Embed Size (px)
Citation preview
Please contact us or your local distributor
for the previous issue of KREATECH NEWS
You can also visit our website:
www.kreatech.com
IN THIS ISSUE…
THE RESURRECTION OF FISH IN NON-SMALL CELL LUNG CANCER TESTING
Prof. Patrick Pauwels, MD, PhD, Department of Pathology, Antwerp University Hospital, Belgium
Lung cancer is considered to be the number one killer among all cancers. In the USA alone, 226,160 new cases and 160,340 deaths from lung cancer have been reported in 2012. The first step in introducing personalized treatment of non-small cell lung cancer (NSCLC) was the discovery that some of these tumors were addicted to an active epidermal growth factor receptor (EGFR) pathway. Small molecules blocking the tyrosine kinase domain of EGFR were introduced in the clinic. Since most NSCLC show overexpression of EGFR (particularly squamous cell carcinomas), it was hoped that a major response would occur in these EGFR overexpressing tumors. Unfortunately, the results were disappointing: the majority of the patients did not respond.
KREATECH NEWSIssue 5
TECHNICAL BULLETIN: ROS1 FUSION PARTNERS AND REPEAT-FREE™ DNA FISH PROBE DESIGN FOR DIAGNOSIS IN LUNG CANCER
ROS1 rearrangements represent around 2% of aberrations in non-small cell lung cancer (NSCLC). Patients with ROS1 rearrangements responding positively to tyrosine kinase inhibitors like crizotinib (Xalkori®) have been reported. This has led to ROS1 being regarded as an emerging biomarker and ROS1 rearrangements therefore are becoming more and more important in the diagnosis of NSCLC.
To date at least 7 fusion partners of ROS1 have been identified. These fusions are: 1. TPM3-ROS1, t(1;6)(q21.2;q22)2. SDC4-ROS1, t(6;20)(q22;q12)3. SLC34A2-ROS1, t(4;6)(q15.2;q22)4. CD74-ROS1, t(5;6)(q32;q22)5. LRIG3-ROS1, t(6;12)(q22;q14.1)6. ROS1-GOPC, del(6)(q22q22.3)7. EZR-ROS1, inv(6)(q22q25.3)
Fluorescent In Situ Hybridization (FISH) is a technology that is especially suited for diagnostic tests of ROS1 rearrangements.
2
Prof. Patrick Pauwels, MD, PhD, Department of Pathology, Antwerp University Hospital, Belgium
Prof. Patrick Pauwels, MD, PhD, Department of Pathology, Antwerp University Hospital, Belgium
THE RESURRECTION OF FISH IN NON-SMALL CELL LUNG CANCER TESTING
Lung cancer is considered to be the number one killer among all cancers. In the USA alone, 226,160 new cases and 160,340 deaths from lung cancer have been reported in 2012. The first step in introducing personalized treatment of non-small cell lung cancer (NSCLC) was the discovery that some of these tumors were addicted to an active epidermal growth factor receptor (EGFR) pathway. Small molecules blocking the tyrosine kinase domain of EGFR were introduced in the clinic. Since most NSCLC show overexpression of EGFR (particularly squamous cell carcinomas), it was hoped that a major response would occur in these EGFR overexpressing tumors. Unfortunately, the results were disappointing: the majority of the patients did not respond. However, a small group of patients did show response. Later on, it was shown that these patients had tumors with an activating EGFR gene mutation. However, EGFR gene amplification seemed also to be a positive predictive factor, although much weaker than the EGFR mutation status. But in fact, most tumors with EGFR amplification that respond to EGFR tyrosine kinase inhibition show also activating EGFR mutations! What matters in the EGFR story is not amplification and/or overexpression but mutation. This seemed to be the end of FISH testing in NSCLC.
