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Chronic lymphocytic leukemia (CLL) is a hematological disorder with complex clinical and biological behavior. TP53 mutational status and cytogenetic assessment of the deletion of the corresponding locus (17p13.1) are considered the most relevant biomarkers associated with pharmaco-predictive response, chemo-refractoriness, and worse prognosis in CLL patients. The implementation of Next Generation Sequencing (NGS) methodologies in the clinical laboratory allows for comprehensively analyzing the TP53 gene and detecting mutations with allele frequencies ≤10%, that is, "subclonal mutations". We retrospectively studied TP53 gene mutational status by NGS in 220 samples from 171 CLL patients. TP53 mutations were found in 60/220 (27.3%) samples and 47/171 (27.5%) patients. Interestingly, subclonal mutations could be detected in 31/60 samples (51.7%) corresponding to 25 patients (25/47, 53.2%). We identified 44 distinct subclonal TP53 mutations clustered in the central DNA-binding domain of p53 protein (exons 5-8, codons 133-286). Missense mutations were predominant (80%), whereas indels, nonsense, and splice site variants were less represented. All subclonal TP53 variants but one [p.(Pro191fs)] were already described in NCI and/or Seshat databases as "damaging" and/or "probably damaging" mutations (38/44, 86% and 6/44, 14%, respectively). Longitudinal samples were available for 37 patients. Almost half of them displayed at least one TP53 mutant subclone, which could be alone (4/16, 25%) or concomitant with other TP53 mutant clonal ones (12/16, 75%); different patterns of mutational dynamics overtimes were documented. In conclusion, utilization of NGS in our "real-life" cohort of CLL patients demonstrated an elevated frequency of subclonal TP53 mutations. This finding indicates the need for precisely identifying these mutations during disease since the clones carrying them may become predominant and be responsible for therapy failures.

Mismatch repair (MMR) testing on all new cases of colorectal cancer (CRC), has for now been preferably performed on surgical specimens as more tissue is available, however, new clinical trials for the use of immune check-point inhibitors in the neoadjuvant setting require MMR testing on biopsy samples. This study aims at identifying advantages, disadvantages and any potential pitfalls in MMR evaluation on biopsy tissue and how to cope with them. The study is prospective-retrospective, recruiting 141 biopsies (86 proficient (p)MMR and 55 deficient (d)MMR) and 97 paired surgical specimens (48 pMMR and 49 dMMR). In biopsy specimens, a high number of indeterminate stains was observed, in particular for MLH1 (31 cases – 56.4%). The main reasons were a punctate nuclear expression of MLH1, enhanced MLH1 nuclear expression compared to internal controls or both (making MLH1 loss difficult to interpret), which can be solved by reducing primary incubation times for MLH1. Cases with adequate immunostains have a mean number of > 5 biopsies compared to ≤ 3 biopsies in inadequate cases. Conversely, surgical specimens rarely suffer from indeterminate reactions while weaker staining intensity (p

Targeted next-generation sequencing (NGS) based on molecular tagging technology allowed considerable improvement in the approaches of cell-free DNA (cfDNA) analysis. Previously, we demonstrated the feasibility of the OncomineTM Lung cell-free DNA Assay (OLcfA) NGS panel when applied on plasma samples of post-tyrosine kinase inhibitors (TKIs) non-small cell lung cancer (NSCLC) patients. Here, we explored in detail the coverage metrics and variant calling of the assay and highlighted strengths and challenges by analyzing 92 plasma samples collected from a routine cohort of 76 NSCLC patients. First, performance of OLcfA was assessed using Horizon HD780 reference standards and sensitivity and specificity of 92.5% and 100% reported, respectively. The OLcfA was consequently evaluated in our plasma cohort and NGS technically successful in all 92 sequenced libraries. We demonstrated that initial cfDNA amount correlated positively with library yields (p &lt, 0.0001) and sequencing performance (p &lt, 0.0001). In addition, 0.1% limit of detection could be achieved even when &lt, 10 ng cfDNA was employed. In contrast, the cfDNA amount seems to not affect the EGFR mutational status (p = 0.16). This study demonstrated an optimal performance of the OLcfA on routine plasma samples from NSCLC patients and supports its application in the liquid biopsy practice for cfDNA investigation in precision medicine laboratories.

