Abstract
The partnership between an ophthalmologist and an ophthalmic pathologist is critical to ensure that the final histopathology report is accurate and intelligible and contains as much relevant information to ensure safe patient management. At all stages of this process, clear verbal and written communication is key; every link in the chain must be robust to ensure the patient comes to no harm. To quote Professor Christopher Fletcher, Director of Surgical Pathology at Harvard Medical School, ‘the reality is that the histology report is the principal determinant of diagnosis, likely clinical course and therapy in any patient found to have a mass’.
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Keywords
- Ocular pathology
- Pathology of ocular tumors
- Ocular tumor pathology
- Ophthalmic pathology
- Ophthalmic pathology request form
- Fluorescence in situ hybridisation
- Chromosomal microarray analysis (aCGH and SNP)
The partnership between an ophthalmologist and an ophthalmic pathologist is critical to ensure that the final histopathology report is accurate and intelligible and contains as much relevant information to ensure safe patient management. At all stages of this process, clear verbal and written communication is key; every link in the chain must be robust to ensure the patient comes to no harm. To quote Professor Christopher Fletcher, Director of Surgical Pathology at Harvard Medical School, ‘the reality is that the histology report is the principal determinant of diagnosis, likely clinical course and therapy in any patient found to have a mass….’ [1].
Ocular pathology specimens travel through three phases: pre-analytical (pre-surgery communication, surgery, request form, specimen handling prior to fixation, fixation and transportation), analytical (processing of specimen, stains, ancillary investigations and histological interpretation by pathologist) and post-analytical (histopathology report, post-report discussion at multidisciplinary meeting, supplementary reporting).
This chapter will cover the following topics:
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Pre-analytical phase
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Pre-surgical planning.
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The ophthalmic pathology request form.
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Handling specimens in theatre before fixing.
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Fixation.
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Transportation of specimens to the histopathology laboratory.
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Post-analytical phase
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Basic histopathology terminology and understanding the histopathology report.
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The use of ancillary investigations in ophthalmic pathology.
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Ocular tumour prognostic factors.
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As this chapter is meant to be a practical guide for clinicians, it will not cover the analytical aspects of how the specimen is grossed/cut up, its processing or staining in the ocular pathology laboratory. Many texts are available that cover these steps in detail and interested readers should consult these.
Pre-analytical Phase
Discussion of Cases Prior to Surgery
Some cases (complex margins), specimens requiring frozen sections or those requiring special requirements (e.g. certain fixatives), are best discussed in advance with the ocular pathology laboratory. This will ensure the pathology laboratory can meet the needs of the surgical procedure and ensure adequate handling without compromising the specimen.
How to Complete the Ophthalmic Pathology Request Form [2]
A pathology request form is a referral letter and a primary source of communication between the ophthalmologist and pathologist. It is also a medico-legal document. For all specimens submitted to the laboratory, a fully completed request form should accompany each case. Request forms are designed to provide:
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Unique identification of the patient.
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A destination for the report and any charging information.
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The laboratory with the clinician contact details if discussion of the case is required.
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Date and time of specimen collection/removal and investigations required (e.g. histology/cytology).
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Type of specimen and anatomical site of origin. This is especially important for multiple map biopsies taken from the conjunctiva to establish the extent of ocular surface squamous, sebaceous and melanocytic malignancies. It is critical that the request form include a diagram indicating the precise origin of each biopsy.
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Clinical information so that the pathologist may handle the specimen appropriately and interpret microscopic findings in the proper context.
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An awareness of any health and safety issues with a given specimen.
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Complete information must be supplied on the request form. Each specimen container, no matter how small, should also be labelled with the appropriate patient identification data (minimum of three identifiers, e.g. first and last name, date of birth/age and gender and preferably patient’s hospital no).
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The information must be consistent with the request form, to prevent errors in specimen and patient identification.
See Fig. 15.1a–e showing examples of well-filled out and suboptimally filled out pathology request forms .
Specimen Handling in Operation Theatre Prior to Fixation
Multiple Specimens from a Surgical Procedure
As a general principle, specimens from different locations should be placed in separate fixation pots. If multiple pieces of tissue from different sites are placed in the same fixation pot and one of the pieces of tissue turns out to be malignant, it is impossible to ascertain where the tissue has been sampled from.
