B. Michael Ghadimi

Effectiveness of Gene Expression Profiling for Response Prediction of Rectal Adenocarcinomas to Preoperative Chemoradiotherapy

PURPOSE There is a wide spectrum of tumor responsiveness of rectal adenocarcinomas to preoperative chemoradiotherapy ranging from complete response to complete resistance. This study aimed to investigate whether parallel gene expression profiling of the primary tumor can contribute to stratification of patients into groups of responders or nonresponders. PATIENTS AND METHODS Pretherapeutic biopsies from 30 locally advanced rectal carcinomas were analyzed for gene expression signatures using microarrays. All patients were participants of a phase III clinical trial (CAO/ARO/AIO-94, German Rectal Cancer Trial) and were randomized to receive a preoperative combined-modality therapy including fluorouracil and radiation. Class comparison was used to identify a set of genes that were differentially expressed between responders and nonresponders as measured by T level downsizing and histopathologic tumor regression grading. RESULTS In an initial set of 23 patients, responders and nonresponders showed significantly different expression levels for 54 genes (P < .001). The ability to predict response to therapy using gene expression profiles was rigorously evaluated using leave-one-out cross-validation. Tumor behavior was correctly predicted in 83% of patients (P = .02). Sensitivity (correct prediction of response) was 78%, and specificity (correct prediction of nonresponse) was 86%, with a positive and negative predictive value of 78% and 86%, respectively. CONCLUSION Our results suggest that pretherapeutic gene expression profiling may assist in response prediction of rectal adenocarcinomas to preoperative chemoradiotherapy. The implementation of gene expression profiles for treatment stratification and clinical management of cancer patients requires validation in large, independent studies, which are now warranted.

Centrosome amplification and instability occurs exclusively in aneuploid, but not in diploid colorectal cancer cell lines, and correlates with numerical chromosomal aberrations†

Measurement of the nuclear DNA content allows classification of human cancers as either diploid or aneuploid. To gain further insight into mechanisms of aneuploidy, we compared the cytogenetic profile of mismatch‐repair–deficient diploid versus mismatch‐repair–proficient aneuploid colorectal carcinoma cell lines using comparative genomic hybridization and spectral karyotyping. Aneuploid carcinomas revealed an average of 19 chromosomal imbalances per cell line. Such numerical aberrations were exceedingly scarce in the diploid tumors. This pattern of chromosomal aberrations is consistent with a mechanism involving the impairment of chromosome segregation fidelity during mitotic cell division. In support of this idea, we demonstrate the exclusive occurrence of centrosome amplification and instability in all of the aneuploid tumor cell lines analyzed. All diploid tumors contained centrosomes that were functionally and structurally indistinguishable from those in normal human fibroblasts. Due to the observed differences in centrosomes between these two classes of tumors, we incubated the cells with the microtubule depolymerizing drugs nocodazole and griseofulvin. Our results indicate that the aneuploid tumor cell lines have an increased sensitivity to these reagents and a delay in aster formation and microtubule regrowth. However, microtubule nucleation was initiated from one or two centers in both the diploid and aneuploid cells. These observations support the notion that the integrity of the centrosome plays a central role in the development of aneuploidy. Genes Chromosomes Cancer 27:183–190, 2000. Published 2000 Wiley‐Liss, Inc.

Specific Chromosomal Aberrations and Amplification of the AIB1 Nuclear Receptor Coactivator Gene in Pancreatic Carcinomas

To screen pancreatic carcinomas for chromosomal aberrations we have applied molecular cytogenetic techniques, including fluorescent in situ hybridization, comparative genomic hybridization, and spectral karyotyping to a series of nine established cell lines. Comparative genomic hybridization revealed recurring chromosomal gains on chromosome arms 3q, 5p, 7p, 8q, 12p, and 20q. Chromosome losses were mapped to chromosome arms 8p, 9p, 17p, 18q, 19p, and chromosome 21. The comparison with comparative genomic hybridization data from primary pancreatic tumors indicates that a specific pattern of chromosomal copy number changes is maintained in cell culture. Metaphase chromosomes from six cell lines were analyzed by spectral karyotyping, a technique that allows one to visualize all chromosomes simultaneously in different colors. Spectral karyotyping identified multiple chromosomal rearrangements, the majority of which were unbalanced. No recurring reciprocal translocation was detected. Cytogenetic aberrations were confirmed using fluorescent in situ hybridization with probes for the MDR gene and the tumor suppressor genes p16 and DCC. Copy number increases on chromosome 20q were validated with a probe specific for the nuclear receptor coactivator AIB1 that maps to chromosome 20q12. Amplification of this gene was identified in six of nine pancreatic cancer cell lines and correlated with increased expression.