Molecular characterization of Circulating Tumor Cells (CTCs) is still challenging, despite attempts to minimize the drawbacks of Whole Genome Amplification (WGA). In this paper, we propose a Next-Generation Sequencing (NGS) optimized protocol based on molecular tagging technology, in order to detect CTCs mutations while skipping the WGA step. MDA-MB-231 and MCF-7 cell lines, as well as leukocytes, were sorted into pools (2&ndash, 5 cells) using a DEPArray&trade, system and were employed to set up the overall NGS procedure. A substantial reduction of reagent volume for the preparation of libraries was performed, in order to fit the limited DNA templates directly derived from cell lysates. Known variants in TP53, KRAS, and PIK3CA genes were detected in almost all the cell line pools (35/37 pools, 94.6%). No additional alterations, other than those which were expected, were found in all tested pools and no mutations were detected in leukocytes. The translational value of the optimized NGS workflow is confirmed by sequencing CTCs pools isolated from eight breast cancer patients and through the successful detection of variants. In conclusion, this study shows that the proposed NGS molecular tagging approach is technically feasible and, compared to traditional NGS approaches, has the advantage of filtering out the artifacts generated during library amplification, allowing for the reliable detection of mutations and, thus, making it highly promising for clinical use.

Background The demonstration of EGFR T790M gene mutation in plasma is crucial to assess the eligibility of Non Small Cell Lung Cancer (NSCLC) patients, who have acquired resistance to first or second generation Tyrosine Kinase Inhibitors (TKIs), to receive a subsequent treatment with osimertinib. Since circulating tumor DNA (ctDNA) is present in very low amounts in plasma, high sensitive and specific methods are required for molecular analysis. Improving sensitivity of T790M mutation detection in plasma ctDNA enables a larger number of NSCLC patients to receive the appropriate therapy without any further invasive procedure. Methods A tag-based next generation sequencing (NGS) platform capable of tagging rare circulating tumor DNA alleles was employed in this study for the identification of T790M mutation in 42 post-TKI NSCLC patients. Results Compared to Real Time PCR, tag-based NGS improved the T790M detection rate (42.85% versus 21.4%, respectively), especially in those cases with a low median mutation abundance (i.e. 0.24, range 0.07–0.78). Moreover, the tag-based NGS identified EGFR activating mutations more efficiently than Real Time PCR (85.7% versus 61.9% detection rate, respectively), particularly of the L858R variant type (0.06–0.75 mutation abundance range). Patients in whom the T790M mutation was detected in plasma, achieved an objective response to osimertinib (9/14, 64.28%). Conclusions Tag-based NGS represents an accurate and sensitive tool in a clinical setting for non-invasive assessment and monitoring of T790M variant in NSCLC patients. Electronic supplementary material The online version of this article (10.1186/s10020-019-0082-5) contains supplementary material, which is available to authorized users.

CTLA-4 blockade with monoclonal antibodies can lead to cancer regression in patients with metastatic melanoma (MM). CTLA-4 gene polymorphisms may influence the response to anti-CTLA-4 antibodies although few data are available regarding this issue. We analyzed six CTLA-4 single nucleotide polymorphisms (−1661A>G, −1577G>A, −658C>T, −319C>T, +49A>G, and CT60G>A) in 14 Italian MM patients and 45 healthy subjects. We found a significant association between the −1577G/A and CT60G/A genotypes and improved overall survival (Pc < 0.006, Bonferroni corrected), further confirmed by the diplotype analysis (−1577&CT60 GG-AA diplotype, p < 0.001). A positive trend toward an association between these genotypes and response to therapy was also observed.

