Ken's Notes

Molecular Alterations in Lung Carcinomas

All lung carcinomas:

Multiple chromosomal aberrations, indicative of genome instability (4)

Balanced translocations are rare (4)
In most instances the alterations results in a genomic imbalance – net gain or loss of genetic material (4)
Often extra chromosomes and unidentified (marker) chromosomes (4)

Mechanism of chromosomal gains / losses not well understood (13)

Telomere dysfunction may be important (13)
Chromosomal breakage-fusion-bridge (BFB) may be important (13)

Knowledge about the order or progression of gains / losses is scarce (13)
gains:

1q (13)

1q31

3q (13)

3q25-27

PIK3CA is in this amplicon

5p (13)

5p13-14

8q (13)

8q23-24

11q13

CCND1 (encoding cyclin D1) (2)

20q by CGH (13)

Amplifications:

Amplifications are common (13)

Double minute chromosomes often (13)

MYCC amplification is seen in both SCLC and NSCLC (8% NSCLC by FISH - PMID: 14705768) (7)

5-15% of SCLC and 8% of NSCLC show amplification of one of the members of the MYC gene family (9)
Rare in primary lung tumours, but more frequently observed in lung cancer cell lines and primary lung cancer following chemotherapy (7)
MYC amplification in treated patient’s tumour cell lines is associated with a shortened survival (9)

DcR3 gene (decoy receptor 3) (50% in small study) (7)
Cyclin D1 (low frequency) (7)
EGFR (low frequency) (7)
HER2/neu (low frequency) (7) (~3%) (9)
MDM2 (low frequency) (7)

losses:

3p21

consistent loss of short arm of chromosome 3 with a peak recurrence at ~50Mb (29 of 79 samples, 36%) (1)
no obvious homozygous deletions which would point to a specific target (1)
this region contains RASSF1, TUSC2, SEMA3B, FHIT, which have shown LOH in NSCLC (1)

8p (13)

8p22

9p (13)

9p21-22

13q (13)

13q22

17p (13)

17p12-13

17p13.1 locus contains p53 gene (2)

homozygous deletions:

3q25 (6)

candidate genes AADAC, SUCNR1 (6)

9p23 (6)

candidate genes PTPRD, BC028038 (6)

LOH at many different loci:

3p14-23 (>90% SCLC, ~70% NSCLC)

Potential tumour suppressor genes include FHIT, RASSF1, and SEMA3B
Methylation of genes on 3p is often present at early stages of lung cancer
LOH frequently seen even in early lesions or histologically normal epithelium of lung cancer patients and smokers (9)
FHIT (Fragile Histidine Triad) (3p14):

is in a highly fragile chromosomal site where it is particularly prone to partial deletion as a result of direct DNA damage from carcinogens in tobacco smoke
encodes a protein with ADP hydrosylase activity
proposed functions include regulation of DNA replication and signaling stress responses
hypermethylation of FHIT is more frequent in smokers than non-smokers

RASSF1:

Involved in control of activity of members of the RAS family of oncogenes

SEMA3B (encodes semaphoring 3B)

Member of a family of secreted proteins involved in development of neuronal and epithelial tissues

8q21-23
9p21 (_) (9)

p16INK4a, p14ARF, p15INK4B genes (2) (9)
frequently observed even in early lesions (9)

13q14 (9)

RB1 (9)
Frequently observed even in early lesions (9)

17p13 (9)

TP53 (9)
Frequently observed even in early lesions (9)

Oncogenes overexpressed:

MYC (virtually all SCLC, 50% of NSCLC) (9)
MDM2 (25-50% of NSCLC) (9)
EGFR (frequent) (9)
AKT constitutively active (>80% of NSCLC cell lines) (9)
ERBB2 (HER2/neu) (30%) (9)
AR (?gene symbol) (amphiregulin – ligand for EGFR) (40%) (9)

Associated with poor prognosis suggested (9)

Tumour suppressor genes loss of expression:

p14ARF (encoed by p16INK4A locus) (9)
FHIT (virtually all SCLC, more frequent in SqCA than AdCA) (9)
RASSF1A (9)

Especially in SCLC (9)

TGFBR2 (9)
PTEN/MMAC1 (low frequency) (9)
TSLC1 (frequent, mostly due to hypermethylation) (9)

Mutations:

