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Targeted deep sequencing of mucinous ovarian tumors reveals multiple overlapping RAS-pathway activating mutations in borderline and cancerous neoplasms.

Mackenzie R, Kommoss S, Winterhoff BJ, Kipp BR, Garcia JJ, Voss J, Halling K, Karnezis A, Senz J, Yang W, Prigge ES, Reuschenbach M, Doeberitz MV, Gilks BC, Huntsman DG, Bakkum-Gamez J, McAlpine JN, Anglesio MS - BMC Cancer (2015)

Bottom Line: Our goals were to establish the overall frequency of cancer-hotspot mutations across a large cohort, especially those tumors previously thought to be "RAS-pathway alteration negative", using highly-sensitive next-generation sequencing as well as further explore a small number of cases with apparent heterogeneity in RAS-pathway activating alterations.Using the Ion Torrent PGM platform, we performed next generation sequencing analysis using the v2 Cancer Hotspot Panel.Proven and potential RAS-pathway activating changes were observed in all but one MC.

View Article: PubMed Central - PubMed

Affiliation: Molecular Oncology, BC Cancer Agency Research Centre, Vancouver, Canada. rmackenzie@bccrc.ca.

ABSTRACT

Background: Mucinous ovarian tumors represent a distinct histotype of epithelial ovarian cancer. The rarest (2-4 % of ovarian carcinomas) of the five major histotypes, their genomic landscape remains poorly described. We undertook hotspot sequencing of 50 genes commonly mutated in human cancer across 69 mucinous ovarian tumors. Our goals were to establish the overall frequency of cancer-hotspot mutations across a large cohort, especially those tumors previously thought to be "RAS-pathway alteration negative", using highly-sensitive next-generation sequencing as well as further explore a small number of cases with apparent heterogeneity in RAS-pathway activating alterations.

Methods: Using the Ion Torrent PGM platform, we performed next generation sequencing analysis using the v2 Cancer Hotspot Panel. Regions of disparate ERBB2-amplification status were sequenced independently for two mucinous carcinoma (MC) cases, previously established as showing ERBB2 amplification/overexpression heterogeneity, to assess the hypothesis of subclonal populations containing either KRAS mutation or ERBB2 amplification independently or simultaneously.

Results: We detected mutations in KRAS, TP53, CDKN2A, PIK3CA, PTEN, BRAF, FGFR2, STK11, CTNNB1, SRC, SMAD4, GNA11 and ERBB2. KRAS mutations remain the most frequently observed alteration among MC (64.9 %) and mucinous borderline tumors (MBOT) (92.3 %). TP53 mutation occurred more frequently in carcinomas than borderline tumors (56.8 % and 11.5 %, respectively), and combined IHC and mutation data suggest alterations occur in approximately 68 % of MC and as many as 20 % of MBOT. Proven and potential RAS-pathway activating changes were observed in all but one MC. Concurrent ERBB2 amplification and KRAS mutation were observed in a substantial number of cases (7/63 total), as was co-occurrence of KRAS and BRAF mutations (one case). Microdissection of ERBB2-amplified regions of tumors harboring KRAS mutation suggests these alterations are occurring in the same cell populations, while consistency of KRAS allelic frequency in both ERBB2 amplified and non-amplified regions suggests this mutation occurred in advance of the amplification event.

Conclusions: Overall, the prevalence of RAS-alteration and striking co-occurrence of pathway "double-hits" supports a critical role for tumor progression in this ovarian malignancy. Given the spectrum of RAS-activating mutations, it is clear that targeting this pathway may be a viable therapeutic option for patients with recurrent or advanced stage mucinous ovarian carcinoma, however caution should be exercised in selecting one or more personalized therapeutics given the frequency of non-redundant RAS-activating alterations.

No MeSH data available.