Estrogen synthesis suppression induced by aromatase inhibitors in breast cancer (BC) patients may be affected by single nucleotide polymorphisms (SNPs) of the gene encoding aromatase enzyme, CYP19A1. We assessed the association between plasma estrone sulfate (ES), letrozole treatment, and four SNPs of CYP19A1 gene (rs10046 C>T, rs4646 G>T, rs749292 C>T, rs727479 T>G) which seem to be related to circulating estrogen levels. Patients were enrolled into a prospective, Italian multi-center clinical trial (Gruppo Italiano Mammella, GIM-5) testing the association of CYP19A1 SNPs with the efficacy of letrozole adjuvant therapy, in postmenopausal early BC patients. SNPs were identified from peripheral blood cell DNA. Plasma ES concentrations were evaluated by Radio Immuno Assay. Blood samples were obtained immediately before letrozole therapy (N = 204), at 6-weeks (N = 178), 6 (N = 152) and 12-months (N = 136) during treatment. Medians (IQR) of ES were 160 pg/mL (85–274) at baseline, 35 pg/mL (12–64) at 6-weeks, 29 pg/mL (17–48) at 6 months and 25 pg/mL (8–46) after 12 months treatment. No statistically significant association was evident between polymorphisms and ES circulating levels during letrozole therapy. Letrozole suppression of the aromatase enzyme function is not affected by polymorphisms of CYP19A1 gene in postmenopausal BC patients.

Paroxysmal nocturnal haemoglobinuria (PNH) is a haematopoietic disorder characterized by expansion of phosphatidylinositol glycan-A-defective progenitor(s). Immune-dependent mechanisms, likely involving a deranged T cell-dependent autoimmune response, have been consistently associated with the selection/dominance of PNH precursors. Natural killer (NK) lymphocytes might participate in PNH pathogenesis, but their role is still controversial. NK activity is dependent on the balance between activating and inhibiting signals. Key component in such regulatory network is represented by killer immunoglobulin-like receptors (KIR). KIR are also involved in the regulation of adaptive cytotoxic T cell response and associated with autoimmunity. This study investigated on the frequency of KIR genes and their known human leukocyte antigen (HLA) ligands in 53 PNH Italian patients. We observed increased frequency of genotypes characterized by ≤2 activating KIR as well as by the presence of an inhibitory/activating gene ratio ≥3.5. In addition, an increased matching between KIR-3DL1 and its ligand HLA-Bw4 was found. These genotypes might be associated with lower NK-dependent recognition of stress-related self molecules; this is conceivable with the hypothesis that an increased availability of specific T cell targets, not cleared by NK cells, could be involved in PNH pathogenesis. These data may provide new insights into autoimmune PNH pathogenesis.

Deficiency of glycosylphosphatidylinositol (GPI)–anchored molecules on blood cells accounts for most features of paroxysmal nocturnal hemoglobinuria (PNH) but not for the expansion of PNH (GPI−) clone(s). A plausible model is that PNH clones expand by escaping negative selection exerted by autoreactive T cells against normal (GPI+) hematopoiesis. By a systematic analysis of T-cell receptor beta (TCR-β) clonotypes of the CD8+ CD57+ T-cell population, frequently deranged in PNH, we show recurrent clonotypes in PNH patients but not in healthy controls: 11 of 16 patients shared at least 1 of 5 clonotypes, and a set of closely related clonotypes was present in 9 patients. The presence of T-cell clones bearing a set of highly homologous TCR-β molecules in most patients with hemolytic PNH is consistent with an immune process driven by the same (or similar) antigen(s)—probably a nonpeptide antigen, because patients sharing clonotypes do not all share identical HLA alleles. These data confirm that CD8+ CD57+ T cells play a role in PNH pathogenesis and provide strong new support to the hypothesis that the expansion of the GPI− blood cell population in PNH is due to selective damage to normal hematopoiesis mediated by an autoimmune attack against a nonpeptide antigen(s) that could be the GPI anchor itself.

