The Value of Pathology Review in Cancer Diagnosis

By Anthony M Magliocco MD

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Pathologists are amongst the most misunderstood of medical specialists.  They are perhaps one of the most important members of the cancer care team, especially now in the age of precision oncology.

Pathologists are MDs who have gone on to undergo focused and specialized training in a 4 yr residency in pathology in either anatomic or clinical pathology.  Anatomic pathology is the study of tissues and includes the subspecialties of surgical pathology, cytopathology, and autopsy pathology. Clinical pathology covers blood based testing and microbiology.

Some pathologists go on to further super subspecialize in focused areas such as cancer, or research. When a surgeon or radiologist removes a tissue specimen from a lesion it is sent to the pathologist for review. This involves examining the specimen carefully and selecting regions for further microscopic examination.  This process involves “fixing” the specimen in formalin, removing the water from it, and embedding it in paraffin. Then thin slices are made to put the tissue on a glass slide for staining and further evaluation by the pathologist. The pathologist may also use advanced diagnostic methods such as immunohistochemistry or molecular analysis to further characterize the tissue.

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Image of high grade breast cancer with mitotic figures

Essentially, pathologists are often the first physicians to make a diagnosis of cancer in a patient and pathologists then perform the key further anlaysis to enable an oncologist to select the right treatment for a patient.

Taking breast cancer as an example, a pathologists role is to make the diagnosis of breast cancer, describe what histological type of cancer it might be- ie ductal or lobular. The pathologist must also evaluate the “grade” of the specimen which gives information of how biologically aggressive it might be, and whether the surgical margins and regional lymph nodes contain metastatic disease. This examination is critical to determine if further surgery is necessary or if other therapy such as endocrine therapy chemotherapy or a targeted therapy such as Trastuzumab.

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breast cancer with HER2 amplification a feature for selecting Trastuzumab

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As the treatments for breast cancer grow, and our knowledge increases we now know that breast cancer is a complicated disease with many subtypes and important features that help guide therapy. Consequently, the demands on pathologists are increasing this also means that non- specialist pathologists may have challenges keeping up with new advances in specialty areas of testing and treatment.

Consequently large cancer centers generally have a policy for all pathology to be reviewed by an expert cancer center subspecialist or team of subspecialists prior to selecting and initiating treatment.

This pathology review frequently adds new information to a case, or actually changes the diagnosis resulting in a change to the treatment plan. In some cases as many as 20% of community pathology diagnoses are changed or amended by central subspecialist pathologist review.

Consequently, a second opinion from a pathologist can be very valuable for a patient and their oncologist to ensure that the treatment course is the most appropriate for the condition.

Having the correct diagnosis and the complete biomarker profile of a cancer is essential to ensure that the most effective therapy is being used and the best chance for a good outcome is achieved.

 

 

 

 

 

 

Understanding the Biology and Treatment Options for Breast Cancer

by Anthony M Magliocco MD

Breast cancer is a common disease with up to 1 in 8 women receiving this diagnosis in her lifetime. It is more common in older women but it certainly can strike the young and even can occur in men at about 1/100 the rate seen in women.

We have learned a lot about the biology of breast cancer over the years and the condition is becoming easier to detect at an earlier stage and fortunately more effective therapies are now being developed.

We now know that breast cancer is complex and has multiple molecular and biological subtypes. The main types are called Luminal A, Luminal B, HER2 positive, and Triple Negative.

Luminal Cancers – Endocrine responsive tumors

The Luminal types of breast cancer are defined by expressing estrogen receptor. They tend to have better differentiation and generally a more indolent course. The standard treatment is surgery, potentially radiation, and endocrine treatment (an anti estrogen agent) for 5 to 10 years. If the tumor appears more aggressive- ie involves lymph nodes, or has higher grade, chemotherapy may be added to the treatment in an adjuvant way. One of the problems with luminal type breast cancer is it can recur many years after the original cancer has been treated. Its thought the cancer cells can spread and remain dormant in distant organs or bone for many years with some mysterious events causing them to become reactivated.

 

The HER2 positive breast cancers

A subset of breast cancers, about 15% seem driven by the oncogene ERBB2 (which produces the protein HER2).  For some reason this gene can become “amplified” – ie many copies are made in a single cell leading to vast over production of the HER2 protein. This over production seems to drive the cancer cell to proliferate and metastasize.

Fortunately, therapies have been designed for HER2 cancer, these include Trastuzumab and Pertuzumab – antibodies that react and block the function of the HER2 gene. These treatments appear to be able to halt the grow of the cancer and prevent metastases from occurring.

