Lung Cancer

Primary Authors Wallander, Michelle, PhD. Heichman, Karen A., PhD.

Key Points

Molecular Markers in Lung Cancer

The presence of molecular markers in non-small cell lung cancer (NSCLC) is associated with responsiveness to certain targeted therapies. The current markers, discussed below, hold the most importance for nonsquamous histology, particularly for adenocarcinoma subtypes.

  • EGFR

    EGFR (epidermal growth factor receptor)

    Pathophysiology

    Receptor tyrosine kinase involved in cell proliferation

    • Inhibition of tyrosine kinase activity diminishes tumor growth

    Two methods for blocking kinase activity (basis of current therapies in lung cancer)

    • Prevent ligand binding to the extracellular domain with a monoclonal antibody OR
    • Inhibit activity with small molecules (tyrosine kinase inhibitors) that block the magnesium-ATP binding pocket of the intracellular TK domain
    Mutation

    Certain EGFR mutations are associated with increased sensitivity to tyrosine kinase inhibitors (TKIs) (eg, gefitinib and erlotinib)

    Lack of EGFR mutations (wild type) is associated with decreased sensitivity to TKIs

    Mutation status of EGFR does not appear to predict response to treatment with EGFR monoclonal antibody (cetuximab)

    T790M mutation associated with decreased sensitivity to TKIs – typical mutation that develops when treatment resistance occurs

    Mutation characteristics

    • 10-15% of NSCLC – almost exclusively adenocarcinoma subtype
    • Highest rate in Asians, adenocarcinoma histology subtype, lifetime nonsmokers or light smokers (<10 packs per year), female sex

    Recommended Testing Strategy

    ARUP available tests: 

    • EGFR Mutation Detection by Pyrosequencing 2002440
    • Lung Cancer Panel 2008894 – includes EGFR, ALK (D5F3), and ROS1
    • Lung Cancer Panel with KRAS 2008895 – includes KRAS, EGFR, ALK (D5F3), and ROS1

    Mutational analysis is preferred over gene amplification (Lindeman, 2013)

    • IHC and FISH are not recommended

    If EGFR testing is negative, testing for ALK may be helpful; if EGFR testing is done initially and is positive, testing for KRAS may be helpful

  • KRAS

    KRAS (v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog I)

    Pathophysiology

    RAS genes regulate signal conduction pathways that control cell growth

    Three distinct genes – HRAS, NRAS, and KRAS

    Mutation

    Almost all RAS mutations in NSCLC are in KRAS gene

    Mutations in KRAS gene are associated with decreased sensitivity to EGFR TKIs and overall poor prognosis

    • Double mutations in EGFR and KRAS are associated with decreased sensitivity to TKIs despite the presence of EGFR mutation

    Mutation characteristics

    • 25-30% of adenocarcinomas
    • Highest rate in adenocarcinoma mucinous subtype, current smokers

    Recommended Testing Strategy

    ARUP available tests:

    • KRAS Mutation Detection 0040248
    • Lung Cancer Panel with KRAS 2008895 – includes KRAS, EGFR, ALK (D5F3), and ROS1

    Mutational analysis for KRAS establishes the reflex pattern of testing that maximizes testing efficiency for EGFR mutations and ALK rearrangements (Lindeman, 2013)

    If KRAS is negative, test for EGFR mutations; if EGFR is negative, test for ALK and ROS1 rearrangements

  • ALK

    ALK (anaplastic lymphoma receptor tyrosine kinase)

    Pathophysiology

    Fusion of ALK gene with echinoderm microtubule-associated protein-like-4 (EML-4) or other fusion partners mediates ligand-independent oligomerization of ALK resulting in ALK kinase activation

    • Functions as potent oncogenic driver
    Mutation

    Certain ALK rearrangements are associated with increased sensitivity to specific TKIs (ALK/MET/ROS1 TKIs [eg, crizotinib]) and decreased sensitivity to EGFR TKIs

    Mutation characteristics

    • Relatively rare mutation in NSCLC (3-7%)
    • Highest rate in young patients, lifetime nonsmokers or light smokers (<10 packs per year), adenocarcinoma with signet rings and mucin histology subtype, male sex

