Hormone Replacement Therapy does not Increase Risk of Breast Cancer in Women with BRCA Mutation after BSO Treatment

By Anthony M Magliocco MD

In a large multicenter international prospective study reported in JAMA Oncology, Kotsopoulos et al found that use of hormone replacement therapy overall did not appear to increase risk the of breast cancer among women with BRCA1-mutation after prophylactic bilateral salpingo-oophorectomy; however, use of estrogen-progesterone hormone replacement therapy appeared to be associated with increased risk vs estrogen alone.

Study Specifics

The study was a prospective, longitudinal cohort study of BRCA1- and BRCA2-mutation carriers from 80 centers in 17 countries which was conducted between 1995 and 2017. There was a mean follow-up of 7.6 years. The study participants had undergone BRCA1 or BRCA2 testing for familial or other reasions.

The current study included a total of 872 BRCA1-mutation carriers with no personal history of cancer with a mean postoophorectomy follow-up of 7.6 years. Patients had a mean age of 43.4 years. the questionnaires were administered every 2 years for information on hormone replacement therapy use.

The investigators concluded,

“These findings suggest that use of estrogen after oophorectomy does not increase the risk of breast cancer among women with a BRCA1 mutation and should reassure BRCA1 mutation carriers considering preventive surgery that [hormone replacement therapy] is safe. The possible adverse effect of progesterone-containing [hormone replacement therapy] warrants further study.”

However the study also revealed that use of estrogen plus progesterone was associated with higher risk vs use of estrogen-alone hormone replacement therapy.

HER2/HER3 and PIK3CA Mutations in Colorectal Cancer are Associated with Microsatellite Instability

By Anthony M Magliocco MD

 

top-10-reasons-to-ask-your-doctor-about-colon-cancer-screening-1080x675

 

A recent study by Loree reported in the  Journal of the National Cancer Institute, found that ERBB2/ERBB3 mutations in colorectal cancer are associated with the presence of MSI microsatellite instability and PIK3CA mutation.

Study Details- Retrospective analysis of Colorectal Cancer Cases from two Cohorts

The study involved retrospective analysis of 419 patients from The University of Texas MD Anderson Cancer Center (MDACC) and 619 patients from the Nurses’ Health Study (NHS)/Health Professionals Follow-Up Study (HPFS) with stage I to IV disease, with tissue sequencing, clinicopathologic, mutational, and colorectal cancer consensus molecular subtype (CMS) profiles of patients with ERBB2/ERBB3 mutations. The circulating tumor DNA profile associated with ERBB2mutation was also investigated in an additional cohort of 1,623 patients with circulating tumor DNA assay results.

epithelial-cell-in-metaphase

Factors Associated With ERBB2/ERBB3 Mutation

Mutations were found in over 5% of cases and were not associated with age, location or stage of tumor. ERBB2 mutations were associated with shortend survival HR 1.82 but not ERBB3

The investigators concluded, “[Microsatellite instability] and PIK3CA mutations are associated with ERBB2/ERBB3 mutations. Co-occurring PIK3CA mutations may represent a second hit to oncogenic signaling that needs consideration when targeting ERBB2/ERBB3.”

 

The findings are interesting in that mutated ERBB2 is potentially an actionable target.

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.

BRCA Mutation Predicts Carboplatinum Response in Women with TNBC

Anthony M Magliocco MD

 

A recent study published in Nature Medicine reports that women with triple negative breast cancer with a BRCA mutation were much more likely to respond to treatment with carboplatin compared to treatment with docetaxel, which is the current treatment recommendation for these patients.

https://www.nature.com/articles/s41591-018-0009-7

Triple negative breast cancer remains a difficult disease to treat as standard anti-estrogen or anti-HER2 treatments are not considered.

In this study there were 376 women with advanced triple-negative breast cancer across the trial, regardless of BRCA gene status, the researchers found the 2 drugs worked similarly well. But among the 43 women in the study who also had BRCA gene mutations those who received carboplatin were twice as likely to respond to therapy as those given docetaxel.

The researchers have reported an observed resesponse of 68% of the patients treated with carboplatin, but only in 33% of the women on docetaxel.

