Predictive biomarkers in lung cancer

In metastatic pulmonary adenocarcinoma (AdC) a platinum-based compound combined with pemetrexed, while for metastatic squamous cell carcinoma (SqCC) a platinum based compound and docetaxol/gemcitabine is chemo regimen of choice.[1]

Histology for prediction chemotherapy

Recently, in a collaborative effort by the IASLC, ATS and ERS a refinement on the WHO classification for AdCs, including guidelines for tumor typing on biopsies was established.[2, 3] In about 35-40% of biopsies the distinction between AdC and SqCC is not possible.[4] For this situation the diagnosis of NSCLC-not otherwise specified (NSCLC-NOS] is used.[2, 3] This should immediately lead to the application of panel of three (immuno)histochemical markers, TTF1 and mucin stain for adeno lineage and p63/p40 for squamous cell lineage. The threshold for positivity is different for TTF1 and p63. TTF1 is considered positive, if weak but certain staining is present (>30 out of max IHC score 300) .[4] However, in most TTF1 positive lung carcinomas the staining is 3+ in the majority of the tumor cell nuclei. p63 may stain weakly (+ / ++) in a heterogenous manner in AdC throughout the tumor with variability amongst individual tumor cells. In contrast in SqCC p63 is strongly (+++) positive in almost all neoplastic nuclei, especially in the periphery of a field of tumor cells, with loss of staining in more differentiated areas, where p63 is (‘ physiologically’ ) negative. The threshold for p63 IHC determining SqCC lineage is high, the majority (>80%) of the nuclei need to be strongly positive.[4] P40 staining parallels this threshold for p63 and may be used for the same purpose.[5, 6] p63+, TTF1-, mucin- tumors should be diagnosed as “NSCLC-NOS, immunohistochemically favouring SqCC” or “probable SqCC”. For p63-, TTF1+ and/or mucin+ tumors the diagnosis is “NSCLC-NOS, immunohistochemically favouring AdC” or “probable AdC”. A small fraction of tumors may be positive for both lineage determining markers, these tumors probably represent adenosquamous carcinomas, or peripheral AdCs with squamous differentiation. Another small fraction of tumors are negative for both lineages, which are recorded as NSCLC- NOS. With this approach about 85-90% of the biopsies are segregated in such a way that EGFR mutations occur in the thus defined AdCs, NSCLC-NOS and probable AdC. Analysis should be performed in these subsets for guidance of chemotherapy. Liberal use of the lineage determining additional stains is advocated, as the H&E does not contain sufficient information.[7][4] Thus, this diagnostic approach provides predictive relevance for chemotherapy.

Markers for targeted therapy

Currently two biomarkers (epidermal growth factor receptor, EGFR and anaplastic lymphoma kinase, ALK) should be tested in all patients with advanced lung cancer. For tumors with EGFR activating mutations first line treatment is indicated with an EGFR tyrosine kinase inhibitor (EGFR TKI; gefitinib, erlotinib, afatinib). [8–12] In Europe for ALK positive lung cancer 2nd line treatment with crizotinib is advised.[13] Both of these biomarkers occur in the same histological categories, AdC, large cell carcinoma, NSCLC-NOS (+/- favouring AdC by IHC), adenosquamous carcinoma and salivary gland type carcinomas. These categories are clinically called “non-SCLC, non-squamous carcinoma”.

In AdCs several different driver mutations are present, implying that inhibition of the driver will inhibit growth of the neoplastic cells, which are dependent on this function. In line with this hypothesis are these driver mutations mutually exclusive.[14–16]

EGFR Mutation analysis
EGFR mutation analysis is the best predictive marker for the use of EGFR TKI therapy in NSCLC.[17] Deletions in exon 19 and point mutatio L858R in exon 21 occur most frequently and are associated with a ~70% response rate on EGFR TKI therapy. In Asian and Japan patients the incidence is higher (30-50%). Overall, gender, ethnicity and smoking status are unsuitable as triage for mutation analysis. [18][19][20]

For the pathologist it is important to be aware of the analytical sensitivity (the minimal percentage of tumor cells within a population required for a positive test result) of the EGFR mutation test. In Sanger sequencing this is generally around 30-50%, while more sensitive techniques reach a sensitivity of 1-10%. In bronchial biopsies in particular low numbers of tumor cells may be present (e.g. lymphangitis carcinomatosa) prohibiting a positive outcome when classical sequencing (and probably also next generation sequencing) would be used. For more detailed information see recent review.[21]

ALK positive lung cancer 
A small inversion within chromosome 2p results in the formation of a fusion gene comprising portions of the EML4- and ALK gene in NSCLC cells.[22] ALK-positive tumours have been detected in all histological subtypes of AdCs, but a preference for a solid signet-ring cell pattern and a mucinous cribriform pattern seems to exist with lack of significant nuclear pleomorphism.[22]

Screening for ALK aberrations by FISH is quite laborious and not easy implemented for regular screening of all AdCs. In Europe approval for ALK positive lung cancer was obtained after publication of a phase III study.[13] For ALK positivity a validated test (IHC, FISH, RT-PCR) may be used. The ALK IHC protocol differs from the ALK lymphoma IHC test, as not all ALK antibodies are useful and the epitope concentration is lower. Currently, two antibodies work for ALK IHC+ lung cancer, 5A4 and D5F3.[23, 24] For more detailed information see recent review[21] or click In the table below genes involved in targeted treatment of NSCLC and method of detection are shown. EGFR and ALK are routinely applied in daily practice. For the others phase I/II and sometimes phase III trials are underway or initiated.

EGFR mut[18]erlotinib, gefitinib, afatinibAdC-/+-+
cMET[25]met mabAdC, SqCC++-
BRAF[27]sorafinib vemurafenibAdC+?-+
PIK3CA[28]everolimus temsirolimusAdC, SqCC--+
HER2[29]trastuzumab/ lapatinib/ dacomatinibAdC++-
EGFR ihc[30]cetuximabAdC, SqCC++/--
PTEN[31]everolimus temsirolimusAdC, SqCC+--
KRAS[33]selumetinib docetaxelAdC--+
NTRK1[34]Crizotinib (?)AdC-++


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