Template:Cytology, tumors, evaluation of suspected malignancies, immunohistochemistry, consultation and reporting

Cytology introduction

Overall evaluation

In addition to a general evaluation, also evaluate the following in cytology samples, or any sample with scattered cells rather than coherent tissue:

• Consider looking for any additional concurrent samples from the same patient, especially excisions, as usually correlate.
• Adequacy of specimen. Cells may be too few or too obscured by other material to make a proper diagnosis.
• Background, mainly if it is clear or dirty
• Overall cellularity

Artifacts

• 

  Smearing of cells across a glass plate may cause smearing artifacts, such as the nuclear smearing of this monocyte, shown as tail-like extension of nuclear material.

• 

  Cervical squamous cells with cornflake artifact, when some mounting medium evaporates before coverslipping. The glass slide can be partially re-processed to remove the artifact.

Tumors, introduction

Gross processing

Gross tumors according to organ and/or tumor type (as per above) whenever possible. In additional to general gross processing guidelines, the following instructions are usually acceptable:

• Identify surgical margins where tumor involvement may be necessary to identify or negate.

• Generally ink such margins.

• Section the specimen so as to get a gross overview of tumor extent.
• (Take a gross photograph of the tumor.)
• Describe the tumor, minimally by color and consistency (firm/rubbery versus semisolid), and possibly also including diffuse versus well-demarcated.
• Measure tumor dimensions and distances to relevant margins.
• Sampling of generally at least one slice per centimeter of tumor (which for larger tumors may be submitted as 2-3 sections per cassette).

Further information: Gross processing

By gross appearance

• 

  Lipomatous tumor

Evaluation

The most important aspects of a tumor is whether it is malignant or not, and staging.

Evaluation of suspected malignancies

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).

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

• 

  Cellular features of malignant cells versus normal cells.

• 

  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:

• 

  Gland-like tumors, with tumor cells around clearings

• 

  Squamoid tumors, typically having abundant eosinophilic cytoplasm.

• 

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

• 

  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:

• 

  Main characteristics of squamous-cell carcinoma

• 

  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

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.

• 

  Endometrial cancer with lymphovascular invasion.

• 

  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

Metastasis

Gross processing

For suspected tumors in bone or other tissue that generally needs decalcification, try to grossly separate some suspicious soft tissue to be processed without decalcification, as decalcification might affect any molecular studies that will later be indicated. See site-specific grossing guidelines for further directions.

Metastasis of unknown primary

Metastases of unknown primary origin

Histopathologic type - see section below for descriptions	Relative incidence among metastases of unknown primary origin
Well and poorly differentiated adenocarcinomas	50%
Undifferentiated carcinoma	30%
Squamous cell carcinoma	15%
Undifferentiated neoplasms	5%

• Memorization-worthy: Do not diagnose a renal cell carcinoma metastasis without radiologic evidence of a renal tumor. To be a plausible primary tumor for a renal cell carcinoma metastasis, a renal tumor should be visible radiologically. In cases of suspected renal cell carcinoma but no renal imaging is available, it is reasonable to ask the ordering doctor to allow you to wait with signing out the pathology report until renal radiology has been performed.

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

Metastatic adenocarcinoma

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.

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.

Generally, classify by degree of differentiation as follows:

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

Degree of differentiation, with example from a colorectal adenocarcinoma.

Example report:

A. Right lung mass, CT-guided needle biopsy: Metastatic adenocarcinoma, moderately differentiated. Comment: Immunohistochemistry was performed, and showed tumor cells positive for CDX2 and CK 20, and negative for CK 7, consistent with metastasis from a gastrointestinal primary tumor.

Further workup

Perform additional biomarker testing as per local guidelines for the corresponding primary tumor at metastatic/advanced stage (such as mismatch repair proteins and next generation sequencing in case of metastatic colon adenocarcinoma).

Immunohistochemistry

Main staining patterns.

