Evaluation of suspected malignancies

Author: Mikael Häggström
For evaluation of suspected malignancies such as tumors, the most important aspect is whether it is benign or malignant. If malignant, then staging is necessary. There are generally specific criteria for various forms of tumors, which should be used whenever applicable, but following are some generalizations.

A general approach is to start looking at a slide which seems to contain non-necrotic tumor, and if possible it should also show surrounding non-tumor tissue, so that the interface can be appreciated (and tumors are generally less necrotic at the periphery).

Contents

Benign or malignant

	Benign	Malignant
Gross examination	Well demarcated from surrounding tissue	Usually no tumor capsule Possibly: Necrosis Infiltration or invasion into surrounding tissue Bleeding
Microscopy	Almost no irregularities of cellular structures	Nuclear atypia: Enlargement Pleomorphism Nuclear polychromasia, which means variability in nuclear chromatin content. Numerous mitotic figures

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  Cellular features of malignant cells versus normal cells.

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  An increased nucleus-cytoplasm ratio is generally an indication of malignancy.

Primary tumor versus metastasis

Major metastasis pathways: Main origins and sites of metastases for some common cancer types. Primary cancers are denoted by "...cancer" and their main metastasis sites are denoted by "...metastases".

Indications of a metastasis rather than primary tumor are mainly:

• Tumors that are unlikely to arise at the location at hand.
• Tumors conforming to more likely metastasis pathways.

If a suspected malignancy is present, generally check the patient history for any history of cancer, especially for tumors in more common metastasis sites, which mainly include lung, bone, liver and/or brain. In case of such history, preferably look at the microscopy slides of the past cancer to help determining whether the current case is of the same origin, versus a primary at the current body location, versus a metastasis of yet another location. If there is no known history of cancer, still consider a metastasis of unknown primary origin, especially for suspected malignancies in lymph nodes, liver, lungs, bones, or skin.
Further information: Metastasis

Histopathologic type

For specific diagnoses by organ system, see anatomic diagram on Patholines Main page. This resource will give the main steps towards reaching a diagnosis, but before making a tumor diagnosis, generally be sure that it fulfills the criteria of the condition according to The WHO Classification of tumors, and generally consult an experienced pathologist as well until you feel confident.

Visually, tumors and other suspected malignancies can usually be classified into one of the following groups:

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  Gland-like tumors, with tumor cells around clearings

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  Squamoid tumors, typically having abundant eosinophilic cytoplasm.

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  Spindle-cell tumors, with elongated cells and/or nuclei

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  Undifferentiated tumors, with few to no visual clues to their origin.

Further pinpointing of a specific tumor type is often attained by thinking of one or more possible diagnoses, and looking up their differential diagnoses, followed by comparing their microscopic descriptions and multiple micrographs with the case at hand. When two or more diagnoses seem to fit with the case at hand, consider performing immunohistochemistry. Find relevant target proteins that are expected to stain substantially differently between the possible diagnoses. If it's not evident from initial sources, you may use Immunoquery.com which will generally suggest the most relevant target proteins to distinguish the suspected conditions at hand.

Gland-like tumors

Typical features of adenocarcinomas. In reality, the appearance varies significantly from case to case. Vacuoles are more prominent in mucinous tumors, but can be seen in serous tumors as well.

Gland-like tumors are mainly evaluated for cellular atypia, architectural dysplasia and invasion, and thereby classified into the following main categories:

• Hyperplastic lesions, lacking significant atypia
• Adenomas, which can range from mild to high-grade dysplastic, yet are generally confined within their anatomic layers, that is, they are not invasive.
• Adenocarcinomas, with the main criterion being invasiveness. Evaluate specifically by location when possible. Some specific locations included in this resource:

• Colorectal adenocarcinoma
• Prostate adenocarcinoma
• Lung adenocarcinoma
• Bladder adenocarcinoma
• Esophageal adenocarcinoma
• Endometrial adenocarcinoma
• Pancreatic ductal adenocarcinoma

Degree of differentiation, with example from a colorectal adenocarcinoma.

Generally, adenocarcinomas are categorized by degree of differentiation as follows:

• Well differentiated: > 95% of tumor has glandular formations
• Moderately differentiated: 50 - 95% glandular
• Poorly differentiated: < 50% glandular

If metastatic adenocarcinoma seems to be the case (always consider this in at least lung adenocarcinoma), look at the history and radiology reports for a most likely primary. Generally, perform immunohistochemistry with one or two stains that are most typical for the suspected primary, and optionally add CK7 and CK20 as a broad screening. Further information: Metastasis

Squamoid tumors

These are more or less looking like a squamous-cell carcinoma:

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  Main characteristics of squamous-cell carcinoma

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  Typical well-differentiated nests have cells with abundant eosinophilic cytoplasm. Keratinizing centers are seen as well.