Orientation of Specimens
Orientation of specimens is required, to ensure the pathologist can report the margins of excision accurately. This is best done by placing sutures at particular margins and then specifying the sutured margin on the pathology request form in writing or as a diagram (Fig. 15.2a).
Conjunctival Mapping Biopsies
Conjunctival biopsies comprise thin strips of epithelium and stroma and are prone to curling if placed straight into fixative. They should be attached to sponge or card before placing into the fixative, so that they arrive in the ocular pathology laboratory in a flattened state. The latter facilitates orientation during embedding and greatly facilitates the histological assessment for the presence or absence of invasion by tumour. If multiple mapping biopsies are performed, the pathology request form must state the precise location of the biopsies. Figure 15.2b shows a very helpful diagram drawn by the surgeon that shows the extent of this patient’s conjunctival lesion (shaded areas) and the precise location of the mapping biopsies.
Sampling Fresh Tissue Prior to Fixation
Some tumours require to be sampled fresh for special genetic/ molecular investigations (diagnostic and prognostic testing) or for research.
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In the case of an enucleation, sample the optic nerve first and place in a separate pot of formalin. This is to prevent fresh tumour contaminating the optic nerve resection margin.
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The globe is transilluminated and using a scalpel, a sclera-choroidal flap is created beneath the tumour, leaving the scleral-choroidal flap hinged along one side. Fresh tumour is harvested with scissors and forceps.
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Fresh tumour is placed in an appropriate medium and vessel.
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Some use a corneal trephine to cut a window into the sclera and choroid.
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After harvesting fresh tumour, the specimen is fixed.
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An alternative strategy is to submit the intact specimen unfixed to the ocular pathology laboratory, where the above steps can be carried out.
Fixation of Ocular Pathology Specimens
Routine Surgical Specimens
Standard 10% phosphate-buffered formalin in a leak-proof container is the fixation of choice (Table 15.1) [3,4,5]. This will allow routine haematoxylin and eosin staining (H&E), other special tinctorial stains and immunohistochemistry. In addition, numerous molecular studies for diagnosis and prognosis can be performed on formalin-fixed tissue (Fig. 15.3a).
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If certain molecular biological diagnostic techniques or research is to be done on unfixed tumour samples, the clinician or assistant must remove fresh tumour from the specimen prior to fixing in formalin.
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The formalin should ideally be 10x the volume of the specimen to allow timely penetration of the tissues at room temperature. To ensure this, an adequate sized plastic container is required. Most well-stocked pathology labs have a variety of sizes of plastic container to facilitate this. Many containers come pre-filled with formalin (Fig. 15.3a).
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Specimens should be placed into the container that has been pre-filled with formalin. If formalin is poured into a dry container that has the specimen in it, there is a chance that the specimen will be ejected by the formalin and will end up on the bench or floor.
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Specimen containers must then be sealed tightly with the lids to avoid spillage of hazardous formalin.
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Smaller containers are then usually placed in a plastic bag which also accommodates a side compartment for the request form. Follow your institutional standard operation protocols for additional packaging.
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The appropriate institutional health and safety protocols must be followed when handling formalin.
Which Specimens Should Not Be Fixed?
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Tissue destined for frozen sections (to assess margins of excision for eyelid, adnexal and orbital tumours).
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Tissue for research or tissue banking.
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Some tumours requiring prognostic information (e.g. sarcomas, paediatric small round blue cells tumours, retinoblastoma) may require special studies on fresh tissue.
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Biopsies destined for flow cytometry (e.g. vitreous and fine-needle aspiration specimens for assessment of lymphoma). In this situation, many practitioners send the cell sample in Roswell Park Medical Institution medium (RPMI) to enhance the survival of the cells.
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Some labs still do Oil Red O staining for suspected cases of sebaceous carcinoma. However, this is being superseded by haematoxylin and eosin-stained (H&E) sections and immunohistochemistry.
Fixation of Cytology Specimens
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Fine-needle aspiration specimens (FNA) are preferably fixed in CytoLyt or Shandon cytofixative (or equivalents) which are methanol-based mild fixatives (Table 15.1). They preserve the cyto-morphology well and permit good-quality DNA to be extracted for molecular diagnostic tests. CytoLyt preserved specimens can be used to make thin-prep slides as well. FNA samples are usually squirted into pre-filled universal tubes that contain the fixative. The syringe used in the collection of the sample may be submitted with the fluid inside. Needles must be removed and the syringe capped. If the specimen is to be fixed, an equal volume of cytology fixative may be drawn up into the same syringe, capped and then agitated gently to mix the sample with the fixative (Fig. 15.3b, c).
