European Society of Pathology
The leading force in European pathology
ECP 2023
|

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 www.alktesting.org. 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.


GeneDrug(s)HistologyIHCFISHmut/ins-del/transl
EGFR mut[18]erlotinib, gefitinib, afatinibAdC-/+-+
ALK[13]crizotinibAdC+++
cMET[25]met mabAdC, SqCC++-
ROS1[26]crizotinibAdC+?+-
BRAF[27]sorafinib vemurafenibAdC+?-+
PIK3CA[28]everolimus temsirolimusAdC, SqCC--+
HER2[29]trastuzumab/ lapatinib/ dacomatinibAdC++-
EGFR ihc[30]cetuximabAdC, SqCC++/--
PTEN[31]everolimus temsirolimusAdC, SqCC+--
NRAS[32]vemurafenibAdC--+
KRAS[33]selumetinib docetaxelAdC--+
NTRK1[34]Crizotinib (?)AdC-++
DDR2[35]dasatinibSqCC--+
FGFR1[36]ponatinibSqCC-+-


References

  1. Vansteenkiste J, De Ruysscher D, Eberhardt WEE, et al. (2013) Early and locally advanced non-small-cell lung cancer (NSCLC): ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Ann Oncol 21:1–10. doi: 10.1093/annonc/mdt241
  2. Travis WD, Brambilla E, Noguchi M, et al. (2011) International association for the study of lung cancer/american thoracic society/european respiratory society international multidisciplinary classification of lung adenocarcinoma. J Thorac Oncol 6:244–85. doi: 10.1097/JTO.0b013e318206a221
  3. Travis WD, Brambilla E, Noguchi M, et al. (2012) Diagnosis of Lung Cancer in Small Biopsies and Cytology: Implications of the 2011 International Association for the Study of Lung Cancer/American Thoracic Society/European Respiratory Society Classification. Arch Pathol Lab Med. doi: 10.5858/arpa.2012-0263-RA
  4. Thunnissen E, Boers E, Heideman DA, et al. (2012) Correlation of immunohistochemical staining p63 and TTF-1 with EGFR and K-ras mutational spectrum and diagnostic reproducibility in non small cell lung carcinoma. Virchows Arch. doi: 10.1007/s00428-012-1324-x
  5. Bishop J a, Teruya-Feldstein J, Westra WH, et al. (2012) p40 (DeltaNp63) is superior to p63 for the diagnosis of pulmonary squamous cell carcinoma. Mod Pathol 25:405–415. doi: 10.1038/modpathol.2011.173
  6. Nonaka D (2012) A study of DeltaNp63 expression in lung non-small cell carcinomas. Am J Surg Pathol 36:895–899. doi: 10.1097/PAS.0b013e3182498f2b
  7. Rekhtman N, Paik PK, Arcila ME, et al. (2012) Clarifying the spectrum of driver oncogene mutations in biomarker-verified squamous carcinoma of lung: lack of EGFR/KRAS and presence of PIK3CA/AKT1 mutations. Clin Cancer Res 18:1167–76. doi: 10.1158/1078-0432.CCR-11-2109.Clarifying
  8. Mok TS, Wu Y-L, Thongprasert S, et al. (2009) Gefitinib or carboplatin-paclitaxel in pulmonary adenocarcinoma. N Engl J Med 361:947–57. doi: 10.1056/NEJMoa0810699
  9. 9. Sequist L V, Yang JC-H, Yamamoto N, et al. (2013) Phase III Study of Afatinib or Cisplatin Plus Pemetrexed in Patients With Metastatic Lung Adenocarcinoma With EGFR Mutations. J Clin Oncol 31:3327–3334. doi: 10.1200/JCO.2012.44.2806
  10. Zhou C, Wu Y-L, Chen G, et al. (2011) Erlotinib versus chemotherapy as first-line treatment for patients with advanced EGFR mutation-positive non-small-cell lung cancer (OPTIMAL, CTONG-0802): a multicentre, open-label, randomised, phase 3 study. Lancet Oncol 12:735–42. doi: 10.1016/S1470-2045(11)70184-X
  11. Mitsudomi T, Morita S, Yatabe Y, et al. (2010) Gefitinib versus cisplatin plus docetaxel in patients with non-small-cell lung cancer harbouring mutations of the epidermal growth factor receptor (WJTOG3405): an open label, randomised phase 3 trial. Lancet Oncol 11:121–8. doi: 10.1016/S1470-2045(09)70364-X
  12. Rosell R, Carcereny E, Gervais R, et al. (2012) Erlotinib versus standard chemotherapy as first-line treatment for European patients with advanced EGFR mutation-positive non-small-cell lung cancer (EURTAC): a multicentre, open-label, randomised phase 3 trial. Lancet Oncol 13:239–46. doi: 10.1016/S1470-2045(11)70393-X
  13. Shaw AT, Kim D-W, Nakagawa K, et al. (2013) Crizotinib versus Chemotherapy in Advanced ALK -Positive Lung Cancer. N Engl J Med 368:130601060020006. doi: 10.1056/NEJMoa1214886
  14. Govindan R, Ding L, Griffith M, et al. (2013) Genomic landscape of non-small cell lung cancer in smokers and never-smokers. Cell 150:1121–1134. doi: 10.1016/j.cell.2012.08.024.GENOMIC
  15. Imielinski M, Berger AH, Hammerman PS, et al. (2013) Mapping the hallmarks of lung adenocarcinoma with massively parallel sequencing. Cell 150:1107–1120. doi: 10.1016/j.cell.2012.08.029.Mapping