ALK AND NSCLC In 2007, for the first time, a new fusion gene was found in a subset of NSCLC: fusion of part of ALK with part of an upstream partner, EML4, was demonstrated. Crizotinib, an ALK-inhibitor, showed a surprisingly high overall response rate of 57% in heavily pretreated patients with ALK rearranged tumors. Ten months after the publication of the study and only four years after the discovery of this new fusion gene, on 26 August 2011, the FDA approved crizotinib for the treatment of advanced NSCLC. About 3-5% of the NSCLC show this rearrangement. Phase III studies with crizotinib in second line but also in first line are still ongoing.
FISH, which was used to identify ALK positive patients in earlier studies on crizotinib, is mandated by the FDA to initiate treatment with the drug and is considered the gold standard for detecting ALK rearrangements. Of particular importance is the fact that the FISH break apart probe detects all ALK rearrangements. Since 2007, in addition to EML4 several other fusion partners of the ALK gene have been described (kinesin family member 5B (KIF5B), TRK-fused gene (TFG)) and this list is growing.
ROS1 AND ALK: BROTHERS IN CRIME ROS1, located on chromosome 6, shows several similarities with ALK. It has been found that there is a 49% amino acid homology between the kinase domains of human ROS1 and ALK and 77% homology of the adenosine triphosphate (ATP)-binding site. To date, seven distinct ROS1 gene fusions have been identified in solid tumors (GOPC, SLC34A2, CD74, EZR, TPM3, SDC4, LRIG3). Recently, a ROS1 break apart FISH probe was used to screen for ROS1 rearrangements: 1.7% of NSCLC (18 of 1073) harbored this rearrangement. ROS1 rearrangements are more likely to be found in younger patients, never smokers with adenocarcinoma subtype, similar to ALK fusions. Of particular importance is the fact that a phase I trial with crizotinib in ROS1-positive advanced-stage NSCLC patients also showed a response rate of 57% with a disease control of 79% at eight weeks.
RET REARRANGEMENTS: A RARE EVENT IN NSCLCRET fusions leading to activated RET have been identified in papillary thyroid carcinomas. Recently, lung adenocarcinomas were reported to harbor novel gene fusions involving the RET tyrosine kinase gene partnered with the KIF5B gene and others like CCDC6. A RET inhibitor vandetanib is already available in the clinic. This inhibitor also targets VEGFR2 and 3, and EGFR. RET fusions have now also been described in NSCLC but they seem to be exceedingly rare: they occur in 0.9% (13 out of 1476) of the NSCLCs. Preclinical studies showed that lung cancer cells bearing RET fusions had their proliferative activity blocked by vandetanib.
KREATECH NEWS
3
450 KB
ROS1
6q22
RH68126
6
SHGC-14420
260 KB
Exon 30Exon 42RH69070
RH104060
GOPC
EXPECTED PATTERN
Normal Cell ROS1 (6q22) translocation
GOPC-ROS1 fusion, fusion to gene regions
distal to GOPC, or deletion of the 6q arm
distal of exon 42
ON ROS1 (6q22) Break - CAT# KBI-10752 ON RET (10q11) Break - CAT# KBI-10753
1 Image kindly provided by Prof. Terris, Dr. Just, Paris.2 Presented at the ADAPT meeting 2012, Washington, DC.
Issue 5
Hybridization of ROS1 (6q22) Break Probe (KBI-10752) to a NSCLC tissue harboring a ROS1 rearrangement.
ALK (2p23) Break probe hybridized to NSCLC tissue showing translocation involving the ALK region at 2p23.1
EXPECTED PATTERN
2p23
SHGC-17159
360 KB
D2S392
340 KBALK
2
D2S405
RH109933
Normal Cell ALK (2p23) translocation
ALK/EML4 t(2;2); inv(2) Fusion probe hybridized to NSCLC tissue showing ALK-EML4 fusion.2
EXPECTED PATTERN
2p23 360 KB
SHGC-17159
D2S392
350 KB
ALK
2
2p21
EML4
SHGC-106023
SHGC-104981
Gap: 12 MB
Normal Cell EML4-ALK Fusion (inv(2))
Hybridization of RET (10q11) Break Probe (KBI-10753) to a NSCLC.