Paroxysmal nocturnal haemoglobinuria (PNH) is a haematopoietic disorder characterized by expansion of phosphatidylinositol glycan-A-defective progenitor(s). Immune-dependent mechanisms, likely involving a deranged T cell-dependent autoimmune response, have been consistently associated with the selection/dominance of PNH precursors. Natural killer (NK) lymphocytes might participate in PNH pathogenesis, but their role is still controversial. NK activity is dependent on the balance between activating and inhibiting signals. Key component in such regulatory network is represented by killer immunoglobulin-like receptors (KIR). KIR are also involved in the regulation of adaptive cytotoxic T cell response and associated with autoimmunity. This study investigated on the frequency of KIR genes and their known human leukocyte antigen (HLA) ligands in 53 PNH Italian patients. We observed increased frequency of genotypes characterized by ≤2 activating KIR as well as by the presence of an inhibitory/activating gene ratio ≥3.5. In addition, an increased matching between KIR-3DL1 and its ligand HLA-Bw4 was found. These genotypes might be associated with lower NK-dependent recognition of stress-related self molecules; this is conceivable with the hypothesis that an increased availability of specific T cell targets, not cleared by NK cells, could be involved in PNH pathogenesis. These data may provide new insights into autoimmune PNH pathogenesis.

The growth and differentiation of normal and neoplastic epithelial cells may be regulated by the presence of adjacent normal tissues and cells, particularly stromal fibroblasts. However, the influence of normal fibroblast-tumor cell interactions on the response of malignant epithelial cells to radiation has not been adequately investigated nor has the possible role played by a 3-D environment in such modulation. We addressed this question by embedding MCF-7 mammary carcinoma cells into a collagen lattice, alone or mixed with HSF human dermal fibroblasts, and kept the gels anchored to the plastic surface or suspended in the culture medium. Some gels served as controls and others were irradiated with 6 MV photons fractionated into 3 daily doses totaling 5 or 10 Gy. After 2 or 7 days from the last treatment (7 or 12 days in culture, respectively), gels were processed in 1 of 2 ways: overall cell survival was determined by the MTT assay, while the survival of MCF-7 cells was selectively detected by a clonogenicity assay. Under these experimental conditions, we found that, in the presence of HSF fibroblasts, the growth of MCF-7 cells was restrained and radiosensitivity increased compared with MCF-7 cells cultured alone. For example, while the average number of MCF-7 foci/gel recovered from control gels with MCF-7 cells alone was 2,460 on day 7 and 3, 290 on day 12 of culture, it was 4 to 5 times lower (p < 0.001) in control gels with mixed MCF-7 and HSF cells. Radiation affected severely the survival of MCF-7 cells in all experimental groups but not sufficiently to mask the differences. For example, following exposure to the low dose of 5 Gy, the average number of MCF-7 foci/gel recovered from MCF-7-containing gels was 590 on day 7 and 329 on day 12 of culture, whereas numbers from the gels containing mixed MCF-7 and HSF cells were only 218 and 73, respectively (p < 0. 003 in both cases). HSF fibroblasts did not grow in our system, but they contracted strongly anchored and floating gels.

Complement C3 ‘slow’ and ‘fast’ allotypes are associated with immune-mediated disorders and may affect the outcome of renal transplantation. We report a tetra-primer amplification refractory mutation system PCR (T-ARMS-PCR) that provides a rapid, reproducible and cost-effective method to genotype both complement C3 ‘slow’ and ‘fast’ alleles by a single tube reaction.

Tetraprimer amplification refractory mutation system PCR (T-ARMS-PCR) is a simple and inexpensive genotyping method for differentiating both alleles of a polymorphism/mutation (both single-nucleotide polymorphisms and small insertions/deletions) with a single-tube PCR (1). In T-ARMS-PCR, a pair of common (outer) primers produces a non–allele-specific control amplicon and in combination with 2 allele-specific (inner) primers (designed to anneal in the opposite orientation) produces 2 allele-specific amplicons. These allele-specific amplicons have different sizes because the polymorphism/mutation is asymmetrically located with respect to the common primers. Thus, the amplicons can be separated by standard gel electrophoresis. T-ARMS-PCR has also been designed in a multiplex fashion to genotype more than one polymorphism/mutation by a single-tube PCR (2). We describe a modified multiplex T-ARMS-PCR, the hexaprimer ARMS-PCR (H-ARMS-PCR), which is for when 2 polymorphisms are close in the sequence. H-ARMS-PCR uses only 6 primers and provides direct information about haplotype structure. The CTLA4 gene (cytotoxic T-lymphocyte–associated protein 4; also known as CD152) is a negative regulator of T-cell function (3). The CTLA4 polymorphisms −318 C>T (rs5742909) and +49 A>G (rs231775) are associated with susceptibility to autoimmune diseases and cancer (3)(4). To genotype these 2 polymorphisms, which are only 365 bp apart in the 5′ region of the CTLA4 gene, we designed an H-ARMS-PCR that combines a single pair of common primers and 2 pairs of allele-specific primers in the same tube (Fig. 1A⇓ ). Figure 1. Polymorphism genotyping by H-ARMS-PCR. (A), H-ARMS-PCR for CTLA4 −318 C>T (rs5742909) and …