  16. Ding L, Getz G, Wheeler DA, et al. (2009) Somatic mutations affect key pathways in lung adenocarcinoma. Nature 455:1069–1075. doi: 10.1038/nature07423.Somatic
  17. Lindeman NI, Cagle PT, Beasley MB, et al. (2013) Molecular Testing Guideline for Selection of Lung Cancer Patients for EGFR and ALK Tyrosine Kinase Inhibitors: Guideline from the College of American Pathologists, International Association for the Study of Lung Cancer, and Association for Molecular Patho. Arch Pathol Lab Med 137:828–860. doi: 10.5858/arpa.2012-0720-OA
  18. Lee CK, Brown C, Gralla RJ, et al. (2013) Impact of EGFR inhibitor in non-small cell lung cancer on progression-free and overall survival: a meta-analysis. J Natl Cancer Inst 105:595–605. doi: 10.1093/jnci/djt072
  19. Gahr S, Stoehr R, Geissinger E, et al. (2013) EGFR mutational status in a large series of Caucasian European NSCLC patients: data from daily practice. Br J Cancer 1–8. doi: 10.1038/bjc.2013.511
  20. Dearden S, Stevens J, Wu Y-L, Blowers D (2013) Mutation incidence and coincidence in non small-cell lung cancer: meta-analyses by ethnicity and histology (mutMap). Ann Oncol 24:2371–6. doi: 10.1093/annonc/mdt205
  21. Thunnissen E, van der Oord K, den Bakker M (2014) Prognostic and predictive biomarkers in lung cancer. A review. Virchows Arch 464:347–58. doi: 10.1007/s00428-014-1535-4
  22. Thunnissen E, Bubendorf L, Dietel M, et al. (2012) EML4-ALK testing in non-small cell carcinomas of the lung: a review with recommendations. Virchows Arch 461:245–57. doi: 10.1007/s00428-012-1281-4
  23. Conklin CMJ, Craddock KJ, Have C, et al. (2013) Immunohistochemistry is a reliable screening tool for identification of ALK rearrangement in non-small-cell lung carcinoma and is antibody dependent. J Thorac Oncol 8:45–51. doi: 10.1097/JTO.0b013e318274a83e
  24. Mino-Kenudson M, Chirieac LR, Law K, et al. (2010) A novel, highly sensitive antibody allows for the routine detection of ALK-rearranged lung adenocarcinomas by standard immunohistochemistry. Clin Cancer Res 16:1561–71. doi: 10.1158/1078-0432.CCR-09-2845
  25. Sadiq A a, Salgia R (2013) MET as a possible target for non-small-cell lung cancer. J Clin Oncol 31:1089–96. doi: 10.1200/JCO.2012.43.9422
  26. Bergethon K, Shaw AT, Ou S-HI, et al. (2012) ROS1 rearrangements define a unique molecular class of lung cancers. J Clin Oncol 30:863–70. doi: 10.1200/JCO.2011.35.6345
  27. Marchetti A, Felicioni L, Malatesta S, et al. (2011) Clinical features and outcome of patients with non-small-cell lung cancer harboring BRAF mutations. J Clin Oncol 29:3574–9. doi: 10.1200/JCO.2011.35.9638
  28. Oxnard GR, Binder A, Jänne P a (2013) New targetable oncogenes in non-small-cell lung cancer. J Clin Oncol 31:1097–104. doi: 10.1200/JCO.2012.42.9829
  29. Mazières J, Peters S, Lepage B, et al. (2013) Lung cancer that harbors an HER2 mutation: epidemiologic characteristics and therapeutic perspectives. J Clin Oncol 31:1997–2003. doi: 10.1200/JCO.2012.45.6095
  30. Pirker R, Pereira JR, von Pawel J, et al. (2012) EGFR expression as a predictor of survival for first-line chemotherapy plus cetuximab in patients with advanced non-small-cell lung cancer: analysis of data from the phase 3 FLEX study. Lancet Oncol 13:33–42. doi: 10.1016/S1470-2045(11)70318-7
  31. Reungwetwattana T, Molina JR, Mandrekar SJ, et al. (2012) Brief report: a phase II “window-of-opportunity” frontline study of the MTOR inhibitor, temsirolimus given as a single agent in patients with advanced NSCLC, an NCCTG study. J Thorac Oncol 7:919–22. doi: 10.1097/JTO.0b013e31824de0d6
  32. Ohashi K, Sequist L V, Arcila ME, et al. (2013) Characteristics of lung cancers harboring NRAS mutations. Clin Cancer Res 19:2584–91. doi: 10.1158/1078-0432.CCR-12-3173
  33. Martin P, Leighl NB, Tsao M-S, Shepherd FA (2013) KRAS mutations as prognostic and predictive markers in non-small cell lung cancer. J Thorac Oncol 8:530–42. doi: 10.1097/JTO.0b013e318283d958
  34. Vaishnavi A, Capelletti M, Le AT, et al. (2013) Oncogenic and drug-sensitive NTRK1 rearrangements in lung cancer. Nat Med. doi: 10.1038/nm.3352
  35. Hammerman PS, Sos ML, Ramos AH, et al. (2011) Mutations in the DDR2 kinase gene identify a novel therapeutic target in squamous cell lung cancer. Cancer Discov 1:78–89. doi: 10.1158/2159-8274.CD-11-0005
  36. Ren M, Hong M, Liu G, et al. (2013) Novel FGFR inhibitor ponatinib suppresses the growth of non-small cell lung cancer cells overexpressing FGFR1. Oncol Rep 29:2181–90. doi: 10.3892/or.2013.2386