 

 

 

Furthermore Carboplatin also appeared to cause fewer side effects along with prolonged tumor progression for longer in women with BRCA mutations—with a progression free survival of  7 months compared with 4 months for those treated with standard docetaxel.

The researchers believe carboplatin is more effective for this patient group because it works by damaging tumor DNA, and BRCA mutations impair the ability of cancer cells to repair the type of DNA damage created by this kind of platinum drug.

This study further highlights the need for availability of BRCA gene testing in women with breast cancer.

This study further highlights the need for NGS gene testing in women with breast cancer for the purpose of appropriate therapy selection

 

One curious feature of the study was women with BRCA1 gene methylation, low BRCA1 mRNA expression, or Myriad HRD analysis was not clearly associated with benefit of treatment with platinum based agents.

 

A study at the Moffitt Cancer Center recently showed many triple negative breast cancers may actually be misclassified due to errors in primary pathology biomarker analysis.

Second opinion analysis should be considered for women diagnosed with triple negative breast cancer as the original biomarker analysis may frequently be flawed and an actionable target such as estrogen receptor or HER2 is identified on reanalysis

 

TRIPLE NEGATIVE BREAST CANCER IS OVER DIAGNOSED

 

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.

 

New Window into Brain Metastasis Using Modified CTC and cfDNA Technologies

Anthony M Magliocco MD

Many cancers metastasize to the central nervous system including the brain and its coverings, the leptomeninges. These cancers are difficult to treat and monitor. The so called “liquid biopsy” is making excellent progress in monitoring disease progression and response using blood samples in patients with disseminated solid tumors. In this technique, either circulating tumor cells (CTCs), or cell free DNA (cfDNA) can be harvested and captured for analysis.

These new “real time” monitoring methods create a new opportunity to monitor cancer progression and evolution while patients are actually under therapy- for example are the number of circulating tumor cells increasing, or is their phenotype evolving into something different.

For example, metastatic breast cancer cells may change their receptor status to go from estrogen receptor positive to negative, or they may acquire a new targetable change, for example acquiring HER2 over expression.

Images of circulating tumor cells captured in the Cell Search System

Intact tumor cells might also allow for examination of activation of signalling cascades and perhaps a pharmacodynamic read out.  One of the challenges of CTCs is their extreme fragility- with current method they generally need to be captured and examined within 48-72 hours. These cells also tend to be exquisitely rare, especially in early stage disease raising questions as to how representative they may be of the systemic disease.

 

Cell Free DNA cfDNA

A complementary assay method,  the measurement of cell free DNA (or cfDNA), provides a new application for monitoring cfDNA in patients. It has found widespread use monitoring lung cancer patients for the development of tyrosine kinase resistance while under therapy.  A common mechanism of resistance is the the switch or evolution to EGFR T790M mutation that portends the looming end of response to first generation TKI drugs and an opportunity to intervene with a switch to the 3rd generation Osimertinib therapy.

Liquid Biopsy is a powerful method to enable real time monitoring of solid tumor evolution and response to therapy using a blood sample to cature CTCs and cfDNA. Unfortunately, this does not typically work for CNS tumors due to the blood brain barrier

Unfortunately tumors occurring or metastasizing to the CNS are not easily followed by blood tests due to the presence of the blood brain barrier. However, these tumors are in contact with the cerebrospinal fluid, a specialized liquid that circulates around the brain cushioning it.

This fluid can be extracted in small amounts for analysis using a spinal tap, or in some cases of patients with malignancy an in-dwelling catheter is placed to enable CSF to be drained off to reduce the central nervous system pressure. This CSF fluid is traditionally sent to pathology for cytopathology analysis. Unfortunately standardized cytopathology methods do not lend themselves to evaluation of rare cells and molecular events in CSF.

 

Because CSF is very similar to blood, we reasoned that liquid biopsy methods used for blood samples might be potentially adapted for use on CSF samples. Recently, the Moffitt teams of the Neuro Oncology group, led by Dr Peter Forsyth and the Morsani Molecular Laboratory teamed up to attempt to modify the liquid biopsy procedures currently used for blood to adapt for CSF.