Phase II Trial of Carcinoembryonic Antigen Radioimmunotherapy With 131I-Labetuzumab After Salvage Resection of Colorectal Metastases in the Liver: Five-Year Safety and Efficacy Results

PURPOSE Although complete resection (R0) of liver metastases (LM) remains the treatment of choice for colorectal cancer (CRC) patients amenable to curative therapy, only approximately one third survive for 5 years. The objective of this phase II study was to evaluate the safety and efficacy of radioimmunotherapy (RAIT) after salvage resection of LM. PATIENTS AND METHODS Twenty-three patients who underwent surgery for LM of CRC received a dose of 40 to 60 mCi/m2 of 131I-labetuzumab, which is a humanized monoclonal antibody against carcinoembryonic antigen. Safety (n = 23), disease-free survival (DFS; n = 19), and overall survival (OS; n = 19) were determined. RESULTS With a median follow-up of 64 months, the median OS time from the first liver resection for RAIT patients was 68.0 months (95% CI, 46.0 months to infinity), and the median DFS time was 18.0 months (95% CI, 11.0 to 31.0 months). The 5-year survival rate was 51.3%. RAIT benefited patients independently of bilobar involvement, size and number of LM, and resection margins. The major adverse effect was transient myelosuppression, resulting mostly in grade < or = 3 neutropenia and/or thrombocytopenia. CONCLUSION Because both the median OS and 5-year survival rates seem to be improved with adjuvant RAIT after complete LM resection in CRC, compared with historical and contemporaneous controls not receiving RAIT, these results justify further evaluation of this modality in a multicenter, randomized trial.

Gene expression profiling reveals a massive, aneuploidy-dependent transcriptional deregulation and distinct differences between lymph node-negative and lymph node-positive colon carcinomas.

To characterize patterns of global transcriptional deregulation in primary colon carcinomas, we did gene expression profiling of 73 tumors [Unio Internationale Contra Cancrum stage II (n = 33) and stage III (n = 40)] using oligonucleotide microarrays. For 30 of the tumors, expression profiles were compared with those from matched normal mucosa samples. We identified a set of 1,950 genes with highly significant deregulation between tumors and mucosa samples (P < 1e-7). A significant proportion of these genes mapped to chromosome 20 (P = 0.01). Seventeen genes had a >5-fold average expression difference between normal colon mucosa and carcinomas, including up-regulation of MYC and of HMGA1, a putative oncogene. Furthermore, we identified 68 genes that were significantly differentially expressed between lymph node-negative and lymph node-positive tumors (P < 0.001), the functional annotation of which revealed a preponderance of genes that play a role in cellular immune response and surveillance. The microarray-derived gene expression levels of 20 deregulated genes were validated using quantitative real-time reverse transcription-PCR in >40 tumor and normal mucosa samples with good concordance between the techniques. Finally, we established a relationship between specific genomic imbalances, which were mapped for 32 of the analyzed colon tumors by comparative genomic hybridization, and alterations of global transcriptional activity. Previously, we had conducted a similar analysis of primary rectal carcinomas. The systematic comparison of colon and rectal carcinomas revealed a significant overlap of genomic imbalances and transcriptional deregulation, including activation of the Wnt/beta-catenin signaling cascade, suggesting similar pathogenic pathways.

The Rectal Cancer microRNAome – microRNA Expression in Rectal Cancer and Matched Normal Mucosa

