A novel histone H4 variant H4G regulates rDNA transcription in breast cancer.

Histone variants, present in various cell types and tissues, are known to exhibit different functions. For example, histone H3.3 and H2A.Z are both involved in gene expression regulation, whereas H2A.X is a specific variant that responds to DNA double-strand breaks. In this study, we characterized H4G, a novel hominidae-specific histone H4 variant. We found that H4G is expressed in a variety of human cell lines and exhibit tumor-stage dependent overexpression in tissues from breast cancer patients. We found that H4G localized primarily to the nucleoli of the cell nucleus. This localization was controlled by the interaction of the alpha-helix 3 of the histone fold motif with a histone chaperone, nucleophosmin 1. In addition, we found that modulating H4G expression affects rRNA expression levels, protein synthesis rates and cell-cycle progression. Our data suggest that H4G expression alters nucleolar chromatin in a way that enhances rDNA transcription in breast cancer tissues.

Expression of the ETV6-NTRK3 gene fusion as a primary event in human secretory breast carcinoma.

We report that human secretory breast carcinoma (SBC), a rare subtype of infiltrating ductal carcinoma, expresses the ETV6-NTRK3 gene fusion previously cloned in pediatric mesenchymal cancers. This gene fusion encodes a chimeric tyrosine kinase with potent transforming activity in fibroblasts. ETV6-NTRK3 expression was confirmed in 12 (92%) of 13 SBC cases, but not in other ductal carcinomas. Retroviral transfer of ETV6-NTRK3 (EN) into murine mammary epithelial cells resulted in transformed cells that readily formed tumors in nude mice. Phenotypically, tumors produced glands and expressed epithelial antigens, confirming that EN transformation is compatible with epithelial differentiation. This represents a recurrent chromosomal rearrangement and expression of a dominantly acting oncogene as a primary event in human breast carcinoma.

Intermittent Fasting in Cancer: a Role in Survivorship?

PURPOSE OF REVIEW: To discuss the historical development of intermittent fasting, its potential underlying mechanisms, and the state of clinical trials, and to reflect on considerations for practice and future recommendations. RECENT FINDINGS: Preclinical studies consistently show the robust disease-modifying efficacy of intermittent fasting in various metabolic diseases which may hold implications for cancer prevention and survivorship. Twenty-one clinical trials have or are being conducted on fasting in cancer, utilizing various fasting regimens across different tumor types as a stand-alone intervention or in adjunct to anticancer treatment, with heterogenous outcome variables. Though there are no known, reproducible diets, to cure or prevent cancer recurrence, preliminary research on the underlying mechanisms, tolerance, and safety of intermittent fasting in cancer warrants further investigation. The inherent flexibility of intermittent fasting to accommodate all types of diets is of necessity in oncology.

Tumor-infiltrating lymphocytes predict response to anthracycline-based chemotherapy in estrogen receptor-negative breast cancer.

INTRODUCTION: Infiltration of breast tumors by tumor-infiltrating lymphocytes (TIL) has been associated with sensitivity to anthracycline-based chemotherapy. However, it is unclear whether this is true within the estrogen receptor-alpha (ER)-negative subset of breast tumors that frequently manifest high TIL levels. METHODS: The association of TIL with short-term and long-term clinical response to anthracycline-based therapy was assessed in two independent ER-negative breast cancer cohorts in which patients were categorized as TIL-high or TIL-low. We defined an eight-gene lymphocyte mRNA expression signature (including CD19, CD3D, CD48, GZMB, LCK, MS4A1, PRF1, and SELL) and used unsupervised hierarchical clustering to examine the association between TIL and short-term response to neoadjuvant chemotherapy in a previously published cohort of ER-negative tumors (n = 113). We also examined the association between TIL and long-term chemotherapeutic efficacy in a second cohort of ER-negative tumors (n = 255) with longer than 6 years of median follow-up by using tissue microarrays and immunohistochemistry (IHC) for detection of CD3, CD8, CD4, CD20, and TIA-1. RESULTS: In patients with ER-negative tumors treated with neoadjuvant anthracycline-based chemotherapy, pathologic complete responses (pCRs) were achieved by 23 (74%) of 31 TIL-high patients and 25 (31%) of 80 TIL-low patients (odds ratio (OR), 6.33; 95% confidence interval (CI), 2.49 to 16.08; P < 0.0001). Multivariate logistic regression with standard clinicopathologic features demonstrated that only tumor size (P = 0.037) and TIL status (P = 0.001) were independent predictors of anthracycline response. In the second cohort, adjuvant anthracycline-based therapy was associated with increased disease-free survival (DFS) only in patients with high levels of intraepithelial CD3+ TIL (P = 0.0023). In contrast, outcomes after CMF treatment (cyclophosphamide, methotrexate, and fluorouracil) showed no association with CD3 status. In both cohorts, cytotoxic T-cells were the primary TIL subtype associated with anthracycline sensitivity. Finally, TIL significantly predicted anthracycline sensitivity for both the Her2-positive and triple-negative tumor phenotypes. CONCLUSIONS: ER-negative breast cancers with high levels of TIL have heightened sensitivity to anthracycline-based chemotherapy, as assessed by the immediate response to neoadjuvant therapy and long-term outcome following adjuvant therapy. Investigations of TIL-based predictive tests to identify patients likely to benefit from anthracycline-based treatments are warranted.

