Tumor infiltrating lymphocytes (TIL) play an essential role in mediating response to chemotherapy and improving clinical outcomes in all subtypes of breast cancer. Triple negative breast cancers (TN) are most likely to have tumors with >50 % lymphocytic infiltrate, termed lymphocyte predominant breast cancer, and derive the greatest survival benefit from each 10 % increase in TIL. The majority of HER2+ breast cancers have similar level of immune infiltrate as TN breast cancer yet the presence of TILs has not shown the same survival benefit. For HER2+ breast cancers, type 1 T-cells, either increased TBET+ tumor infiltration or increased type 1 HER2-specific CD4+ T-cells in the peripheral blood, are associated with better outcomes. Hormone receptor positive HER2 negative tumors tend to have the least immune infiltrate yet are the only breast cancer subtype to show worse prognosis with increased FOXP3 regulatory T-cell infiltrate. Notably, all breast cancer subtypes have tumors with low, intermediate, or high TIL infiltrate. Tumors with high TILs may also have increased PD-L1 expression which might be the reason that TN breast cancer seems to demonstrate the most robust clinical response to immune checkpoint inhibitor therapy but further investigation is needed. Tumors with intermediate or low levels of pre-treatment immune infiltrate, on the other hand, may benefit from an intervention that may increase TIL, particularly type 1 T-cells. Examples of these interventions include specific types of cytotoxic chemotherapy, radiation, or vaccine therapy. Therefore, the systematic evaluation of TIL and specific populations of TIL may be able to both guide prognosis and the appropriate sequencing of therapies in breast cancer.

The magnitude of TIL is variable within and between breast cancer subtypes. Levels of lymphocytic subpopulations may identify breast cancers more amenable to immunomodulation and indicate additional strategies to enhance immunity in patients with low to moderate levels of TILs.

Human cancers arise by a combination of discrete mutations and chromosomal alterations. Loss of heterozygosity (LOH) of chromosomal regions bearing mutated tumor suppressor genes is a key event in the evolution of epithelial and mesenchymal tumors. Global patterns of LOH can be understood through allelotyping of tumors with polymorphic genetic markers. Simple sequence length polymorphisms (SSLPs, or microsatellites) are reliable genetic markers for studying LOH, but only a modest number of SSLPs are used in LOH studies because the genotyping procedure is rather tedious. Here, we report the use of a highly parallel approach to genotype large numbers of single-nucleotide polymorphisms (SNPs) for LOH, in which samples are genotyped for nearly 1,500 loci by performing 24 polymerase chain reactions (PCR), pooling the resulting amplification products and hybridizing the mixture to a high-density oligonucleotide array. We characterize the results of LOH analyses on human small-cell lung cancer (SCLC) and control DNA samples by hybridization. We show that the patterns of LOH are consistent with those obtained by analysis with both SSLPs and comparative genomic hybridization (CGH), whereas amplifications rarely are detected by the SNP array. The results validate the use of SNP array hybridization for tumor studies.

Recent studies of tumor lymphocytic immune infiltrates in breast cancer have suggested an improved prognosis associated with increasing levels of tumor-infiltrating lymphocytes (TIL). Triple-negative breast cancer (TNBC) is the breast cancer subtype that has the greatest incidence of patients with a robust tumor immune infiltrate, although it is still a minority of patients. Elevated levels of either intratumoral or stromal T cells are associated with an improved overall survival (OS) and disease-free survival (DFS) in TNBC as compared with other breast cancer subtypes. TNBC may be immunogenic for several reasons. Subtypes of TNBC have a significant number of genetic mutations, and the immune system may see the aberrant proteins encoded by these mutations as foreign. Moreover, TNBC is associated with a prognostic gene signature that also includes B cells. Antibodies secreted by B cells may bind to tumor antigens and amplify the adaptive immune response that has already been initiated in the tumor. New immune modulatory agents, including immune checkpoint inhibitors, have shown activity in immunogenic tumors such as melanoma and bladder cancer and have recently been tested in TNBC. The clinical response rates observed, patterns of response, and adverse event profiles are similar to what has been described in melanoma where this class of agents has already been approved for clinical use in some cases. Lessons learned in assessing the immunogenicity of TNBC, potential mechanisms of immune stimulation, and response to immune modulatory drugs lay the foundation for the development of immune-based therapies in all subtypes of the disease.

