Molecular diagnostics … made easy!

Dr Kim Linton

Consultant Medical Oncologist

The Christie NHS Foundation Trust

Clinical

Morphology IHC

Evolution of diagnostic criteria

• Confirms 90% of diagnoses using current WHO classification

Clinical

Morphology IHC Flow cytometry

Evolution of diagnostic criteria

• Where facilities exist and fresh tissue is available, flow cytometry complements IHC findings for diagnostic confirmation

Clinical

Morphology IHC Flow cytometry

Cytogenetics PCR FISH GEP NGS

Evolution of diagnostic criteria

• Molecular diagnostics clinches the diagnosis when uncertainty exists

More than just diagnostics

Diagnosis PrognosisTreatmentselection

Response Relapse

• Molecular information is increasingly been used to sub-classify and re-classify lymphoma

• Laboratory-based molecular techniques are transitioning into the clinic …

• … and within the next few years expected to be an integral part of routine diagnosis and assessment of lymphoma

Core principle

Knowledge of DNA structure

Hybridisation technique+ =

Analysis of biological markers

in the genome and proteome

Core principle of molecular diagnostics

• This talk will describe the different molecular techniques

• And illustrate how molecular diagnostics is influencing lymphoma management now and in the future

Traditional cytogenetics

2. Centrifuge blood to obtain

mononuclear cells

3. Culture with phytohaemagglutinin

4. Add a mitosis inhibitor e.g. colchicine to arrest in metaphase

1. Blood, marrow or disaggregated fresh tissue

5. Add hypotonic solution, fix and drop onto slide

6. Partially digest with trypsin, stain

with Giemsa (G-band)

• Traditional cytogenetics is laborious and impractical:

o fresh tissue

o highly skilled interpretation

o metaphases - so not well suited to poorly dividing cells

o and has limited sensitivity– can easily miss subtle changes

• Being replaced in the modern era by molecular cytogenetics (FISH, next generation sequencing)

Cytogenetics in modern practice

Polymerase chain reaction (PCR)

• A gene analysis technique to amplify DNA targets/genes using DNA polymerase and primers that are complementary oligonucleotide sequence to the target region

• Used when the target gene is known

• 3 steps define a PCR thermal cycle

• Repetition of these cycles leads to millions of copies of the target sequence (1000 bases/minute)

• After 30-40 cycles more than 1x109 copies are made

Conventional PCR readout

• The PCR product is visualised in the form of bands using agarose gel electrophoresis or in the form of peaks using gene scanning

A case of ALCL

Wide clinical applications of PCR

• To confirm clonality: B cell - IgH gene rearrangements and Igƙ/Igλ light chain monoclonal expansion; T cell – TCR. Clonality studies help distinguish reactive lymphoproliferation and lymphoma e.g. MALT/Helicobacter gastritis. Can also document clonal identity at different anatomical sites

• To detect translocation specific lymphoma cells e.g. for molecular diagnosis e.g. DHL, in situ lymphoma e.g. FL and MCL or early lesions e.g. mycosis fungoides

• Detect virus associated lymphoma e.g. EBV+ lymphoma of elderly

• To clone DNA for sequencing

• To detect molecular remission (MRD in FL/MCL/gastric MALT) or early relapse

MRD analysis GALLIUM trial

Fluorescence in situ hybridisation (FISH)

• Used when the cytogenetic abnormality is known, but the target gene is not identified completely

Chromosomal spread or nuclei preparation on slide

Complimentary DNA sequence

Fluorescent label –makes it a probe

Probe applied to slide

Denature at 750

Hybridise at 370

Careful washing steps

Analyse with fluorescent microscope

Advantages and limitations of FISH

• Advantages

o Allows direct visualisation of selected genetic targets in the conserved tissue (in situ)

o Can use on cells in interphase or metaphase – mitosis not essential

o Generally easy to interpret (although some pitfalls)

o Can be combined with classical cytogenetics

• Limitations

o only very specific parts of the genome can be examined (unless SKY, a form of spectral karyotyping using multiple fluorophores to see all chromosomes )

o And it may miss translocations with alternative breakpoints (unless CGH, a variation of FISH which can detect very small additions or deletions

Clinical applications of FISH

• Detection of chromosomal translocations to confirm diagnosis of FL, MCL or gastric MALT

• Detection of MYC in DLBCL (prognostic)

• Evaluation of MRD on therapy completion (prognostic)

t(11;14) of classical mantle cell

lymphomaBarrans et al, J Clin Oncol 2010; 28:3360

Galimberti et al, Clin Cancer Res 2014; 20(24); 6398–405

• DNA microarray technology can simultaneously determine expression levels for the whole (known) transcriptome

• Array is a solid support e.g. glass slide onto which a gene-specific DNA sequence is printed or chemically synthesised (a probe)