    Recommended Testing Strategy

    ARUP available tests:

    • ALK (D5F3) with Interpretation by Immunohistochemistry 2007324
    • Lung Cancer Panel 2008894 – includes EGFR, ALK (D5F3), and ROS1
    • Lung Cancer Panel with KRAS 2008895 – includes KRAS, EGFR, ALK (D5F3), and ROS1

    Immunohistochemistry detects protein expression and is a surrogate marker for ALK gene fusion; more cost effective and efficient compared to FISH and RT-PCR

    RT-PCR may be used to detect the most common ALK rearrangements but may not detect all ALK fusion partners, and is therefore not recommended (Lindeman, 2013)

    FISH interpretation is challenging due to small inversions involving chromosome 2p (2p21 and 2p23)

    Presence of ALK rearrangements are generally mutually exclusive of EGFR and KRAS mutations

    ROS1

    ROS1 (c-ros oncogene 1, receptor tyrosine kinase)

    Pathophysiology

    Receptor tyrosine kinase involved in cell proliferation

    ROS1 gene rearrangement and fusion with multiple known partners mediates constitutive ROS1 kinase activation

    Inhibition of tyrosine kinase activity diminishes tumor growth

    Mutation

    ROS1 rearrangements

    • Increased sensitivity to ALK/MET/ROS1 TKIs (eg, crizotinib)
    • Decreased sensitivity to EGFR TKIs

    Mutation characteristics

    • Rare mutation in NSCLC – 1-2%
    • Highest rate in Asians, young patients, lifetime nonsmokers or light smokers (≤10 packs per year history), and adenocarcinomas

    Recommended Testing Strategy

    ARUP available tests: 

    • ROS1 with Interpretation by Immunohistochemistry with Reflex to FISH if Equivocal 2008414
    • Lung Cancer Panel 2008894 – includes EGFR, ALK (D5F3), and ROS1
    • Lung Cancer Panel with KRAS 2008895 – includes KRAS, EGFR, ALK (D5F3), and ROS1

    Immunohistochemistry detects protein expression and is a surrogate marker for ROS1 gene fusion

    • More cost effective and efficient when compared to FISH, but FISH is indicated for equivocal results by IHC

    Presence of ROS1 rearrangements are generally mutually exclusive of EGFR and KRAS mutations

Diagnosis

Indications for Testing

  • New pulmonary mass

Laboratory Testing

  • Serum testing is not helpful in diagnosing lung cancer; however, baseline testing (eg, CBC with differential, liver function) may be performed as a general screen for metastases
  • Other testing
    • Neuron specific enolase (NSE), serum – may have diagnostic value for small cell lung cancer (SCLC)  

Imaging Studies

  • Chest x-ray, CT scan, MRI provide basis for initial testing

Histology

  • Invasive testing to obtain tissue necessary for diagnosis
    • Bronchoscopic biopsy
    • Mediastinal node sampling
    • Fine needle aspiration using CT guidance
    • Open lung biopsy
    • Fine needle aspiration using endobronchial ultrasound (EBUS)
  • Immunohistochemistry in diagnosis
    • Should be used adjunctly and interpreted in clinical context
    • ALK immunohistochemistry – more cost effective and efficient than ALK by RT-PCR, FISH
      • Uses ALK clone D5F3
        • D5F3 clone – more sensitive than ALK1 clone
    • Primary adenocarcinoma and bronchioalveolar carcinoma
      • Positive for CK 7, napsin A, and thyroid transcription factor-1 (TTF-1); negative for CK 20
      • Primary mucinous adenocarcinoma – positive for CK 7 and CK 20, negative for TTF-1
    • Squamous cell carcinoma (SCC)
      • Usually negative for CK 7, CK 20, TTF-1
      • Does not typically require immunohistochemical staining for diagnosis
    • SCLC and neuroendocrine carcinomas
      • Typically negative for CK 7, CK 20
      • Positive for neuroendocrine markers such as chromogranin A, CD56 (NCAM), synaptophysin, NSE
      • Differentiating poorly differentiated SCC from SCLC
        • p63 and CK 5,6 positive and TTF-1 negative supports SCC
    • Differentiating non-small cell lung cancer (NSCLC) adenocarcinoma from mesothelioma
      • Positive in NSCLC/negative in mesothelioma – CEA-P, b72.3, Ber-EP4, MOC-31, TTF-1
    • Differentiating NSCLC from SCLC
      • MicroRNA (mRNA) expression – may be used to distinguish NSCLC from SCLC
    • Differentiating metastatic adenocarcinomas
      • Pulmonary – CK 7+/CK 20-
      • Colorectal – CK 7-/CK 20+
    • Differentiating primary lung tumors from metastatic gastrointestinal tumors – CDX2 specific for gastrointestinal tumors
    • Dysregulation in tumor progression, therapeutic target – c-MET