BackgroundVariations in single nucleotide polymorphisms (SNPs) have been associated with enhanced drug efficacy and toxicity in cancer therapy. SNP variations in the ErbB2 gene have been identified that alter the protein sequence of the HER2-neu protein, but how these polymorphisms affect prognosis and response to HER2 targeted therapy is unknown. We examined eleven ErbB2 SNPs that alter the HER2-neu amino acid sequence to determine whether any of these particular polymorphisms were associated with increased trastuzumab cardiotoxicity in a case–control study.Methods140 subjects were enrolled from a single institution under Weill Cornell Medical College IRB protocol #0804009734. Patients were eligible if they had histologically or cytologically proven HER2-neu positive breast cancer and more than 3 months of trastuzumab therapy. Cases had either symptomatic CHF or a decline in LVEF of 15% (or if the LVEF <55%, a decline in LVEF of 10%) that resulted in at least temporary discontinuation of trastuzumab, whereas controls had no decline in their LVEF. Eleven ErbB2 single gene SNPs that resulted in an alteration in the HER2-neu protein amino acid sequence were studied. Single gene SNP analysis was carried out using SNP genotyping assays from genomic DNA obtained from peripheral blood or buccal swab.ResultsOnly two of the ErbB2 SNPs (Ile 655 Val and Pro 1170 Ala) were found to have variation. There was no association between codon 665 and cardiotoxicity; however the proline variant of amino acid 1170 was more likely than the alanine variant to be found in cases with trastuzumab cardiotoxicity (35% of case patients as compared to 17% of controls, p = 0.04). This association remained significant in multivariable analysis taking into account age, race, and history of hypertension (adjusted OR = 2.60, 95% CI = 1.02, 6.62, p = 0.046).ConclusionsThe Her2/neu Pro 1170 Ala polymorphism can be used to identify a subset of patients who are at increased risk of cardiotoxicity from trastuzumab therapy. Her2/neu single nucleotide polymorphisms may be useful in conjunction with other biomarkers to risk stratify patients in order to optimize clinical management.

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Rybp (DEDAF) is a member of the Rybp/Yaf2 protein family and has been shown to encode pro-apoptotic functions and to be essential for mouse embryogenesis. The related Yaf2 protein has not been studied extensively at the cellular or organismal levels. Here we describe zebrafish yaf2 (zyaf2) and show that it is widely expressed during early embryogenesis, with subsequent enrichment of transcripts in the anterior head region. Depletion of zYaf2 during embryogenesis using specific morpholinos activates a wide-spread program of apoptosis and causes developmental arrest before the one somite stage. Partial depletion of Yaf2, achieved by injecting lower dosages of morpholino, circumvents the early arrest but leads to CNS degeneration associated with excessive apoptosis. These phenotypes can be rescued by co-injection of human YAF2 mRNA with the morpholinos or by treatment with a pan-caspase inhibitor or a caspase 8-specific inhibitor. Finally, the observed activation of caspase 8 in the morphants is in accord with the ability of Yaf2 to inhibit caspase 8-mediated apoptosis in cultured cells. Our findings implicate Yaf2 as a survival factor during early zebrafish development and organogenesis. This may suggest that Yaf2 and Rybp can encode opposing functions in the regulation of apoptosis.

Rybp (DEDAF) has been shown to interact with DED-containing proteins and to encode pro-apoptotic functions. Here we characterize a novel interaction between Rybp and Hippi, a protein implicated in neuronal apoptosis as well as in the pathogenesis of Huntington's disease. Rybp can synergize with Hippi to enhance Caspase 8-mediated apoptosis and also appears to be essential for Hippi-mediated apoptosis. Moreover, Rybp may mediate or regulate the interaction between Hippi and Caspase 8. Finally, Rybp and Hippi co-localize in a subset of neurons in the developing mouse brain. Together, these findings suggest that Rybp and Hippi may functionally interact in the apoptotic processes that accompany normal murine neural development.