Differential diagnoses depend on location, such as:

• Squamous-cell carcinoma of the skin, with multiple differential diagnoses
• Squamous-cell carcinoma of the lung
• Urinary bladder: Urothelial versus squamous-cell carcinoma
• Cervix uteri: Cervical dysplasia

Spindle-cell tumors

For Spindle-cell tumors, the shape of the nuclei is a clue to the diagnosis, with the following tendency:

• Pointed on both ends: True fibroblastic tumors
• Pointed on one end and blunted on the other ("bullet-shaped"): Neural
• Blunted on both ends ("cigar-shaped"): Smooth muscle
• Triangular: Myofibroblastic

Evaluate specifically by location when possible:

• Spindle-cell tumors of the midgut
• Soft tissue tumor

Further histopathologic subtyping and grading

Beyond determining overall malignancy diagnosis (such as adenocarcinoma), probable origin and staging, classification of tumors into a specific histopathologic type or grade is generally of relatively less value. In cases of clearly non-malignant tumors where it is difficult to determine the specific histopathologic type or grade, it is generally acceptable to conclude the evaluation and report it as such, unless the clinician specifically requests otherwise. For potentially malignant or high-risk tumors, typing and grading often still affects the management, but generally much less so than staging.

Undifferentiated malignancy

Histopathology of a biopsy of a lymph node with cancer of unknown primary origin, showing undifferentiated high-grade tumor cells.

An initial panel of cytokeratin (CK, such as by CK AE1/AE3 cocktail), S100, vimentin and LCA (CD45) can be used (see source article for subsequent work-up).

Alternatively, a more comprehensive panel can be performed upfront, with the most pertinent panel suggested below (with main associated primary origins in parentheses), but it should be tailored to additional clues from each individual case:

Mucicarmine stain showing signet ring cell carcinoma, with the mucin within tumor cells colored pink/red.

• Mucicarmine or Cytokeratin CAM 5.2 (adenocarcinoma)

• CK7 and CK20 can give a broad indication of the primary site. Still use more specific immunohistochemistry stains instead or in addition where applicable. See table below for main patterns.
• NKX3.1 in males (prostate adenocarcinoma), or PAX-8 or estrogen and progesterone receptor in females (female reproductive tract).
• TTF-1 (thyroid or lung tumor)
• GATA3 (urothelial carcinoma, breast tumor)
• CDX2 (gastrointestinal tumor)
• p63 (squamous cell carcinoma)

• S100 (neural, invasive melanoma)
• LCA (CD45) (hematopoietic)
• Synaptophysin (neuroendocrine)

Most common CK7 and CK20 patterns in carcinomas of unknown primary site (CUPs)

		CK20
		Positive	Negative
CK7	Positive	Urothelial carcinoma Pancreatic adenocarcinoma Ovarian mucinous carcinoma Bladder adenocarcinoma Gastric adenocarcinoma Cholangiocarcinoma	Breast carcinoma Lung adenocarcinoma Endometrial adenocarcinoma Endocervical adenocarcinoma Ovarian (serous) carcinoma Cholangiocarcinoma Small cell lung carcinoma Mesothelioma Thyroid carcinoma Salivary gland tumours Kidney (papillary) Urothelial carcinoma (subset) Pancreatic adenocarcinoma Gastric adenocarcinoma Esophageal adenocarcinoma
	Negative	Colorectal adenocarcinoma Merkel cell carcinoma Gastric adenocarcinoma	Prostate adenocarcinoma Renal (clear cells) Hepatocellular carcinoma Adrenocortical carcinoma Non-seminoma germ cell tumours Mesothelioma Small cell lung carcinoma Gastric adenocarcinoma

Non-neoplastic

If a neoplasm has been ruled out for what clinically appeared like a tumor, seek a diagnosis that can be consistent with the clinical findings that caused the suspicion. If no explanation is found on the slides, generally take additional levels on the paraffin block, or more sections from any leftover tissue.

For example, for a breast biopsy of what appeared to look like a mass, and there is no neoplasia, look mainly for dense fibrosis or other fibrous changes, so that you can report it and thereby explain the finding, rather than merely writing "benign breast tissue".

Heterogeneity

After having characterized a suspected malignancy, still screen through it for any significant areas that are different and may need own mentioning, or even change the overall type or grade.

Additional levels or slices

Situations requiring additional material include mainly where tumor is expected but nevertheless not seen on existing slides. Such cases include:

• The gross report or other observation describes a tumor or polyp, but none is seen on microscopy.
• Re-excision does not identify tumor cells in a clearly non-radical primary excision or biopsy.

Also consider more material if the most aggressive pattern is seen in the last available section, in which case more sections are indicated (from the same paraffin block if additional tissue is not available).

Depending on availability and greatest suspicion, additional material is either acquired by taking addition step sections of remaining tissue in a paraffin block, or taking additional slices from the original specimen.

