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.

 

Loxo Oncology work with Illumina to develop Cdx NGS assay for Larotrectinib (NTRK) and LOXO-292 (RET)

Anthony Magliocco MD

In an important announcement Industry sequencing leader Illumina and LOXO Oncology, released that they are working on developing a companion diagnostic for larotrectinib, a NTRK inhibitor and LOXO-292 which targets ret.

 

It appears that they intend to use the TST170 as a basis and perhaps a DX version of the Nexseq 500

This is an important development for 2 reasons. The first is the molecular alterations in question are relatively rare, but they can occur in any tumor type regardless of the tissue of origin. The second is the identification of a standard instrument platform and multi-gene assay panel that is already in clinical use (Moffitt has recently deployed a version of TST170 for patient care as Moffitt STAR) will undoubtedly expedite the capability to scale this test for widespread deployment accross laboratories

The fact that TST170 is so comprehensive potentially offers the opportunity for other oncology drug developers to consider using this versatile assay as a companion diagnostic as well.

https://ir.loxooncology.com/press-releases/loxo-oncology-and-illumina-to-partner-on-developing-next-generation-sequencing-based-pan-cancer-companion-diagnostics

STAMFORD, Conn. and SAN DIEGO, April 10, 2018 (GLOBE NEWSWIRE) —  Loxo Oncology (Nasdaq:LOXO) and Illumina, Inc. (Nasdaq:ILMN) today announced a global strategic partnership to develop and commercialize a multi-gene panel for broad tumor profiling, resulting in a distributable, next-generation sequencing (NGS) based companion diagnostic (CDx) with a pan-cancer indication. The co-development partnership will seek approval for a version of the Illumina TruSight Tumor 170 as a companion diagnostic (CDx) for Loxo Oncology’s larotrectinib, which targets NTRK gene fusions, and LOXO-292, which targets RET gene alterations, across tumor types.

TruSight Tumor 170 is a comprehensive, state-of-the-art, next-generation sequencing test that interrogates point mutations, fusions, amplifications and splice variants in 170 genes associated with common solid tumors. The CDx version of TruSight Tumor 170 will allow local laboratories to provide referring physicians with comprehensive genomic information, so that patients can be matched to the most appropriate therapeutic options. This version of TruSight Tumor 170 will run on the NextSeq 550Dx platform.

“We are leveraging our leadership in next-generation sequencing to deliver in-vitro diagnostic solutions to improve the management of cancer patients in the clinic,” said Garret Hampton, Ph.D., executive vice president of clinical genomics at Illumina. “To this end, we are partnering with leading biotechnology companies, such as Loxo Oncology, to develop companion diagnostics for best-in-class therapeutics. Distributable diagnostic solutions, such as a CDx version of TruSight Tumor 170, in combination with the NextSeq 550Dx platform, will enable labs to perform precision medicine testing in-house.”

Under the partnership, the companies will collaborate to validate a CDx version of TruSight Tumor 170 for NTRK fusions and RET fusions/mutations as a Class III FDA-approved diagnostic in conjunction with larotrectinib and LOXO-292, respectively. The companies are also planning to broaden the clinical utility of the full panel by obtaining regulatory approval for the other assay content, to be marketed as a tumor profiling test. Illumina will lead regulatory activities related to the Class III plans for NTRK and RET, the Class II plans for the tumor profiling content, and CE marking.

“We are very excited to announce this collaboration with Illumina, the world’s leader in NGS technology,” said Jacob Van Naarden, chief business officer of Loxo Oncology. “We have piloted numerous NGS assays, and the Illumina TruSight Tumor 170 assay has consistently demonstrated robust performance with its assessment of both DNA and RNA, including highly sensitive gene fusion detection. The broad 170-gene assay content has the potential to deliver meaningful insights from a single tumor specimen, identifying patients with NTRK fusions, RET fusions, RET mutations, and many other actionable tumor alterations. Furthermore, we believe that this collaboration will improve patient access to high-quality NGS testing because pathologists will be able to run TruSight Tumor 170 locally and receive reimbursement.”

MOFFITT NGS STAR* Enters Clinical Service

Moffitt’s latest NGS sequencing assay the Moffitt STAR (Solid Tumor Actionable Result) panel was validated by the Moffitt Morsani Molecular Laboratory and launched into service this month at the busy Florida Comprehensive Cancer Center in Tampa.

The assay is based on Illumina’s TruSight Tumor 170 assay which is a next-generation sequencing assay designed to cover 170 genes that are commonly designated as drivers in solid tumors. The assay evaluates both DNA and RNA and focuses on detecting actionable mutations which include SNV, dels, insertions, amplifications, and translocations. Such alterations are the target for many new targetable therapies including anti-EGFR agents, anti BRAF therapies and treatments targeting the Tropomyosin Receptor Kinase fusions (TRK) such as Larotrectinib.

Many key actionable mutations only occur rarely, making detection by single marker tests problematic and wasteful. However, the Moffitt STAR assay now allows the Moffitt molecular laboratory to screen patient tumors for multiple targetable mutations efficiently in a single test using a relatively small amount of nucleic acid extracted from routine formalin fixed, paraffin embedded tissues (FFPE). This important advance enables the Moffitt molecular diagnostic laboratory to effectively evaluate a patient for eligibility to receive treatment with a FDA approved targeted therapy, or be considered for clinical trial enrollment. Moffitt STAR is essentially an “All in one” test that can provide multiple functions.

Moffitt NGS STAR* is an exciting new “all in one” technology advance for Moffitt Cancer Center patients enabling rapid assessment of their tumors for presence of key mutations directing selection of effective approved targeted therapies or for qualification to enroll in the latest generation of clinical trials

Evidence is also emerging the assay, despite its mid size, Moffitt STAR could also reliably measure tumor mutational load and microsatellite instability. These molecular features are often associated with potential response to the latest immune check point inhibitors such as Pembrolizumab which has recently received FDA approval for use in tumors with high microsatellite instability.

Moffitt NGS STAR also provides information on tumor mutational burden and microsatellite instability- key features which may drive patient response to the latest immuno-oncology check point inhibitor therapies

Moffitt NGS STAR can also detect mutations in BRCA genes, a molecular feature that may predict response to parp inhibitors such as olaparib.

Moffitt NGS STAR can be performed on as little as 40ng of input nucleic acid.

Development and launch of Moffitt NGS STAR was made possible through collaboration with industry partners PierianDx and Illumina Inc.

The Moffitt Cancer center is one of the largest in the United States, is consistently ranked in the top cancer centers by U.S. News & World Report. Moffitt Cancer Center has a mission to “contribute to the prevention and cure of cancer” and the vision ” to transform cancer care through service, science, and partnership”

For further details contact anthony.magliocco@moffitt.org