10q11535 KB
RET
RH51277
SHGC-148579
10
D10S1176
500 KB
GDB:579598
EXPECTED PATTERN
Normal Cell RET (10q11) translocation
ON ALK (2p23) Break - CAT# KBI-10747 ON ALK/EML4 t(2;2); inv(2) Fusion - CAT# KBI-10746
4
MET AS POTENTIAL THERAPEUTIC TARGET The MET proto-oncogene encodes for the receptor tyrosine kinase MET. The most frequent genetic alteration is gene amplification. MET amplification is relatively rare in previously untreated NSCLC patients (4-7%) but is an important mechanism in EGFR tyrosine kinase inhibitor (TKI) resistance. Rebiopsy in patients with progressive disease under EGFR TKI treatment showed MET amplification in up to 20% of cases. Several MET inhibitors are under development.
AND WHAT ABOUT SQUAMOUS CELL CARCINOMA?The aforementioned “targetable” genetically aberrations were found almost exclusively in adenocarcinomas. However, approximately 30% of the NSCLC are squamous cell carcinomas (SCC). These tumors are clearly induced by smoking and are increasingly recognized as harboring different molecular profiles and requiring different treatment strategies. Until recently, no targetable genetic aberration was described in SCC, but things are changing. One of these recently described targets is fibroblastic growth factor receptor 1 (FGFR1), located at 8p11. FGFR1 amplification is found in up to 20% of SCC, but rarely in adenocarcinomas. Several FGFR inhibitors are being tested. One of these, AZD4547 has already shown the ability to induce tumor regressions in patient–derived models of FGFR1-amplified squamous cell lung cancers. AZD4547 is currently being evaluated in phase I/II clinical trials.
CONCLUSIONThe FISH story in NSCLC started disappointingly. There was no place for FISH testing in the EGFR TKI story. Now we look for MET amplification in EGFR TKI resistant tumors. But with the discovery of ALK rearrangements in NSCLC – and with a very promising drug, crizotinib – things changed more dramatically. ALK FISH is now acknowledged to be the gold standard for looking for ALK rearrangements. Other trials targeting ROS1 and RET rearranged NSCLC tumors are coming up. Fortunately, also for our patients with SCC, we can offer hope: FGFR1 amplification is targetable. FISH in NSCLC is clearly back!
REFERENCES[1] Blackhall F, Thatcher N, Booton R, Kerr K. The impact on the multidisciplinary team of molecular profiling for personalized therapy in non-small cell lung cancer. Lung Cancer. 2013;79:101-103.[2] Camidge DR, Theodoro M, Delee A.M. et al. Correlations between the percentage of tumor cells showing an anaplastic lymphoma kinase (ALK) gene rearrangement, ALK signal copy number, and response to crizotinib therapy in ALK fluorescence in situ hybridization-positive nonsmall cell lung cancer. Cancer. 2012; 18:4486-4494.[3] Bergethan K, Shaw AT, Ou SH et al. ROS1 rearrangements define a unique molecular class of lung cancers. J Clin Oncol 2012; 30:863-870.[4] Wang R, Hu H, Pan Y et al. RET fusions define a unique molecular and clinicopathologic subtype of non-small-cell lung cancer. J. Clin Oncol 2012; 30:4352-9.
KREATECH NEWS
ON FGFR1 (8p11) / SE 8 (D8Z1) - CAT# KBI-12754
FGFR1 (8p11)/ SE 8 probe hybridized to SCC NSCLC tissue..3
540KBFGFR1
8p11
RH46977
D8S414
8
EXPECTED PATTERN
Normal Cell FGFR1 (8p11)amplification
[5] Tsuta K, Kozu Y, Mimae T et al. C-MET/Phospho-MET protein expression and MET gene copy number in non-small cell lung carcinomas. J Thorac Oncol. 2012; 7:331-339.[6] Weiss J, SOS ML, Peifer M. et al. Frequent and focal FGFR1 amplification associates with therapeutically tractable FGFR1 dependency in squamous cell lung cancer. Sci Transl Med 2010; 2:62-93.
