies, it is then crucial to submit the whole sample, with the surgical margins that were removed en bloc. If the sample is larger than 2 cm in diameter, transverse incisions can be performed to obtain slices of 1 cm thickness, and allow for better fixation in formalin. The incisions are performed on the side of the lesion where margin information is not needed which, for a cutaneous or subcutaneous tumor, would be the outermost surface (with skin) of the sample, and not the deep surface.

A proper sample:formalin ratio of 1:10 must be respected for good tissue fixation to occur. For very large samples, larger containers are required and should always be available for that eventuality. If the sample is simply too big for submission in its entirety, representative samples of the mass may be submitted. If this is done, samples of margins at higher risk of being incomplete should be obtained, and placed in separate and properly labeled containers. Furthermore, the portion of the original mass that is not submitted should be kept aside until the final report has been obtained. On a large sample where specific margin information is important, they should be ideally identified via inking (or sutures) for easier identification and to direct the pathologist's attention to these crucial surgical margins.

The submitted sample must be properly identified with the patient information on both the container and the cover. Filling the submission form as thoroughly as possible will ensure that correct answers are obtained. Signalment, history, and a clinical description of the lesion sampled all constitute vital information. This step is often partly neglected, especially on busy days, and often will negatively impact on the information obtained. Voluntarily omitting some information to "not influence the pathologist" in the interpretation is a mistake. All pertinent clinical information is helpful to obtain the best possible answers from the submitted sample. Information that should be on the submission form includes: age, sex, breed, appearance of the lesion, size, localization on the patient, duration and speed of growth, invasion in neighboring tissues, other pertinent clinical details, appearance on diagnostic imaging, biopsy type, clinical suspicion, response to empirical therapy, etc. Omitting pertinent clinical information on the submission form is one of the most common causes of incorrect interpretation of the sample by pathologists.

SECOND Step: reading and understanding the report

A good histopathology report will contain a detailed microscopic description, with information on margins for excisional biopsies, histologic grade when a grading system exists that influences prognosis or treatment plan, other information occasionally important for specific tumor types (ex: vascular invasion, mitotic index, % necrosis), and a final diagnosis. The histologic grading system varies with tumor types, but generally is affected by variables such as mitotic index, % necrosis, invasion in adjacent normal tissues, vascular or lymphatic invasion, nuclear or nucleolar criteria, nuclear:cytoplasmic ratio, anisocytosis, lymphocytic infiltrate, reaction of surrounding stromal cells, etc. The report will also discuss the quality of the submitted sample, and may comment on the necessity of obtaining additional samples to get a firmer diagnosis (ex: The most probable diagnosis is X, but considering the small sample size and presence of artifacts, we recommend obtaining additional samples to reach an unequivocal diagnosis.).

Certain histopathology reports will be short, and still contain all pertinent information. While many laboratories offer the option of lower cost "mini-reports", without a microscopic description, such reports should generally be considered unacceptable, and are not recommended. When oncology patients present with "mini-report" diagnoses, we invariably ask for complete reports or pathology reviews to obtain all the microscopic information. It is not rare to then find additional information that will clearly alter the prognosis, as well as the specific treatment plan.

A good histopathology report does not need to contain clinical information, especially not information on the recommended therapeutic approach, although many like to see such information under the "Comments" section. Pathologists are rarely seen treating cases in the clinic; if clinical information is included, a list of up-to-date references should follow, ideally from the primary literature (not books). It is not possible to include all relevant information in a few lines, and providing the reader with a few numbers often results in summarized information that may not be complete enough when it comes to decide of a patient's fate. This is amplified if the initial submission form was inadequately filled! Ideally, the clinical information will come from clinicians from the specialty of interest (ex: dermatologist if dermatologic condition; internist if immune-mediated, respiratory or digestive; surgeon if orthopedic condition; oncologist if neoplastic condition; etc.). Naturally, having good reliable and recent resources (printed, online, CE) will be very helpful.

THIRD Step: what else should be asked for?

Examples of three situations when additional information should be requested:

     A. The histopathologic diagnosis does not fit the clinical picture. Example: diagnosis of a benign tumor according to histopathology, but the mass is growing very fast, is invasive or destructive, or has already spread to draining lymph nodes or distant sites.

This situation may result from a sample that is non diagnostic, from a mix up between two samples submitted to this laboratory (in the clinic, during transport, in the lab, by the pathologist of staff), from an erroneous interpretation of the pertinent clinical information, or from an atypical presentation of the condition properly diagnosed.

     B. Crucial information is missing on the report. Example: surgical margins are not mentioned on an excisional biopsy sample, no histologic grade, no comment on vascular or lymphatic invasion, etc.

It is then imperative to communicate with the laboratory or the pathologist and ask for that information. With mammary carcinomas, for example, it is important to know if vascular or lymphatic invasion is present. If the report does not mention it, it does not necessarily mean that there is none. On the contrary, if the report mentions that "No vascular or lymphatic invasion was observed on the examined sections.", then we can be certain that the pathologist looked for it but could not find it.

     C. The information is present, but is not satisfactory. Examples: "The margins are clean but close."

This is a common situation in oncology. In general, margins that have more than 2 to 3 cm of normal tissue before tumor cells are observed are called "radical". Wide is generally 1-3 cm. "Close" is definitely less then 1 cm, and I would call less than 1 mm "very close". However, not every pathologist has the same interpretation as others on this, and specific information ("clean by X millimeters") is always preferred. Furthermore, for some tumor types, millimeters are enough, but for others radical margins are required. If a tumor is said to be "high grade", it sometimes will be helpful to know what led the pathologist to give this grade, since some information may influence therapy. For example, if the grade is high specifically because the mitotic index was very high, cell cycle specific drugs could be favored in this patient (ex: antimetabolites, mitotic spindle poisons, etc.).

