Tumor Mutational Burden a New Pan Cancer Marker for Immuno-Oncology?

By Anthony M Magliocco MD

A new molecular marker “Tumor Mutational Burden” is rapidly emerging in the immuno-oncology world. New trials are showing that TMB may be superior to PDL1 IHC analysis to determine a patients probability of responding to costly and potentially toxic immuno-therapy treatments such as immune checkpoint inhibitors.

Tumor Mutational Burden

The Cancer Genome Atlas (TCGA) has shown that cancers have significant variation in the burden of genomic mutations they carry.  Some tumors such as melanoma have extremely high burdens whereas others such as thyroid cancer have very low loads.

 

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FREQUENCY OF TMB ACROSS TUMOR HISTOLOGY TYPES

 

Some tumors have exceptionally high mutational loads which probably represents an underlying DNA repair deficiency such as POLE or MSI abnormalities. It may also reflect the mechanism of oncogenesis as UV induced tumors such as melanoma have very high burdens.

 

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THE NEOANTIGEN BURDEN IS DIRECTLY RELATED TO TMB

It is thought that TMB actually results in the development of neo-antigens, which are essentially immunogenic.  The probability of neo-antigens emerging is proportional to the total tumor mutational burden. However, this is still a probability measurement, its possible that tumors with even low mutational loads might still generate neo-antigens of interest to the immune system.

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CANCER CELL WITH NEOANTIGENS STIMULATE IMMUNE CELLS

 

Recent Clinical Trials Point to TMB as an important pan-cancer marker

There have recently been three interesting trials in advanced lung cancer reported with a significant association between tumor mutational burden (TMB) and response to the PD-L1 inhibitor nivolumab (Opdivo).  CheckMate 012 trial, was a single-arm evaluation of the combination of the PD-1 inhibitor nivolumab (Opdivo) and the CTLA-4 inhibitor ipilimumab (Yervoy), reveled benefit in patients with high TMB independent of PDL-1 expression.

At AACR The CheckMate 568 trial,   used a TMB cutoff of ≥10 mutations per megabase of DNA (mut/Mb) as the definition of high TMB. Comparing TMB and response rate in 98 patients with untreated stage IV non-small cell lung cancer (NSCLC), investigators found a 44% response rate in association with TMB ≥10 mut/Mb and no further improvement in response with a higher TMB. The response rate fell dramatically with TMB <10 mut/Mb.  This finding is interesting as it may there is a “shelf” or a bimodal distribution of TMB

According to Dr Ramalingam  of Emory “PD-L1 and TMB identify distinct and independent populations of non-small cell lung cancer that independently are associated with enhanced objective response rate and progression-free survival.”

Investigators in the randomized CheckMate 227 trial prospectively applied the TMB cutoff of ≥10 mut/Mb.  There was aa threefold improvement in 12-month PFS (42.6% versus 13.2%) in the subgroup of patients with TMB ≥10 mut/Mb.

The PFS difference persisted across analyses of high and low PD-L1 expression and squamous versus nonsquamous histology.

“CheckMate 227 validates TMB as an important and independent biomarker to be routinely tested in treatment-naive, advanced non-small cell lung cancer,” said Matthew Hellmann, MD, of Memorial Sloan Kettering Cancer Center in New York City

“TMB should be a standard of care in the initial evaluation of the patient with non-small cell lung cancer,” said Naiyer Rizvi, MD, of Columbia University Medical Center in New York City. “PD-L1 as a biomarker remains as a standard of care in concert with TMB. a validated TMB platform needs to be used.”

Problems with TMB

TMB is definitely showing promise, but what are the drawbacks?

NGS is required

First, TMB calculations require that a significant portion of DNA be sequenced, to generate enough sequence information to determine the tumor mutational load. However, some recent studies suggest that even targeted sequencing panels may provide enough sequence information to determine if the load is high. Access to NGS sequencing remains a challenge. Due to low reimbursements and difficulty of implementing the technology many oncologists may have difficulty  accessing the technology

The calculation of TMB is currently non-standardized and non-trivial

Second, TMB calculation is not standardized. It is not a trivial bioinformatics process as the bioinformatifcs process needs to determine if a  DNA variation is “real” or an artifact of sequencing- this is non-trivial as filters need to be defined to define the criteria to make a “call” ie what the confidence of the read is, what the alleic fraction is and whether the mutation is somatic or germline. In addition it must be determined if the sequence affects the coding region of a gene.  Further complicating this is what the denominator might be in an assay- ie does the NGS sequence only coding regions or are there significant non-coding regions. If the non-coding regions are included in the calculation the number may be artifactually low.

Third, thinking from a biological and mechanistic approach, it may matter whether the mutation actually produces a neo-antigen. Again this cannot be easily measured. It involves factors such as whehter the mutation is actually transcribed into protein, and whether the protein conformation is actually altered and neo-antigenic. Further issues include whether the sequence is secreted or made available when the cells degenerate.

TMB are not the only source of neo-antigens

DNA mutations may only account for some of the neo-antigens that a cancer can create. Other sources of neo-antigens in neoplasia include microbes (ie HPV virus in HPV driven cancers such as cervical or head and neck cancer. Other sources include post-translational modifications in cancer such as glycosolation events etc.

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HPV VIRUSES ADD ANTIGENS TO TUMORS

 

Host Factors

Further complicating the impact of neo-antigens include the condition of the host immune system and its capacity to recognize and react to neo-antigens. For example in immune deficiency conditions, neo-antigens may be present but ignored by the immune systems. Or genetic variants in cellular receptors or MHC may affect how neo-antigens are presented to the immune system

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Neo-Antigens may be induced in tumors as a therapeutic strategy

Another interesting angle affecting therapy is the possibility that neo-antigens could be induced in a cancer to trigger immune response. This effect may be a side effect of some therapies such as temozolamide or radiation therapy.

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RADIATION THERAPY MAY INDUCE NEO-ANTIGENS

 

Dr Magliocco is Chair of Pathology and director of the Morsani Molecular Laboratories at the Moffitt Cancer Center

 

 

 

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.

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.