Single nucleotide polymorphisms (SNP’s) of hypoxia-related genes correlate with pathological complete response following neoadjuvant chemoradiation for locally advanced rectal cancer.

Motofumi Tanaka1, Isabelle Bedrosian2, George J. Chang2, Y. Nancy You2, Prajnan Das3, Donghui Li1, Miguel A. Rodriguez-Bigas2, John M. Skibber2, James L. Abbruzzese1, Cathy Eng1 The Department of Gastrointestinal Medical Oncology1, Surgical Oncology2, and Radiation Oncology3, The University of Texas M. D. Anderson Cancer Center, Houston, Texas.

REFERENCES

CONCLUSIONS

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2009;74:673-88.5. Kim HO, et al. Oncol Rep. 2008;20:1181-7. 6. Prior SJ, et al. Physiol Genomics. 2003;15:20-6. 7. Toiyama Y, et al. Clin Oncol 2010;22:272-80. 8. Rasheed S, et al. Br J Cancer 2009;100:1666–73.9. Tanimoto K, et al. Carcinogenesis 2003;24:1779-8310. Ruas JL, et al. J Biol Chem. 2010;285:2601-9.

METHODS

AIM• The purpose of this study was to investigate the association between SNP’s of hypoxia-related genes and pathologic complete response (pCR) to preoperative chemoradiation for locally advanced rectal cancer.

BACKGROUND• Preoperative chemoradiation for advanced rectal cancer has been shown to improve local regional recurrence and sphincter preservation (1).• Tumor hypoxia has been correlated with radiation (XRT) resistance (2,3). • Definitive biomarkers to predict efficacy of fluoropyrimidine based chemoradiation (chemoXRT) have not been established(4). • Hypoxia inducible factor 1-α is a subunit of the HIF-1 transcription factor which causes transcription of several genes involved in the cellular response to hypoxia. • Nucleotide polymorphisms of HIF-1α have been identified (5, 6),

but have not previously been correlated to efficacy of radiation therapy.

Table 4. Pathological response and MVD

Table 3. Genotypes and association with pCR

Figure 2. MVD and CBP expression (×200)

High Low

High Low

• The hypoxia related genotypes of HIF-1α P582S and/or CBP A214G are associated with pCR following preoperative fluoropyrimindine-based concurrent chemoradiation for locally advanced rectal cancer.

• HIF-1α expression is associated with radiosensitivity of rectal cancer and patient survival (7, 8).

• The HIF-1α P582S variant allele T has been associated with high expression level of HIF-1α (5) and enhanced transcriptional capacity (9).

• Development of pCR correlated with low MVD.

• CBP is reported to have important function in HIF-1α- dependent activation of transcription for angiogenesis, apoptosis, and cell proliferation (10).

• Though no association between CBP A214G genotype and expression level was noted, CBP A214G G allele was associated with low MVD. • SNP’s associated with the hypoxia related genes of HIF-1α and CBP were identified and correlated with pCR to neoadjuvant chemoradiation therapy. This trial continues patient accrual for further tissue analysis.

Figure 1. Schema of HIF-1α pathway

*Values are mean number of microvessels counted in a ×200 field.†T test‡Fisher’s exact test.§ HIF1α 85CT and CBP 214AA

Table 5. Genotype and MVD

*Values are mean number of microvessels counted in a ×200 field.†T test‡Fisher’s exact test.

*Fisher’s exact test.†Odds ratio adjusted for age, gender, tumor size, and AJCC stage.‡HIF1α 85CT and CBP 214AA

• NTAD (N-terminal transactivation domain)• CTAD (C-terminal transactivation domain) • ODD (oxygen-dependent degradation domain)

RESULTS

Supported by a American College of Surgeons Faculty Research Scholarship (I. Bedrosian) and a

philanthropic grant from the Urbieta Family Colorectal Cancer Research Fund (C. Eng).

• The rate of pCR was 26% (16 of 61 pts).• After median follow-up of 35.4 months, none of the pts (with pCR) had evidence of local recurrence. • HIF1α C85T (P582S) and CBP A214G genotypes were associated with pCR following chemoradiation (Table 3).• IHC analysis demonstrates pCR was associated with

lower MVD (Table 4).• The genotype of CBP A214G alone or in combination with HIF1α C85T was associated with MVD (p = 0.035 and 0.029, respectively, Table 5). • CBP expression was not associated with CBP A214G genotype (p = 0.682).

Table 1. SNP’s evaluated

MVD

CBP

Study Design and Population • 61 patients with AJCC stage II (T3-4N0M0) and III (TXN1-2M0) were enrolled in a prospective study of preoperative chemoXRT followed by TME from 2005-2010. All patients underwent 50.4Gy XRT with concurrent fluoropyrimidine-based therapy. Genotyping• DNA was extracted from fresh-frozen pretreatment tumor biopsies, and TaqMan SNP Genotyping Assay was performed. • 11 SNPs of 7 genes associated with tumor hypoxia were examined: HIF1α, VHL (von Hippel-Lindau), CBP (CREB-binding protein),), leptin, THBS1 (Thrombospondin 1), and BIRC5 (baculoviral IAP repeat-containing 5). (Table 1)Immunohistochemistry (IHC) of microvessel and CBP• PPFE tumor tissue was stained for anti-CD34 (QBEnd/10, 1:100; Santa Cruz) and anti-CBP (AC238, 1:250; Santa Cruz). • IHC was performed by automated immunostainer (Lab Vision Autostainer 360-2D; LabVision).Assessment of microvessel density (MVD)• The 3 most hypervascular areas were selected under low magnification. • Single endothelial cells or cluster of endothelial cells were counted as a single microvessel. • The mean value for three high-power (×200) fields was regarded as the MVD for each tumor. MVD was also categorized into high and low by using mean value in all cases as cutoff value. Statistical Analysis• Associations between pCR and clinicopathological features or genotypes were compared using the χ2 test (Fisher’s exact), and logistic regression (Table 2).• The association of MVD with pCR were analyzed using the T test.

Table 2. Clinicopathological characteristics and pathological complete response (pCR)

*Fisher’s exact test.