InCyte Pathology's Blog

Thyroid Diagnosis – Can Be Tricky Business

incytepathology — Thu, 26 Jan 2012 16:03:40 +0000

A recent Wall Street Journal article, titled “What if the Doctor is Wrong?,” reported the story of a woman who requested a second opinion after receiving a diagnosis of probable ovarian cancer. In the article, the patient presented with multiple tumors in her pelvis, abdomen and spine. When her initial biopsy came back with inconclusive results, clinicians felt that she had ovarian cancer and should be treated with a hysterectomy. The patient decided to postpone surgery and get a second opinion. During the course of the second opinion, a second biopsy was performed which determined that in fact she had a rare form of lymphoma, which can be difficult to diagnose.

Some malignancies including lymphomas as well as rare cancers of the thyroid and salivary glands can be very tricky to diagnose. Test results may return as inconclusive or in some cases provide false results. Recently, the president of Argentina had her thyroid removed after being diagnosed with cancer from a biopsy. It was revealed after the surgery that she had a benign condition.

There are other considerations that may lead to an inconclusive diagnosis. Testing for thyroid cancers requires a procedure called a fine needle aspiration (FNA). These cases provide their own unique set of challenges because the quality of the specimen is highly dependent on the skill of the clinician performing the procedure. The ability of that individual to create readable, well preserved smears containing sufficient numbers of diagnostic cellular material and lacks obscuring debris, excessive blood, air drying and other artifacts is critical to how well the pathologist can diagnose disease.

InCyte pathologists interpret over 1,700 thyroid specimens annually, approximately 70% of these are fine needle aspirations (FNA). InCyte Pathology recognizes the importance of a second opinion for difficult thyroid biopsy interpretations. All of our pathologists are board certified, with many having subspecialty training in cytopathology.

One of the benefits of having 24 board certified pathologists at InCyte Pathology, is that difficult cases can be reviewed by the group. InCyte pathologists follow the practice of having all cancer cases and unusual cases reviewed by at least one other pathologist. Sometimes, when appropriate, multiple pathologists are asked to review the same case.

Additional consults with recognized experts outside of InCyte Pathology may also occur in very difficult or unusual cases. When such a consultation is sought, the referring physician is notified because an outside consultation may delay the diagnosis for up to two weeks, which may cause undue stress for the patient and their family. Therefore, knowing when it is appropriate to use an outside consult demonstrates good practice, brings additional expertise to the diagnostic process, and provides clinicians enough information to create a treatment plan appropriate for each patient.

InCyte Pathologist in 2012 Top Dentists List

incytepathology — Wed, 25 Jan 2012 00:20:04 +0000

Robert Achterberg, D.D.S., M.S. was recently selected by his peers for inclusion in topDENTISTS 2012 in the field of Oral Pathology. Dr. Achterberg joined InCyte Pathology in 2003 and is currently the Oral & Maxillofacial Pathologist at InCyte Pathology. He is board certified in both oral medicine and maxillofacial pathology and is a past member of the American Dental Association Council on Scientific Affairs and the Washington Dental Quality Assurance Commission. Dr. Achterberg has been listed in topDentists since 2009.

“InCyte Pathology congratulates Dr. Achterberg for his inclusion in topDENTISTS 2012 again this year,” said Dr. David Hoak, Medical Director, InCyte Pathology.

The Molecular Pap: E6 and E7 Hold Promise for Cervical Cancer Screening

incytepathology — Fri, 30 Dec 2011 00:33:50 +0000

Molecular testing offers great promise in detecting cellular abnormalities in women whose DNA has been transformed and, therefore, in identifying women at greatest risk of having or developing cervical cancer. How might a new molecular test for E6, E7 affect current cervical cancer screening strategies?

Currently, our basic strategies for cervical cancer screening can be summarized as follows:

Exclude CIN 2-3 (HSIL)
Determine the risk
Rule out invasion
Establish the next screening interval

The first two strategies listed here are often interchangeable in their occurrence, and our means of accomplishing these steps are summarized in Table 1.

Screening Step	Action/Means/ Considerations	Notes
Determine the risk	Age Pap Test (HPV test)	Women under 21 are not tested for HPV by ASCCP guidelines Clinical risk factors (multiple sexual partners, smoking, etc.) are not incorporated into ASCCP guidelines Positive HPV test neither confirms nor excludes possibility of HSIL (CIN 2-3) hrHPV testing evaluates for HPV subtypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68 Typing for HPV subtypes 16, 18 is a separate means of risk stratification testing HPV testing for LSIL not recommended by ASCCP
Exclude CIN 2-3	Routine Pap testing (or colposcopy) Age	Pap tests results are very specific, but lack sensitivity HPV+ women over 30 y/o fall into higher risk category
Rule out invasion	Colposcopy	Biopsies of abnormal areas to exclude CIN 2-3 and invasion
Establish the next screening interval	Inform patient of when next cervical collection will occur	ASCCP guidelines used for determining next interval

Table 1

Hypothetical 25 y/o Patient

Let’s use a hypothetical 25 year old patient with ASC-US Pap results to illustrate the process outlined in Table 1.

