Zhigang Charles Wang

Ovarian and triple-negative breast cancers with BRCA1 or BRCA2 loss are highly sensitive to treatment with PARP inhibitors and platinum-based cytotoxic agents and show an accumulation of genomic scars in the form of gross DNA copy number aberrations. Cancers without BRCA1 or BRCA2 loss but with accumulation of similar genomic scars also show increased sensitivity to platinum-based chemotherapy. Therefore, reliable biomarkers to identify DNA repair-deficient cancers prior to treatment may be useful for directing patients to platinum chemotherapy and possibly PARP inhibitors. Recently, three SNP array-based signatures of chromosomal instability were published that each quantitate a distinct type of genomic scar considered likely to be caused by improper DNA repair. They measure telomeric allelic imbalance (named NtAI), large scale transition (named LST), and loss of heterozygosity (named HRD-LOH), and it is suggested that these signatures may act as biomarkers for the state of DNA repair deficiency in a given cancer. We explored the pan-cancer distribution of scores of the three signatures utilizing a panel of 5371 tumors representing 15 cancer types from The Cancer Genome Atlas, and found a good correlation between scores of the three signatures (Spearman’s ρ 0.73–0.87). In addition we found that cancer types ordinarily receiving platinum as standard of care have higher median scores of all three signatures. Interestingly, we also found that smaller subpopulations of high-scoring tumors exist in most cancer types, including those for which platinum chemotherapy is not standard therapy. Within several cancer types that are not ordinarily treated with platinum chemotherapy, we identified tumors with high levels of the three genomic biomarkers. These tumors represent identifiable subtypes of patients which may be strong candidates for clinical trials with PARP inhibitors or platinum-based chemotherapeutic regimens.

Increased number of single nucleotide substitutions is seen in breast and ovarian cancer genomes carrying disease-associated mutations in BRCA1 or BRCA2. The significance of these genome-wide mutations is unknown. We hypothesize genome-wide mutation burden mirrors deficiencies in DNA repair and is associated with treatment outcome in ovarian cancer. The total number of synonymous and non-synonymous exome mutations (Nmut), and the presence of germline or somatic mutation in BRCA1 or BRCA2 (mBRCA) were extracted from whole-exome sequences of high-grade serous ovarian cancers from The Cancer Genome Atlas (TCGA). Cox regression and Kaplan-Meier methods were used to correlate Nmut with chemotherapy response and outcome. Higher Nmut correlated with a better response to chemotherapy after surgery. In patients with mBRCA-associated cancer, low Nmut was associated with shorter progression-free survival (PFS) and overall survival (OS), independent of other prognostic factors in multivariate analysis. Patients with mBRCA-associated cancers and a high Nmut had remarkably favorable PFS and OS. The association with survival was similar in cancers with either BRCA1 or BRCA2 mutations. In cancers with wild-type BRCA, tumor Nmut was associated with treatment response in patients with no residual disease after surgery. Tumor Nmut was associated with treatment response and with both PFS and OS in patients with high-grade serous ovarian cancer carrying BRCA1 or BRCA2 mutations. In the TCGA cohort, low Nmut predicted resistance to chemotherapy, and for shorter PFS and OS, while high Nmut forecasts a remarkably favorable outcome in mBRCA-associated ovarian cancer. Our observations suggest that the total mutation burden coupled with BRCA1 or BRCA2 mutations in ovarian cancer is a genomic marker of prognosis and predictor of treatment response. This marker may reflect the degree of deficiency in BRCA-mediated pathways, or the extent of compensation for the deficiency by alternative mechanisms.

Autophagy is a fundamental salvage pathway that encapsulates damaged cellular components and delivers them to the lysosome for degradation and recycling. This pathway usually conducts a protective ...