Mapping the human T cell repertoire to recurrent driver mutations in MYD88 and EZH2 in lymphoma.

Oncogenic "driver" mutations are theoretically attractive targets for the immunotherapy of lymphoid cancers, yet the proportion that can be recognized by T cells remains poorly defined. To address this issue without any confounding effects of the patient's immune system, we assessed T cells from 19 healthy donors for recognition of three common driver mutations in lymphoma: MYD88L265P, EZH2Y641F , and EZH2Y641N . Donors collectively expressed the 10 most prevalent HLA class I alleles, including HLA-A*02:01. Peripheral blood T cells were primed with peptide-loaded dendritic cells (DC), and reactive T cells were assessed for recognition of naturally processed mutant versus wild type full-length proteins. After screening three driver mutations across 17-26 HLA class I alleles and 3 × 106-3 × 107 T cells per donor, we identified CD4+ T cells against EFISENCGEII from EZH2Y641N (presented by HLA-DRB1*13:02) and CD8+ T cells against RPIPIKYKA from MYD88L265P (presented by HLA-B*07:02). We failed to detect RPIPIKYKA-specific T cells in seven other HLA-B*07:02-positive donors, including two lymphoma patients. Thus, healthy donors harbor T cells specific for common driver mutations in lymphoma. However, such responses appear to be rare due to the combined limitations of antigen processing, HLA restriction, and T cell repertoire size, highlighting the need for highly individualized approaches for selecting targets.

NRG1 gene rearrangements in clinical breast cancer: identification of an adjacent novel amplicon associated with poor prognosis.

Rearrangements of the neuregulin (NRG1) gene have been implicated in breast carcinoma oncogenesis. To determine the frequency and clinical significance of NRG1 aberrations in clinical breast tumors, a breast cancer tissue microarray was screened for NRG1 aberrations by fluorescent in situ hybridization (FISH) using a two-color split-apart probe combination flanking the NRG1 gene. Rearrangements of NRG1 were identified in 17/382 cases by FISH, and bacterial artificial chromosome array comparative genomic hybridization was applied to five of these cases to further map the chromosome 8p abnormalities. In all five cases, there was a novel amplicon centromeric to NRG1 with a minimum common region of amplification encompassing two genes, SPFH2 and FLJ14299. Subsequent FISH analysis for the novel amplicon revealed that it was present in 63/262 cases. Abnormalities of NRG1 did not correlate with patient outcome, but the novel amplicon was associated with poor prognosis in univariate analysis, and in multivariate analysis was of prognostic significance independent of nodal status, tumor grade, estrogen receptor status, and human epidermal growth factor receptor (HER)2 overexpression. Of the two genes in the novel amplicon, expression of SPFH2 correlated most significantly with amplification. This amplicon may emerge as a result of breakpoints and chromosomal rearrangements within the NRG1 locus.

Introduction of combined CHOP plus rituximab therapy dramatically improved outcome of diffuse large B-cell lymphoma in British Columbia.