TP53 mutations (50% of NSCLC, 70% of SCLC) (_) (40-70% of NSCLC, 90% of SCLC) (9):

Mostly missense (inactivating) mutations
Can occur very early in cancer progression
More frequent in cancers of smokers than in non-smokers
May be associated with poor prognosis (9)
Excess of transversions at G bases (G to T), particularly in smokers

Preferentially occur in a limited number of codons, corresponding to sites of adduction for metabolites of benzo(a)pyrene

Considerable differences in mutation patterns according to histological type and gender
Vast majority of lung cancers with TP53 mutations in non-smokers are ADCs in women

Low frequency of G to T transversions compared to smoking-related cancers

BUB and MAD mutations (low frequency) (9):

Mitotic spindle checkpoint (9)
Result of mutation is chromosomal instability / imbalances (9)

PPP1R3 (5%) (9)

Pathways:

P16INK4A-RB pathway inactivation:

RB1 (retinoblastoma gene, 13q11) (tumour suppressor)

RB alterations detected in >90% SCLC and large cell with neuroendocrine features (9)
Relatively infrequent in NSCLC (9)

P16INK4A also involved in this pathway (9)

Keeps RB in the hypophosphorylated form by inhibiting CyclinD-CDK4/6 activity (9)
Inactivation of p16INK4A is more common in NSCLC (9)

End result in either case is activation of E2F/DP signaling (9)
Encodes Rb protein – “gatekeeper” for G1 to S transition
Multiple ways to knock out this pathway:

Loss of RB1 expression
Silencing of INK4 (CDKN2a) encoding p16

LOH (9p21)
Promoter hypermethylation

Overexpression of CCND1 (encoding cyclin D1)

Sometimes due to gene amplification (11q13)

p53-MDM2-p16ARF pathway inactivation (9):

MDM2 is at 12q14.3 (9)

Oncogene – down-regulates p53 protein expression (9)
AKT is a downstream effector of growth factor signals, and positively regulates MDM2 (9)

p14ARF is encoded by analternative coding frame of the p16INK4A locus (9)

growth inhibition by inhibiting activity of MDM2 (9)

TGF-Beta signal pathway:

TGFBR2 (TGF-Beta type II receptor) – 3p22

Expression frequently lost due to epigenetic mechanism (9)

MADH2 (Smad2) – 18q21.1
MADH4 (Smad4) – 18q21.1
TGF-Beta strongly inhibits proliferation of normal epithelial cells (9)
TGF-Beta signaling is impaired by activation of MAPK cascade and NF-Kappa-B pathway (9)

RAS-RAF-MAPK pathway:

RAS family (KRAS, HRAS, NRAS)

GTP binding
Mutations (KRAS in lung cancer) result in constitutive activation (9)
Activates RAF1 (9)

RAF1:

Induces transcription of several growth-promoting genes via the MAPK cascade (9)

CCND1, MYC, growth factors

PI3K-AKT pathway:
PLC-PKC pathway:
ERBB family:

EGFR (ERBB), ERBB2 (HER-2/neu), ERBB3, ERBB4

Receptor-type tyrosine kinases (9)
ERBB2 enhances signaling of other members of the family (9)

EGF, TGF-alpha amphiregulin (AR)

EGFR-specific ligands (9)

Neuregulins (NRG), heregulins

Bind to ERBB3 or ERBB4

Stimulate several signaling pathways (9):

RAS-RAF-MAPK
PI3K-AKT
PLC-PKC

o NSCLC:

§ Aneuploid; often near-triploid karyotypes (4) (5)

· Multiple unbalanced rearrangements generally (4)

· Most common breakpoints:

o At or near the heterochromatic centromere regions of chromosomes 1, 3, 5, 6, 7, 8, 9, 11, 13, 14, 15, 17 (4)

o Regions of chromosomes 1, 3, 6, 7, 9p, 11, 19 (4)

§ Microsatellite instability (5)

§ Gains:

· 1q (karyotyping) (4)

o 1q31 (CGH) (peak of 37%, background 30%) (4)

§ 1q21-31 (4)

o 1q22-1q32.1 (153Mb-197Mb) (40%) (3)

o isochromosome 1q (4)

· 2p21.1-p14 (39Mb-65Mb) (52%) (3)

o 2p (cell lines) (8)