When learning pathology, the percentages by which immunohistochemistry (IHC) results are positive or negative for various diseases are generally easily looked up when needed, so what a pathologist needs to learn is mainly how to select the optimal immunohistochemistry panels in the first place for various presentations where the diagnosis is unknown.

Immunohistochemistry ordering

The main approaches to immunohistochemistry ordering are:

• If there is insufficient tissue left for immunohistochemistry after standard (usually H&E) staining, you can potentially ask a histotechnologist to destain a glass slide and subsequently perform IHC on that slide. For small specimens, if you predict that there will be several stainsin the near future, order extra unstained slides upfront (since there is always a bit of a waste of tissue at the microtome if the paraffin block needs to be remounted and cut later).
• Look for a specified panel for the presentation at hand. For example, for an undifferentiated tumor with no clear lineage differentiation, an initial panel of cytokeratin (CK), S100, vimentin and LCA (CD45) can be used. Further information: Tumor evaluation
  In cancer cases, also be familiar with local routines for reflex tests by various cancer types, which may include both immunohistochemistry and other molecular tests.
• Coming up with the most relevant differential diagnoses for the case at hand, and find the immunohistochemistry stains that best distinguish them. Immunohistochemistry profiles for diseases and conditions, as well as their main differential diagnoses, is generally found at Pathology Outlines, or you can pay for a subscription to ImmunoQuery:

Using Immunoquery

The main page of ImmunoQuery gives you the "Diagnoses" option, where you enter up to 3 differential diagnoses to generate the optimal immunohistochemistry panel to differentiate them (you may need to click ∨ Suggested Panel to show it if it is collapsed). It also displays an automatic message when the included antibodies/immunostains are not sufficient for a satisfactory panel, in which case you can:

• Make a specific search only including the two conditions where suggested immunostains were insufficient, if you had previously compared 3 diagnoses.
• Consider additional stains from the "Comprehensive panel" displayed below the suggested one.
• Switch the "Sensitivity" setting (seen at top) from 1 (which means that diffuse, focal as well as not specified staining count as positive) to 3 (which means that only diffuse staining counts as positive whereas both focal and absent staining count as negative, and references without any specified staining pattern are omitted from the analysis). This has less data to support the suggested stains (since many references do not specify whether positivity was diffuse or focal), but can sometimes state a better distinction between conditions. When including a stain based on its distinguishing features on a sensitivity setting of 3, you need to keep the practice of classifying only diffuse staining counts as positive, and focal to absent staining as negative.