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Vitrectomy specimens: It is preferable to receive a pars plana vitrectomy specimen in a vitreous cassette or bag. If samples are required for microbiology, virology, PCR, etc., please remove these from the cassette/bag before fixation, although, ideally, these should have been sent from the initial core vitreous tap. Vitrectomy samples need to be preserved/fixed in an equal or excess volume of RPMI or cytology fixative. It is important to seal the bag or cassette to prevent leaks in transit (Fig. 15.3d).
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Impression cytology plastic rings can be dropped into a small pot of formalin for fixation (Fig. 15.3e).
Fixation of Specimens Requiring Transmission Electron Microscopy
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Buffered 2.5–3% glutaraldehyde is used to fix fresh specimens for transmission electron microscopy. This would be done for cases that are intended for research or in which the original interpretation of an initial tumour biopsy was equivocal and clarification is sought from ultrastructure. It is best to place only part of the fresh tissue into buffered glutaraldehyde, as whilst it is an excellent fixative for ultrastructure, it adversely affects antigen retrieval for immunohistochemistry and extraction of DNA for molecular studies (Fig. 15.3f).
Transporting Specimens to the Pathology Laboratory
High Risk/Danger of Infection Specimens
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It is the ophthalmologist’s responsibility to indicate on the request form and the specimen pot if the patient is known or suspected to be a ‘High Risk/Danger of Infection’ category (e.g. HIV, TB, hepatitis B, hepatitis C), to facilitate appropriate handling in the laboratory.
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It is the ophthalmologist’s responsibility to ensure that all specimens are submitted to the laboratory in suitable and approved containers.
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Approved specimen containers have leak-proof lids and the appropriate hazard warning signs for the fixative, e.g. formalin.
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Specimen containers should be closed securely and placed inside a sealed specimen bag.
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Specimens received leaking or damaged are a danger to all those who come into contact with them, including theatre staff, porters and laboratory staff and can also compromise the diagnosis.
Mailed or Couriered Specimens
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Specimens mailed or couriered should be packaged in approved containers in accordance with the requirements of the delivery service. To confirm receipt of specimen(s) in the department, it is good practice that a ‘confirmation of receipt fax-back’ form, providing the senders’ confidential fax number, is enclosed with the specimen(s).
Post-analytical Phase
Understanding the Ophthalmic Histopathology Report
The ophthalmic histopathology report contains a lot of specialised pathology terminology that can seem rather bewildering. To understand histopathologic terminology related to tumours, it is best to return to basics. Table 15.2 outlines the tissue of origin and their related tumours that are commonly encountered [6] (Fig. 15.4a–j).
Knowledge of this basic table does go a long way to understanding the various tumours encountered in ocular oncology practice and their behaviour and prognosis. It also allows for a conversation with your local pathologist, who is often equally keen to learn specialised ophthalmology terminology to enhance the quality and clarity of the professional interaction.
Examples of Histopathology Reports with Explanations of Specialised Terminology
Set out below are examples of ophthalmic histopathology reports that may be encountered in a busy ocular oncology practice with specialised terms defined and figures provided for clarity [6].
Eyelid Tumour Histopathology Report Example
This full thickness wedge excision contains an invasive neoplasm. It is arising from the Meibomian gland and is composed of pleomorphic cells, with vacuolated cytoplasm, indenting the nuclei. Numerous mitotic figures are present. The individual tumour cells exhibit hyperchromatic chromatin and nucleoli are prominent. Foci of intraepithelial pagetoid spread are seen over the invasive tumour, involving the tarsal conjunctival epithelium and the epidermis of the skin. The invasive tumour shows focal comedo-like necrosis and shows vascular and perineural invasion.
Immunohistochemistry shows that the invasive tumour is positive for adipophilin, EMA and shows nuclear androgen receptor positivity.
The features are those of an invasive sebaceous carcinoma , derived from the meibomian gland. The excision of the invasive component is complete (nearest margin is 3 mm). However, the pagetoid spread contaminates the tarsal conjunctival margin and is therefore incompletely excised.