• If the sample contains the gene of interest, the target sequence hybridises to the complementary probe to allow detection and quantitation

• Each gene usually has multiple probes

Gene expression profiling

How it works

2 colour array

Hybridise target cDNA to immobilised probes on the array

Scanning & image processing

Heatmapillustrating differential expression levels

1 colour array

Diagnosis of B-cell NHL subtypes

Dave et al (2006) NEJM

Diagnosis and prognosis of DLBCL

Rosenwald et al NEJM (2002)

WHO 2016 now separates DLBCL: • Germinal centre B-cell type• Activated B-cell type

‘Druggable’ targets for ABC-DLBCL

ReMODL-B trial

• In the UK real time gene expression profiling is feasible in a clinically relevant timeframe from FFPE material. Failure rates are low.

Barrans SL, et al. Brit J Haematol 2012 159 (4) 441-453 Care M, et al. PLoS ONE 8(2) : e55895

Slide courtesy of Andy Davies, ASH 2015

Next generation sequencing (NGS)

Convert RNA to cDNA

Generate expression profile

Align multiple short reads to reference genome / transcriptome

Obtain short reads

Add sequencing adaptors

From Wang et al, Nature Reviews 2009

Directly sequences mRNA/DNA without a hybridisation step i.e. no prior knowledge of gene/transcript necessary (unbiased)

1194625 1204350

AB Sequence Alignment Browser and PICR X:Map browser

Applications

• NGS has led to major advances in understanding and diagnosing lymphoma

• Discovery of novel biologically relevant mutations, SNPs polymorphisms and transcripts including rare mutations (deep sequencing)

• Quantitative gene expression data: count reads from a transcript (RNASeq)

NOTCH mutations in CLL

• NGS and Sanger sequencing detects NOTCH1 mutations in CLL

• Deletion of AG seen in the mutated case

• 50% decrease in coverage shows the mutation is present in half the reads

From: Swerdlow et al, 2016 WHO classification of lymphoid neoplasms, Blood (2016)

Activating mutations and drug targets

“Whole exome sequencing (WES) of >200 DLBCLs has completely redefined the genetic landscape by identifying recurrent single nucleotide variants and providing new therapeutic opportunities in DLBCL molecular subtypes”

Bohers et al, Leukemia & Lymphoma, May 2015; 56(5): 1213–1222

• Mr A, a 67 year old man, presents with rapid enlargement of cervical lymph node + thyroid involvement

• High grade B NHL by morphology: is this DLBCL or Burkitt’s?

Case 1

Burkitt Lymphoma confirmed by FISH

• CD20 +

• CD10 +

• BCL2 -

• MUM1-

• IGH@MYC +

• IGH@BCL2 -

• BCL6-IGH@MYC

Burkitt lymphoma does badly if treated like DLBCL…

Smeland et al, Annals of Oncology 15: 1072–1078, 2004

Mm CHOP (CHOP + IT and IV MTX)

30.8%

70.6%

73.7%

Mm CHOP + HDT

BFM

• Mr A receives the right diagnosis (BL) and the right treatment (R-CODOX-M/R-IVAC) and is alive and free of disease after 4 years of follow-up

Case 2

• Mr B, a 66 year old retired maintenance fitter presented 2 years earlier with a Bell’s palsy.

• Abnormal blood test – myeloma screening negative – Bone marrow biopsy: small B cells with an atypical phenotype

most in keeping with SLL

• Observed for 2 years• 3 month history of increasing fatigue, dyspnoea on exertion

and drenching sweats• Blood count showed elevated white cell count >200 blasts• CT scan showed widespread small volume lymphadenopathy• Bone marrow negative• Left neck biopsy performed

CD20 CD5

Cyclin D1 Ki-67

Morphology and IHC

46,XX,t(11;14)(q13;q32)

Cytogenetics

IGH (Green)/CCND1 Red Merged signals = IGH-CCND1 gene fusion

FISH

Making this a diagnosis of …

Mantle cell lymphoma

Not CLL!

Conclusions

• Most lymphomas are diagnosed on traditional clinical, histomorphological and immunohistochemical grounds

• Molecular diagnostics support these findings and clinch the diagnosis in ~10% of cases not diagnosed by traditional methods

• The scope of molecular diagnostics extends beyond diagnostic confirmation alone, providing clinically useful insights into prognosis, treatment selection, response assessment and the evaluation of disease recurrence

• The use of molecular diagnostics is expected to rise as molecular information is increasingly underpinning the modern re-classification of lymphoma and with the emergence of molecularly targeted treatment

• This will require changes in current practice - collection of fresh tissue and referral to centralised labs for molecular ‘work-up’

Conclusions

And finally…

• Thanks to Graham Collins, Andy Davies, Lia Menasce, Patrick Shenjere, Nick Telford and Roche Medical for providing some of the slides used in this talk

• Questions