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.”

FDA Approved PARP Inhibitor Rucaparib For Maintainance Treatment for Recurrent Ovarian Cancer Boosting Need for Clinical NGS Testing

By Anthony M Magliocco MD

On April 6, 2018, the Food and Drug Administration approved rucaparib (Rubraca®, Clovis Oncology Inc.), a poly ADP-ribose polymerase (PARP) inhibitor, for the maintenance treatment of recurrent epithelial ovarian, fallopian tube, or primary peritoneal cancer who are in a complete or partial response to platinum-based chemotherapy.      

Approval was based on ARIEL3 (NCT01968213), a randomized, double-blind, placebo-controlled trial in 561 patients with recurrent epithelial ovarian, fallopian tube, or primary peritoneal cancer who had been treated with at least two prior treatments of platinum-based chemotherapy and were in complete or partial response to the most recent platinum-based chemotherapy. Patients were randomized (2:1) to rucaparib 600 mg orally twice daily (n=372) or placebo (n=189) and were treated until disease progression or unacceptable toxicity. 

Tumor tissue samples were examined with a next-generation sequencing assay to determine whether DNA contained a deleterious somatic or germline BRCA mutation (tBRCA). This test was also used to determine the percentage of genomic loss of heterozygosity (LOH). Positive homologous recombination deficiency (HRD) status was defined as tBRCA-positive and/or LOH high. Three patient outcomes analyses were performed on the following groups: all patients, HRD subgroup, and tBRCA subgroup.

NGS ASSAY WAS REQUIRED TO IDENTIFY BRCA MUTATIONS

 

ARIEL3 demonstrated a statistically significant improvement in estimated median progression-free survival (PFS) assessed by investigator for patients randomized to rucaparib compared with placebo in all patients (median PFS 10.8 vs. 5.4 months, HR 0.36; 95% CI:0.30, 0.45; p<0.0001), in the HRD subgroup (median PFS 13.6 vs. 5.4 months, HR 0.32; 95% CI: 0.24, 0.42; p<0.0001), and in the tBRCA subgroup (median PFS 16.6 vs. 5.4 months, HR 0.23; 95% CI: 0.16, 0.34; p <0.0001).

 

The FDA also concurrently approved the complementary diagnostic test, FoundationFocusTM CDx BRCA LOH, for tumor samples to determine HRD status. 

In ARIEL3, the most common adverse reactions in at least 20% of patients treated with rucaparib included nausea, fatigue (including asthenia), abdominal pain/distension, rash, dysgeusia, anemia, ALT/AST elevation, constipation, vomiting, diarrhea, thrombocytopenia, nasopharyngitis/URI, stomatitis, decreased appetite, and neutropenia. Myelodysplastic syndrome and/or acute myeloid leukemia occurred in 7 of 372 (1.9%) patients treated with rucaparib and in 1 of 189 (0.5%) patients assigned to placebo. Discontinuation due to adverse reactions occurred in 15% of patients receiving rucaparib and 2% of those assigned to placebo. 

The recommended rucaparib dose is 600 mg (two 300 mg tablets) taken orally twice daily with or without food.

FDA granted this application priority review. A description of FDA expedited programs is in the Guidance for Industry: Expedited Programs for Serious Conditions-Drugs and Biologics, available at:

 

This approval is another example of the exploding need to broad access to next generation sequencing in routine clinical care of cancer patients forming the foundational basis of personalized oncology

” This most recent approval of yet another therapeutic advance that is reliant on NGS for patient selection further increases the urgency for access to routine NGS for all cancer patients”  – Anthony M Magliocco MD

The Moffitt Morsani Laboratories have developed and launched a new NGS assay ‘Moffitt STAR(TM)” that covers the key actionable target genes including BRCA1 and BRCA2

Moffitt STAR NGS panel covers the key mutations

PierianDx Customer, Moffitt Cancer Center, Launches Clinical NGS Assay, STAR

VISIT MOFFITT CANCER CENTER WEBSITE

http://www.moffitt.org

 