ON C-MET (7q31) / SE 7 - CAT# KBI-10719
Hybridization of MET Amplification Probe (KBI-10719) hybridized to a NSCLC tissue showing MET amplification.
7q31
420 KB
D7S3086
RH110508
7
C-MET
D7Z1
EXPECTED PATTERN
Normal Cell MET (7q31) amplification
3 Image kindly provided by Dr. Otte, Vienna.
5
KREATECH NEWS
Figure 2: Expected patterns of probe designs for SDC4-ROS1 fusion. This pattern is also to be expected for fusions with TPM3, SLC34A2, CD74 and LRIG3.
ROS1 rearrangements represent around 2% of aberrations in non-small cell lung cancer (NSCLC) [1]. Patients with ROS1 rearrangements responding positively to tyrosine kinase inhibitors like crizotinib (Xalkori®) have been reported. This has led to ROS1 being regarded as an emerging biomarker and ROS1 rearrangements therefore are becoming more and more important in the diagnosis of NSCLC [2].
To date at least 7 fusion partners of ROS1 have been identified [1,3,4]. These fusions are: 1. TPM3-ROS1, t(1;6)(q21.2;q22)2. SDC4-ROS1, t(6;20)(q22;q12)3. SLC34A2-ROS1, t(4;6)(q15.2;q22)4. CD74-ROS1, t(5;6)(q32;q22)5. LRIG3-ROS1, t(6;12)(q22;q14.1)6. ROS1-GOPC, del(6)(q22q22.3)7. EZR-ROS1 inv(6)(q22q25.3)
Fluorescent In Situ Hybridization (FISH) is a technology that is especially suited for diagnostic tests of ROS1 rearrangements. However, it is of paramount importance that such a DNA FISH assay is able to detect all of these rearrangements in order to minimize the number of false negatives as this could result in inefficient therapy management.
The REPEAT-FREE™ POSEIDON ROS1 (6q22) Break probe (KBI-10752) is the only FISH assay available to date that is designed to capture all these events. Especially detection of the ROS1-GOPC fusion can lead to difficulties in interpretation with alternative DNA FISH probes available as the ROS1 and GOPC genes are only separated by a 240 kb region on chromosome 6.
Figures 1 to 4 describe some specific details of the REPEAT-FREE™ POSEIDON ROS1 (6q22) Break probe (KBI-10752) and show examples of the patterns that can be expected for the 7 different rearrangements described in the literature. To be able to detect all currently known ROS1 rearrangements we have designed the RF POSEIDON ROS1 (6q22) Break probe in such a way that the GOPC gene located at 6q22.3 is not covered by the distal 5’ region probe (see figure 1). Therefore, the fusion with GOPC identified in NSCLC [3,4] will result in a deletion of the red signal. Alternative designs (figure 1) available today cover the GOPC gene region at 6q22.3. This will result in (significant) residual signal of the region covering the GOPC gene leading to false negatives.
ROS1 (6q22) Break Designs
ROS1 Translocations to other ChromosomesExample: SDC4-ROS1 t(6;20)(q22;q12). The same principal accounts for other translocations
KREATECH Expected Pattern
6 6
20 20
6 6
20
20
6 6
20
20
Normal cell; 2 Fusion on 6q22
6 6
20 20
6 6
20
20
6 6
20
20
SDC4-ROS1: 1F1G1R
Alternative design Expected Pattern
6 6
20 20
6 6
20
20
6 6
20
20
Normal cell; 2 Fusion on 6q22
6 6
20 20
6 6
20
20
6 6
20
20
SDC4-ROS1: 1F1G1R
TECHNICAL BULLETIN: ROS1 FUSION PARTNERS AND REPEAT-FREE™ DNA FISH PROBE DESIGN FOR DIAGNOSIS IN LUNG CANCER
Figure 1: Comparison between the KREATECH ROS1 probe design and possible alternative designs.