Sebaceous neoplasms can be associated with the Muir-Torre syndrome. Is there a history of internal malignancies in this patient or is there a family history of malignancies?
Terminology Explained
Invasive
Malignant tumours invade through basement membranes into the surrounding tissue and can metastasise by invading into blood vessels and lymphatics.
Neoplasm
Literally means ‘new growth’ (derived from Greek).
Pleomorphic
Variation is size and shape of the nucleus and cytoplasm of the individual tumour cells. This is a feature of malignant cells that have lost their normal constraining signals (Fig. 15.4k).
Mitotic Figures
Prophase, metaphase, anaphase, telophase and cytokinesis are the five phases of mitosis (refer to any intermediate biology textbook). Mitotic figures are seen in normal and repairing tissues. An example of a normal tissue containing mitotic figures is the proliferative phase of endometrial glands and stroma. When injured tissue is regenerating, it is common to see many mitotic figures, e.g. regenerating epithelium in skin after trauma. Benign and malignant tumours contain mitotic figures. Malignant tumours tend to have more mitotic figures than benign tumours. Of the malignant tumours, poorly differentiated tumours tend to have higher mitotic figure counts compared to better differentiated tumours. In malignant tumours, the mitotic figures can have an abnormal morphology that is not seen in benign, normal or regenerating tissues. These include tripolar or multipolar forms (Fig. 15.4l).
Vacuolated
A bubbly appearance to the cytoplasm due to the presence of various substances. In this case, it is lipid as the diagnosis is sebaceous carcinoma. It could be mucin (mucin forming carcinomas) or glycogen (e.g. renal cell carcinoma). The products made by tumour cells can be very helpful for classification (Fig. 15.4m).
Hyperchromatic Chromatin
Simply means dark staining DNA in the nucleus. This is a feature of malignant cells (Fig. 15.4n).
Intraepithelial
Within the confines of the epithelium.
Pagetoid
Horizontal and vertical spread of malignant tumour cells as individuals or groups within epithelium. Named after Sir James Paget, an English surgeon and pathologist who described Paget’s disease that shows this pattern of spread. Tumours that show this pattern of spread include malignant melanoma and sebaceous carcinoma (Fig. 15.4o).
Comedo
Literally means like a ‘zit’. Malignant tumour cells forming sheets are hypoxic in the middle and undergo necrosis. The appearance is that of a necrotic centre surrounded by a viable rim of malignant tumour cells. If the tissue is squeezed gently during handling, the necrotic tumour centre oozes out of the cut surface like zits being squeezed.
Necrosis
Cell death with loss of cell contents to the exterior. Malignant tumours that outstrip their blood supply often become necrotic (Fig. 15.4p).
Vascular and Perineural Invasion
Malignant tumours can invade into vessels and metastasise to distant locations. They also follow the paths of least resistance and spread along natural tissue scaffolds such as nerves (Fig. 15.4q, r).
Immunohistochemistry
A common ancillary technique employed in histopathology to detect signature proteins using antibodies in various disease processes. In malignant tumours, it can help to type the tumour (e.g. carcinoma, lymphoma, melanoma, sarcoma) and can also reveal vital prognostic and therapeutic information.
Carcinoma
A malignant epithelial neoplasm .
Further Comments
The last comment in the report relates to the surgical margins. In this case, the contamination of the conjunctival margin may necessitate mapping biopsies to ascertain the extent of the pagetoid spread that would inform subsequent therapy choices.
Conjunctival Tumour Histopathology Report Example
The bulbar conjunctiva contains a focus of in situ squamous carcinoma, arising in a background of various degrees of dysplasia . No invasion is identified in the planes examined. The surgical margins are clear. The background stromal tissue shows solar elastosis.
Terminology Explained
Dysplasia
Derived from Greek meaning ‘disordered growth’. Dysplasia is a term that is applied to epithelium. Histologically, the individual epithelial cells show pleomorphism (defined above), can have hyperchromatic nuclei and have a disorganised distribution within the epithelium. Mitotic figures may be seen. Pathologists can grade epithelial dysplasia according to how much of the epithelial thickness is occupied by the dysplastic cells. For example, common classifications of epithelial dysplasia are mild, moderate and severe or low-grade and high-grade. When the full thickness of the epithelium is consumed by dysplastic cells and the basement membrane has not been breached, it is called carcinoma in situ and is said to be pre-invasive. Invasion is not present. Therapeutic intervention at this stage of the disease is potentially curative (Fig. 15.4s).