PURPOSE: For more than two decades, cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) has been the standard therapy for diffuse large B-cell lymphoma (DLBCL). The addition of rituximab to CHOP has been shown to improve outcome in elderly patients with DLBCL. We conducted a population-based analysis to assess the impact of this combination therapy on adult patients with DLBCL in the province of British Columbia (BC). METHODS: We compared outcomes during a 3-year period; 18 months before (prerituximab) and 18 months after (postrituximab) institution of a policy recommending the combination of CHOP and rituximab for all patients with newly diagnosed advanced-stage (stage III or IV or stage I or II with "B" symptoms or bulky [> 10 cm] disease) DLBCL. RESULTS: A total of 292 patients were evaluated; 140 in the prerituximab group (median follow-up, 42 months) and 152 in the postrituximab group (median follow-up, 24 months). Both progression-free survival (risk ratio, 0.56; 95% CI, 0.39 to 0.81; P = .002) and overall survival (risk ratio, 0.40; 95% CI, 0.27 to 0.61, P < .0001) were significantly improved in the postrituximab group. After controlling for age and International Prognostic Index score, era of treatment remained a strong independent predictor of progression-free survival (risk ratio, 0.59; 95% CI, 0.41 to 0.85; P = .005) and overall survival (risk ratio, 0.43; 95% CI, 0.29 to 0.66; P < .001). The benefit of treatment in the postrituximab era was present regardless of age. CONCLUSION: The addition of rituximab to CHOP chemotherapy has resulted in a dramatic improvement in outcome for DLBCL patients of all ages in the province of BC.

Toward Personalized Lymphoma Immunotherapy: Identification of Common Driver Mutations Recognized by Patient CD8+ T Cells.

PURPOSE: A fundamental challenge in the era of next-generation sequencing (NGS) is to design effective treatments tailored to the mutational profiles of tumors. Many newly discovered cancer mutations are difficult to target pharmacologically; however, T-cell-based therapies may provide a valuable alternative owing to the exquisite sensitivity and specificity of antigen recognition. To explore this concept, we assessed the immunogenicity of a panel of genes that are common sites of driver mutations in follicular lymphoma, an immunologically sensitive yet currently incurable disease. EXPERIMENTAL DESIGN: Exon capture and NGS were used to interrogate tumor samples from 53 patients with follicular lymphoma for mutations in 10 frequently mutated genes. For 13 patients, predicted mutant peptides and proteins were evaluated for recognition by autologous peripheral blood T cells after in vitro priming. RESULTS: Mutations were identified in 1-5 genes in 81% (43/53) of tumor samples. Autologous, mutation-specific CD8(+) T cells were identified in 23% (3/13) of evaluated cases. T-cell responses were directed toward putative driver mutations in CREBBP and MEF2B. Responding T cells showed exquisite specificity for mutant versus wild-type proteins and recognized lymphoma cells expressing the appropriate mutations. Responding T cells appeared to be from the naïve repertoire, as they were found at low frequencies and only at single time points in each patient. CONCLUSIONS: Patients with follicular lymphoma harbor rare yet functionally competent CD8(+) T cells specific for recurrent mutations. Our results support the concept of using NGS to design individualized immunotherapies targeting common driver mutations in follicular lymphoma and other malignancies. Clin Cancer Res; 22(9); 2226-36. ©2015 AACR.

Primary cutaneous diffuse large B-cell lymphoma: a clinicopathologic study of 15 cases.

Primary cutaneous diffuse large B-cell lymphoma (DLBCL) is an uncommon lymphoma. Some authors have suggested that large B-cell lymphoma can be segregated based on anatomic site, with tumors of the lower extremity being unique. We report 15 cases of primary cutaneous DLBCL. Each case was analyzed immunohistochemically using antibodies specific for CD3, CD5, CD10, CD20, bcl-2, bcl-6, and p53. Polymerase chain reaction analysis for t(14;18)(q32;q21) also was performed. There were 13 men and 2 women (median age, 64 years). Thirteen tumors were composed predominantly of centroblasts, and 2 were immunoblastic. There was a median follow-up of 72 months. Of the 4 patients with primary cutaneous DLBCL of the lower extremity (thigh, knee, leg), 2 (50%) experienced a recurrence and 1 patient died of disease. In the non-lower extremity cases, 18% (2/11) recurred and no patients died of disease. We conclude that primary cutaneous DLBCL usually occurs in elderly patients with a male predominance. Recurrences are common, but death of disease is rare.