New Mechanism Involving NFkappaB Drives Metastasis in Cancer with Chromosomal Instability

By Anthony M Magliocco MD

An interesting and important new study recently published in Nature gives new insights into possible targetable molecular mechanism linking chromosomal instability observed in some cancers and progession and metastasis.

https://www.nature.com/articles/nature25432

A subset of cancers develop chromosomal instability via disjunction errors during mitosis. This can lead to highly aneuploid and abnormal karyotypes more prevalent in certain cancers than other. For example, ovarian cancer is notorious for developing a  disrupted and unstable karyotype.

Metastatic Cancer

It appears that chromosomal instability in itself does not promote metastasis, however, in an intriguing observation it was noted that tumors with chromosomal instability also developed micronuclei when under mechanical stress.

These micronuclei are more prone to cytoplasmic rupture. Free DNA in the cytoplasm triggers a non-canonical NFkappaB cascade which may lead to mesenchymal transformation and a propensity to metastasis.

Interestingly cancers with high aneuploidy do not generally develop a high mutational burden such as cancers like melanoma. Nor do they tend to have targetable driver mutations.

“Cytosolic DNA from micronuclear rupture appears to trigger non-cannonical NFKappaB signaling which could lead to tumor metastasis”

 

DNA in the cytoplasm triggers NFKappaB as part of a host response to viral infection, but cancer may subvert this mechanism. In addition,  NFkappaB non-cannonical activation by cytosolic DNA from ruptured micronuclei may lead to a variety of effects immune activation which could assist with tumor metastasis.

These new insights into the consequences of chromosomal instability create opportunities for developing new therapeutic strategies for these types of cancer that lack specific driver mutations and significant neoantigen loads

 

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.

Bringing Digital Droplet PCR into the Cancer Clinic for Treating Progressive Lung Cancer

By Anthony M Magliocco MD

Digital Droplet PCR, or ddPCR is a phenomenally sensitive, specific, and most importantly, precise method to measure target DNA.

One important application for this technology that is now reaching the clinical laboratory is the development of tests for monitoring circulating cell free DNA that is released from cancer cells.

The challenge of detecting cfDNA is monumental.  A tube of blood is loaded with a rich variety of complex biomolecules and cells including lymphocytes, neutrophils, platelets, and of course red corpuscles.  These cells, including RBCs, are loaded with nucleic acids and whats worse, they can easily rupture during blood draws or pre analytical handling.

Advanced lung adenocarcinoma, (NSCLC) is probably the single tumor type that has caused the largest advances in personalized oncology of solid tumors. Investigations into the molecular biology of this disease has uncovered the fact that NSCLC is a heterogeneous disease defined by distinct molecular subtypes and clear molecular driving pathways. Further the discovery of targetable driver mutations including presence of EGFR mutations among others has led to pivotal clinical trials proving the efficacy of the Tyrosine Kinase inhibitor Drugs (TKIs).  Further it seems that lung tumors with EGFR mutations are more likely to arise in women and non-smokers.

Despite the remarkable efficacy of TKI therapy in many patients with advanced NSCLC, the disease is relentless and frequently overcomes treatment by developing resistance mechanisms. The commonest mechanism of resistance is acquisition of EGFR T790M mutation which further enhances the activity of the EGFR pathway, driving growth of the lung cancer cells.

 

 

Fortunately, third generation non-competitive inhibitors of EGFR were developed that can overcome the development of T790M resistance -osimertinib or Tagresso from AZD.

When Osimertinib first became available it was necessary to develop an ultra sensitive assay to detect the mutation in tissues and blood.

The Moffitt Cancer Center Morsani Laboratories, when faced with this challenge, decided to use a digital PCR approach because of its superior sensitivity, specificity and reasonable cost of operation.

Dr John Puskas worked diligently to develop and validate a cfDNA assay for EGFR T790M mutation using ddPCR for CLIA at the Moffitt Cancer Center.  Different PCR platforms were evaluated but the Bio-Rad QX200 was eventually selected for use.

