Highlights in Pathology: CPD review (September 2019)

Dr. Shamini Selvarajah, University Health Network

As the summer winds down and the excitement of a new academic year returns to the air, it is time to resume the monthly Highlights in Pathology for LMP’s CPD corner. 

For this issue, four articles are presented, which highlight the ever changing state of molecular biomarkers in clinical practice.

Technological advances in genomic research have been paving the way to personalized medicine.

With the unraveling of the genomic “big data”, a critical challenge we face is the refinement of this knowledge into assays that have clinical utility.

The studies highlighted below illustrate some of the key steps in the incremental process of refinement and translation of existing knowledge into clinical validity.

1. Association of Breast and Ovarian Cancers With Predisposition Genes Identified by Large-Scale Sequencing

JAMA Oncol. 2019 Jan 1; 5(1):51-57 [PMID: 30128536] HM Lu, S Li, MH Black, S Lee, R Hoiness, S Wu, W Mu, R Huether, J Chen, S Sridhar, Y Tian, R McFarland, J Dolinsky, B Tippin Davis, S Mexal, C Dunlop, A Elliott

Among the women with hereditary breast and ovarian cancer syndrome (HBOC) an appreciable portion carry a pathogenic variant in the breast cancer susceptibility genes, BRCA1 and BRCA2.  However, there are a number of other inherited gene mutations that significantly or moderately increase the risk of developing breast and ovarian cancer. 

The authors of this paper - a mix of scientists from Ambry Genetics (a genetic testing company) and academic researchers - endeavoured to use whole-exome sequencing and genotype-phenotype associations to evaluate the clinical significance of HBOC predisposition genes. 

An increased risk of breast cancer was associated with PALB2, ATM, CHEK2, and MSH6 genes, whereas MSH6, RAD51C, TP53, and ATM genes were associated with an increased risk of ovarian cancer. 

The study also identified MSH6 and ATM as possible moderate-risk HBOC predisposition genes, findings which are not reflected in the current literature.  

Their data also challenge some previously described associations. For example, their study found no link between breast cancer risk and MRE11A and NBN genes, which are currently included on some HBOC susceptibility gene panels.

A number of strengths of the study include the large cohort size that can sufficiently power the statistical analysis of NGS data designed for clinical diagnostic use.    

The study provides an objective insights regarding which of these candidate genes to include into existing diagnostic panels (when there is currently limited data regarding clinical utility), determining recommendations for risk management for these novel genes, and identifying individuals who may test positive for mutations but do not meet certain testing criteria. 

This study provides an enhanced resolution of the genotype-phenotype correlation of a panel of biologically related candidate genes in HBOC and will ultimately help in the interpretation of results from multigene panel testing.

2. MGMT Promoter Methylation Cutoff With Safety Margin for Selecting Glioblastoma Patients Into Trials Omitting Temozolomide

Clinical Cancer Research, March 2019, Volume 25, Issue 6 [PMID: 30514777] Monika E. Hegi, Els Genbrugge, Thierry Gorlia, Roger Stupp, Mark R. Gilbert, Olivier L. Chinot, L. Burt Nabors, Greg Jones, Wim Van Criekinge, Josef Straub, and Michael Weller

O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation is an established predictive as well as prognostic biomarker in patients diagnosed with gliomas. However, a particularly challenging point in the methylation analysis is defining a technically and clinically relevant cut-off between positive and negative test results. 

The existing gap between the technical and clinical sensitivity in current testing methods is problematic as a considerable minority of unmethylated patients with low expression of MGMT may be overlooked for potentially effective treatment.

In this study, the authors analyzed the MGMT promoter methylation status of cohorts from four randomized control trials, as determined by quantitative methylation-specific PCR (qMSP).

The analysis revealed a technical or "unsupervised" threshold which allowed dichotomization of the results into methylated and unmethylated patients. 

In addition, a supervised analysis using the overall survival defined a “grey zone” associated with the technical threshold that expanded the potential cohort that may benefit from the alkylating agents.

Application of these thresholds to a test cohort revealed that patients in the grey zone (weakly methylated) had significantly better survival compared with truly unmethylated patients, suggesting that they benefit from alkylating agents despite only low-level MGMT methylation.

This article illustrates the challenge often encountered in clinical assay development, namely the reconciliation of the technical and clinical data into a test that is tailored the meet the actual clinical needs. 

In this study, the clinical data justifies the maintenance of a lower safety margin of MGMT promoter methylation to identify patients who may benefit from alkylating agents.

