Molecular pathology of non-small cell lung cancer Diagnostic Histopathology Mini-symposium: Molecular testing of solid tumours

Why do it? NSCLC accounts for 80% of lung tumours. Classes of drugs licensed for treatment of NSCLC. Activity of these is dependent on presence of specific molecular or protein changes on cancer cells

NSCLC Adenocarcinomas arise from epithelial cells in the terminal respiratory unit and display variable morphological appearances (acinar, lepidic and papillary). TTF1 and CK7 positive. Squamous cell carcinoma (SCC) are more central lesions arising from basal bronchial epithelial cells and often arise in the context of squamous metaplasia. SCC is p63 and p40 positive.

NSCLC Most lung cancers are associated with tobacco smoke and other carcinogens. Lung cancers in ‘never-smokers’ have a distinct molecular phenotype

Oncogenes Activating mutations in Epidermal growth factor receptor (EGFR). Translocations in anaplastic lymphoma kinase (ALK), ROS1, hepatocyte growth factor receptor (MET), RET and human epidermal growth factor receptor 2 (HER2).

Oncogenes associated with adenocarcinoma Adenocarcinoma in non-smokers. Higher frequency of mutations in EGFR, ALK and ROS1. Adenocarcinoma in smokers. KRAS mutations Different pathogenic mechanisms of tumour development in smokers and non-smokers. EGFR and ALK driver abnormalities seen in 10-15% of white European patients and 60% of patients of Asian origin.

Oncogenes associated with SCC Discoidin domain containing receptor 2 (DDR2). Fibroblast growth factor receptors FGFR1-3 TP53 PIK3CA

Oncogenes associated with small cell carcinoma Inactivation of tumour suppressor gene TP53 and Retinoblastoma 1(RB1).

EGFR Transmembrane receptor tyrosine kinase (RTK) is constitutively activated by variety of somatic mutations. These occur in tyrosine kinase domain of EGFR gene on chromosome number 7. Exon 19 deletion and exon 18 and exon 21 substitutions. Trigger several downstream signalling pathways including RAS, PI3K and STAT3. Adenocarcinoma-greatest advances in the application of therapy-defining tumour profiling

Together there regulate gene transcription, cell differentiation, proliferation, migration and apoptosis. EGFR mutation significantly improved overall survival whilst patients without an EGFR mutation had a significantly worse prognosis.

EGRF-tyrosine kinase inhibitor (TKI)- Resistance develops in two settings. Exon 20 insertions and T790M mutation. 3rd line TKIs are currently entering clinical practise.

ALK ALK rearrangements occur in less than 5% of adenocarcinomas. Often associated with young, non-smokers with clinically advanced disease. ALK-positive lung cancers are highly responsive to ATP-competitive kinase inhibition by ALK-TKI therapies which target human ALK and human growth factor (HGF)receptors.

ALK Crizotinib is approved as first line therapy. Drug resistance develops after 11 months of treatment either due to alterations in ALK or activation of bypass signalling pathway including EGFR, KRAS or KIT. Development of second line agents is on-going.

ROS -1 ROS1 gene (chromosome 6q) also encodes an RTK within the insulin receptor subfamily and shares and shares many similarities to ALK. ROS1-associated fusions are present in approximately 2% of all adenocarcinomas and are present in young, non-smoking patients. Crizotnib results in progression free survival of 19.2 months.

PD-L1 Not specific for NSCLC. Programmed death 1/programmed death ligand 1 (PD-1/PD-L1) as in immunotherapeutic agent highlights the influence of the tumour micro-environment both on tumour development and an avenue for therapeutic intervention.

Certain subtypes of solid tumours express PD-L1 on their cell surface Certain subtypes of solid tumours express PD-L1 on their cell surface. When the PD-L1 binds to the PD1 present on CD8 positive T cells inhibiting T cell proliferation and clonal expansion thereby preventing identification of ‘non-self’ antigen. Checkpoint receptors such as PD1 and cytotoxic T-lymphocyte associated protein 4 (CTLA4) are importing for discrimination ‘self’ from ‘non-self’ antigens in the development of immune tolerance.

Laboratory aspects of molecular testing in NSCLC Handing of specimens One core biopsy in one cassette. Cutting spares at the time of initial sectioning for H&E. Use of double immunohistochemistry staining (eg CK7 and TTF1). Formalin or alcohol based fixatives appear to be equally good for molecular testing for EGFR mutations as well as IHC/FISH for ALK/ROS1. PD-L1 testing by IHC is currently only validated on formalin fixed biopsy specimen.

Role of the pathologist

Role of the pathologists Accurate diagnosis and determination of cell type. IHC is undertaken using antibodies that are carefully selected. Biopsy specimens –identify areas that can my macroscopically dissected from the slide.

What do we do locally? Lung biopsies/EBUS specimens have spares cut at the time of initial sectioning for H&E. Sections for EGFR, ALK, PD-L1 testing can be cut on any microtome after cleaning it. Even if we receive two core biopsies they are put in the same cassette. Advantages of reflex testing.

What to test and when? EGFR and ALK/ROS1 should be carried out on adenocarcinomas, NSCLC- not otherwise specified. Some data suggests that TTF1 negative adenocarcinomas will most likely be negative for EGFR mutation.

Have a local approach. Reflex testing of appropriate cases (high stage disease where patients are eligible for TKI therapy) Or Discuss at MDT and request through oncology.

Sample analyses Mutation detection methods should have a minimum limit of detection (the minimum proportion of mutated DNA which must be present for reliable detection) of 10% mutated DNA; equal to at least 20% tumour cells.

Formalin fixed and paraffin tissue Poor quality DNA. Fragmented DNA. False positive mutations.

Cytology preparations Better quality DNA. Although depending on the site these may contain a high proportion of non-neoplastic cells.

EGFR – Sanger sequencing. ALK – IHC/ FISH break apart probes. ROS1 – break-apart FISH. PD-L1 – IHC on FFPE tissue. Alcohol fixatives are believed to affect the level of staining. Expression is variable and need to be done using criteria for each antibody. Reports must state primary antibody/kit used and percentage of tumour cells expressing PD-L1.

CONCLUSION Test for EGFR, ALK and PD-L1 according to local protocol. Use tissue sparingly when using an IHC panel. Use tissue for making a diagnoses before molecular tests.

Thank you!