1) Exclude CIN 2-3+

Our hypothetical patient has an abnormal Pap test (ASC-US) for which CIN 2-3 is not excluded.

The ASC-US – LSIL Long Term Follow-Up Study (ALTS) found that women having ASC-US Pap results AND who have a positive high risk HPV (hr HPV) test were found to have a significant CIN 2-3 cervical lesion on colposcopic follow-up in approximately 10-20% of patients. Therefore, the American Society for Colposcopy and Cervical Pathology (ASCCP) guidelines specify colposcopic evaluation to rule out the presence of high grade squamous intraepithelial lesion or invasive carcinoma in patients having ASC-US Pap results AND a positive HPV test.

2) Determine the Risk

If our patient, age 25, had a normal Pap test, she would return in 1 or 2 years for her next routine Pap depending on which interval an office chooses to follow.

For our hypothetical patient, however, the Pap test has Atypical Squamous Cells of Undetermined Significance (ASC-US), and a reflex HPV test is now performed to assess the patient’s risk category.

An abbreviated algorithm followed for this patient looks like this:

It is important to understand that the HPV test is a risk stratification test, not a test for cervical cancer or its precursors. Positive HPV results signify nothing more than placement of the patient into a higher risk category. It is the patient’s risk category that helps stratify the approach to the next steps in her care.

Negative HPV results would place the patient into a category having the same risk as patients with a normal Pap test. Hence, a patient with negative hrHPV testing would return to the “normal pool,” almost as if she had never had an abnormal Pap result to begin with.

3) Rule Out Invasion

The Pap test can report squamous carcinoma when specific cytologic criteria are present, but the gold standard to exclude invasive disease is colposcopy with directed biopsy of abnormal-appearing regions of the cervix.

4) Establish the Next Interval

For our patient, if her hrHPV test was negative, she would return at the routine interval for her next Pap test. If our patient had positive reflex hrHPV testing, colposcopy is recommended to rule out 2-3+. Cervical biopsies positive for high grade dysplasia or invasive carcinoma would initiate therapeutic intervention.

For our hypothetical patient, her colposcopy and biopsy are negative and she is instructed to return in one year for repeat Pap test and hrHPV testing per ASCCP algorithmic guidelines.

Discussion

It is important to note that the HPV test is a risk stratification test that, by helping establish risk category, adds sensitivity to the routine Pap testing protocol and helps determine which patients should undergo colposcopy. There are evidence-based algorithms for ASC-US and the results of subsequent reflex hrHPV testing.

Not all patients with HPV infection will develop invasive cervical cancer.

Important question: If not every woman with HPV infection will develop invasive cancer, why do we currently approach LSIL and ASC-US abnormalities as if the patient could have invasive cancer?

Answer: Given evidence-based studies and existing technology, this approach represents our current state-of-the-art strategy. Colposcopy is performed following LSIL/ASCUS, +HPV Pap results to rule out CIN 2-3+ and invasive disease.

The Wish For A Better Test

Future testing will need to focus on identifying women who are most likely to have potentially lethal cervical disease. Stated differently, we need a test that will separate those women having mere transient HPV infection and its associated non-lethal Pap and biopsy abnormalities from those having HPV-driven neoplasia.

The Better Test May Be Here

Integration by HPV into the host’s DNA is a key step in neoplastic transformation (see Glossary). An exciting new molecular approach in identifying transformed cells uses the principle of identifying two HPV gene products, E6 and E7, that are overexpressed in cells where HPV has integrated into the host genome and has initiated neoplasia (see Glossary).

Review of the Role of HPV Infection in Cervical Cancer

Cellular Infection

HPV invades the skin or mucosa by entering tiny breaks in the surface (even those not visible to the naked eye). Once inside, HPV infects host epithelial cells, tricking them into producing new viruses. Then, in the process of normal cell replacement, the infected cells die, releasing viral particles.

High risk strains of HPV can integrate their viral DNA into the host’s DNA. This is not a normal part of the HPV life cycle and is felt to occur in only a minority of HPV infections. Viral integration, however, may give infected host cells a selective advantage, leading to a longer infection time and, in the case of malignant transformation, cellular immortality. The longer the infection lasts, the more time there is for integration and abnormal cellular events to develop.

The HPV genome contains several genes that encode proteins. In the HPV genome, three of these genes — E2, E6, and E7 — are of particular interest because of their roles in the development of cervical cancer. The E2 protein functions by binding to both the E6 and E7 proteins. When E6 and E7 are bound to E2, they are blocked from their normal actions in the cell.

Oncoproteins E6 & E7 Block Production of Two Suppressor Proteins: p53 & Rb

Overview

After HPV integration into the host’s DNA, the viral genes E6 and E7 may be over-expressed, creating proteins that prevent the activity of key tumor suppressors. Here, E6 can inhibit p53, a protein that controls responses to different types of cellular stress, including DNA damage and viral infection (see Glossary). The E7 protein can inhibit Rb, a protein that can prevent cell division by blocking the activity of transcription factors.

The combined effects of E6 and E7, therefore, can put cells at risk for undergoing uncontrolled division that can lead to cancer.

