Morsani Molecular Lab at Moffitt Cancer Center is Central to its Personalized Oncology Mission

By Anthony M Magliocco MD

Moffitt Cancer Center, the largest and only NCI designated comprehensive cancer center in Florida and one of the largest in the country has been rapidly expanding its capabilities in molecular diagnostics with a clear focus on supporting its burgeoning personalized oncology programs which are led by internationally recognized expert Dr Howard McLeod.

It became clear several years ago that a huge revolution was coming in the area of personalized oncology and cancer treatments. That revolution is now here.

Explosive advances and numerous FDA approvals for new targeted therapies have emerged for treating and controlling once universal killers such as metastatic melanoma and advanced lung cancer.

These targeted therapy medicines, include vemurafenib targeting mutant BRAF, Erlotinib for EGFR mutant lung cancer, and crizontinib targeting ALK translocations. Further truly dramatic results have been seen with immunotherapy treatments such as the anti PDL-1 immune checkpoint inhibitor pembrolizumab for treatment of a variety of advanced cancers that over express the protein PDL-1 or carry MSI. More recently approvals of NTRK inibitors Larotrectinib and the PARP inhibitor Olaparib for BRCA mutant cancer have further increased demand for specialized testing as both of these treatments have companion diagnostic requirements

 

Immune cells (red and blue) surround a invasive cancer cells (green)

The approval of many new targeted treatments requiring companion biomarker evaluation is providing impetus to develop more advanced diagnostic technology including new cellular imaging methods

These rapid advances in treatment options hinge on the availability of routine, high quality, clinical grade, molecular analysis and diagnosis to properly identify patients who will be most likely to benefit from these costly and frequently toxic treatments.

The Morsani Molecular Laboratories were created to facilitate the rapid development and implementation of new molecular diagnostic assays into the routine CLIA laboratory at Moffitt

Moffitt leadership and its generous philanthropic doners and foundation laid the important cornerstone of the Moffitt Morsani Molecular Laboratories. Under the direction of Dr Magliocco, the laboratories had a singular mission to rapidly develop and deploy the advanced clinical grade diagnostic services necessary to support Moffitts rapidly maturing personalized medicine program.

The Laboratories were initially opened in 2012 and were first equipped with Mass array instruments and conventional Sanger sequencing, pyrosequencing and routine PCR ability. The decision was made to recruit PhD scientists who would help develop the new assays to a CLIA standard and then launch them into a “routine” production laboratory.

The demand from the Moffitt clincal services was high, especially from thoracic oncology, a ground breaking team offering a multitude of clinical trail options for Moffitt patients. This demand required that the assays be CLIA grade, complex and delivered in a rapid way.

This was very challenging. Launching a highly multiplex assay into a CLIA lab is not a trivial matter. The assay must be calibrated to show sensitivity, specificity, analytical performance, range, precision, accuracy and many other technical components. Further, any new assay must also be put through its paces to show its robustness and reproducibility when handled by different scientists and technologists.

Launching new assays into CLIA labs is not trivial and requires extensive expertise and investments

We initially chose to launch the LungCarta (TM) Mass Array Panel from Sequenom. This was very challenging to validate as it contained individual assays run in multiplex to assay over 213 distinct mutations. as these were separate mutations and assays, the decision was made to only validate the most clinically important ones, namely BRAF V600E, KRAS, EGFR, and PIK3CA. The next issue was obtaining appropriate clinical reference materials to validate these assays. Fortunately with Moffitt’s very high clinical volume, previous experience with single-plex testing, and availability of Total Cancer Care Protocol tumor bank which houses data and specimens from over 400,000 patients it was possible to obtain the necessary control and case materials to validate and launch the assays into clinical service.

The development of a CLIA assay requires access to appropriately characterized reference materials to enable clinical validation of the process and assay results.

Although the complete 213 mutation panel was run, only the CLIA validated assay results were reported to the treating physician. The remaining results were ported into Moffitt’s data warehouse for storage and use in properly approved research studies. Following the launch of the LungCarta assay, the Morsani Molecular laboratory also launched assays for melanoma, and a specially constructed Glioma panel assay.

 

In 2014, clinical demands for even more complex sequencing arose for proper management of Myelodysplastic Syndrome (MDS) mounted. The hematology team needed access to over 30 genes and potentially thousands of mutations. It was time for the big guns, time for next generation sequencing. At the same time Moffitt molecular pathologists also saw increasing needs for more complex sequencing for solid tumors as well. After careful discussions and further evaluations it was decided to begin next generation sequencing. Following some debate, the consensus was to start with Illumina, given their long history in next gen sequencing and also the experience with the technology in Moffitt’s core genetic research laboratory. It was decided to develop an off the shelf 26 gene panel TST26 that covered most of the key mutations in lung cancer and in melanoma. In addition Moffitt CLIA scientists, molecular pathologists, and clinicians worked with Illumina to design a 32 gene myeloid NGS panel.

Bringing on NGS brought new challenges of a complex wet lab and also a very complex bioinformatics dry lab. At Moffitt we worked with PierianDx who helped design bioinformatics pipelines and an efficient validation program for both the wet and dry lab as well as an information system – a genome workbench- which allows molecular pathologists to rapidly review cases and access a knowledge database to enable rapid sign out. We also required access to numerous control samples to validate the assays. Again these came from Moffitts vast biospecimen resources and also Horizon Discovery, a company specializing the the provision of reference materials for clinical validations. To date Moffitt has run over 10,000 NGS assays.

By 2017, clinical demands continued to mount for even more complex testing. There were new drugs approved that needed to evaluate fusions, “exon skipping” mutations, and even MSI and tumor mutational burden. With these demands, we turned to illuminas TST170 assay, a new type of sequencing assay that had both DNA and RNA. In addition the assay was designed with “actionable targets” in mind. Meaning, that targeted agents in clinical trials were scrutinized to determine the collection of genes and mutations that would likely be most informative for treatment selection in solid tumor oncology. This approach makes the assay very useful and practical for deployment in a busy cancer center where multiple trials are underway and complex patients with unusual cancers are presenting. Moffitts Morsani molecular team worked hard and spent several months validating the assay to bring it to acceptable CLIA standard finally launching it as “Moffitt STAR” an assay to screen for actionable mutations.

Since its launch, demand has been exceptionally high with hundreds of physician orders in the first week alone.

 

Moffitt laboratories continue to work closely with the worlds leading oncologists and pharma and technology companies to ensure that Moffitt Patients always have access to the latest diagnostic tools to enable them access to the most current treatment options.

That is what makes Moffitt an exceptional hospital for cancer patients seeking innovative treatments and explains why Moffitt has some of the best cancer outcome response rates in the country.

 

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.