Amplification of chromosome 8q22, which includes the gene for lysosomal associated transmembrane protein LAPTM4B, has been linked to de novo anthracycline resistance in primary breast cancers with poor prognosis. LAPTM4B overexpression can induce cytosolic retention of anthracyclines and decrease drug-induced DNA damage. In this study, we tested the hypothesis that LAPTM4B may contribute to tumor cell growth or survival in the absence of a chemotherapeutic exposure. In mammary cells, LAPTM4B protein was localized in lysosomes where its depletion increased membrane permeability, pH, cathepsin release, and cellular apoptosis. Loss of LAPTM4B also inhibited later stages of autophagy by blocking maturation of the autophagosome, thereby rendering cells more sensitive to nutrient deprivation or hypoxia. Conversely, enforced overexpression of LAPTM4B promoted autophagic flux and cell survival during in vitro starvation and stimulated more rapid tumor growth in vivo. Together, our results indicate that LAPTM4B is required for lysosome homeostasis, acidification, and function, and that LAPTM4B renders tumor cells resistant to lysosome-mediated cell death triggered by environmental and genotoxic stresses. Cancer Res; 71(24); 7481–9. ©2011 AACR.

Introduction: The majority of breast cancers that occur in BRCA1 mutation carriers (BRCA1 carriers) are estrogen receptor-negative (ER-). Therefore, it has been suggested that ER negativity is intrinsic to BRCA1 cancers and reflects the cell of origin of these tumors. However, approximately 20% of breast cancers that develop in BRCA1 carriers are ER-positive (ER+); these cancers are more likely to develop as BRCA1 carriers age, suggesting that they may be incidental and unrelated to BRCA1 deficiency. The purpose of this study was to compare the prevalence of loss of heterozygosity due to loss of wild type (wt) BRCA1 in ER+ and ER- breast cancers that have occurred in BRCA1 carriers and to determine whether age at diagnosis or any pathologic features or biomarkers predict for loss of wt BRCA1 in these breast cancers. Methods: Relative amounts of mutated and wt BRCA1 DNA were measured by quantitative polymerase chain reaction performed on laser capture microdissected cancer cells from 42 ER+ and 35 ER- invasive breast cancers that developed in BRCA1 carriers. BRCA1 gene methylation was determined on all cancers in which sufficient DNA was available. Immunostains for cytokeratins (CK) 5/6, 14, 8 and 18, epidermal growth factor receptor and p53 were performed on paraffin sections from tissue microarrays containing these cancers. Results: Loss of wt BRCA1 was equally frequent in ER+ and ER- BRCA1-associated cancers (81.0% vs 88.6%, respectively; P = 0.53). One of nine cancers tested that retained wt BRCA1 demonstrated BRCA1 gene methylation. Age at diagnosis was not significantly different between first invasive ER+ BRCA1 breast cancers with and without loss of wt BRCA1 (mean age 45.2 years vs 50.1 years, respectively; P = 0.51). ER+ BRCA1 cancers that retained wt BRCA1 were significantly more likely than those that lost wt BRCA1 to have a low mitotic rate (odds ratio (OR), 5.16; 95% CI, 1.91 to ∞). BRCA1 cancers with loss of wt BRCA1 were more likely to express basal cytokeratins CK 5/6 or 14 (OR 4.7; 95% CI, 1.85 to ∞). Conclusions: We found no difference in the prevalence of loss of wt BRCA1 between ER+ and ER- invasive BRCA1-associated breast cancers. Our findings suggest that many of the newer therapies for BRCA1 breast cancers designed to exploit the BRCA1 deficiency in these cancers may also be effective in ER+ cancers that develop in this population.