· 2q11.2-q32.2 (97Mb-191Mb) (30%) (3)

· 3p14.3-p26.33 (58-182Mb) (60%) (3)

· 3q (karyotyping) (4), 50-70% (5)

o 3q25-27 (CGH) (peak of 50%, background 30-40%) (4)

§ 3q26-qter (3)

o isochromosome 3q (4)

o high copy numbers in many cases (4)

o peak at regions including PIK3CA, BCL6, SST (5)

· chromosome 4:

o almost entire chromosome: 4p16.1-q34.3 (7-178Mb) (80%) (3)

· 5p (karyotyping) (4)

o 5p13-14 (CGH) (peak of 45%, background 40%) (4)

o 5p15.33-p13.3 (1-32Mb) (35%) (3) 5p15 (10)

§ contains TERT (10)

o isochromosome 5p is common (5)

o high copy numbers in many cases (4)

· isochromosome 6p (4)

· Chromosome 7 (4)

o Trisomy 7 may be very early change in NSCLC (4)

o 7p (>60% by karyotyping) (4)

§ 7p (cell lines) (8)

§ isochromosome 7p (4)

o 7q (>60% by karyotyping) (4)

§ 7q22.3-q31.32 (106-121Mb) (55%) (3)

§ 7q gains have been associated with higher stage tumours and either positive nodal involvement or higher tumour grade (4)

· 8q (3) 20% (5)

o 8q23-24 (CGH) (peak at 45%, background 30-40%) (4)

§ 8q23-qter (4)

o 8q11.21-q24.3 (48-143Mb) (35%) (3)

o isochromosome 8q is common (5)

o high copy numbers in many cases (4)

o have been associated with higher stage tumours and either positive nodal involvement or higher tumour grade (4)

· 11q (karyotyping) (4)

o 11q14.1-q22.3 (77-109Mb) (17%) (3)

o 11q13 (10)

§ contains CCND1 (10)

· 12p13.2-p11.22 (11-29Mb) (65%) (3)

· 12q (karyotyping) (4)

· isochromosome 13q (4)

· isochromosome 14q (4)

· 20q13 gain has been linked with invasiveness in lung ACs (4)

o 20q (cell lines) (8)

§ amplifications:

· MYC (8-16%) (4) (8) (8% by FISH - PMID: 14705768) (5% by PCR - PMID: 17385831) (50% by FISH - PMID: 11578259) (50% by FISH - PMID: 10339988)

o MYCC seen in both SCLC and NSCLC, MYCN and MYCL1 usually limited to SCLC (7)

o Rare in primary lung tumours, but more frequently observed in lung cancer cell lines and primary lung cancer following chemotherapy (7)

o Association with poorer survival (14705768, 11578259, 10339988)

· EGFR (4)

o Copy number alterations correlate with presence of mutation (11)

o Better prognosis, similar to presence of mutation (level II evidence) (12)

· CCND1 (6) (5-20%) (11679179)

o Associated with poor prognosis in one study (ref in 11679179)

o But not in this study – low numbers… (10190311)

· 3q26 most frequent site of DNA amplification (CGH) (4)

· 5p15.33 (cell lines) (8)

o hTERT

· 3q13 (4)

· 3q28-qter (4)

· regions of chromosome 7 (8)

o 7q11.2 (4)

· 8p11-12 (4)

· 8q24 (4)

· 11q22 (rare ~5%) (7)

o contains cIAP1 and CIAP2 (?gene names), antiapoptotic oncogenes (7)

· 12p12 (4)

· 14q13.2-q13.3 (cell lines) (8)

o minimal region contains 9 known genes (PSMA6, NFKBIA, INSM2, BRMS1, MBIP, TITF1, NKX2-8, PAX9, SLC25A21)

· 19q13.1-13.2 (4)

· high copy numbers:

o 3q (4)

o 5p (4)

o 8q (4)

· Only region of genome consistently shown to differ between SCC and AC is 3q26-29 (1, ref in 1) / 3q25-qter (5)

o This region is amplified in SCC (50-85%)

o Located at 180 to 199Mb on chromosome 3 (3q26-29)

o PIK3CA is a potential oncogene (5)

§ losses:

· 1p: (20%) (5)

o 1p36.33-p32.3 (1-51Mb) (80%) (3)