Solitary Pulmonary Nodule

  • Usually found incidentally on chest x-ray (0.09-0.2% of all x-rays)
    • 35% are malignant
  • Histologic diagnosis required in the following situations
    • Patient is ≥35 years
    • Nodule is >10 mm diameter
    • Growth of lesion
    • Lack of calcification
    • Adenopathy
    • Positive PET scan
    • No previous imaging to review in order to determine if nodule has changed in size

Prognosis

  • Markers
    • EGFR, KRAS, ALK (D5F3), ROS1 – refer to Key Points section
      • Establish eligibility for therapies such as tyrosine kinase inhibitors and ALK inhibitors
  • Promising markers
    • Cytokeratin-19 fragment (CYFRA 21-1)
      • Most sensitive tumor marker for NSCLC (particularly squamous)
      • Uses
        • Prognostication
          • Elevated pretreatment level – associated with unfavorable prognosis
        • Monitoring
          • Decreasing levels predict objective response to treatment
    • NSE
      • High specificity for SCLC
      • May be useful in assessing prognosis in NSCLC and SCLC
      • Currently in clinical use, but prognostic value has not been validated in high-level study
    • p53 – tumor suppressor gene
      • Presence prognostic of short survival and potential benefit from adjuvant chemotherapy in patients with NSCLC
    • Pro GRP – SCLC
    • CA 125 (available as an immunohistochemical stain) – NSCLC
    • STK11 – adenocarcinoma
    • KRAS, NRAS, HRAS – adenocarcinoma
    • HER-2 – NSCLC
    • Tumor M2-PK (TU M2-PK) – all types
    • BCL-2 (available as an immunohistochemical stain) – SCLC
    • ERCC1 expression
      • High level – prognostic of better survival in NSCLC independent of therapy
      • High level – predictive of poor response to platinum-based chemotherapy; low level associated with sensitivity
    • RRM1 expression
      • High level – prognostic of better survival in NSCLC independent of therapy
      • High level – predictive of poor response to gemcitabine-based chemotherapy

Differential Diagnosis

Screening

  • Trials evaluating screening
    • U.S. trials
      • National Lung Screening Trial – lung cancer mortality reduced by annual screening with low-dose computed tomography (LDCT) in high-risk populations
        • LDCT screening not recommended due to low specificity, cost, and radiation exposure
    • European trials
      • NELSON Trial – Netherlands and Belgium
      • Danish Lung Screening Trial
      • LUSI Trial – Germany
      • ITALUNG – Italy
      • MILD Trial – Italy
  • Screening recommendations
    • ACCP and ASCO – recommend lung cancer screening for high-risk patients who meet criteria of the NLST (high-risk smokers and former smokers age 55-74 years with a 30 packs per year smoking history and no history of lung cancer)
      • American Thoracic Society approved this recommendation
    • NCCN (2012) – screening recommended for high-risk individuals
      • High risk defined as age 55-74 AND ≥30 packs per year history of smoking and smoking cessation <15 years OR
      • Age ≥50 years AND ≥20 packs per year history of smoking AND one additional risk factor (other than second-hand smoke)
        • Additional risk factors – smoking history (present or past); radon exposure; occupational exposure; cancer history; family history; COPD or pulmonary fibrosis; second-hand smoke exposure
    • Controversial with all screening recommendations
      • Cost effectiveness
      • Radiation exposure from CT

Monitoring

  • Recommended monitoring (postcurative approach)
    • History and physical, chest x-ray, CBC and chemistries every 3-6 months for first 2 years (National Comprehensive Cancer Network [NCCN] recommends CT every 6 months in non-small cell lung cancer [NSCLC])
  • Small cell lung cancer (SCLC)
    • Serial NSE may be useful to monitor tumor recurrence in SCLC
  • NSCLC
    • CK 19 – potential role for monitoring therapy in advanced NSCLC

Clinical Background

Lung cancer is the leading cause of cancer-related mortality in the U.S. and worldwide.