KREATECH Design
ROS1
6GOPC
450 KB
ROS1
6q22
RH68126
6
SHGC-14420
260 KB
Exon 30Exon 42RH69070
RH104060
GOPC
Alternative Design
6
Issue 5
ROS1
6GOPC
6 6 6 6
6
6
6 6 6 6 6
6 6 6 6
6
6
6 6 6 6 6
6 6 6 6
6
6
6 6 6 6 6
No difference in signal or slightly diminished green
Normal cell; 2 Fusion on 6q22
6 6 6 6
6 6 6 6
Deletion 240 kb
Deletion 240 kb
6 6 6 6
6 6 6 6
Deletion 240 kb
Deletion 240 kb
Normal cell; 2 Fusion on 6q22
SDC4-ROS1: 1F1G1R
SDC4-ROS1: 1F1G1R
6 6 6 6
6
6
6 6 6 6 6
6 6 6 6
6
6
6 6 6 6 6
6 6 6 6
6
6
6 6 6 6 6Normal cell; 2 Fusion on 6q22 ROS1-SDC4: 1F1G1R
ORFusion ROS1-EZR deletion by inversion: 1F1G
ORFusion ROS1-EZR deletion by inversion: 1F1R
6 6 6 6
6
6
6 6 6 6 6
450 KB
ROS1
6q22
RH68126
6
SHGC-14420
260 KB
Exon 30Exon 42RH69070
RH104060
GOPC
6 6 6 6
6 6 6 6
Deletion 240 kb
Deletion 240 kb
6 6 6 6
6 6 6 6
Deletion 240 kb
Deletion 240 kb
Normal cell; 2 Fusion on 6q22
Fusion ROS1-GOPC: 1F1G. Deletion of Red
Figure 3: Expected patterns of probe designs for ROS1-GOPC fusion.
Figure 4: Expected patterns of probe designs for ROS1-EZR fusions.
ROS1-GOPC fusionDeletion of ± 240 kb region between exon 42 of ROS1(6q22) and GOPC (6q22.3)
Alternative design Expected Pattern
Alternative design Expected Pattern
ROS1-EZR inversion/deletionThis inversion, inv(6)(q22q25.3), of a ± 40 MB region (that can also result in deletion by inversion) is an example of rearrangements of ROS1 on chromosome 6 DISTAL to GOPC.
KREATECH Expected Pattern
KREATECH Expected Pattern
REFERENCES[1] Takeuchi et. al., Nature Med (18); 378, 2012[2] Case study ROS1: Patient responding to Xalkori®
[3] Rimkunas et. al., Clin Cancer Res. 2012 Aug 15;18(16):4449-57[4] Suehara et. al., Clin Cancer Res. 2012 Nov 14
7
©2013 KREATECH DiagnosticsPublished May 2013
KREATECH NEWS
DESCRIPTION COLOR CONTENT CAT.#
ON ALK (2p23) Break Red/Green 10 Tests KBI-10747
ON ALK/EML4 t(2;2); inv(2) Fusion Red/Green 10 Tests KBI-10746
ON ROS1 (6q22) Break Red/Green 10 Tests KBI-10752
ON C-MET (7q31) / SE 7 Red/Green 10 Tests KBI-10719
ON RET (10q11) Break Red/Green 10 Tests KBI-10753
ON FGFR1 (8p11) / SE 8 (D8Z1) Red/Green 20 Tests KBI-12754
ON FGFR1 (8p11) / SE 8 (D8Z1) Red/Green 50 Tests KBI-14754
Other relevant Lung Cancer Probes
ON EGFR, Her-1 (7p11) / SE7 Red/Green 10 Tests KBI-10702
ON hTERT (5p15) / 5q31- for Tissue Red/Green 10 Tests KBI-10709
ON ERCC1 (19q13) & ZNF443 (19p13) Red/Green 10 Tests KBI-10739
FGFR2 amplification probe Red/Green Inquire Inquire
FGFR4 amplification probe Red/Green Inquire Inquire
Product information