The current CTC platform in use at the Morsani Molecular Laboratory at Moffitt is the CellSearch system which is designed for magnetic capture of EPCAM expressing carcinoma cells in blood followed by evaluation of keratin expression in an automated scanning step with exclusion of non- specific cells using stains for nuclei and lymphocytes.

The adaptation of this system for analysis of melanoma in CSF was not trivial, as the volume of CSF is significantly less requiring adjustments on the liquid handling approaches. Further, the Neuroncology team was particularly interested in metastatic and primary melanomas of the central nervous system. This required changing capture antibodies to CD146 which targets melanoma and visualization with MelPE.

The Moffitt Morsani Molecular Laboratory has Developed New Methods to Monitor Melanoma in the CSF using both CTCs and cfDNA approaches

We also determined that DNA could be extracted and sequenced from CSF using both the NGS sequencing methods and MassArray systems. Further, we determined that melanoma cells captured from CSF can be grown ex-vivo and cultured for further analysis.

These advances create new opportunities to apply advances from personalized oncology to patients with metastatic melanoma in the CSF and central nervous system enabling potential real time adaptation of treatment strategies based on directly monitoring tumor molecular responsiveness to therapy in real time \\.

REFERENCES

Fedorenko IV, Evernden B, Kenchappa RS, et al. A rare case of leptomeningeal carcinomatosis in a patient with uveal melanoma: case report and review of literature. Melanoma research. 2016;26(5):481-486. doi:10.1097/CMR.0000000000000274.

Neuro-Oncology, Volume 19, Issue suppl_6, 6 November 2017, Pages vi46,https://doi.org/10.1093/neuonc/nox168.183 Published: 06 November 2017

 

A Tale of 2 Biomarkers: CTCs and cfDNA are Key to Managing the Plethora of New Trial Options

By Anthony M Magliocco MD

 

We are currently living in revolutionary times when it comes to cancer therapy and treatment options. There are literally hundreds of clinical trials under way evaluating dozens of new therapeutic compounds and combinations of therapy. In fact there are so many trials that its difficult to always find enough patients for them and also to design them to produce the level of evidence expected in traditional multi-armed phase III trials.

There are hundreds of open clinical trials testing dozens of compounds and combinations of therapy

 

Indeed, we seem to be arriving at the point where cancer therapy really is being tailored and delivered to the individual. This shift from evidence based conventional cancer therapy clinical trials, to newer, matched and “N of One” trials creates new challenges for the oncologist and diagnostic laboratory industry.

In a traditional trial, selection and enrollment would depend on results of a key biomarker such as HER2 overexpression for Trastuzumab therapy

Traditionally a biomarker would be required to select a patient for a specific therapy. For example, in breast cancer, presence of HER2 over expression or amplification was a marker to help select patients for enrollment in a trial. Because the frequency of the biomarker was relatively common in a common disease, it was possible to build a well powered phase III trial to collect convincing evidence that on a population based setting that this was an effective strategy. This led to FDA approval and the development of companion diagnostics such as the Herceptin Test.

Fast forward twenty years or so. Now we have so called “basket trials” where cancers can be tested with complex genomic tests that will evaluate hundreds of genes, for example the Moffitt STAR assay that covers 170 actionable mutations and alterations resulting in detection of relatively rare, but still actionable mutations in many tumors. However the mutations are variable and the choices for treatment complex meaning that more than one combination of therapy could be considered. Whats an oncologist to do?

Going forward, it appears that if patients cannot be managed or enrolled in large trials, there will need to be an approach to effectively manage the so called “N of One” patient. In fact, it could be considered that almost every patient is a now unique in some way and could be classified as a rare disease

With a movement away from classical evidence based clinical trials toward n of one trials, off label therapy, and basket trials new approaches to companion diagnostics are urgently needed

In this situation, it appears that physicians will need to act on “best available evidence” or actual bioinformatics or other predictive models of possible response based on understanding the underlying molecular circuitry in the cancer in question. In this situation a “best guess” is made for assignment of therapy (either in a basket trial or in an off-label situation).