Epidemiology

  • Incidence – 62.5/100,000
    • 160,000 deaths from lung cancer estimated in 2012 (NCCN, 2012)
  • Age – peak incidence is 65-74 years; median is 71 years
  • Sex – M>F, minimal
    • Female prevalence has increased; male prevalence has stabilized
  • Ethnicity – highest incidence in African American males

Risk Factors

  • Tobacco use – 85-90% of all lung cancers attributed to tobacco use
    • 13-fold increase in risk for primary user
    • Secondhand exposure – 20-30% increased risk for those who live/lived with smokers
  • Radon/uranium exposure
  • Asbestos exposure – cumulative risk; estimated to cause 3-4% of cases
    • Risk increased if patient also smokes
  • Occupational exposure to carcinogens (eg, bis(chloromethyl)ether, polycyclic aromatic hydrocarbons, chromium, nickel, organic arsenic)
  • Previous chest irradiation
  • Genetic – positive family history combined with tobacco use increases the risk

Pathophysiology

  • Any tumor arising from respiratory epithelium or pneumocytes
  • Two main types
    • Non-small cell lung cancer (NSCLC) – ~85% of all lung cancers
      • Adenocarcinoma (non-squamous cell) – most common
        • Occurs in glandular tissue of lung lining
      • Squamous (epidermoid) cell carcinoma (SCC)
      • Large cell (large cell anaplastic)
    • Small cell lung cancer (SCLC) – ~15% of all lung cancers
      • Epithelial tumor of small cells
        • 95% arise in lung; may also arise from extrapulmonary sites (eg, nasopharynx, gastrointestinal tract, genitourinary tract)
      • Nearly all cases result from smoking
      • Two subtypes
        • Small cell carcinoma (oat cell)
        • Combined small cell carcinoma
  • Other tumors (rare)
  • Undifferentiated bronchial-gland tumors, sarcomas, neuroendocrine tumors

Clinical Presentation

  • 20% of cases incidentally identified while asymptomatic by chest x-ray for other reasons
  • Symptoms based on area of tumor growth
    • Central – cough, wheeze, hemoptysis, stridor, dyspnea, postobstructive pneumonia
    • Peripheral – pleural/chest wall pain, cough, dyspnea
    • Invasion and obstruction of adjacent structures
      • Tracheal obstruction – dyspnea, wheezing
      • Esophageal compression – dysphagia
      • Recurrent laryngeal nerve invasion – hoarseness
      • Phrenic nerve invasion – diaphragmatic paralysis
      • Sympathetic nerve invasion – Horner syndrome
        • Ptosis
        • Miosis
        • Enophthalmos
        • Unilateral loss of sweating
      • Invasion of lung apex – Pancoast tumor, superior vena caval syndrome
    • Distant metastases
      • Superior vena caval syndrome
      • Pericardial tamponade
      • Pleural effusions
      • Pathologic bone fractures
      • Adrenal insufficiency (rare) 
    • Paraneoplastic syndromes – common; may be first presenting symptoms of lung cancer

Indications for Laboratory Testing

  • Tests generally appear in the order most useful for common clinical situations
  • Click on number for test-specific information in the ARUP Laboratory Test Directory
Test Name and Number Recommended Use Limitations Follow Up
Cytology, Pulmonary 8209702
Method: Microscopy

May be used as initial screening test in detection of lung cancer

Negative screening does not mean cancer is not present

Bronchial brushings, washings, tissue biopsy (transbronchial and open lung) from site of lung involved

Cytology, Bronchoalveolar Lavage, Malignancy 8280005
Method: Microscopy

May be used as initial screening test in detection of lung cancer

Negative result does not mean cancer is not present

Bronchial brushings, washings, tissue biopsy (transbronchial and open lung) from site of lung involved