E6 – Can Bind p53

When E6 is not bound to E2, it is free to bind to the p53 tumor suppressor protein. Tumor suppressors are normal occurring anti-cancer proteins. When E6 binds to p53, p53 is destroyed and cannot function. The p53 protein is a key element in cell cycle control and is not functional in over 50% of all human cancers. Without the p53 protein, a cell has reduced ability to respond to DNA damage and may continue to divide even if it is damaged (see Glossary).

The E6 protein causes the expression of telomerase, a protein that is not normally produced by most cells in adult humans. When present, telomerase maintains the ends of the chromosomes. This prevents the breakdown of chromosomes and helps cancer cells to divide unchecked — essentially forever.

Since the expression of E6 is strictly required for maintenance of a malignant phenotype in HPV-induced cancers, the E6 proteins are a target of therapeutic HPV vaccines.

E7 – Can Bind Rb

The HPV protein E7 also plays a key role in helping HPV manipulate control of infected cells by its antagonism of a cellular transcription factor, E2F, which causes the cell to proceed along the cell cycle and to divide. When not bound by E2, E7 binds to the retinoblastoma protein (Rb). When E7 is bound to Rb, Rb cannot carry out its normal function, which is to bind to E2F, the transcription factor that causes cell cycle progression.

When bound to Rb, E2F cannot function as a transcription factor and, therefore, cannot help the cell to divide. But, if E7 binds to Rb, E2F then cannot bind to Rb, and is thereby free to act unchecked as a transcription factor, a critical step in cancer development.

In essence, E7, by binding to Rb, “inhibits an inhibitor” of cell division, thereby preventing cell death (apoptosis) and, subsequently, promoting cell division (see Glossary). E7 also participates in immortalization of infected cells by activating cellular telomerase.

Simply stated, the E6 and E7 proteins help HPV hijack cell division to become immortal and to help drive cellular neoplasia.

E6 E7 mRNA – A Molecular Probe Test

One example of a new molecular test developed to detect transformed cells is HPV OncoTect^TM E6, E7 mRNA Kit, a product of the InCell Dx company. Although not an endorsement by InCyte Pathology, the following promotional literature from InCell Dx highlights the exciting capabilities of molecular testing which is available to be performed on Pap collections.

The HPV OncoTect^TM E6, E7 mRNA Kit is a unique detection method that measures both the number of transforming cells and the quantity of E6, E7 mRNA in each cell. These two measurements precisely assess the overexpression of E6, E7 mRNA in routine patient samples collected in ThinPrep® and Surepath^TM vials, thereby further refining accuracy and specificity of HPV testing.

Many women with positive test results from a HR HPV DNA Test will have normal biopsy. The HPV OncoTectTM E6, E7 mRNA Test is only positive if there is overexpression of E6, E7 — a sign of neoplastic transformation. In the life cycle of the human papillomavirus, the overexpression of E6, E7 mRNA in a cell is the molecular switch leading to cervical cancer.

__________________________________________________________________________

SUMMARY

Molecular testing offers great promise in detecting cellular abnormalities in women whose DNA has been transformed and, therefore, in identifying women at greatest risk of having or developing cervical cancer. This new testing employs detection of gene products E6 & E7, a molecular approach that provides greater specificity to current cervical cancer screening strategies.

Our Hypothetical Patient

How might a test for E6, E7 affect our hypothetical patient with the ASC-US Pap test? If molecular testing for transformed cells lives up to its promise in the future, an ASC-US result for our patient may dictate E6, E7 testing performed instead of hrHPV testing, and there are those who are discussing that this type of testing (or similar molecular approaches) could replace the Pap test altogether. Seems premature, but there is no question that molecular testing will have impact on the ever-evolving strategies in cervical cancer testing. In the meantime, we employ ASCCP guidelines and the four basic strategies that we have discussed. Stay tuned.

GLOSSARY OF TERMS

Term	Definition
Early HPV proteins	The HPV genome is composed of six early (E1, E2, E3, E4, E6, and E7) and two late (L1 and L2) genes. Majority components of the exterior shell that surrounds the circular HPV DNA are coded for by the ‘E’ genes of the HPV genome.
Late HPV proteins	The HPV genome is composed of six early (E1, E2, E3, E4, E6, and E7) and two late (L1 and L2) proteins are keystone corners of the decahedron shell that surrounds the circular HPV DNA and are coded for by the ‘L’ genes of the HPV genome.
upstream regulatory region (URR)	HPV genomes isolated from cervical cancers often contain nucleotide sequence alterations in the HPV upstream regulatory region (URR) that controls viral-gene transcription and drives viral replication.
neoplasia	Neoplasia is abnormal and irreversible proliferation of cells. The growth of neoplastic cells exceeds and is not coordinated with that of surrounding normal tissues. Abnormal growth persists in the same excessive manner even after cessation of stimuli. Neoplasia often causes a lump or tumor. Neoplasms may be benign, pre-malignant (carcinoma in-situ), or malignant (cancer).
telomerase	Telomerase is an enzyme that adds DNA sequence “repeats” to the end of DNA strands in the telomere regions, which are found at the ends of eukaryotic chromosomes. Telomerase consists of repeated nucleotides containing non-coding DNA material. Telomerase prevent constant loss of important DNA from chromosome ends.
transformation	Malignant transformation is the process by which cells acquire the properties of cancer. This may occur as a primary process in normal tissue, or secondarily as malignant degeneration of a previously existing benign tumor.
integration	In viral infection, integration refers to incorporation of viral genetic material into the host’s DNA.
tumor promoters	Tumor promotion is a process in which existing tumors are stimulated to grow. It is the second phase in tumor development. Tumor promoters by themselves are not able to cause tumors to form. In molecular biology, tumor promoter regions are genes that code for proteins that promote cellular or neoplastic growth.
tumor suppressors	A tumor suppressor gene, or anti-oncogene, is a gene that protects a cell from steps on the path to malignant transformation. When a tumor suppressor gene is mutated to cause a loss or reduction in its function, the cell can progress to neoplasia, usually in combination with other genetic changes.