Test question ImmunoQuery for a squamous cell carcinoma Squamous cell carcinoma, with large cells with abundant eosinophilic cytoplasm and large, often vesicular, nuclei. The attending gives you a lung biopsy case to preview. You are first uncertain about the type of tumor, so you ask a fellow resident, who finds a diagnostic area of the tumor and tells you that this is a typical squamous cell carcinoma. You also look through the patient's history, and find that the patient has had a squamous cell carcinoma of the anus in the past, and you now want to find out whether the tumor originated in the lung, or if it is a metastasis from the anus, or possibly the skin. You therefore do an ImmunoQuery lookup, with the following results: Suggested Panel Insufficient antibodies for a satisfactory panel to differentiate Lung squamous cell carcinoma and Anus squamous cell carcinoma Antibodies Lung SCC Anus SCC Skin SCC EpCAM 81% Positive Membrane, Cytoplasm 75% Positive Membrane, Cytoplasm 0% Positive Membrane, Cytoplasm GATA3 5% Positive Nucleus 20% Positive Nucleus 84% Positive Nucleus p16 17% Positive Cytoplasm, Nucleus 87% Positive Cytoplasm, Nucleus 45% Positive Cytoplasm, Nucleus You go talk with the attending, who agrees that EpCAM, GATA3 and p16 should be in the panel, but just as ImmunoQuery also tells, the attending thinks that the panel is not satisfactory to differentiate lung SCC from anus SCC, and wants you to add one more stain to improve the panel. You go back to ImmunoQuery and increase the Sensitivity from 1 to 3 (which means that only diffuse staining counts as positive whereas both focal and absent staining count as negative, and references without any specified staining pattern are omitted from the analysis), and get the following results: Suggested panel Antibodies Lung SCC Anus SCC Skin SCC EpCAM 74% Positive Membrane, Cytoplasm 50% Positive Membrane, Cytoplasm 0% Positive Membrane, Cytoplasm DLK 28% Positive Membrane, Cytoplasm N/A Membrane, Cytoplasm 100% Positive Membrane, Cytoplasm You also perform a repeated search by only entering Lung and Anus SCC, and you get the following results: Suggested panel Antibodies Lung SCC Anus SCC p16 17% Positive Cytoplasm, Nucleus 87% Positive Cytoplasm, Nucleus Comprehensive panel Top results: Antibodies Lung SCC Anus SCC p16 Cytoplasm, Nucleus 17% 87% GRPR Cytoplasm 56% 100% You switch sensitivity from 1 to 3 for this result as well, showing: Suggested panel No antibodies to differentiate Lung squamous cell carcinoma and Anus squamous cell carcinoma Comprehensive panel Top result: Antibodies Lung SCC Anus SCC GRPR Cytoplasm 46% 91% You go talk with a technician at the histology lab, and your hospital offers all the stains in the alternatives, at similar costs, so you don't have to think about expenses and logistics of sending the case out to external labs. In addition to EpCAM, GATA3 and p16, what is the best alternative, given the information you retrieved above, to differentiate lung versus anus squamous cell carcinoma? Add DLK to the panel, and favor anus origin if having a diffusely membranous staining rather than diffusely cytoplasmic. Add DLK to the panel, and favor anus origin if having a diffusely cytoplasmic staining rather than diffusely membranous. Add GRPR to the panel, and favor anus origin if having either a diffuse or focal cytoplasmic staining. Add GRPR to the panel, and favor anus origin if having a diffuse but not a focal cytoplasmic staining. The ImmunoQuery results above are not sufficient to suggest an additional stain besides EpCAM, GATA3 and p16. Correct answer GRPR is the top result when specifically comparing lung versus anus squamous cell carcinoma, both at sensitivity settings 1 and 3, with no major difference between them, and thus anus origin is favored in case of either diffuse or focal cytoplasmic staining. DLK showed as N/A for anus SCC.

Chromogen color

Request a red rather than brown chromogen in heavily pigmented lesions, such as in some melanomas.

Evaluation

First be familiar with which cells on the slide are being evaluated, such as first looking at a slide with standard staining (usually H&E) to avoid counting background cells.

When the relevant cells take up stains, confirm whether their staining pattern actually counts as positive. For example, a cytoplasmic staining for p63 still counts as "negative (cytoplasmic)" when suspecting squamous, myoepithelial and prostatic basal cells (where only nuclear stain counts as positive). On the other hand, the same cytoplasmic staining for p63 counts as "positive (cytoplasmic)" when suspecting muscle differentiation.

Interpretation

The main methods for interpreting immunohistochemistry results are:

• Looking up each differential diagnosis at for example Pathology Outlines and comparing their expected staining to see which entity is most likely.
• Paying for a subscription to ImmunoQuery, where you can enter immunohistochemistry results and generate a list of most likely conditions with that profile.

Preferably, immunohistochemistry results will be very specific or sensitive for a suspected condition, thereby confirming it if positive, or excluding it if negative, respectively. Even when that is not the case, immunohistochemistry can at least alter the likelihoods of different differential diagnoses. In practice, clinicians or pathologists do not state exact or even approximate numbers of likelihoods of differential diagnoses (Further information: Reporting see the Reporting chapter for phrasing uncertainty), since reality is too complex for that, but to demonstrate the general principle of how immunohistochemistry results can be calculated, the following formula can be used:

• Gross likelihood of a disease/condition = (Pre-test probability) x (Probability that the condition shows the immunohistochemistry results at hand).