Background: We describe a simple tetra-primer amplification refractory mutation system PCR (T-ARMS-PCR) for detecting MUTYH mutations, which are associated with colorectal adenomas and colorectal cancer. Methods: We designed specific T-ARMS-PCR assays for 6 mutations (Y165C, G382D, 1395_7delGGA, Y90X, 1103delC, and R231H) selected on the basis of the frequency of their occurrence. We also designed a set of 3 multiplex T-ARMS PCR assays, each for detection of 2 mutations. We tested DNA samples from patients with attenuated or classic adenomatous polyposis coli and no detectable APC germline mutations. Results: All mutations were easily detected with both the specific and multiplex T-ARMS-PCR assays. Results were confirmed by DNA HPLC analysis in all 54 patients, and each mutation was confirmed by direct DNA sequencing. Conclusions: T-ARMS-PCR does not require any special equipment, and it provides rapid, reproducible, and cost-effective detection of common MUTYH mutations. Multiplex T-ARMS-PCR allows the detection of 6 common MUTYH mutations with use of as few as 3 single tube PCR reactions. It could be useful to carry out large population-based epidemiologic studies.

Patients with paroxysmal nocturnal hemoglobinuria (PNH) have a large clonal population of blood cells deriving from hematopoietic stem cells (HSCs) deficient in glycosylphosphatidylinositol (GPI)-anchored surface molecules. A current model postulates that PNH arises through negative selection against normal HSCs exerted by autoreactive T cells, whereas PNH HSCs escape damage. We have investigated the inhibitory receptor superfamily (IRS) system in 13 patients with PNH. We found a slight increase in the proportion of T cells expressing IRS. In contrast to what applies to healthy donors, the engagement of IRS molecules on T cells from patients with PNH elicited a powerful cytolytic activity in a redirected killing assay, indicating that these IRSs belong to the activating type. This was confirmed by clonal analysis: 50% of IRS+ T-cell clones in patients with PNH were of the activating type, while only 5% were of the activating type in healthy donors. Moreover, the ligation of IRS induces (1) production of tumor necrosis factor α (TNF-α) and interferon γ (IFN-γ) and (2) brisk cytolytic activity against cells bearing appropriate IRS counter-ligands. In addition, these IRS+ T cells show natural killer (NK)-like cytolytic activity to which GPI- cells were less sensitive than GPI+ cells. Thus, T cells with NK-like features, expressing the activating isoforms of IRS, may include effector cells involved in the pathogenesis of PNH.

8588 Background: It has been suggested that specific CTLA-4 gene polymorphisms may have an important role in advanced melanoma patients (pts) receiving CTLA-4 blockade therapy (Breunis W. et al. J ...

2606 Background: The efficacy of aromatase inhibitors in breast cancer patients might be related to various SNPs of CYP19A1 gene (encoding aromatase enzyme), determining a different suppression of ...