Term	p53 & Rb Proteins - Definitions and Notes
p53 protein	p53 is a tumor suppressor protein. It is crucial in multicellular organisms, where it regulates the cell cycle and functions as a tumor suppressor. As such, p53 has been described as “the guardian of the genome”, the “guardian angel gene”, and the “master watchman”, referring to its role in conserving stability by preventing genome mutation. The name p53 is a reference to its molecular mass. When bound to E6, p53 is destroyed and cannot function.
Rb protein	Retinoblastoma (Rb) protein is a tumor suppressor protein that is dysfunctional in many types of cancer. One highly studied function of Rb is to prevent excessive cell growth by inhibiting cell cycle progression until a cell is ready to divide. It is also a recruiter of several chromatin remodeling enzymes such as methylases and acetylases. In humans, the protein is encoded by the Rb1 gene. Early studies of Rb1 showed that, if both alleles of this gene are mutated early in life, the protein is inactivated, thereby contributing to the development of retinoblastoma cancer, and hence, the name Rb. It is not known why an eye cancer results from a mutation in a gene that is important everywhere in the body. Rb prevents cells from replicating damaged DNA by preventing progression along the cell cycle through G1 (first gap phase) into S (synthesis) phase. Rb binds and inhibits transcription factors of the E2F family, which are composed of dimers of an E2F protein and a dimerization protein termed “DP”. The transcription activating complexes of E2 promoter-binding-protein-dimerization partners (E2F-DP) can push a cell into S phase. As long as E2F-DP is inactivated, the cell remains stalled in the G1 phase. When Rb is bound to E2F, the complex acts as a growth suppressor and prevents progression through the cell cycle. The RB-E2F/DP complex also attracts a histone deacetylase (HDAC) protein to the chromatin, reducing transcription of S phase promoting factors, further suppressing DNA synthesis. If E7 (instead of E2F) is bound to Rb, E2F acts unchecked as a promoter of cell growth.

Term

p53 & Rb Proteins - Definitions and Notes

p53 protein

p53 is a tumor suppressor protein. It is crucial in multicellular organisms, where it regulates the cell cycle and functions as a tumor suppressor. As such, p53 has been described as “the guardian of the genome”, the “guardian angel gene”, and the “master watchman”, referring to its role in conserving stability by preventing genome mutation. The name p53 is a reference to its molecular mass. When bound to E6, p53 is destroyed and cannot function.

Rb protein

Retinoblastoma (Rb) protein is a tumor suppressor protein that is dysfunctional in many types of cancer. One highly studied function of Rb is to prevent excessive cell growth by inhibiting cell cycle progression until a cell is ready to divide. It is also a recruiter of several chromatin remodeling enzymes such as methylases and acetylases. In humans, the protein is encoded by the Rb1 gene. Early studies of Rb1 showed that, if both alleles of this gene are mutated early in life, the protein is inactivated, thereby contributing to the development of retinoblastoma cancer, and hence, the name Rb. It is not known why an eye cancer results from a mutation in a gene that is important everywhere in the body.

Rb prevents cells from replicating damaged DNA by preventing progression along the cell cycle through G1 (first gap phase) into S (synthesis) phase. Rb binds and inhibits transcription factors of the E2F family, which are composed of dimers of an E2F protein and a dimerization protein termed “DP”. The transcription activating complexes of E2 promoter-binding-protein-dimerization partners (E2F-DP) can push a cell into S phase. As long as E2F-DP is inactivated, the cell remains stalled in the G1 phase. When Rb is bound to E2F, the complex acts as a growth suppressor and prevents progression through the cell cycle. The RB-E2F/DP complex also attracts a histone deacetylase (HDAC) protein to the chromatin, reducing transcription of S phase promoting factors, further suppressing DNA synthesis. If E7 (instead of E2F) is bound to Rb, E2F acts unchecked as a promoter of cell growth.

InCyte Pathologist Receives CAP Certificates of Recognition

incytepathology — Fri, 02 Dec 2011 22:27:12 +0000

David C. Hoak, M.D.