Intraocular Tumour Histopathology Report Example
This enucleation contains a malignant melanoma of uveal type. It is located in the ciliary body and choroid and has a diffuse component. It is composed of spindle and epithelioid cells and has up to 15 mitotic figures per 10 hpf. Closed loops and networks matrix patterns are seen. Focal tumour necrosis is present. The tumour does not breach Bruch’s membrane. There is no scleral invasion. The vortex veins are tumour-free. FISH analysis on the tumour has shown monosomy 3 and a gain of iso-chromosome 8q in 90% of the cells.
Summary: A pathological stage pT3b uveal melanoma of the ciliary body and choroid. The presence of monosomy 3 and iso-chromosome 8q indicates a poor prognosis.
Terminology Explained
Spindle and Epithelioid Cells
This refers to the shape of the cells found in uveal melanoma, referred to as the Callender classification. This is of prognostic significance; more epithelioid cells carry a worse prognosis [7] (Fig. 15.4t, u).
Per 10hpf
This is a mitotic count performed in 10 ‘high-power fields’ (each with an area of 0.152 mm2 and usually corresponding to a ×40 lens on a high-quality light microscope).
Closed Loops and Networks’ Matrix Patterns
This refers to the presence of specific basement membrane patterns stained with a periodic acid-Schiff (PAS) stain, present around expanding nests of melanoma cells. These matrix patterns are related to a poorer prognosis in uveal melanoma [8] (Fig. 15.4v).
FISH: Fluorescence In Situ Hybridisation
FISH has been used in this case to gain genetic data on the status of chromosomes 3 and 8 in this uveal melanoma. Uveal melanoma genetics data is a powerful predictor of prognosis [9,10,11,12,13] (Fig. 15.4w).
Pathological Stage pT3b
This refers to the TNM pathological classification of the stage of the uveal melanoma [14].
Further Comments
Ophthalmic pathologists will use the TNM14 or AJCC [15] cancer staging manuals to include all of the relevant tumour prognostic information required for prognosis and treatment planning for uveal melanoma.
Also note how the report has combined histopathological and genetic data. This is increasingly common and is called integrated reporting, in the context of individualised oncology practice.
Orbital Tumour Histopathology Report Example
This orbital fat biopsy contains numerous reactive lymphoid aggregates . On this background, there is a larger expansile nodule composed of small lymphocytes, with a monocytoid appearance, associated with cells exhibiting lymphoplasmacytic differentiation. These monocytoid cells are positive for B-cell markers using immunohistochemistry (CD20 and CD79a positive) and are negative for CD5, CD10, Cd23 and Cyclin D1. The Ki67 fraction is 5%. PCR for IgH rearrangement shows clonal peaks in FR1, FR2 and FR3 confirming an unequivocal clonal population of B lymphocytes.
The appearances are those of an indolent B-cell lymphoma , sub-classification: extranodal marginal zone lymphoma of MALT type (MALToma-WHO classification). This patient requires referral to your haemato-oncology service for further management.
Terminology Explained
Reactive Lymphoid Aggregates
These are non-lymphomatous collections of lymphocytes comprising a germinal centre of larger lymphocytes undergoing apoptosis surrounded by a mantle of smaller lymphocytes . They indicate chronic antigenic stimulation and contain polyclonal groups of B lymphocytes.
Monocytoid Appearance
It is common for a pathologist to describe the appearance of certain tumour cells with peculiar adjectives. Monocytoid means a lymphocyte nucleus within a clear cytoplasm, resembling a fried egg.
B-cell Markers
This refers usually to immunohistochemistry markers for staining B-cells. Common markers include CD20 and CD79a. It is vitally important that any lymphoma should be differentiated into B or T cell type for diagnostic, prognostic and treatment purposes (Fig. 15.4x).
The Ki67 Fraction
This refers to the % of cells that express Ki67. This protein is expressed in cells in all non-G0 phases of the cell cycle. It gives an indication of how fast tumour cells are proliferating. A higher Ki67 fraction tends to predict aggressive tumour behaviour (Fig. 15.4y).