The pre-test probability is a product of for example the incidence of the condition in the patient's epidemiologic type such as age and sex, as well as the probability that the condition would have caused the clinical course, including signs and symptoms, as well as the microscopic impression. For example, if you want to differentiate a pleomorphic liposarcoma from a pleomorphic rhabdomyosarcoma in soft tissue, you may find in ImmunoQuery that the following stains are most efficient in distinguishing the two, with the following percentages of being positive:

	Soft tissue pleomorphic liposarcoma	Soft tissue pleomorphic rhabdomyosarcoma
Desmin	17%	95%
Actin HHF-35	0%	71%

Let's say for example that your pre-test probability was about 30% for pleomorphic liposarcoma and 70% for pleomorphic rhabdomyosarcoma, that desmin stains positive, and actin HHF-35 stains negative in this case. Assuming that 0% positive rate means that 100% stain negative, the probability that pleomorphic liposarcoma shows these immunohistochemistry results at hand is:

• 17% x 100% = 17%

The gross likelihood of pleomorphic liposarcoma therefore becomes:

• 30% x 17% = 5.1%

In the same way, the corresponding gross likelihood of pleomorphic rhabdomyosarcoma is calculated as:

• 70% x 95% x (100% - 71%) = 19%

As a result in this case, immunohistochemistry resulted in pleomorphic rhabdomyosarcoma going from about twice as likely compared to pleomorphic liposarcoma to about 4 times as likely. Although results like these do not make major differences individually, detailed calculations of results from a large panel of immunohistochemistry stains can have a major impact when taken together, even if each stain has relatively low sensitivity and specificity.

Reporting

Following are general notes on reporting in pathology.

• Save your digital work frequently, and also before you leave a computer, even if you think you'll be back shortly. If you have many small specimens to write up in the same report, you may want to save every 2 to 3 specimens. It doesn't matter how much time and effort you spend on something if you're just going to let it disappear in the next glitch.
• Double-check your report, especially if you copy-pasted and adapted a previous report rather than using a template with blank fields or making your report from scratch.

Components

Selection and trimming

From the stage of selection and trimming, a histopathology report should preferably include:

• Case:

• Patient identification and/or sample number
• Type of tissue sample as described on container

• Gross processing:

• Dimensions of original tissue
• Directions or other features of any inked surfaces.
• Generally the weight of larger samples
• Dimensions of pathologic components
• Whether the entire specimen or representative sections were submitted.

Microscopic evaluation

• Specimen chronology, often A, B, C, etc., at least where there are multiple specimens from the same case. With multiple specimens, preferably write out the chronology for all of them first, so that you don't miss reporting any of them later.
• Specimen type and/or surgery type, such as "appendix, appendectomy", for clarification. This is not necessary at all departments. For the procedure, use the same term as the operating report whenever possible.
• Microscopic description. This is not always necessary, but should be included if the diagnosis is uncertain. One systematic approach is to describe findings from largest to smallest ones. For example, a description of a tumor can start with the demarcation of the tumor, followed by texture, cell shapes, nucleus shapes and chromatin appearance.
• Diagnosis or most probable diagnoses.

• If the diagnosis does not clearly account for all conditions that were requested, suspected or asked to be ruled out by the referring clinician (such as stated on the requisition form), you need to classify the specimen as "positive for" versus "negative for" for each such condition, or give a reason for why an evaluation thereof could not be made.

• In case of malignancy or suspected malignancy:

• Depth or most distant invasion of malignant findings. Depending on location, it may need to exclude important pathways, such as vascular, neural and/or through capsules or other layers.
• Whether the resection is radical or not.