3. Detection of Tumor NTRK Gene Fusions to identify patients who may benefit from Tyrosine Kinase (TRK) Inhibitor Therapy

J Mol Diagn. 2019 July;21(4):553-571. [PMID: 31075511] Hsiao SJ, Zehir A, Sireci AN, Aisner DL

The tropomyosin receptor kinase (TRK) family is a group of three neurotrophic receptor tyrosine kinase proteins (TRKA, TRKB and TRKC) encoded by the NTRK1, NTRK2 and NTRK3 genes.  

NTRK gene fusions, which have been identified in a broad range of cancers, lead to constitutive activation of the kinase domain similarly to many other oncogenic drivers such as BCR-ABL translocation and EGFR amplification/ mutation. 

While the gene fusion was historically first isolated from a colon carcinoma, recurrent fusions have now been identified in rare cancers such as infantile (or congenital) fibrosarcoma, congenital mesoblastic nephroma, and secretory breast carcinoma.

It has also been identified in a small percentage of common adult cancers (non-small cell lung cancer, salivary gland, colorectal, head and neck, thyroid, bladder cancers as well as malignant melanomas, soft tissue sarcomas and brain tumors (gliomas). 

The fusions have garnered clinical attention in recent years due to the remarkable efficacy of small molecule inhibitors such as larotrectinib and entrectinib, the former being notable as the first drug fully approved independent of the tumor site of origin. 

These small molecule inhibitors work by blocking the downstream signaling pathways that are activated and drive the cancer. Currently, there are ongoing Phase 2 trials with these drugs involving patients whose cancers harbor NTRK gene fusions, irrespective of tumor histology (“basket trials”) as well as Phase 1 trials with other NTRK inhibitors. 

This article provides a concise review of the molecular characterization and incidence of these tumor agnostic fusions. 

The authors also outline the different approaches available for detecting these fusions including the diagnostic challenges and drawbacks associated with each method. 

They conclude by proposing a diagnostic algorithm that may help identify patients with NTRK fusion cancers.

Although NTRK fusions may only be present in a small proportion of tumors, identifying these patients will be crucial for providing them with the benefit of targeted precision therapy already shown to have profound and prolonged anti-cancer effects.

4. Double-Hit Gene Expression Signature defines a distinct subgroup of Germinal Center B-Cell-Like Diffuse Large B-Cell Lymphoma

J Clin Oncol. 2019 Jan 20; 37(3):190-201. [PMID: 30523716] Ennishi D, Jiang A, Boyle M, Collinge B, Grande BM, Ben-Neriah S, Rushton C, Tang J, Thomas N, Slack GW, Farinha P, Takata K, Miyata-Takata T, Craig J, Mottok A, Meissner B, Saberi S, Bashashati A, Villa D, Savage KJ, Sehn LH, Kridel R, Mungall AJ, Marra MA, Shah SP, Steidl C, Connors JM, Gascoyne RD, Morin RD, Scott DW.

Approximately 8% of diffuse large B-cell lymphoma (DLBCL) are high-grade B-cell lymphoma with MYC and BCL2 and/or BCL6 rearrangements (HGBL-DH/TH) as identified by fluorescence in situ hybridization (FISH). 

The cell of origin for HGBL-DH/TH almost exclusively belongs to the germinal center B-cell-like (GCB) molecular subgroup, which is ironically also the entity with a better prognosis (compared to the activated B-cell DLBCL subgroup). Thus, GCB DLBCL has superior prognosis unless the patient has the double or triple hit (DH/TH) version, which often prompts intensive treatments. 

The authors of this article investigated the molecular and biological differences between the GCB DLBCL and “DH” DLBCL by targeted resequencing, whole-exome sequencing, RNA sequencing, and immunohistochemistry data. 

They uncovered a clinically and biologically distinct subgroup of patients who did not meet the classical DH DLBCL. Based on their data, this almost doubled the proportion of patients with a DH signature.

Several interesting findings were highlighted. 

Firstly, a distinct mutational landscape comprising several chromatin modeling genes was observed. 

An overexpression of MYC and E2F target genes, as well as those associated with oxidative phosphorylation, were noted alongside decreased expression of genes with immune and inflammatory function. 

Interestingly, despite the MYC signatures by gene expression profiles and mutational analysis, MYC rearrangements were not present by FISH testing, which point to other mechanisms of deregulation.

If the findings of this “DH signature DLBCL” can be validated by other groups, the authors suggest that the signature could form the basis of a more inclusive category that encompasses DLBCLs both with and without MYC, BLC2 and/or BCL6 rearrangements.

Grouping tumors with shared biology together would expand a group of patients with a need for more intensive therapies, while removing them from the remaining GCB-DLBCL group would delineate a cohort with a superior prognosis.