13049 Background: The irinotecan active metabolite, SN38, is inactivated through glucuronidation mainly by uridine diphosphate glucuronosyltransferase (UGT) 1A1. The (TA)7 allele of a polymorphism in UGT1A1 promoter has been associated with reduced SN38-glucuronidation rate and more severe toxicity. Since other UGT1A family isoforms, the extrahepatic UGT1A7 and the hepatic UGT1A9, are involved in SN38 glucuronidation, it has been suggested that also polymorphic variants of these genes may affect irinotecan toxicity. Methods: 84 patients with advanced colorectal cancer received an irinotecan-based (180 mg/m2 q.2wks) combination treatment. Polymorphisms of UGT1A1 [(TA)6>7], UGT1A7 [387T > G, 391–2CG > AA, 622T > C] and UGT1A9 [-118(T)9>10, -87G > A, 98T > C, I152G > A] were identified by sequencing on DNA from blood samples obtained under an IRB approved protocol. Toxicity has been graded according to NCI-CT criteria. Results: Patient median age was 64 y (range: 31–82); median PS was 0 (range: 0–2). Severe toxicity (diarrhea or/and neutropenia ≥G3) has been observed in 32 patients: diarrhea in 11, neutropenia in 25 (4 patients had both). The estimated allele frequencies were as follows: UGT1A1, (TA)7 (0.35); UGT1A7, 387T and the completely linked 391–2CG (0.39), 622C (0.39); UGT1A9, -118(T)10 (0.39), -87A (0.05), T98C (0.02), I152A (0.21). Severe toxicity was significantly associated only with: (a) the UGT1A1 (TA)7 allele [severe toxicity in 25% of (TA) 6/6, in 46% of (TA) 6/7, in 55% of (TA) 7/7 (Mantel-Haenszel trend test, P < 0.03)]; and (b) the low activity UGT1A7 622C allele [severe toxicity in 26% of 622 T/T, in 39% of 622 T/C, in 67% of 622 C/C (Mantel-Haenszel, P < 0.02)]. In addition, the analysis of the combined genotypes [(TA)6>7 and 622T > C] has shown severe toxicity in 21% of patients with none of (TA)7 and of 622C alleles, in 43% of patients in which the sum of the number of (TA)7 and of 622C alleles is 1 or 2, in 57% of patients in which this sum is ≥3 (the latter includes all patients with both toxicities) (Mantel-Haenszel, P < 0.02). Conclusions: These data show that both the low activity UGT1A1 (TA)7 and UGT1A7 622C alleles are significantly associated with irinotecan severe toxicity. It is to evaluate if their combined genotype may be a better predictor of toxicity. No significant financial relationships to disclose.

2016 Background: Inherited variation in the activity of enzymes involved in drug metabolism are likely to be responsible for most inter-individual variability in the toxicity and the efficacy of the drugs themselves. The topoisomerase-I inhibitor irinotecan may have unpredictable severe toxicity: mainly diarrhea and myelosuppression. Its active metabolite, SN38, is inactivated through glucuronidation by uridine diphosphate glucuronosyltransferase 1A1 (UGT1A1). The UGT1A1 promoter includes, in the TATA box region, a well known polymorphism containing either 7 or, more commonly, 6 TA repeats. The (TA)7 allele has been associated with a reduced rate of SN-38 glucuronidation and more severe toxicity in patients treated with irinotecan. Methods: UGT1A1 was genotyped by PCR on samples obtained under an institutional review board-approved protocol from 66 patients with advanced colorectal cancer treated, 24 in first and 42 in second line, with irinotecan-containing chemotherapy. Toxicity has been graded accordin...