Related in: MedlinePlus

ERBB2 immunohistochemical heterogeneity in two MC and sequencing results from each distinct component. ERBB2+ regions were microdissected and sequenced independently from the ERBB2- components to compare mutation events. Identical KRAS mutations were observed in the ERBB2+ and ERBB2- regions for both cases. ERBB2 high-intensity staining regions was used as a proxy for gene amplification status, as regions previously defined by this high-level IHC staining correlated perfectly with FISH and/or CISH data suggesting amplification of the ERBB2 gene [13]
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Fig4: ERBB2 immunohistochemical heterogeneity in two MC and sequencing results from each distinct component. ERBB2+ regions were microdissected and sequenced independently from the ERBB2- components to compare mutation events. Identical KRAS mutations were observed in the ERBB2+ and ERBB2- regions for both cases. ERBB2 high-intensity staining regions was used as a proxy for gene amplification status, as regions previously defined by this high-level IHC staining correlated perfectly with FISH and/or CISH data suggesting amplification of the ERBB2 gene [13]

Mentions: Two cases of MC (VOA695 and VOA439) were previously described to be heterogeneous for ERBB2 amplification/overexpression [13]. As greater access was available for these local cases, a full series of clinical blocks was examined for ERBB2 3+ and negative IHC. Positive and negative regions were then fine-needle microdissected with both front and back ERBB2-stained sections as a guide to ensure consistency in IHC positive (3+) and negative (0) regions. Sequencing of the disparate regions of VOA439 confirmed the previously observed KRAS p.Gly12Asp mutation at similar allelic frequency in both ERBB2+ and ERBB2- regions: 46.1 % and 43.5 % respectively (Fig. 4). Two TP53 and one CDKN2A mutations were also found in both regions at similar allelic frequencies. Similar results were observed in case VOA695 across ERBB2+ and ERBB2- regions: KRAS p.Gly12Asp mutation at 18.1 % and 16.6 %, and TP53 p. Ser127Pro mutation at 10.5 % and 19.6 % allelic frequency, respectively. Double-hit RAS-pathway alterations were confirmed in six additional MC cases (total 21.6 %). Double-hits were observed in both MBOT (two cases; 7.7 %) and MC, but were more prevalent in MC. In general, examination of allelic ratios of RAS-pathway alterations in comparison to cellularity estimates suggested that RAS-pathway mutations may be more likely to be hemizygous or homozygous (Additional file 1) although copy number analysis was not available to validate this.Fig. 4


Targeted deep sequencing of mucinous ovarian tumors reveals multiple overlapping RAS-pathway activating mutations in borderline and cancerous neoplasms.

Mackenzie R, Kommoss S, Winterhoff BJ, Kipp BR, Garcia JJ, Voss J, Halling K, Karnezis A, Senz J, Yang W, Prigge ES, Reuschenbach M, Doeberitz MV, Gilks BC, Huntsman DG, Bakkum-Gamez J, McAlpine JN, Anglesio MS - BMC Cancer (2015)

ERBB2 immunohistochemical heterogeneity in two MC and sequencing results from each distinct component. ERBB2+ regions were microdissected and sequenced independently from the ERBB2- components to compare mutation events. Identical KRAS mutations were observed in the ERBB2+ and ERBB2- regions for both cases. ERBB2 high-intensity staining regions was used as a proxy for gene amplification status, as regions previously defined by this high-level IHC staining correlated perfectly with FISH and/or CISH data suggesting amplification of the ERBB2 gene [13]
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4494777&req=5

Fig4: ERBB2 immunohistochemical heterogeneity in two MC and sequencing results from each distinct component. ERBB2+ regions were microdissected and sequenced independently from the ERBB2- components to compare mutation events. Identical KRAS mutations were observed in the ERBB2+ and ERBB2- regions for both cases. ERBB2 high-intensity staining regions was used as a proxy for gene amplification status, as regions previously defined by this high-level IHC staining correlated perfectly with FISH and/or CISH data suggesting amplification of the ERBB2 gene [13]
Mentions: Two cases of MC (VOA695 and VOA439) were previously described to be heterogeneous for ERBB2 amplification/overexpression [13]. As greater access was available for these local cases, a full series of clinical blocks was examined for ERBB2 3+ and negative IHC. Positive and negative regions were then fine-needle microdissected with both front and back ERBB2-stained sections as a guide to ensure consistency in IHC positive (3+) and negative (0) regions. Sequencing of the disparate regions of VOA439 confirmed the previously observed KRAS p.Gly12Asp mutation at similar allelic frequency in both ERBB2+ and ERBB2- regions: 46.1 % and 43.5 % respectively (Fig. 4). Two TP53 and one CDKN2A mutations were also found in both regions at similar allelic frequencies. Similar results were observed in case VOA695 across ERBB2+ and ERBB2- regions: KRAS p.Gly12Asp mutation at 18.1 % and 16.6 %, and TP53 p. Ser127Pro mutation at 10.5 % and 19.6 % allelic frequency, respectively. Double-hit RAS-pathway alterations were confirmed in six additional MC cases (total 21.6 %). Double-hits were observed in both MBOT (two cases; 7.7 %) and MC, but were more prevalent in MC. In general, examination of allelic ratios of RAS-pathway alterations in comparison to cellularity estimates suggested that RAS-pathway mutations may be more likely to be hemizygous or homozygous (Additional file 1) although copy number analysis was not available to validate this.Fig. 4