Lung Cancer Panel 2008894
Method: Polymerase Chain Reaction/Pyrosequencing/Immunohistochemistry

Screening panel to determine eligibility for TKI therapy

EGFR, ALK, and ROS1 genes tested simultaneously

Results of these tests must be interpreted in the context of morphological and other relevant data

Tests should not be used alone to diagnose malignancy

 
Lung Cancer Panel with KRAS 2008895
Method: Polymerase Chain Reaction/Pyrosequencing/Immunohistochemistry

Screening panel to determine eligibility for TKI therapy

KRAS, EGFR, ALK, and ROS1 genes tested simultaneously

Results of these tests must be interpreted in the context of morphological and other relevant data

Tests should not be used alone to diagnose malignancy

 
KRAS Mutation Detection 0040248
Method: Polymerase Chain Reaction/Pyrosequencing

Suggested initial single-gene assay to determine eligibility for tyrosine kinase inhibitor (TKI) therapy

Results must be interpreted in the context of morphological and other relevant data

Test should not be used alone to diagnose malignancy

If KRAS is negative, test for EGFR

If EGFR is negative, test for ALK and ROS1

EGFR Mutation Detection by Pyrosequencing 2002440
Method: Polymerase Chain Reaction/Pyrosequencing

Order if KRAS mutation not detected

Single-gene assay to determine eligibility for TKI therapy

Detects mutations in EGFR exons 18, 19, 20, and 21

Results must be interpreted in the context of morphological and other relevant data

Test should not be used alone to diagnose malignancy

If EGFR is negative, test for ALK and ROS1

If EGFR is positive, consider KRAS

ALK (D5F3) with Interpretation by Immunohistochemistry 2007324
Method: Immunohistochemistry

Determine eligibility for TKI therapy for individuals with lung cancer

Order if no mutations detected in either KRAS or EGFR genes

Cost effective, efficient screen for all ALK fusion proteins

D5F3 clone more sensitive than ALK1 clone for detection of ALK protein expression in lung cancer

Results must be interpreted in the context of morphological and other relevant data

Test should not be used alone to diagnose malignancy

 
ROS1 with Interpretation by Immunohistochemistry with Reflex to FISH if Equivocal 2008414
Method: Immunohistochemistry

Determine eligibility for TKI therapy for individuals with lung cancer

Cost-effective, efficient screen for ROS1 fusion proteins

Use if no mutations detected in either KRAS or EGFR genes

Results must be interpreted in the context of morphological and other relevant data

Test should not be used alone to diagnose malignancy

 
CYFRA 21-1 (Cytokeratin 19 Fragment), Serum 0081344
Method: Quantitative Enzyme-Linked Immunosorbent Assay

Use for prognostication and monitoring in NSCLC

Clinical sensitivity – varies by disease stage

Analytical sensitivity – limit of detection is 0.5 ng/mL

Do not use for screening

Results obtained with different methods or kits cannot be used interchangeably

 
Neuron Specific Enolase 0098198
Method: Quantitative Enzyme-Linked Immunosorbent Assay
Use for prognostication and monitoring in NSCLC

Do not use for screening

Results obtained with different methods or kits cannot be used interchangeably

 
Cytokeratin 7 (CK 7) by Immunohistochemistry 2003854
Method: Immunohistochemistry

Aid in histologic diagnosis of lung cancer

Stained and returned to client pathologist for interpretation; consultation available if needed

   
Cytokeratin 20 (CK 20) by Immunohistochemistry 2003848
Method: Immunohistochemistry

Aid in histologic diagnosis of lung cancer

Stained and returned to client pathologist for interpretation; consultation available if needed

   
Thyroid Transcription Factor (TTF-1) by Immunohistochemistry 2004166
Method: Immunohistochemistry

Aid in histologic diagnosis of lung cancer

Stained and returned to client pathologist for interpretation; consultation available if needed

   
Chromogranin A by Immunohistochemistry 2003830
Method: Immunohistochemistry

Aid in histologic diagnosis of lung cancer

Stained and returned to client pathologist for interpretation; consultation available if needed