InCyte Pathology’s Laboratory Medical Director, David C. Hoak, M.D., recently received the College of American Pathologists (CAP) Laboratory Medical Director Advanced Practical Pathology Program, as well as the Breast Predictive Factors Testing (BPFT)Advanced Practical Pathology Program Certificate of Recognition. The BPFT program was designed specifically for pathologists with intermediate or higher skill level in breast predictive factors testing. This selective training program covers the accurate performance and interpretation of breast cancer predictive factors in accordance with the guidelines of the American Society of Clinical Oncology and the College of American Pathologists (ASCO/CAP).

The College of American Pathologists is the leading organization of board-certified pathologists, serving patients, pathologists, and the public by fostering and advocating excellence in the practice of pathology and laboratory medicine. CAP advanced training programs focus on providing pathologists with practical skills, tools, and techniques for implementation in their laboratories to ensure more effective results for patients, as well as compliance with current laboratory guidelines.

A majority of Dr. Hoak’s training was on the performance and interpretation of prognostic and predictive factors such as estrogen receptor (ER), progesterone receptor (PgR), and Human Epidermal growth factor Receptor 2 (HER2) in breast cancer diagnostic pathology. These factors determine the clinical course of disease for the patient and their suitability for specific treatments. Given the critically important implications that these factors have in terms of subsequent selection of therapy and ultimately the clinical outcomes for a patient, ensuring testing accuracy is a high priority for InCyte Pathology.

“The greatest and most important benefit is providing patients the assurance that their breast biopsy results are accurate,” explains Dr. Hoak. “Patients can feel confident that the clinician’s treatment decisions will also be accurate and effective.”

ASC-US vs. ASC-H? What is the difference?

incytepathology — Mon, 21 Nov 2011 19:31:48 +0000

HISTORY

The current nomenclature now used to report Pap findings in the United States began with a meeting of a small group of experts in December of 1988 in Bethesda, Maryland. The results of this conference were termed the 1988 Bethesda System (TBS). Of all the TBS changes that were introduced by this conference, none was as problematic and controversial as “atypical squamous cells of undetermined significance” or “ASC-US”.

Figure 1

The silver lining of the black cloud created by ASC-US was the initiation of a large clinical trial, the ASC-US/LSIL Triage Study (ALTS), which was sponsored by the NCI and conducted under the auspices of the American Society for Colposcopy and Cervical Pathology (ASCCP). ALTS provided data for the development of evidence-based guidelines for management of women with abnormal results reported using TBS terminology.

In the end, an algorithmic management approach was developed, with examples seen in Figures 2 & 3 (below). It was a landmark event that an ancillary test for human papilloma virus (HPV) was employed to determine which women should go to colposcopy after ASC-US Pap findings.

ATYPICAL SQUAMOUS CELLS (ASC)

Atypical squamous cells represent cellular abnormalities more marked than simple reactive changes, but which do not meet the criteria for squamous intraepithelial neoplasia (SIL). These cells are not of typical appearance and are, therefore, atypical. (See Figure 1).

CATEGORIES OF ASC

The Pap diagnosis of Atypical Squamous Cells (ASC) is the most common abnormal finding during cervical cancer screening and is reported in about 5 percent of all cervical screening tests.

There are two subtypes of ASC:

Atypical Squamous Cells of Undetermined Significance (ASC-US)
Atypical Squamous Cells, Cannot Rule Out High-Grade Squamous Intra-epithelial Lesion (ASC-H).

For a diagnosis of ASC-US, a Pap smear has to have a specific type of abnormal cells (see Illustration 1).

Illustration 1. ASC-US

Most nuclear enlargement in Pap tests is due to reactive change, and reactive changes and LSIL must be excluded by the pathologist when an ASC-US diagnosis is provided.

At InCyte Pathology, our ASC-US rate is 3.7%. ASC-US results initiate reflex HPV testing, with positive HR HPV test results prompting colposcopic examination. ASC-US is the most common of the ASC diagnoses.

ASC-H REQUIRES COLPOSCOPY

Since a high grade squamous intraepithelial lesion (HSIL) cannot be excluded in ASC-H, triage directly to colposcopy is recommended by the ASCCP for ASC-H diagnosis. ASC-H is rare in our laboratory (0.2%). Cells of ASC-H are depicted in Illustration 2.

Illustration 2. ASC-H

For a diagnosis of ASC-H, the cytopathologist must first exclude moderate/severe cervical intraepithelial neoplasia or carcinoma in-situ (CIN3/HSIL). In some cases, women having ASC-H findings may harbor HSIL or invasive cervical cancer in their follow up.

Of all women with HSIL results, 2% or less have invasive cervical cancer at the time of diagnosis. About 20% can however, progress to have invasive cervical cancer if not treated or followed appropriately. Relative risk for ASC-H Pap diagnosis is certainly less than 20%, but HSIL must first be excluded by colposcopy and biopsy after ASC-H findings. If no colposcopic lesion is visible in follow up of ASC-H, vigorous ECC should be undertaken.

At this time, there is often confusion (and even controversy) among healthcare providers regarding the proper evaluation and management of ASC, especially ASC-H.

The ASCCP 2006 consensus guidelines for the evaluation of women with cervical cytology (ASC-US and ASC-H) are provided below (Figures 2 & 3).