PCR for IgH Rearrangement
PCR, polymerase chain reaction. IgH-heavy chain of the immunoglobulin molecule. This is a common molecular diagnostic test performed in cases of suspected lymphoma.
Clonal Population of B Lymphocytes
This means a population of B-cells all expressing the same heavy chain of immunoglobulin. Monoclonality usually means lymphoma but not always. Reactive lymphoid populations can contain monoclonal lymphocyte populations. The pathologist will always interpret the results of ancillary diagnostic/supportive molecular tests in the context of the clinical and histopathological findings (Fig. 15.4za, zb).
Further Comments
Lymphoma diagnosis often requires histopathological, immunohistochemical and molecular diagnostic tests to secure a firm diagnosis. An ophthalmic pathologist will often discuss the case with a specialist lymphoma pathologist and the case reviewed at the local multidisciplinary team meeting to plan further investigations (e.g. staging) and treatment. Again, note how the report contains integrated items of histopathological and molecular data and a suggestion for referral to an appropriate clinical team.
Common Ancillary Investigations Used in Ophthalmic Histopathology
Ancillary investigations are used to assist in making a diagnosis and to give prognostic information. The results of ancillary tests must always be interpreted alongside the light microscopic histological features in order to avoid an over or under diagnosis.
Immunohistochemistry
This is the application of antibodies to antigens in formalin-fixed, paraffin-embedded sections, visualised with various coloured chromogens. The antibodies are directed toward certain proteins that are expressed by specific cell types. Tumour cells express specific proteins allowing the tumour type to be established, which otherwise may be difficult on light microscopic examination alone.
Diagnostic Application
Example 1
Figure 15.5a shows a biopsy of the conjunctiva. The morphology shows atypical cells occupying the full thickness of the epithelium. The morphological differential diagnosis includes in situ melanoma , in situ squamous carcinoma and in situ sebaceous carcinoma. These are tumours with varying behaviour, prognosis and treatments so the diagnosis must be refined. Application of a small panel of antibodies allows discrimination. In this case, the tumour is positive for Melan A antibody (Fig. 15.5b) and negative for cytokeratin and androgen receptor. Melan A is a protein specific to melanoma cells. Cytokeratins are expressed by epithelial malignancies (in situ squamous and sebaceous carcinoma), and androgen receptor is expressed strongly by in situ sebaceous carcinoma. The final diagnosis is in situ melanoma.
Example 2
This orbital biopsy shows a lymphocytic infiltrate that histologically is a lymphoma (Fig. 15.5c). However, every lymphoma must be precisely sub-typed as each lymphoma has a specific morphology, immunohistochemical phenotype, molecular signature, prognosis and treatment (WHO classification). Application of a panel of lymphoid markers shows that this lymphoma is of B-cell phenotype (positive for CD20, Fig. 15.5d) and furthermore is positive for Cyclin D1 (Fig. 15.5e) which indicates the precise diagnosis of mantle cell lymphoma [16].
Prognostic Application
Figure 15.5f shows a uveal melanoma that shows nuclear positivity for BAP1. Figure 15.5g shows nuclear BAP-1 absence in another uveal melanoma, an indication that the tumour is of probable monosomy 3 genotype, associated with a poor prognosis [17].
Polymerase Chain Reaction (PCR)
Diagnostic Application
Sometimes, it is difficult to ascertain on light microscopy whether a lymphoid infiltrate is reactive or neoplastic. Formalin-fixed, paraffin-embedded extracted DNA can be tested for lymphocyte clonality, to establish whether a population of lymphoid cells are polyclonal (reactive-not lymphoma) or monoclonal (likely lymphoma) (Fig. 15.6a).
Prognostic Application
The states of chromosomes 1, 3, 6 and 8 allow uveal melanoma to be stratified into good and bad prognostic categories. A technique called MLPA (multi-ligation-dependent probe amplification), which is a variation of PCR, can be used to test for these chromosomal changes [18] (Fig. 15.6b).
Flow Cytometry
Diagnostic Application
This technique is used in some centres to ascertain whether a neoplastic population of B or T cells are present in vitreous or aqueous biopsies, in limited material. It is a powerful technique for the diagnosis of lymphoma [19]. Multiple antibodies directed to various lymphoid cell proteins, including kappa and lambda light chains, are applied as a cocktail to an unfixed suspension of lymphoid cells.