Purpose: miRNAs play a prominent role in a variety of physiologic and pathologic biologic processes, including cancer. For rectal cancers, only limited data are available on miRNA expression profiles, whereas the underlying genomic and transcriptomic aberrations have been firmly established. We therefore, aimed to comprehensively map the miRNA expression patterns of this disease. Experimental Design: Tumor biopsies and corresponding matched mucosa samples were prospectively collected from 57 patients with locally advanced rectal cancers. Total RNA was extracted, and tumor and mucosa miRNA expression profiles were subsequently established for all patients. The expression of selected miRNAs was validated using semi-quantitative real-time PCR. Results: Forty-nine miRNAs were significantly differentially expressed (log2-fold difference >0.5 and P < 0.001) between rectal cancer and normal rectal mucosa. The predicted targets for these miRNAs were enriched for the following pathways: Wnt, TGF-beta, mTOR, insulin, mitogen-activated protein kinase, and ErbB signaling. Thirteen of these 49 miRNAs seem to be rectal cancer-specific, and have not been previously reported for colon cancers: miR-492, miR-542-5p, miR-584, miR-483-5p, miR-144, miR-2110, miR-652, miR-375, miR-147b, miR-148a, miR-190, miR-26a/b, and miR-338-3p. Of clinical impact, miR-135b expression correlated significantly with disease-free and cancer-specific survival in an independent multicenter cohort of 116 patients. Conclusion: This comprehensive analysis of the rectal cancer miRNAome uncovered novel miRNAs and pathways associated with rectal cancer. This information contributes to a detailed view of this disease. Moreover, the identification and validation of miR-135b may help to identify novel molecular targets and pathways for therapeutic exploitation. Clin Cancer Res; 18(18); 4919–30. ©2012 AACR.

Lymph Node Status and TS Gene Expression Are Prognostic Markers in Stage II/III Rectal Cancer After Neoadjuvant Fluorouracil-Based Chemoradiotherapy

PURPOSE According to the CAO/ARO/AIO-94 trial of the German Rectal Cancer Study Group, preoperative combined fluorouracil (FU) -based long-term chemoradiotherapy (CT/RT) is recommended for patients with International Union Against Cancer (UICC) stage II/III rectal cancer. However, despite the local benefit of neoadjuvant treatment, the overall prognostic value remains uncertain in comparison with adjuvant CT/RT. Furthermore, the prognostic value of molecular biomarkers, such as thymidylate synthase (TS), thymidine phosphorylase (TP), and dihydropyrimidine dehydrogenase (DPD), all of which are involved in the FU metabolism, is unknown in neoadjuvant settings. We assessed the impact of standardized preoperative CT/RT and intratumoral TS, TP, and DPD levels on patient outcome. PATIENTS AND METHODS Forty patients with rectal cancer pretherapeutic UICC stage II/III, receiving preoperative FU-based CT/RT (CAO/ARO/AIO-94 trial) followed by standardized surgery, including total mesorectal excision, were investigated. Downsizing, downstaging, tumor regression, as well as TS, TP, and DPD gene expression of post-treatment surgical specimens were correlated with disease-free survival (DFS) and overall survival (OS). RESULTS Significant downsizing (P < .001) and downstaging (P = .001) were achieved with preoperative CT/RT. During a median follow-up of 49 months (95% CI, 43 to 58 months), the cancer recurrence rate was 28.2%. DFS and OS were significantly increased in patients with downstaging (P < .001 and P = .003, respectively), compared with patients without downstaging. All patients who developed cancer recurrence had a persistent positive lymph node status after preoperative CT/RT (P < .001) and a significantly higher TS gene expression (P = .035) compared with those patients without recurrence. CONCLUSION Persistent positive lymph node status and high intratumoral TS expression after preoperative CT/RT are predictive of an unfavorable prognosis in rectal cancer UICC stage II/III.

Amplification of 4q21–q22 and the MXR gene in independently derived mitoxantrone‐resistant cell lines