The assay was superbly sensitive and precise as well as convenient to run.  The assay can detect mutant EGFR T790M down to 0.1% and can easily monitor blood concentration over time to give assessment of disease progression

 

 

The development of accurate and specific cfDNA assays to precisely monitor cancer progression in the metastatic setting is an important tool to potentially enable oncologists the opportunity to determine more rapidly f tumors are responding to treatment or not and make adjustments.

This novel opportunity to trace tumor evolution in real time, at a molecular level, could play a role in trials and treatments of the future where treatments are carefully adjusted to avoid the inadvertent development of treatment resistance due to over treatment.

 

Moffitt Cancer Center Morsani Molecular Lab Researchers Create New Laboratory Test to Better Diagnose Metastatic Bladder Cancer

Moffitt researchers reported the discovery of a new 19 gene expression signature that appears to have value in improving the accuracy of urothelial cancer diagnosis at the USCAP 2018 meeting in Vancouver.

The correct diagnosis of urothelial cancer, especially when metastatic can be very challenging for the surgical pathologist Urothelial carcinoma (UC) can mimic a poorly squamous cell carcinoma (SCC) and it may be difficult to distinguish the two especially in metastatic sites.

Metastatic cancer imaged with PET scan

 

It is important to distinguish the two cancers for proper patient management. These two cancers have an overlapping immunohistochemical profile (both positive for CK5/6, CK7, p63 and p40). GATA3 although relatively specific for UC, can also be expressed in SCC.

It is important to distinguish urothelial carcinoma from other epithelial cancers to ensure proper diagnosis and patient managment

Gene expression analysis is a powerful method that can measure the expression level of thousands of mRNA messages simultaneously in a tissue biopsy specimen. Analysis of data from this technique, when coupled with bioinformatics analysis can produce powerful classification algorithms which could be used in a CLIA laboratory to improve the accuracy of current diagnostic approaches.

Affymetrix chips for rapid gene expression

The Moffitt researchers analyzed 161 UC, 38 head and neck cases, and 268 lung cancer SCC cases from Moffitts Total Cancer Care (TCC) database to create the initial algorithm. The data was collected on the a specially modified HuRSTA chip containing 60607 probe sets covering 26356 genes. An algorithm was developed from the 19 top most differentially expressed genes using principal component analysis (PCA). The PCA model performed at above 98% sensitivity and specificity correctly identifying the histological origin of the specimens on self validation.

PCA analysis separating tumor types using 19 gene signature

The signature was further validated on publicly available external datasets at GEO datasets, with 96 cases of H&N SCC from GSE31056, 18 lung SCCs from GSE10245 and 93 UC from GSE31684, total of 207 cases.This signature correctly identified 112 of the 114 publicly available SCCs with gene data (96 H&N and 16 lung) as SCC and all of 93 publicly available UCs with gene data as UC

The identification of a gene classification algorithm will enable improved classification of these challenging lesions, particularly in the metastatic setting.

The Moffitt investigators will continue to evaluate this new signature and propose to develop it into a routine diagnostic assay. In addition, deeper understanding of the molecular basis of urothelial cancer and how it differs at the molecular level potentially opens new approaches to therapy.

An assay like this could be far superior than classical IHC as the complexity of gene expression will give multiple insights into not only tissue of orign but also other aspects of the cancer biology such as immune status.

Understanding the molecular basis of urothelial cancer will provide insights into novel treatment opportunity

The work was supported by the Moffitt Morsani Molecular Laboratories

Presented at the 2018 USCAP meeting in Vancouver Canada

Gene Biomarker Signature for Distinguishing Urothelial Carcinoma from Squamous Cell Carcinoma. Jasreman Dhillon, MD Yin Xiong, Ph.D. Anthony Magliocco, MD, Soner Altiok, MD, PhD, H. Lee Moffitt Cancer Center, Tampa FL

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

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.