SUMMARY

Since 1988, the diagnosis of Atypical Squamous Cells has evolved from a poorly-defined, wastebasket category of diagnosis into an evidence-based triage test which employs a very sensitive risk stratification assay (i.e., the HPV test) to determine which patients need colposcopy. Current algorithms have helped greatly to standardize clinical management of patients with ASC abnormalities.

Figure 2.

Figure 3

InCyte Pathology Supports 2011 Race for the Cure in Coeur d’Alene

incytepathology — Wed, 09 Nov 2011 17:44:05 +0000

This Fall, InCyte Pathology employees fielded a team of 53 participants to support the 2011 Susan G. Komen Race for the Cure in Coeur d’Alene, Idaho. This community event raises funds and awareness for the fight against breast cancer, celebrates breast cancer survivorship, and honors those who have lost their battle with the disease. Team InCyte was fourth in the company division for number of registrants.

According to their website (www.komencda.org), up to 75 percent of the funds raised by the Race remains here in the Komen CDA Affiliate service area (Benewah, Bonner, Boundary, Kootenai & Shoshone Counties) to provide diagnostics, screening, treatment, services and education of breast cancer. The remaining 25 percent goes to fund national research to discover the causes of breast cancer and, ultimately, its cures.

Use of E-Cadherin and p120ctn IHC for Breast Carcinoma

incytepathology — Tue, 01 Nov 2011 16:17:23 +0000

David C. Hoak, M.D.

Invasive ductal carcinoma of the breast is the most common type of breast cancer. Approximately 55% of patients will develop this form of breast cancer. Invasive lobular carcinoma is less common with only about 10% of breast cancer patients diagnosed with this type of cancer. These two tumor subtypes are distinguished on the basis of their histology. Ductal tumors arise from the milk ducts and tend to form glandular structures, while lobular tumors arise from the breast lobules where breast milk is made during lactation. The cancer cells of invasive lobular carcinoma are less cohesive and tend to invade tissue as single cells. Lobular tumors are often slower growing than ductal tumors, more often estrogen and progesterone receptor positive and the tumor is more frequently larger in size than suspected clinically.

When pathologists examine a patient’s breast cancer under the microscope, it can sometimes be difficult to distinguish invasive ductal carcinoma from invasive lobular carcinoma. In other words, some ductal carcinoma can mimic lobular carcinoma and vice versa.

Invasive ductal carcinoma: E-cadherin & p120ctn reveals red-brown staining of membrane

E-cadherin is useful in distinguishing between ductal and lobular cancer because it was recently discovered that lobular carcinomas have a genetic mutation resulting in the loss of E-cadherin, while ductal carcinomas retain E-cadherin. E-cadherin is a molecule that projects from the membrane of epithelial cells to ensure that the cells “adhere” together, like “velcro”. The loss of E-cadherin “allows” the lobular carcinoma cells to invade as single cells. In the laboratory, pathologists can test for the presence or absence of E-Cadherin by immunohisto-chemistry. Although treatment for stage-matched ductal versus lobular tumors is similar, some studies suggest that metastatic patterns differ between lobular and ductal tumors, and lobular tumors may be less responsive to neoadjuvant therapy.

In addition to the absence of immunodetectable E-cadherin in lobular carcinoma, there is diffuse localization of the molecule P120 catenin throughout the cytoplasm in lobular cancers. Catenins are proteins found in complexes with cadherin cell adhesion molecules at the cell membrane of many animal cells.

Invasive lobular carcinoma: E-Cadherin negative (brown stain) & p120ctn positive (red stain)

In contrast, ductal carcinoma cells retain the membrane immunostaining pattern of P120 catenin, reflecting the normal construction of the E-cadherin complex.

E-cadherin and p120 catenin are markers used to distinguish between ductal carcinoma in-situ (DCIS) and lobular carcinoma in-situ (LCIS). Sometimes DCIS cells can extend into the breast lobules and mimic LCIS. In some patients, LCIS can extend into the major ducts and mimic DCIS. Ductal carcinoma in-situ will show positive membranous immunostaining for E-cadherin and p120-catenin. Lobular carcinoma in-situ will not express E-cadherin and p120 catenin will be located in the cytoplasm rather than on the membrane.

Immunohistochemistry for E-cadherin is now widely used as an adjunct antibody for differentiating ductal from lobular pre-invasive and invasive lesions. This is particularly useful for lesions with indeterminate morphology and is important clinically because of the distinct management implications of ductal and lobular cancers.

Lobular carcinoma in situ: p120ctn staining positive

Tubulolobular carcinoma is a less common type of mammary carcinoma that displays a mixture of invasive tubules and lobular cells. It is a rare subtype of breast carcinoma intermediate between ductal and lobular in differentiation, representing less than 3% of all breast carcinomas. A criterion of 75% of the tumor showing a tubulolobular pattern has been determined.

A tubulolobular tumor consists of a mixture of small tubules and cell cords. The tubules are round, lacking the angularity and apical snouts typical of tubular carcinoma, one type of invasive ductal carcinoma. A minor component may be pure tubular or pure lobular (< 25%). Tubulolobular carcinoma of the breast is, thus, a distinct type of mammary carcinoma that displays both tubular and lobular patterns histologically but displays the membranous E-cadherin/catenin complex characteristic of the ductal immunophenotype.