Molecular cytogenetic studies were conducted on three multidrug‐resistant cancer sublines which are highly resistant to the chemotherapeutic agent mitoxantrone, an anthracenedione. The three independently selected sublines were derived by exposure to mitoxantrone or Adriamycin and do not overexpress MDR1 or MRP. Two sublines, MCF‐7 AdVp3000 and MCF‐7 MX, showed an amplification peak at 4q21–q22, as demonstrated by comparative genomic hybridization (CGH), while the third, S1‐M1–80, did not. FISH using a whole chromosome 4 paint demonstrated multiple rearrangements involving chromosome 4 in MCF‐7 AdVp3000 and MCF‐7 MX, while S1‐M1–80 contained only a simple reciprocal translocation. The parental cell lines had no chromosome 4 rearrangements and no copy number gain or amplification of chromosome 4. Spectral karyotyping (SKY) analysis revealed a balanced translocation, t(4;17)(q21–q22;p13) in S1‐M1–80 and multiple clonal translocations involving chromosome 4 in MCF‐7 AdVp3000 and MCF‐7 MX. A novel cDNA, designated MXR, which encodes an ABC half‐transporter and is highly overexpressed in the three sublines, was localized to chromosome 4 by somatic cell hybrid analysis. Southern blot analysis demonstrated amplification of the MXR gene in MCF‐7 AdVp3000 and MCF‐7 MX, but not in S1‐M1–80. FISH studies with a BAC probe for MXR localized the gene to 4q21–22 in the normal chromosome 4 and revealed in both MCF‐7 AdVp3000 and MCF‐7 MX amplification of MXR at one translocation juncture, shown by SKY to be t(4;5)(4qter→4cen→4q21–22::5q13→5qter) in MCF‐7 AdVp3000 and t(6;4;6;3)(6pter→6q15::4q21–q22::hsr::6q?::3q?27→3qter) in MCF MX; neither of the breakpoints in the partner chromosomes showed amplification by CGH. The data are consistent with the hypothesis of a transporter, presumably that encoded by the MXR gene, mediating mitoxantrone resistance. The MXR gene encodes a half‐transporter and the absence of cytogenetic evidence of coamplification of other regions suggests that a partner may not be overexpressed, and instead the MXR half‐transporter homodimerizes to mediate drug transport. Genes Chromosomes Cancer 27:110–116, 2000. Published 2000 Wiley‐Liss, Inc.

A genomic approach to colon cancer risk stratification yields biologic insights into therapeutic opportunities

Gene expression profiles provide an opportunity to dissect the heterogeneity of solid tumors, including colon cancer, to improve prognosis and predict response to therapies. Bayesian binary regression methods were used to generate a signature of disease recurrence in patients with resected early stage colon cancer validated in an independent cohort. A 50-gene signature was developed that effectively distinguished early stage colon cancer patients with a low or high risk of disease recurrence. RT-PCR analysis of the 50-gene signature validated 9 of the top 10 differentially expressed genes. When applied to two independent validation cohorts of 55 and 73 patients, the 50-gene model accurately predicted recurrence. Standard Kaplan–Meier survival analysis confirmed the prognostic accuracy (P < 0.01, log rank), as did multivariate Cox proportional hazard models. We tested potential targeted therapeutic options for patients at high risk for disease recurrence and found a clinically important relationship between sensitivity to celecoxib, LY-294002 (PI3kinase inhibitor), retinol, and sulindac in colon cancer cell lines expressing the poor prognostic phenotype (P < 0.01, t test), which performed better than standard chemotherapy (5-FU and oxaliplatin). We present a genomic strategy in early stage colon cancer to identify patients at highest risk of recurrence. An ability to move beyond current staging by refining the estimation of prognosis in early stage colon cancer also has implications for individualized therapy.

Mutated KRAS results in overexpression of DUSP4, a MAP‐kinase phosphatase, and SMYD3, a histone methyltransferase, in rectal carcinomas

Mutations of the KRAS oncogene are predictive for resistance to treatment with antibodies against the epithelial growth factor receptor in patients with colorectal cancer. Overcoming this therapeutic dilemma could potentially be achieved by the introduction of drugs that inhibit signaling pathways that are activated by KRAS mutations. To identify comprehensively such signaling pathways, we profiled pretreatment biopsies and normal mucosa from 65 patients with locally advanced rectal cancer—30 of which carried mutated KRAS—using global gene expression microarrays. By comparing all tumor tissues exclusively to matched normal mucosa, we could improve assay sensitivity, and identified a total of 22,297 features that were differentially expressed (adjusted P‐value <0.05) between normal mucosa and cancer, including several novel potential rectal cancer genes. We then used this comprehensive description of the rectal cancer transcriptome as the baseline for identifying KRAS‐dependent alterations. The presence of activating KRAS mutations is significantly correlated to an upregulation of 13 genes (adjusted P‐value <0.05), among them DUSP4, a MAP‐kinase phosphatase, and SMYD3, a histone methyltransferase. Inhibition of the expression of both genes has previously been shown using the MEK1‐inhibitor PD98059 and the antibacterial compound Novobiocin, respectively. These findings suggest a potential approach to overcome resistance to treatment with antibodies against the epithelial growth factor receptor in patients with KRAS‐mutant rectal carcinomas.