Pathologists should not feel insulted when questions arise concerning certain portions of their report. With difficult or equivocal cases, they often discuss or consult with peers to get their opinion and reach a consensus. If this has not been done, the submitting clinician can request it, and most pathologists will happily ask for another opinion. Some tumor types more often will raise diverging opinions between pathologists, and could cause some confusion. Again, the sample quality and clinical information provided may help in such situations.

Various tumor types have specific prognostic information that is required on the histopathology report. For example, a mast cell tumor should always have a histologic grade. Similarly, if the tumor is a melanoma, the report should contain a number of mitotic figures per 10 high power fields, as this is crucial prognostic information. Increasingly, certain tumor types will not be described by a broad terminology (ex: lymphoma), but in a more specific manner (ex: marginal zone lymphoma), although certain classification systems have yet to be widely accepted, and are in constant evolution. How well differentiated a tumor is can be important for certain tumor types, such as canine mammary and pulmonary carcinomas, and that information should always be included in a report describing such a tumor.

The histopathologic grade should be included if a grading system exists with established criteria, is accepted, and predicts the prognosis. If this information does not appear on the report, the clinician should request it. Ideally, the grading system in use should be mentioned, especially when more than one system exists in the literature. The best example is again with mast cell tumors. The histologic grading system in use most frequently is the one developed by Patnaik, where a grade I is well differentiated and has a better prognosis, grade II is intermediate, and grade III is a high-grade predicting a more aggressive biologic behavior. Another system exists (Bostock) that is inverted, with grade I being a high grade, and grade III being well differentiated. The latter may be found more commonly in older publications from the UK.

Established grading systems exist for the following tumor types: canine and feline lymphomas, canine and feline cutaneous mast cell tumors, canine and feline mammary carcinomas, canine and feline pulmonary carcinomas, canine synovial cell sarcoma (this system requires revision since histiocytic sarcomas have been better described), canine multilobular tumor of bone, canine melanoma, canine soft-tissue sarcomas, canine appendicular and mandibular osteosarcoma, canine splenic and cutaneous hemangiosarcoma, feline cutaneous and canine lingual squamous cell carcinomas, canine bladder transitional cell carcinoma, canine splenic fibrohistiocytic nodules, and canine splenic sarcomas.

If an entire tumor was submitted with margins (en bloc resection) and the histopathology report does not mention information on surgical margins, communication with the pathologist or laboratory is mandatory. If the sample submission form mentions that the submitted sample is an excisional biopsy with margins, the margin information always should be found on the report. When certain margins were more concerning to the clinician during surgical excision or based on advanced imaging, they should be marked appropriately, ideally with ink, in order to help the pathologist in identifying cancer cells approaching this crucial area, and perform more sections of the inked tissue to improve the yield.

Special stains and immunohistochemistry

The use of special stains and poly- or monoclonal antibodies has increased tremendously in the last few years, and is quite helpful for more specific identification of certain tumor types that otherwise would be difficult to diagnose because of poor morphologic differentiation.

Examples of special stains used to label poorly granulated mast cell tumors include toluidine blue and Giemsa. Masson trichrome helps identifying collagen fibers produced by certain mesenchymal tumors. Periodic Acid Schiffs (PAS) will help in identifying mucus produced by certain poorly differentiated adenocarcinomas. Other staining techniques are used to identify proliferation markers associated with higher grade or biologic behavior in certain tumors. Such a marker is AgNOR (argyrophilic nuclear organizer regions), which stains certain ribosomal proteins and gives additional prognostic information for some tumors (ex: canine mast cell tumors).

Amongst the commonly used polyclonal or monoclonal antibodies, without which a definitive diagnosis is oftentimes hard to reach, are the various clusters of differentiation, or CD, expressed with relative specificity on the surface of certain cellular lineages. For example, CD79a and CD20 will be used to identify B lymphocytes, while CD3 is commonly used to label T lymphocytes. The von Willebrand factor (factor VIII-related antigen) and CD31 are useful to identify endothelial cells. Certain less specific antibodies identify intermediate cellular filaments including cytokeratin (epithelial cells), vimentin (mesenchymal cells), and desmin or actin (muscle cells). Finally, other common antibodies detect CD18 (dendritic cell origin), CD117 (c-Kit receptor on mast cells or on gastrointestinal stromal tumors), S-100 (cells of neuroectodermal origin: glial cells, melanocytes), Melan-A and HMB-45 (melanocytes), chromogranine and synaptophysin (neuroendocrine tumors), thyroglobulin (thyroid cells), kappa or lambda light chain IgG (plasma cells or B lymphocytes), PCNA and Ki67 (proliferation markers), etc.

No laboratory will perform immunohistochemistry with all known markers, and it is important to know that false negatives and false positives may arise. Many factors come into play when interpreting the results of immunodiagnostic techniques, and the validity of the results is somewhat user-dependant, technique-dependant (antigen retrieval, etc.) and may be affected by the quality control (positive and negative controls).

Conclusion

Good communication between the pathologist and clinician is essential to obtain all the information possible from tissue biopsy samples. An appropriate diagnosis with as much prognostic information as possible will lead to better understanding of the disease process in a given patient, more realistic expectations by the clinician and owner, and appropriate therapeutic planning.

References

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