E-cadherin displays membranous staining in tubular and tubulolobular carcinomas, and is negative in lobular carcinomas. P120ctn displays membranous staining in tubulolobular and tubular carcinomas and cytoplasmic staining in lobular carcinomas.

In summary, the combined use of E-cadherin and p120ctn immunostaining on a single slide is very helpful in subclassifying certain breast carcinomas. ❧

PAS Stain: A Sensitive Test for Diagnosis of Onychomycosis

incytepathology — Tue, 18 Oct 2011 15:13:36 +0000

Dr. Felix Martinez, Jr.

The PAS stain is a special histological technique utilized by most pathology laboratories for routine diagnostic purposes. Its utility in podiatric medicine is well established, with the primary application being the identification of fungal elements in toenails, skin, and deep tissues. Other techniques using microscopy available for fungal identification in toenails/fingernails include the potassium hydroxide (KOH) preparation, a technique requiring immediate evaluation after scraping. Although culture was long considered the gold standard, the PAS stain has been shown to be a sensitive method for the diagnosis of onychomycosis.

PAS (Periodic Acid Schiff) works through the oxidation of glycol groups by periodic acid to form dialdehydes, which combine with Schiff’s reagent to form an insoluble magenta compound, seen as a beautiful rose-colored reaction on a glass slide (Figure 1). Substances rich in glycol groups include polysaccharides, basement membrane material, and mucosubstances. The normally colorless fungi possess abundant carbohydrates in their cell walls and, therefore, the PAS reaction is an effective means to highlight fungal hyphae within tissue (Figure 1).

Abnormal nails can be caused by a variety of underlying diseases other than fungi (Figure 2), including nonfungal infections, various inflammatory dermatologic diseases of the nail unit, and tumors. Changes in nails can also be a sign of systemic diseases; they can occur secondarily to systemic drug use and many other ill-defined factors or rare conditions. The term onychodystophy generically refers to this group of abnormalities, and dyschromia refers descriptively only to changes in nail color (Figure 3).

Onychomycosis is a generic term for any fungal infection of the nails. Sixty percent are attributed to yeasts/molds, particularly Candida albicans. Candida infections, however, usually involve the nails (paronychia) only as a secondary reaction to a primary fungal infection of the surrounding skin or soft tissue, especially on the feet. Principle dermatophytes other than Candida are Trichphyton rubrum and Trichophyton mentagrophytes var. interdigitable. The fungal elements reside mostly in the deeper portions of the nail plate and in the hyperkeratotic nail bed, rather than on the surface of the nail plate. This would explain possible negative results obtained on KOH scrapings in some cases of onychomycosis.

Effective oral medications for onychomycosis are available; however, they are not cheap, and they require several weeks of treatment. Side effects of oral anti-fungal drugs must be monitored with blood tests. Therefore, a sensitive and specific test is needed to avoid overtreating noninfectious causes of nail abnormalities. Moreover, some payers now require documentation of fungal infection prior to approving payment for these medications.

Numerous studies have compared the diagnostic strength of the three most widely used tests: biopsy/PAS, KOH and culture. Although there is some variability in the data, the biopsy/PAS test was uniformly the most sensitive single test for the diagnosis of onychomycosis. Sensitivities ranged from 80.8% to 93.3%. The sensitivity of KOH and culture were in the 76.5% to 82.5% and 44% to 59% range, respectively. Specificity was addressed in one study with both KOH and biopsy/PAS showing 72% specificity, while culture was slightly more specific at 82%. It must be stated, however, that such comparisons may over-estimate the specificity of culture because of the false assumption that all organisms isolated in culture are disease-causing, as opposed to possible contaminates.

Sometimes, pathologists choose to use a silver impregnation stain (Gomori methenamine silver stain) either in conjunction with or instead of PAS. Often the difference is only preferential; both are equally sensitive and provide very similar results.

In summary, the PAS stain is clearly indicated if other diagnostic methods are negative and the clinical suspicion for infection is high. Moreover, it can arguably be used as the single method of choice for the evaluation of onychomycosis. Nail clippings should be submitted dry in an empty specimen container and the turn-around time is that of a typical tissue biopsy (usually within than 48 hours following tissue receipt in laboratory).

References:

Weedon D. Dermatophyte infections. In: Skin Pathology. Harcourt Publishers 1998: 557.
Oppel T, Korting HC. Onychodystrophy and its management. German Medical Science 2003;1:Doc02
Weinberg JM, Koestenblatt EK, Najarian L. Comparison of diagnostic methods in the evaluation of onychomycosis. Am Acad Dermatol 2003;49:193-197.
Karimzadegan-Nia M, Mir-Amin-Mohammadi A, Bouzari N, Firooz A. Comparison of direct smear, culture and histology for the diagnosis of onychomycosis. Aust J Dermatol 2007;48:18-21.
Grover C, Reddy BS, Chaturvedi KU. Onychomycosis and the diagnostic significance of nail biopsy. J Dermatol 2003;30(2):116-22.