   
CD56 (NCAM) by Immunohistochemistry 2003589
Method: Immunohistochemistry

Aid in histologic diagnosis of lung cancer

Stained and returned to client pathologist for interpretation; consultation available if needed

   
Synaptophysin by Immunohistochemistry 2004139
Method: Immunohistochemistry

Aid in histologic diagnosis of lung cancer

Stained and returned to client pathologist for interpretation; consultation available if needed

   
p63 by Immunohistochemistry 2004073
Method: Immunohistochemistry

Aid in histologic diagnosis of lung cancer

Stained and returned to client pathologist for interpretation; consultation available if needed

   
Cytokeratin 5,6  (CK 5,6) by Immunohistochemistry 2003851
Method: Immunohistochemistry

Aid in histologic diagnosis of lung cancer

Stained and returned to client pathologist for interpretation; consultation available if needed

   
Carcinoembryonic Antigen, Polyclonal (CEA P) by Immunohistochemistry 2003827
Method: Immunohistochemistry

Aid in histologic diagnosis of lung cancer

Stained and returned to client pathologist for interpretation; consultation available if needed

   
Breast Carcinoma b72.3 by Immunohistochemistry 2003445
Method: Immunohistochemistry

Aid in histologic diagnosis of lung cancer

Stained and returned to client pathologist for interpretation; consultation available if needed

   
Anti-Human Epithelial Antigen, Ber-EP4 by Immunohistochemistry 2003463
Method: Immunohistochemistry

Aid in histologic diagnosis of lung cancer

Stained and returned to client pathologist for interpretation; consultation available if needed

   
Epithelial-Related Antigen, MOC-31 by Immunohistochemistry 2003875
Method: Immunohistochemistry

Aid in histologic diagnosis of lung cancer

Stained and returned to client pathologist for interpretation; consultation available if needed

   
Neuron Specific Enolase, Polyclonal (NSE P) by Immunohistochemistry 2004052
Method: Immunohistochemistry

Aid in histologic diagnosis of lung cancer

Stained and returned to client pathologist for interpretation; consultation available if needed

   
CDX2 by Immunohistochemistry 2003821
Method: Immunohistochemistry

Aid in differentiating primary lung tumors from metastatic GI tumors

Stained and returned to client pathologist for interpretation; consultation available if needed

   
c-MET by Immunohistochemistry 2008652
Method: Immunohistochemistry

Aid in histologic diagnosis of lung cancer

Stained and returned to client pathologist for interpretation; consultation available if needed

   
Napsin A by Immunohistochemistry 2008716
Method: Immunohistochemistry

Aid in histologic diagnosis of lung cancer

Stained and returned to client pathologist for interpretation; consultation available if needed

   
Additional Tests Available
 
Click the plus sign to expand the table of additional tests.
Test Name and NumberComments
EGFR Gene Amplification by FISH 2008605
Method: Fluorescence in situ Hybridization

Not recommended for lung cancer mutation screening

ALK Gene Rearrangements in NSCLC for Crizotinib Eligibility by FISH 2006102
Method: Fluorescence in situ Hybridization

Preferred test is ALK (D5F3) by IHC; interpretation is challenging in FISH for rearrangements due to small inversions involving chromosome 2p

Lung Cancer Assessment and Early Detection (EarlyCDT) 2007513
Method: Semi-Quantitative Enzyme-Linked Immunosorbent Assay

May be useful as screening for patients deemed high risk for lung cancer

Solid Tumor Mutation Panel by Next Generation Sequencing 2007991
Method: Massively Parallel Sequencing

48 gene panel for detection of lung cancer mutations

Includes KRAS, EGFR, BRAF, and ERBB2 genes

BRAF Codon 600 Mutation Detection by Pyrosequencing 2002498
Method: Polymerase Chain Reaction/Pyrosequencing

May aid in lung cancer therapy decisions

Carcinoembryonic Antigen 0080080
Method: Quantitative Electrochemiluminescent Immunoassay

Prognostication and monitoring in lung cancer

Squamous Cell Carcinoma Antigen, Serum 0081054
Method: Quantitative Enzyme-Linked Immunosorbent Assay

Prognostication and monitoring in lung cancer