Fluorescence In Situ Hybridisation (FISH)
This technique uses fluorescent-tagged DNA or RNA probes to detect the presence or absence of a specific chromosome or the identification of two genes involved in a translocation. It can be used on cytology and paraffin-embedded tissue.
Diagnostic Application
Many sarcomas have distinctive, diagnostic recurrent genetic translocations. The example in Fig. 15.6c shows a FISH analysis of an orbital alveolar rhabdomyosarcoma with the diagnostic 1:13 translocation, which is also important as a prognostic marker in this tumour [20].
Prognostic Application
Uveal melanoma can have its chromosome 3 and 8 status tested by FISH [10, 12, 13] (Fig. 15.6d).
Chromosomal Microarray Analysis (aCGH and SNP)
Techniques such as array comparative genomic hybridisation (aCGH) [21] and single nucleotide polymorphism (SNP) chromosomal arrays [22] are powerful tools to detect genome-wide copy number changes in chromosomes. Furthermore, SNP arrays can detect copy-neutral changes and loss of heterozygosity. These techniques can be applied to fresh and paraffin-embedded tumour tissue successfully.
Diagnostic Applications
Can be used to help differentiate benign nevus from melanoma of the skin.
Prognostic Application
Can be used in uveal melanoma prognostic chromosomal analysis (Fig. 15.6e, f).
Tumour Prognostic Factors
Histopathology reports contain critical bits of information that will reflect the biological behaviour of the tumour; hence, its prognosis will trigger specific clinical management. What follows are brief descriptions of prognostic factors for some ocular tumours impacting on clinical management.
Every prognostic factor for every ocular tumour cannot be reproduced here. The relevant chapters in the TNM classification of Malignant Tumours (8th edition -UICC) [14] and the AJCC Cancer Staging Manual (8th edition) [15] have comprehensive lists of prognostic factors for all major ophthalmic tumours. Additionally, the tumour-reporting guidelines/datasets from the College of American Pathologists [23, 24] and the Royal College of Pathologists UK [25, 26] are a very comprehensive guide to macroscopic and microscopic prognostic factors for ocular tumours. Tumour prognostic factors from a pathological perspective can be macroscopic, microscopic and molecular/genetic .
Examples of Tumour Prognostic Factors Encountered in Ophthalmic Histopathology Reports
Eyelid Basal Cell Carcinoma [14, 15, 27]
The growth pattern of basal cell carcinoma will be mentioned in a report. Infiltrating and/or sclerosing/morphoea and/or micronodular growth patterns are high-risk growth patterns. These growth patterns correlate with a significantly increased risk for local recurrence and very occasionally metastasis, hence high risk. The clearance of the tumour in ‘mm’ from specific margins will be mentioned in the report. Contaminated margins will trigger further treatment to ensure clearance.
Conjunctival Melanoma [14, 15]
For conjunctival melanoma , the site and depth of invasion determine the ‘pT’ staging of these tumours. Tumours of the palpebral, forniceal and caruncular conjunctiva are higher risk compared to bulbar conjunctival tumours. The higher risk relates to recurrence and metastatic rates. The deeper the invasive component, the higher the pT stage.
Retinoblastoma [14, 15, 23, 24]
The presence of ‘high-risk’ histopathological factors on a histopathology report of a retinoblastoma enucleation includes ‘massive choroidal invasion’, ‘extra-scleral tumour extension’, ‘optic nerve invasion’ and ‘anterior chamber extension’. These features will lead to post-enucleation chemotherapy.
Uveal Melanoma [14, 15, 25, 26]
Iris melanomas have a much better prognosis that ciliary body and choroidal located tumours. Ciliary body and choroidal uveal melanomas with monosomy 3 and iso-chromosome 8q gain will have a poor prognosis, compared to tumours with balanced genetics.
Orbital Alveolar Rhabdomyosarcoma
Histology reports will make the distinction between the different subtypes of orbital rhabdomyosarcoma as they have different prognoses. An alveolar growth pattern which shows a 1:13 or 2:13 translocation [28] has is more aggressive and of higher risk for recurrence and metastasis compared to the embryonal variant.
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Mudhar, H.S. (2019). Ocular Pathology. In: Chaugule, S., Honavar, S., Finger, P. (eds) Surgical Ophthalmic Oncology. Springer, Cham. https://doi.org/10.1007/978-3-030-18757-6_15
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