Two amplified genes from chromosome 8q22—YWHAZ and LAPTM4B—are associated with metastatic breast cancer recurrence by promoting resistance to anthracyclines. YWHAZ codes for an antiapoptotic protein and LAPTM4B encodes a previously undescribed lysosomal protein. Adjuvant chemotherapy for breast cancer after surgery has effectively lowered metastatic recurrence rates1. However, a considerable proportion of women suffer recurrent cancer at distant metastatic sites despite adjuvant treatment. Identification of the genes crucial for tumor response to specific chemotherapy drugs is a challenge but is necessary to improve outcomes2. By using integrated genomics, we identified a small number of overexpressed and amplified genes from chromosome 8q22 that were associated with early disease recurrence despite anthracycline-based adjuvant chemotherapy. We confirmed the association in an analysis of multiple independent cohorts. SiRNA-mediated knockdown of either of two of these genes, the antiapoptotic gene YWHAZ and a lysosomal gene LAPTM4B, sensitized tumor cells to anthracyclines, and overexpression of either of the genes induced anthracycline resistance. Overexpression of LAPTM4B resulted in sequestration of the anthracycline doxorubicin, delaying its appearance in the nucleus. Overexpression of these two genes was associated with poor tumor response to anthracycline treatment in a neoadjuvant chemotherapy trial in women with primary breast cancer. Our results suggest that 8q22 amplification and overexpression of LAPTM4B and YWHAZ contribute to de novo chemoresistance to anthracyclines and are permissive for metastatic recurrence. Overexpression of these two genes may predict anthracycline resistance and influence selection of chemotherapy.

Background: A major challenge facing DNA copy number (CN) studies of tumors is that most banked samples with extensive clinical follow-up information are Formalin-Fixed Paraffin Embedded (FFPE). DNA from FFPE samples generally underperforms or suffers high failure rates compared to fresh frozen samples because of DNA degradation and cross-linking during FFPE fixation and processing. As FFPE protocols may vary widely between labs and samples may be stored for decades at room temperature, an ideal FFPE CN technology should work on diverse sample sets. Molecular Inversion Probe (MIP) technology has been applied successfully to obtain high quality CN and genotype data from cell line and frozen tumor DNA. Since the MIP probes require only a small (~40 bp) target binding site, we reasoned they may be well suited to assess degraded FFPE DNA. We assessed CN with a MIP panel of 50,000 markers in 93 FFPE tumor samples from 7 diverse collections. For 38 FFPE samples from three collections we were also able to asses CN in matched fresh frozen tumor tissue.Results: Using an input of 37 ng genomic DNA, we generated high quality CN data with MIP technology in 88% of FFPE samples from seven diverse collections. When matched fresh frozen tissue was available, the performance of FFPE DNA was comparable to that of DNA obtained from matched frozen tumor (genotype concordance averaged 99.9%), with only a modest loss in performance in FFPE.Conclusion: MIP technology can be used to generate high quality CN and genotype data in FFPE as well as fresh frozen samples.

BACKGROUND: Women with recurrent pregnancy loss (RPL) and T-helper (Th)1-type immunity to trophoblast antigens have an increased frequency of the IL1B-511*1 promoter variant. Since CD46 gene products also regulate maternal immune responses including Th1 immunity, we investigated whether CD46 gene polymorphisms are also associated with RPL in women with and without Th1 immunity to trophoblast, and the possibility of a synergistic effect with the IL1B-511*1 promoter variant. METHODS: A case-controlled study was performed to document HindIII site polymorphism in intron 1 of the CD46 gene in 131 women with RPL and 72 fertile controls. Clinical information, Th1-type immune responsiveness to trophoblast in women with RPL history, and IL1B promoter allelotypes for this cohort were documented in a previous study. RESULTS: The frequency of the CD46H*2 allele and CD46H*2 homozygosity were significantly increased in women with RPL compared with fertile controls (P < 0.028 and P < 0.011). CD46H*2 homozygosity was highly associated with RPL-Th1(+) (32.4 versus 9.7% in fertile controls, P < 0.0045). Logistic regression analysis revealed that women homozygous for both the IL1B-511*1 and CD46H*2 alleles had an extremely high risk of RPL-Th1(+) [exponential coefficients (EC) = 24]. Among women with RPL, homozygosity at both alleles, but not each alone, significantly increased the risk of Th1 immunity to trophoblast antigens (EC = 16), suggesting a possible genetic interaction between these two alleles in the development of Th1 immunity. CONCLUSIONS: The combination of homozygosity for both IL1B-511*1 and CD46H*2 alleles is a high risk factor for RPL-Th1(+).