· 3p (75% by karyotyping) (4) 25-40% (5)

o 3p14-21 (4)

o 3p21 (CGH) (peak at 27%, background 20%) (4)

o 3p21.31-p21.1 (47-52Mb) (55%) (3)

o 3p25.3 (9-10Mb) (25%) (3)

o 3p21 minimally deleted (4)

o LOH 3p is thought to be an early genetic change (4)

o Candidate TSGs:

§ RASSF1A (4)

§ FHIT (4)

· 5q:

o 5q (CGH) (some areas 20-25%) (4)

o 5q23.3-q35.3 (131-180Mb) (20%) (3)

· 6p22.1-p21.1 (27-44Mb) (47%) (3)

· 6q (>60% by karyotyping) (4)

o 6q (CGH) (some areas 20-25%) (4)

· 8p (>60% by karyotyping) (4) (13)

o 8p21-23 (3) (4)

o 8p22 (CGH) (peak at 28%, background 20%) (4)

· chromosome 9 (4)

o 9p (84% by karyotyping) (4) (20-30%) (5) (13)

§ 9p21-22 (CGH) (peak at 27%, background 25%) (4)

§ proposed as a critical change in NSCLC (4)

§ candidate TSGs:

1. CDKN2A (INK4a) / ARF locus (9p21)

2. p16INK4a gene (rarely in SCLCs)

o 9q (>60% by karyotyping) (4)

§ 9q33.3-q34.3 (122-137Mb) (40%) (3)

· 10q22.1-q26.3 (70-135Mb) (26%) (3)

· 11p11.2 (45-47Mb) (20%) (3)

· 11q:

o 11q12.2-q13.4 (60-73Mb) (65%) (3)

o 11q14.1-q22.3 (77-109Mb) (17%) (3)

· 12q:

o 12q13.12-q14.1 (47-61Mb) (73%) (3)

o 12q24.11-q24.33 (107-132Mb) (65%) (3)

· chromosome 13 (4)

o 13q (>60% by karyotyping) (4) (13)

o 13q22 (CGH) (peak at 28%, background 20%)

· 15q: (20%) (5)

o 15q24.1-q24.2 (72-73Mb) (43%) (3)

· 16p13.3-q22.2 (0-70Mb) (70%) (3)

· 17p (>60% by karyotyping) (4) (13)

o 17p12-13 (CGH) (peak at 18%) (4)

o 17p13.3-q25.3 (0-77Mb) (73%) (3)

o candidate TSGs:

§ TP53 (4)

· 18q (>60% by karyotyping) (4)

· 19p (>60% by karyotyping) (4)

· 19p13.3-q13.43 (1-62Mb) (80%) (3)

· 21q (>60% by karyotyping) (4)

· 22q (>60% by karyotyping) (4)

o 22q11.1-q13.33 (16-49Mb) (70%) (3)

· chromosome Y in males (4)

· short arms of acrocentric chromosomes (>60% by karyotyping) (4)

· isochromosomes (loss of the other arm)

o 1q, 3q, 5p, 8q, 6p, 7p, 13q, 14q (4)

§ LOH:

· 1p (ref in 8)

· 3p (ref in 8) (70%) (9)

· 4p (ref in 8)

· 4q (ref in 8)

· 5q (ref in 8)

· 8p (ref in 8)

· 9p (ref in 8)

· 9q (ref in 8)

· 10p (ref in 8)

· 10q (ref in 8)

· 13q (ref in 8)

· 15q (ref in 8)

· 17p (ref in 8)

· 18q (ref in 8)

· 19p (ref in 8)

· Xp (ref in 8)

· Xq (ref in 8)

§ homozygous deletions:

· 3p21 (4)

· 9p21 (CDKN2A / INK4a)

§ oncogenes overexpressed:

· MYC (3)

· KRAS (3)

· EGFR (3) (70% of SqCA and 40% of AdCA) (9)

· ERBB3 (20% of NSCLC) (9)

o Associated with poor prognosis (9)

· Cyclin D1 (3)

· BCL2 (3)

· MDM2 (25-50% of NSCLC) (9)

o Favourable prognosis associated (9)

· TGF-alpha (60% of NSCLC) (9)

· VEGF (50%) (9)

o Association with higher microvessel density (9)

· Telomerase activation (100% of SCLC, 80% of NSCLC) (9)

o Also detectable in pre-cancerous tissues suggesting early involvement

§ Tumour suppressor genes underexpressed:

· P53 (3)

· P16 (3) (CDKN2a?)