PNH is an acquired clonal disorder of the hematopoietic stem cell (HSC) characterized by intravascular hemolysis, venous thrombosis, and variable degrees of bone marrow failure. In PNH a somatic mutation of the X-linked PIG-A gene in HSC results in complete or partial deficiency of all proteins anchored by the glycosylphosphatidylinositol (GPI) on the membrane of the mutated HSC and in its mature progeny. The close association between PNH and Idiopathic Aplastic Anemia (IAA), and other lines of evidence support the hypothesis that auto-reactive T cells might be responsible for the expansion of hematopoietic PNH clone(s), which is required to cause clinical PNH. Stemming from our observation of a unique patient with PNH and with a large granular lymphocyte (LGL) leukemia with NKT phenotype (Karadimitris et al, Br J Haematol115:1010, 2001), we have measured systematically the percentage of NKT [CD3+ CD8+(bright) CD57+] cells in the peripheral blood of PNH patients. The proportion of NKT cells was quite variable and very similar in 18 patients (6.9±5.9; range: 0.8 – 22.3%) and in 18 healthy individuals (6.5±5.2; range: 0.9 – 21.2; P>0.5). However, when we analyzed the size distribution of the complementarity-determining region 3 (CDR3) of the TCR-beta chain genes in sorted NKT cells, there was a sharp difference. In healthy individuals we observed a normally distributed ladder of bands of different sizes. By contrast, in 14 out of 15 PNH patients we found a markedly non-random (“oligoclonal”) pattern; and in each patient some clones were predominant. In 6 out of 6 patients followed-up longitudinally over 6–12 months the “oligoclonal” pattern was consistent and persistent. In each of 10 patients in whom we carried out systematic sequencing of the TCR-beta CDR3 of sorted NKT cells we have observed an average of 25 different TCR-beta CDR3 sequences (out of an average of 80 total sequences obtained): but only one or two sequences were predominant. Interestingly, an identical or quasi-identical (single amino acid difference) sequence was found in 4 patients; and in two of these the sequence belonged to one of the predominant clones. In addition, in 5 cases a sequence found in one patient was subsequently found also in another patient. These data are reminiscent of recent findings reported in patients with IAA (Risitano et al, Lancet364:355, 2004): in these patients, however, no identity of sequence was detected. We surmise that in both groups of patients specific T cells clones may be responsible for damage to normal HSCs. However, it is possible that in IAA a number of different antigens are recognized on HSCs in individual patients; whereas in PNH the range of potential target antigens is much more restricted, because they must be present on normal HSC but not on PNH HSCs, thus enabling them to survive the auto immune attack and to expand.

Background Trastuzumab is a humanized monoclonal antibody (mAb) currently used for the treatment of breast cancer (BC) patients with HER-2 overexpressing tumor subtype. Previous data reported the involvement of FcγRIIIA/IIA gene polymorphisms and/or antibody-dependent cellular cytotoxicity (ADCC) in the therapeutic efficacy of trastuzumab, although results on these issues are still controversial. This study was aimed to evaluate in vitro the functional relationships among FcγRIIIA/IIA polymorphisms, ADCC intensity and HER-2 expression on tumor target cells and to correlate them with response to trastuzumab. Patients and methods Twenty-five patients with HER-2 overexpressing BC, receiving trastuzumab in a neoadjuvant (NEO) or metastatic (MTS) setting, were genotyped for the FcγRIIIA 158V>F and FcγRIIA 131H>R polymorphisms by a newly developed pyrosequencing assay and by multiplex Tetra-primer-ARMS PCR, respectively. Trastuzumab-mediated ADCC of patients’ peripheral blood mononuclear cells (PBMCs) was evaluated prior to therapy and measured by 51Chromium release using as targets three human BC cell lines showing different levels of reactivity with trastuzumab. Results We found that the FcγRIIIA 158F and/or the FcγRIIA 131R variants, commonly reported as unfavorable in BC, may actually behave as ADCC favorable genotypes, in both the NEO (P ranging from 0.009 to 0.039 and from 0.007 to 0.047, respectively) and MTS (P ranging from 0.009 to 0.032 and P = 0.034, respectively) patients. The ADCC intensity was affected by different levels of trastuzumab reactivity with BC target cells. In this context, the MCF-7 cell line, showing the lowest reactivity with trastuzumab, resulted the most suitable cell line for evaluating ADCC and response to trastuzumab. Indeed, we found a statistically significant correlation between an increased frequency of patients showing ADCC of MCF-7 and complete response to trastuzumab in the NEO setting (P = 0.006). Conclusions Although this study was performed in a limited number of patients, it would indicate a correlation of FcγR gene polymorphisms to the ADCC extent in combination with the HER-2 expression levels on tumor target cells in BC patients. However, to confirm our findings further experimental evidences obtained from a larger cohort of BC patients are mandatory. Electronic supplementary material The online version of this article (doi:10.1186/s12967-015-0680-0) contains supplementary material, which is available to authorized users.