Bottom Line: Our goals were to establish the overall frequency of cancer-hotspot mutations across a large cohort, especially those tumors previously thought to be "RAS-pathway alteration negative", using highly-sensitive next-generation sequencing as well as further explore a small number of cases with apparent heterogeneity in RAS-pathway activating alterations.Using the Ion Torrent PGM platform, we performed next generation sequencing analysis using the v2 Cancer Hotspot Panel.Proven and potential RAS-pathway activating changes were observed in all but one MC.

View Article: PubMed Central - PubMed

Affiliation: Molecular Oncology, BC Cancer Agency Research Centre, Vancouver, Canada. rmackenzie@bccrc.ca.

ABSTRACT

Background: Mucinous ovarian tumors represent a distinct histotype of epithelial ovarian cancer. The rarest (2-4 % of ovarian carcinomas) of the five major histotypes, their genomic landscape remains poorly described. We undertook hotspot sequencing of 50 genes commonly mutated in human cancer across 69 mucinous ovarian tumors. Our goals were to establish the overall frequency of cancer-hotspot mutations across a large cohort, especially those tumors previously thought to be "RAS-pathway alteration negative", using highly-sensitive next-generation sequencing as well as further explore a small number of cases with apparent heterogeneity in RAS-pathway activating alterations.

Methods: Using the Ion Torrent PGM platform, we performed next generation sequencing analysis using the v2 Cancer Hotspot Panel. Regions of disparate ERBB2-amplification status were sequenced independently for two mucinous carcinoma (MC) cases, previously established as showing ERBB2 amplification/overexpression heterogeneity, to assess the hypothesis of subclonal populations containing either KRAS mutation or ERBB2 amplification independently or simultaneously.

Results: We detected mutations in KRAS, TP53, CDKN2A, PIK3CA, PTEN, BRAF, FGFR2, STK11, CTNNB1, SRC, SMAD4, GNA11 and ERBB2. KRAS mutations remain the most frequently observed alteration among MC (64.9 %) and mucinous borderline tumors (MBOT) (92.3 %). TP53 mutation occurred more frequently in carcinomas than borderline tumors (56.8 % and 11.5 %, respectively), and combined IHC and mutation data suggest alterations occur in approximately 68 % of MC and as many as 20 % of MBOT. Proven and potential RAS-pathway activating changes were observed in all but one MC. Concurrent ERBB2 amplification and KRAS mutation were observed in a substantial number of cases (7/63 total), as was co-occurrence of KRAS and BRAF mutations (one case). Microdissection of ERBB2-amplified regions of tumors harboring KRAS mutation suggests these alterations are occurring in the same cell populations, while consistency of KRAS allelic frequency in both ERBB2 amplified and non-amplified regions suggests this mutation occurred in advance of the amplification event.

Conclusions: Overall, the prevalence of RAS-alteration and striking co-occurrence of pathway "double-hits" supports a critical role for tumor progression in this ovarian malignancy. Given the spectrum of RAS-activating mutations, it is clear that targeting this pathway may be a viable therapeutic option for patients with recurrent or advanced stage mucinous ovarian carcinoma, however caution should be exercised in selecting one or more personalized therapeutics given the frequency of non-redundant RAS-activating alterations.

No MeSH data available.


Related in: MedlinePlus