Treatment of elderly Hodgkin's lymphoma patients with a novel 5-drug regimen (ODBEP): a phase II study.

Elderly patients with Hodgkin's lymphoma (HL) have a worse outcome than young patients. In an effort to improve the outcome in elderly HL patients, we used a 5-drug chemotherapy regimen called ODBEP (vincristine, doxorubicin, bleomycin, etoposide, prednisone) from 1986-1995. We hoped that by increasing dose intensity through delivery of treatment without delays, and increasing the number of non-cross-resistant chemotherapeutic drugs that were selected for minimal cumulative myelotoxicity, we might improve the cure rate in elderly patients with Hodgkin's lymphoma. Comparison was made with a similar group of patients treated from 1981-1986 with MOPP/ABV-variant chemotherapy. Ninety-nine patients who were 65 years or older, were diagnosed with HL from 1981-1995. Seventy-one patients had advanced disease and 55 of this group were treated with curative intent using multi-agent chemotherapy (ODBEP = 38; MOPP/ABV-variant = 17). ODBEP and MOPP/ABV-type treatment gave a median survival of 43 and 39 months, with 5-year overall survival (OS) of 42 and 32%, respectively. There was no statistically significant difference in OS or disease specific survival between the treatments. Both treatments were well tolerated, but ODBEP was less myelotoxic. ODBEP patients had a relative risk of 0.47 of developing febrile neutropenia compared to the MOPP/ABV-variant patients. In conclusion, treatment of elderly Hodgkin's lymphoma patients with ODBEP resulted in a similar OS and disease-specific survival compared to those treated with MOPP/ABV type chemotherapy, but appeared to be less toxic.

Therapeutic strategies using the aromatase inhibitor letrozole and tamoxifen in a breast cancer model.

BACKGROUND: The antiestrogen tamoxifen has potent activity against estrogen receptor-positive breast cancer, but two nonsteroidal aromatase inhibitors, letrozole and anastrozole, show considerable advantages over tamoxifen with respect to patient survival and tolerability. To determine the optimal way to use letrozole and tamoxifen, we studied their effects on a breast tumor xenograft model, MCF-7Ca, that is responsive to both antiestrogens and aromatase inhibitors. METHODS: Female ovariectomized BALB/c athymic nude mice carrying xenograft tumors were treated daily subcutaneously with one of the following first-line therapies for varying durations: no drug (control), tamoxifen (100 microg/day) alone, letrozole (10 microg/day) alone, both drugs at the same time, or alternating 4-week courses of each drug (beginning with a course of tamoxifen or beginning with a course of letrozole). Tumor volumes and weights were estimated using linear mixed-effects models. The time to tumor doubling was calculated, and tumor weights in the treatment groups were compared, with adjustments for multiple comparisons being made with either Tukey's or Dunnett's procedure. Second-line therapies (with tamoxifen, letrozole, or fulvestrant) were initiated when tumors doubled in size under first-line therapies. All statistical tests were two-sided. RESULTS: The times for doubling of tumor volume were as follows: control, 3-4 weeks; tamoxifen alone, 16 weeks; tamoxifen alternating with letrozole, 17-18 weeks; tamoxifen plus letrozole, 18 weeks; letrozole alternating with tamoxifen, 22 weeks; letrozole alone, 34 weeks. First-line treatment with letrozole was superior to treatment with tamoxifen alone or with the two drugs combined (at week 16, both P<.001). Alternating tamoxifen and letrozole and alternating letrozole and tamoxifen were also not as effective as letrozole alone (at week 16, P =.002 and P<.001, respectively). Tumors progressing on tamoxifen remained sensitive to second-line therapy with letrozole compared with those remaining on tamoxifen at the end of treatment (week 28, P<.001), whereas tumors progressing on letrozole were unaffected by second-line treatment with the antiestrogens tamoxifen or fulvestrant. CONCLUSIONS: First-line letrozole therapy extends time for tumor progression in this model relative to the other treatment regimens tested. However, further studies are needed to determine the most effective second-line therapy for tumors that progress on letrozole.