The Triple Negative Breast Cancers (TNBC)

The third main type of breast cancer are the Triple Negative Cancers or TNBC. This group is actually a mixed group of tumors defined by lack of expression of estrogen receptor or HER2.  They frequently occur in younger women, they may be familial, are over represented in African American women and often progress more quickly. Some of them seem to have a high immune infiltrate, some carry the BRCA gene mutation, and some show strange growth characteristics such as bone formation or “metaplasia”.

 

 

The problem with TNBC is they are a generally a diagnosis by exclusion. However there are certain tests a pathologist can order to help prove that a cancer is TNBC such as Nestin.

Because the TNBC is diagnosed by exclusion there is a significant possibility of error. For example some tumors are heterogenous, and only a small portion might be sampled. If the portion is ER negative or HER2 negative the tumor might be erroneously classified as TNBC.

In other cases, TNBC cancers with very low false expression of HER2 or ER may end up being misclassified as HER2 positive or ER positive which could lead to erroneous use of potentially toxic and ineffective anti -HER2 therapy

Use of Second opinion Expert Review Pathology 

A second opinion pathology review by an expert pathologist can help a patient and her oncology be confident that a tumor is indeed a TNBC. The pathologist may order a repeat immunohistochemical stain on additional case material which frequently changes a diagnosis of TNBC to ER positive or HER2 positive. Occasionally a ER positive or HER2 positive will be reclassified as TNBC also.

TNBC is a unique form of breast cancer with subtypes. further classification of TNBC into tumors with BRCA mutation will help with selection of Parp inhibitor or chemotherapy with carboplatinum.

Further, some TNBC tumors have been shown to be sensitive to immunotherapy.

Some TNBC also seem to express androgen receptor, which could be a therapeutic target.

 

Use of Liquid Biopsy

In some patients a liquid biopsy could also be helpful. If a patient has metastatic disease the cancer cells can be isolated and analyzed from a blood sample.  ER and HER2 status can be measured in these circulating tumor cells. In addition fragments of DNA from disintegrating cancer cells can also be measured and classified providing further evidence as to the amount of cancer and whether the cancer is changing or responding to therapy.

Of interest, it seems that breast cancers may undergo biomarker change as they progress or metastasize. for example an ER positive tumor might change and become ER negative or switch to HER2 overexpression.

Patients should seek second opinion pathology review if they have concern regarding the accuracy of their diagnosis or want to ensure that all treatment opportunities are properly considered.

 

 

As treatment options continue to expand, and testing methods improve, it is important that patients with breast cancer have access to the highest quality pathology services and they should also not hesitate to seek second opinion if there is concern regarding the accuracy of diagnosis.

Moffitt Cancer Center Pushing Frontier of Pathology with Advanced Multiplex IHC in Its Morsani CLIA Laboratory

By Anthony M Magliocco MD

Next generation sequencing NGS has begun to revolutionize the capabilities of molecular pathologists to analyze the molecular foundation of cancer specimens offering improved diagnosis, classification and treatment selection.

Not to be outdone, similar remarkable advances are impacting tissue pathology as well. Immunohistochemistry is a powerful technique that allows pathologists to visualize and measure protein in routine formalin fixed, paraffin embedded tissue sections.

Moffitt Cancer Center is pushing the boundaries of tissue analysis using digital image analysis and multispectral immunohistocemistry in the Morsani Molecular Laboratories directed by Dr Magliocco.  The Advanced CLIA Analytical Microscopy laboratory at Moffitt is led by Susan McCarthy, an expert in molecular analysis and histotechnology. The fact that the laboratory is also certified by CLIA and CAP is important, as the new complex imaging methods can then easily be translated to patient care under a CLIA LDT regulatory mechanism.

Classically, Immunohistochemistry involves applying a primary antibody which will bind to an antigen and then detecting it with a tagged secondary antibody (essentially an antibody against the first antbody). Typically this binding is visualized using a brown dye (or chromogen) such as DAB.

The result allows the pathologist to assess the presence of a target protein and approximately its concentration. This method is very common and hundreds of different proteins can be analyzed. in the example above, an invasive breast cancer is stained with an antibody against estrogen receptor. The intense brown staining of the nuclei of cancer cells indicate that the cancer is strongly expressing the estrogen receptor and is likely being driven by estrogenic processes. The blue staining cells in the background are mostly lymphocytes stained with a blue counter stain to allow visualization of the tissue.  Breast cancers like this are considered to be estrogen receptor positive. It is known that estrogen receptor positive cancers are most likely to respond to hormonal treatments such as tamoxifen or aromatase inhibitors.

While single marker IHC is a powerful tool it presents challenges in that only one marker at a time and accurate quantification tools are lacking. Further, the assays are usually pushed to enable high sensitivity without proper dynamic range, so it can be difficult to accurately quantify expression in strongly expressing cells.