This approach can be problematic and has numerous complications such as the possibility of providing futile or toxic treatment. Fortunately, there are plenty of new advances in technology that might address this problem. The most help may come from the so-called “liquid biopsy”. Which is essentially usually a simple blood test evaluated with a exotic new technology.

Liquid Biopsy

The main components of a liquid Biopsy include circulating normal cells (WBC, Platelets, RBCs) possibly circulating living cancer cells (CTCs) and bits of dying cancer cells (cell free DNA, miRNA etc).

A Story of Two Biomarkers CTCs and cfDNA

CTCs Circulating Tumor Cells

The CTCs are very fragile, rare and hard to detect but give a window into the living cancer in the patient as treatment progresses. These cells can be further evaluated to determine if they are proliferating or if certain signalling pathways are active. In fact they can also be harvested, sequenced, and in some cases grown and expanded in culture.

Cell Free DNA cfDNA

Cell Free DNA or cfDNA gives a more stable read out of the tumor load, and mutation composition of the DNA, or at least the DNA leaking into the blood. It might be expected that when a new treatment begins, cancer cells in the host may undergo death and leak DNA into the blood. Consequently there could be an initial “Spike” in the amount of circulating DNA – this could be a positve signal. In other instances, cfDNA might indicate if there is residual cancer left in a patient after a surgery was completed that was initially intended to remove all disease giving a type of molecular staging. If a therapy is working well we would expect that CTCs and cfDNA would decrease and perhaps become immeasurable. On development of resistance there would be detection of new clones and expansion of the concentration of CTCs and cfDNA fragments.

Liquid biopsy provides a means to monitor tumor response to therapy in a dynamic and real time manner giving unprecedented opportunities to modulate treatment and truly personalize therapy for cancer patients

I expect that the twin technologies of CTC and cfDNA analysis will become more valued by oncologists, patients and payers as these tools will provide a way to dynamically monitor tumor response to treatment and provide immediate evidence of efficacy of a therapy or also of impending relapse potentially allowing a window of opportunity to adjust treatment.

 

FDA approves osimertinib for first line use in lung cancer with EGFR mutation

By Anthony Magliocco MD

TAGRISSO delivered unprecedented median progression-free survival of 18.9 months versus 10.2 months for EGFR-TKIs (erlotinib or gefitinib) in 1st-line EGFRm NSCLC

 

the US Food and Drug Administration (FDA) has approved TAGRISSO® (osimertinib) for the 1st-line treatment of patients with metastatic non-small cell lung cancer (NSCLC) whose tumors have epidermal growth factor receptor (EGFR) mutations (exon 19 deletions or exon 21 L858R mutations), as detected by an FDA-approved test. The approval is based on results from the Phase III FLAURA trial, which were presented at the European Society of Medical Oncology 2017 Congress and published in the New England Journal of Medicine.

 

The results of the phase III FLAURA trial were impressive with dramatic improvements to progression free survival.

The FLAURA trial compared TAGRISSO to current 1st-line EGFR tyrosine kinase inhibitors (TKIs), erlotinib or gefitinib, in previously untreated patients with locally advanced or metastatic EGFR-mutated (EGFRm) NSCLC. TAGRISSO met the primary endpoint of progression-free survival. PFS results with TAGRISSO were consistent across all pre-specified patient subgroups, including in patients with or without central nervous system (CNS) metastases. Overall survival data were not mature at the time of the final PFS analysis.

 

Osimertinib was previously approved for use as second line therapy in patients who had progressed on a TKI treatment or for those whose tumors developed a T790M mutation confering resistance to the first generation TKI therapies.

Osimerinibs mechanism of action is thought to be irreversible binding to the EGFR receptor.  Perhaps this explains the improvement in PFS compared to other TKIs. These findings are potentially practice changing.

Despite these impressive improvements in PFS almost all patients eventually fail targeted therapies with TKI agents. Consequently more work is needed to understand the biological mechanisms of resistance and progression in lung cancer patients to enable more effective therapies to be developed.

https://www.onclive.com/web-exclusives/fda-approves-frontline-osimertinib-for-nsclc

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

 

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.