Depth

Factors supporting a relatively more comprehensive report, particularly in the inclusion of negated findings:

• Lack of explanation from existing evidence. For example, an inflamed appendix that fits the medical history does not need detailed mention of harmless incidental findings.
• Prospective review: If your report is likely to undergo double-reading by another pathologist before sign-out, it should either be more detailed, because the doctor who will do the double-reading then gets an idea of your thought process, including what you have looked for versus what may still need to be evaluated. If you know who will do the prospective review for a report of yours, you may alternatively convey your thought process by other means such as directly talking to that person.
• Highly suspected locations, such as given from the referral.
• Difficulty in obtaining the specimen, such as a CT-guided biopsy versus a skin shave.
• Defensive precautions, which appears to be more common among doctors in the Unites States compared to for example Europe.

Multiple instances of the same type of pathology (such as lung nodules) can often simply be reported as such, at least with a particular mention of the largest or the most severe example thereof.

Uncertainty

Words, from
most likely to
least likely

(is) positive for

probably likely

suggesting

suspicious for

possibly

(benign condition) cannot be excluded

not likely

(malignant condition) cannot be excluded

negative for effectively ruling out

When something looks very much like a specific entity but you are not sure, preferably use "-like" (or when feasible, "-oid" such as squamoid for squamous-like cells).

When the clinical picture strongly favors a certain condition, and the pathology favors it as well, findings are generally described as "consistent with". Sometimes, "bordering on" can be described when the picture almost fits specified criteria of a specific diagnosis.

It is alright to consider the diagnosis of a pathology report to be a combination of the clinical picture and what can be seen on the specimen. For example, if the microscopy picture is uncertain, you may to a certain degree tend towards the diagnosis that best fits the clinical picture. However, mention differential diagnoses if they are still significantly possible, and would confer a different treatment or another substantially different consequence.

For both findings and diagnoses, is preferable to write "negative for..." rather than "no..." to emphasize the possibility of false negative findings.

Synoptic reports

For cancers, generally include a synoptic report, such as per College of American Pathologists (CAP) protocols at cap.org/protocols-and-guidelines. However, synoptic reports are generally not needed for tumor metastases.

Sizes

Whenever possible, give numerical quantities of sizes, rather than descriptions that are subjective (such as "small" or "large") or variable (such as "apple-sized").

Tailoring

The information contained in the reporting sections in this resource assume that the clinician has requested the exam for the topic at hand, but should still be tailored to any particular questions or requests by the clinician. Any relevant findings beyond the issues or questions raised by the clinician should also be mentioned.

As there are generally local practices about how to write reports, it is recommended to have either a personal or practice-specific repository of report templates that you can copy-paste for various cases. When doing so, however, use marks or empty spaces for relevant findings that are frequently changed in the template. Such marks should be conspicuous, so that the report clearly looks unfinished if you haven't tailored it to the case at hand (such as "...measuring _____."). Thereby, you avoid omissions or even wrongly entered information from templates.

The most important findings are preferably moved to near the top of the report if feasible.

Example of report format:

A. Organ, side/site, procedure: Diagnosis 1. Diagnosis 2.

Wording

If a certain grammatical rule has a risk of making the report less clear to the reader, ignore that rule in that situation.

Restrict acronyms/abbreviations to those who are certainly well known among all doctors, such as "cm".

Generally describe what can be seen rather than processes (such as preferring "an abundance of" rather than "proliferation of").

If using a dictation device, avoid "no", and instead use "negative for" (and "positive for" in opposite cases), since there's a risk of "no" not being transcribed and thereby creating the opposite meaning.

Avoid "evidence of" unless you feel comfortable with the legal implications of its meaning in the report.

Whenever there is text needing formatting (text size, font type and/or UPPER vs lower case), it is generally most efficient to do it all at once after all the text is written.

Skin excisions

In skin cancers, use "peripheral" or "radial" margins (whereas "lateral" margin should be reserved for the margin opposite to the medial margin).

Main page

• Main page of Patholines