“Next Generation IHC” or multiplex IHC is a technique where more than one antibody can be applied to a tissue section at the same time and then visualized with secondary antibodies attached to different fluorescing tags. This produces a multiplex image which can be “deconvoluted” into separate channels for direct analysis

 

In this image the separate proteins Progesterone receptor and Cytokeratin can be isolated using filters to isolate the red and green fluorescence (the colors are actually false but help visualization for the human eye) the DAPI is a stain the stains nuclei. 

Once the various channels of image are acquired using a digital scanning microscope such as Aperio FL, computer software such as AQUA can isolate components of the images to define areas of interest on the tissue also called “masks”. These areas could be tumor nuclei, tumor cytoplasm,  lymphocytes, etc. Once masks are defined the signal from the target protein can then be accurately measured.

This ability to accurately localize and measure the protein semi-automatically produces very reproducible and precise data. Much like running an elisa on the slide

Some assays like estrogen receptor can be problematic to quantify, and this can lead to misclassification and mistreatment of patients causing serious risks to patients. A region of Canada unfortunately experienced problematic testing for many years which affected the quality and treatment of hundreds of breast cancer patients

http://www.cbc.ca/news/canada/newfoundland-labrador/lab-mistakes-poor-oversight-flagged-in-n-l-breast-cancer-inquiry-1.793504

ALso different approved method to measure hormone receptor in breast tissue may produce different results

https://www.nature.com/articles/modpathol2016151?WT.feed_name=subjects_cancer

Perkin Elmer is pushing multispectral imaging and quantification even further with their Vectra imaging systems and new Opal chemistries facilitating high multiplex staining.

http://www.perkinelmer.com/category/quantitative-pathology-research?gclid=CjwKCAjw75HWBRAwEiwAdzefxD-_DAE27g95aS1n9Jdyv3uLksY4AKWwRVIJs-W0fqyaJxjJbQ3JuBoCoSsQAvD_BwE

This elegant system allows 6 or more separate antibodies to be evaluated on a single tissue section.

 

This powerful, and stunningly beautiful, method enables multiple cell types to be visualized in a single tissue section.

In the above image  of a lung cancer produced by Susan McCarthy at Moffitt the orange areas are PDL1 and PD1 expression, the green are CD3 cells, the yellow CD8 and the red FOXP3. The cancer cells are highlighted in light blue and the dark blue identify nuceli.

Analysis like this preserve the rich complexity of the tumor microenvironment and allow direct visualization of not only the types of cells present, but their localization in respect to cancer cells and other immune cells.  This method has produced excitement amongst oncologists and immunologists seeking improved tools and clues into which tumors may be most susceptible to immune checkpoint treatment.

Challenges still remain in bringing assays like this into routine care. Its critical that the assay can meet quality and CLIA standards which include development of standard operating procedures, and delineation of the performance characteristics of the assay such as sensitivity, precision, performance, detectable range, and robustness.

Another layer of complexity is approach to the digital analysis of the data or “dry lab” as there can be many approaches such as counting cells or measuring areas of signal. Sources of variation could include different approaches to segmenting images and normalization. Despite these challenges I remain optimistic that these technologies will provide powerful new insights into the biology of cancer, and provide pathologists with compelling new way to microscopically examine human tissue.

TRIPLE NEGATIVE BREAST CANCER IS OVER DIAGNOSED

By Dr. Anthony Magliocco

Getting a second opinion for a cancer diagnosis is highly recommended, but even more so if you face triple negative breast cancer, which can be aggressive and difficult to treat.

A new study led by Moffitt Cancer Center pathologist Dr. Marilin Rosa shows that triple negative breast cancer may be frequently overdiagnosed and reclassified after expert review and biomarker retesting. Moffitt investigators presented the data at the 2018 United States & Canadian Academy of Pathology Annual Meeting in Vancouver.

Researchers reviewed over 560 cases of breast cancer referred to Moffitt and found that 113 were initially classified as triple negative by external evaluation. After biomarker retesting, about 28 percent of the triple negative cases were reclassified as hormone receptor positive.

Moffitt’s study demonstrates the value of biomarker retesting for triple negative breast cancers before selecting an appropriate treatment plan. A second opinion that changes your diagnosis can have a huge impact on survival.

In triple negative breast cancer, the three most common types of receptors known to fuel most breast cancer growth — estrogen, progesterone and the HER-2 gene — are not present. This makes common treatments such as hormone therapy and drugs that target the three missing receptors ineffective.

Up to 20 percent of breast cancer diagnoses are triple negative and are more likely to affect younger patients, blacks, Hispanics and those with a BRCA1 gene mutation. This disease is also more likely to spread and recur.

The takeaway: Having an accurate cancer diagnosis is critical to planning appropriate treatment. If you are diagnosed with triple negative breast cancer, consider getting a second opinion before starting a treatment plan.