Staging

Staging is generally done by TNM classification. Specific TNM systems should be used whenever applicable, mainly the manual by the American Joint Committee on Cancer (AJCC) if you can access it. Further information: Secrets . Otherwise, a general system may be used:

T: size or direct extent of the primary tumor Tx: tumor cannot be assessed Tis: carcinoma in situ T0: no evidence of tumor T1, T2, T3, T4: size and/or extension of the primary tumor N: degree of spread to regional lymph nodes Nx: lymph nodes cannot be assessed N0: no regional lymph node metastasis N1: regional lymph node metastasis present; at some sites, tumor spread to closest or small number of regional lymph nodes N2: tumor spread to an extent between N1 and N3 (N2 is not used at all sites) N3: tumor spread to more distant or numerous regional lymph nodes (N3 is not used at all sites) M: presence of distant metastasis Further information: Metastasis M0: no distant metastasis M1: metastasis to distant organs (beyond regional lymph nodes)

For positive lymph nodes, generally also note any extranodal extension, and the size thereof.

Put your main focus on features that will determine the final stage. For example, if you see a lymph node involved by cancer, the presence or absence of lymphatic invasion is no longer critical, but rather the presence or absence of additional involved nodes or distant metastasis. If a stage-defining finding is uncertain, generally fall back to the lower stage.

For the size, the greatest dimension of the tumor is most important. Second and third dimensions (preferably at right angles compared to each other) are optional. Use the largest measure for each dimension. For example if one slide shows a tumor measuring 2.0 x 1.0 cm, and another slide shows the same tumor measuring 1.5 x 1.5 cm, then the tumor can be reported as measuring 2.0 x 1.5 cm. The third dimension can be estimated from gross measurement, or, if the tumor is entirely submitted, adding together the presumed thickness of each slice where tumor is found microscopically. For excisions with less than a centimeter of tumor, and where there has been a previous biopsy, consider looking at the size of tumor on the previous biopsy, which may be the largest measurement, and therefore the measurement of choice for staging.

Nx: lymph nodes cannot be assessed also applies when you only find lymph nodes that are not within the physiologic drainage path of the cancer, and they are benign.

Radicality

If tumor is seen at edge of the sample, but it is not inked, consider confirming the finding with adjacent microtomy levels, especially if no ink is seen on an inked surgical margin. In this case, a separation artifact in top image has removed a surgical margin of connective tissue, seen on adjacent microtomy section in bottom image.

Determine if malignant cells are located close to, or even in, any surgical resection margins.

Lymphovascular invasion

Lymphovascular invasion should always be mentioned. When present at margins, it does not count as tumor extension.

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  Endometrial cancer with lymphovascular invasion.

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  If unsure whether a case is true lymphovascular invasion or a retraction artifact (imaged), use immunohistochemistry for D2-40 to highlight lymphatic vessels or CD-31 for blood vessels.

Molecular workup

Ensure that any cancer undergoes reflex testing where applicable by local guidelines. When they are not applicable, a rule of thumb is to order individual genetic testing if there is a need to test up to 2 genetic targets, and to order genomic sequencing if more targets need genetic testing (not counting immunohistochemitry or other more easily performed tests).

When needing to send out tissue to an outside laboratory, follow their requirements and guidelines for each test. Generally, choose the tissue block with the greatest amount/area of the highest grade malignancy. For molecular proliferation tests (other than immunohistochemistry) such as Ki67, avoid samples with previous biopsy site or inflammation (as these will cause a false high proliferation rates). On the other hand, for next-generation sequencing, hemorrhage, necrosis and adipose tissue does not need to be minimized, as these contain only little genetic material.

Reporting

Also note "treatment effect", seen as fibroelastotic tissue, here with scattered remaining tumor cells.

For cancers, generally include a synoptic report, such as per College of American Pathologists (CAP) protocols at cap.org/protocols-and-guidelines. If there is no CAP synoptic available for the cancer type at hand, attempt to find one in the latest AJCC Cancer Staging Manual (see the Secrets article), but make sure to use pathologic staging (with a p preceding the T, N or M) rather than clinical staging.

If a surgery produces a specimen with cancer, as well as re-excisions from certain directions, you should preferably give the closest distance to margins in each specimen, as well as the closest distance overall in a synoptic, for example:

A. (Specimen with most of the cancer) (...) Invasive carcinoma is present at inked medial margin (see specimen B for final medial margin), and is located 0.3 cm from the lateral margin. B. (Re-excision in the direction of the medial margin) (...) Invasive carcinoma is located 0.5 cm from the new medial margin. Synoptic report (...) Distance from invasive carcinoma to closest margin: 0.3 cm Closest margin(s) to invasive carcinoma: Lateral

If possible, generally include features of any previous biopsies or smaller excisions in synoptic reports and staging. For example, if staging is based on tumor size, and the tumor size in a previous excisional biopsy corresponds to a higher stage than the size of residual tumor in a subsequent wider excision, then the report of the latter should give the higher stage, with a comment thereof, such as:

pT__

- Based on previous excisional biopsy (specimen ID: ______)

  See also: General notes on reporting

General notes

Further reading: Starting pathology (handbook) Learning pathology Emergency pathology Fixation Gross processing Evaluation: Suspected malignancies Immunohistochemistry Consultation Reporting Secrets

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