· RB (3) (15-30% - much lower than SCLC – association with late stage NSCLC) (9)

· FHIT (3) (4)

o More frequently in SqCA than AdCA (9)

o Association with smoking history (9)

· RASSFF1A (3) (4)

· SEMA3B (3)

· PTEN (3)

· CDKN2A (8) (9)

· CDKN1B (p27KIP1 protein) (9) (70%)

o Associated with a poor prognosis (9)

· ERCC1

o Not an oncogene but involved in repair of DNA damage by cisplatin

o Overexpresssion -> Better prognosis (12)

o Overexpression -> Imparts relative resistance to cisplatin (12)

· p27

o cell cycle regulator – slows cell cycle and prevents apoptosis

o IHC positive -> better prognosis (12)

o IHC positive -> less benefit from cisplatin (12)

§ Mutations:

· KRAS mutations (activating) (9) (15-20% NSCLC, 20-30% AdCA) (11):

o Almost exclusively in AdCA, particularly goblet-cell type (9)

o Present in 40% of AAH lesions in one study (9)

o Usually codon 12 mutations (9)

o Associated with poor survival (9) (12)

o Predicts for lack of benefit from adjuvant cisplatin + vinorelbine (JBR10)

· EGFR mutations (10% of caucasians, 30% of East Asians) (11)

o EGFR/KRASmutations do not occur in pure biomarker verified SQCC [213]

o these mutations are restricted to squamous histology that represents a component of ADSQC whereas they are not a feature of pure SQCC [213]

o See Adenocarcinoma notes

· also no mutations were detected in BRAF, NRAS, ERBB2, and MAP2K1 (MEK1) in SQCC(213)

· BRAF mutations (2% of AdCA) (11)

o Common to many malignancies (11)

o Exclusive of tumours with KRAS mutations (11)

· PIK3CA (1/24 NSCLCs) (11)

o Activates the B/Akt pathway (11)

· p53 mutations (5)

o more frequent in ADCA than SQCC (5)

o G to T transversions, known to be associated with smoking, were marginally more common among patients who developed a second-primary lungcancer or recurrence/metastasis (Progressive Disease)200

o G:C to T:A transversions are characteristic finding in tobacco-associated lungcancer192

o p53 mutations in LCINS are distinct from those seen in tobacco-induced lungcancer192

o The p53 mutations in women never smokers with adenocarcinoma were predominantly transitions (83%). However, in smokers, the mutations were predominantly transversions (60%) and deletions (20%) in one study.192

o TP53 or KRAS transversion mutations in NSCLC of smokers occur prevalently at G bases and are commonly the sites of adduct formation by metabolites ofpolycyclic aromatic hydrocarbons, one of the main family of tobacco carcinogens[4-6].200

§ Pathways:

RB pathway:

INK4 inactivation (CDKN2A – 9p21) (65% of NSCLC)

Encodes for p16
Hypermethylation of INK4 is more frequent in smokers than non-smokers
Hypermethylation is often present in early stages of lung cancers
also may be inactivated by homozygous deletions or mutations

RB – loss of protein expression (15% of NSCLC)

ErbB (EGFR, HER2/neu, KRAS) pathway abnormalities (common in NSCLC, absent in SCLC):

EGFR positive (84% of SCC, significantly more frequent in SCC than other types)

EGFR somatic mutations reported to be frequent in AdenoCA (2)
Also amplification of the gene is observed (2)

HER2/neu expression rare in SCC (frequent in adenocarcinomas)
KRAS activating mutations rare in SCC (frequent in adenocarcinomas)

Often mutually exclusive from EGFR mutations (2)

ERBB2 somatic mutations reported to be frequent in adenoCA (2)

Act pathway activation (11)

?through PIK3CA mutation (11)

genomic profiles of AC and SCC are highly overlapping such that neither supervised nor unsupervised clustering of the global CGH profiles was able to classify these tumours according to their histopathological subtypes (1)

References:

Robbins 2005
Travis WD. Pathology of Lung Cancer. Clinics in Chest Medicine 2002; 23(1): 65-81.
WHO blue book 2004
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