Patient Centered Care is a Shared Responsibility

incytepathology — Thu, 13 Oct 2011 22:24:57 +0000

Tom Rehwald, CFO

Making a patient’s visit to the doctor’s office as painless as possible is one thing we strive for as healthcare professionals. At InCyte Pathology, our in-house billing department can sense the frustration felt by patients, when they call to find out why they received a bill for pathology lab services that should have been covered by their insurance. It is an unwelcomed hassle for patients to contact their insurance companies in order to find out what the problem is with their claim. Understanding the complicated medical terminology and navigating automated phone trees at an insurance company can be a daunting task.

Oftentimes this frustration can be avoided for Pap testing in particular, if we remember the importance of providing the proper screening or diagnostic codes at the time of specimen submission. A few minutes on the front end saves frustration for the patient, as well as for you or your coworkers in having to address a problem weeks after the visit. The appropriate signs, symptoms, diagnoses or associated ICD-9 codes supplied on the requisition are very important. Insurance companies base payment on whether the Pap smear was performed as part of a patient’s routine care or for diagnostic purposes such as follow-up to an abnormal pap or other concerning female conditions like dysfunctional uterine bleeding. Most insurance coverage will pay 100% for routine care when it is performed no more than once a year, while they will only pay a percentage (varies based on policy) for diagnostic Pap tests.

In some instances, a checklist might be useful to make sure important criteria is included on each patient’s Pap requisition:

Does the patient have postmenopausal bleeding?
Is the patient pregnant?
Did she have a prior abnormal Pap test requiring increased monitoring?

The responsibility of providing quality healthcare to our patients goes beyond the office visit. It includes proper submission of screening, signs and symptom codes with every Pap sample submitted, thereby avoiding unnecessary stress and frustration having to straighten out problems that manifest as a result of working inefficiently.

Distinguishing Adenocarcinoma From Squamous Cell Carcinoma of the Lung

incytepathology — Mon, 03 Oct 2011 15:56:16 +0000

David C. Hoak, M.D.

Prior to a patient beginning lung cancer treatment, it is important for an experienced lung cancer pathologist to review each case. This is a critical step because small cell lung cancer responds well to chemotherapy, is generally not treated surgically and can be confused with non-small cell carcinoma on initial microscopic examination. In addition, the response to certain drug regimens in patients with non-small cell carcinoma (NSCLC) with adenocarcinoma is different from squamous cell carcinoma patient response.

In an article in the May 2011 issue of CAP Today titled, “Use of Cytology and Small Biopsy Specimens in Diagnosing and Treating Lung Cancer,” the importance of distinguishing adenocarcinoma from squamous cell carcinoma early on is discussed. In addition, the article states it was previously believed that only histologic material was appropriate for sub-typing of non-small cell carcinoma. However, today’s IHC stains work well in small biopsy and cytology material, allowing the separation of adenocarcinoma from squamous cell carcinoma with an accuracy of up to 100 percent.

At the time of biopsy, the pathologist and especially the cytopathologist, play a pivotal role in the appropriate triage of these often tiny specimens. The significant clinical implications of determining adenocarcinoma from squamous cell carcinoma include:

Selection of the most effective drug regimen
Avoidance of a potentially harmful drug regimen
Triage of NSCLC for molecular mutation analysis.

At InCyte Pathology, we have a panel of immunohistochemical markers for NSCLC which includes TTF-1, napsin A, CK5, CK7 and p63. Positive staining for TTF-1 and Napsin A supports a diagnosis of adenocarcinoma. TTF-1 (thyroid transcription factor-1) is known as a thyroid-specific enhancer binding protein and is used to determine if a tumor arises from the lung or thyroid.

A positive TTF-1 stain supports a diagnosis of adenocarcinoma originating from the lung. The use of p63 stain is the preferred choice to support squamous differentiation. There can be some overlap in the staining between adenocarcinoma and squamous cell carcinoma, however it is exceedingly rare for squamous cell carcinomas to lack p63 expression. Any amount of p63 positivity along with a negative TTF-1 stain would indicate squamous differentiation.

If a tumor stains positive for both TTF-1 and p63, it is more likely to be an adenocarcinoma than a squamous cell carcinoma because TTF-1 is a more sensitive marker than p63. Cytokeratin 5 (CK5) positivity can be seen in undifferentiated large cell carcinoma as well as squamous carcinoma of the lung. Cytokeratin 7 (CK7) is valuable to determine some adenocarcinomas. These stains also work well in cytology cell blocks which can be valuable when there is a limited amount of material to test. Correct application of TTF-1 and p63 can avoid any misclassification of the tumor.

Lung cancer remains the leading cause of cancer-related mortality in the United States with 157,300 deaths and 222,520 new cases diagnosed in 2010 alone. It’s diagnosis is challenging for all pathologists, and has become more so as additional special testing is requested on even smaller samples of non-small carcinoma of the lung. Careful pathologic handling of all tumor material, whether it is surgically resected or minimally obtained, may result in earlier clarification of tumor type. This will lead to better patient care management and improved morbidity rates.