Claudia Palermo

Patient-Tailored Treatments with Anti-EGFR Monoclonal Antibodies in Advanced Colorectal Cancer: KRAS and Beyond

Personalized medicine emphasizes the practice of considering individual patient characteristics as opposed to that centered on standards derived from epidemiological studies which, by definition, do not take into account the variability of individuals within a given population. When applied to oncology, personalized medicine is an even more complex concept because it extends the variability beyond the individual patient to the individual tumor. Indeed, the great genotypic and phenotypic variability (both in primary and metastatic sites of cancer) the development of targeted therapies, and the growing availability of biological assays complicate the scenario of personalized medicine in the oncological field. In this paper we review the results of anti-epidermal growth factor receptor (EGFR) monoclonal antibody (mAb) therapy in metastatic colorectal cancer (mCRC) in the context of tumor biology, delineating the future prospects of patient-tailored medicine in this area. In particular, we deal with EGFR inhibition by Cetuximab, a chimeric mouse human IgG1 mAb, and panitumumab, a fully human IgG2 mAb. We discuss the clinical impact of anti-EGFR mAbs on wild-type (WT) KRAS mCRC, also taking into account the feasibility of novel multi-marker approaches to treatment decision-making, aimed at increasing the predictive power of pre-therapy biomarkers. Experimental topics and fields of ongoing research, such as targeting microRNAs (miRNAs) with novel anticancer drugs and epigenetics in CRC are also addressed.

Kinase domain mutations of BCR-ABL identified at diagnosis before imatinib-based therapy are associated with progression in patients with high Sokal risk chronic phase chronic myeloid leukemia

Acquired resistance to imatinib in the advanced phase of chronic myeloid leukemia (CML) has been associated with mutations in the kinase domain (KD) of BCR-ABL. On the contrary, the prognostic implication of KD mutations in early chronic phase (CP) patients at diagnosis before imatinib-based therapy has not yet been established. We have reviewed the status of mutations in 43 patients with early CP-CML on the samples collected at diagnosis. Mutations were identified by direct sequencing (DS) with BidDye Terminator v 1.1. cycle sequencing kit and analyzed with a 3130 ABI capillary electrophoresis system. Eight out 13 (61.5%) high Sokal risk patients showed the following mutations: Y253C, S265R, E255K, F359Y, N374S, E255V, E255V, E255V. Three patients progressed during imatinib and second-line inhibitors and died of blastic phase CML at 23, 33, and 69 months. Another patient with intermediate Sokal risk showed D363G mutation at diagnosis, progressed under imatinib, was allografted and he is now alive in major molecular remission (MMR). No low-risk patient carried KD mutation at diagnosis. In conclusion, KD mutations conferring high-level imatinib resistance are present in patients with de novo CML and in some of them lead to disease progression.

Osteogenesis in vitro in rat tibia-derived osteoblasts is promoted by the homeopathic preparation, FMS*Calciumfluor.

We studied the effects of in vitro treatment of differentiating osteogenic cells with FMS*Calciumfluor, to determine whether it caused changes in proliferative or differentiation potential of osteoblasts. FMS*Calciumfluor was developed for the therapy of post‐menopausal and age‐related osteoporosis on the basis of the principles of resonance homeopathy and VTR Vega test. Its daily prescribed therapeutical usage is about 30,000‐fold less in fluoride concentration than that recommended for NaF associated with calcium monophosphate. Rat tibial osteoblast (ROB) primary cultures represent populations of early osteoblasts and their derivative cultures of more than 60 cumulative population doubling (CPD) represent more mature osteogenic cells. Both these populations were shown to undergo in vitro differentiation, as monitored by the sequential expression of markers that define the stages of the osteogenic progression. Here we report that continual treatment of ROB during osteogenesis with FMS*Calciumfluor modulated the expression of critical osteogenic markers: alkaline phosphatase (AP), an indicator of osteoblast maturation, and45Ca incorporation into the matrix and nodule formation, events of the last phase of osteogenesis and a measure of osteoid mineralization. Treatment did not affect proliferation, or expression and activation of metalloproteinases (MMP). AP activity and levels of AP mRNA were increased by treatment with FMS*Calciumfluor; the incorporation of radiolabelled Ca into the matrix was also increased and the formation of nodules occurred in a shorter time and with higher frequency than in untreated control cultures. The effects of FMS*Calciumfluor were concentration dependent and specific for its modalities of preparation, and were observed at a concentration about three orders of magnitude lower than similar effects reported in the literature by treatment of osteoblast cultures in vitro with NaF.

Evaluating Treatment Response of Chronic Myeloid Leukemia: Emerging Science and Technology

Chronic myeloid leukemia (CML) is a hematological disease accounting for about 15-20% of all adult leukemias. The clinical and biologic advances achieved in such a malignancy, represent one of the best successes obtained by translational medicine. Indeed, identification of the fusion oncogene BCR-ABL has allowed using of small molecule inhibitors of its tyrosine kinase activity which, in turn, have literally revolutionized the treatment of CML. Importantly the successfully clinical management was also realized on appropriate diagnosis, disease monitoring as well as early identification of such mutations causing drug resistance. Notably the recent availability of refined laboratory equipments represented by the Next Generation Sequencing (NGS) and genomic analyses has further contributed to gain ground towards the cure of this tumor. These issues are discussed here together with an overview on how patients treated with tyrosine kinase inhibitors should be monitored.

Quantitative Real Time PCR assessment of hormonal receptors and HER2 status on fine-needle aspiration pre-operatory specimens from a prospectively accrued cohort of women with suspect breast malignant lesions

OBJECTIVES Reliable assessment of estrogen, progesterone (ER and PR), and HER2 receptor status are essential in breast cancer (BC) treatment. Immunohistochemical methods are limited by intra- and inter-laboratory variability. Furthermore, current methods are not the ideal approach for reproducing the biological continuum of ER, PR, and HER2 receptor levels, due to their intrinsic, semi-quantitative nature, relying in part on subjective interpretation. METHODS In the present study, we tested a molecular approach to define ER, PR, and HER2 status in fine-needle-aspirate (FNA) samples from patients with early BC. We performed flow cytometry analysis on 88 FNA specimens from suspect BC patients to determine cellularity. We used quantitative Real Time PCR (QRT-PCR) to assess ER, PR, HER2 status, and qPCR for HER2 gene copy number (GCN). RESULTS ER and PR mRNA levels showed a highly significant correlation with IHC data on surgical samples. qPCR showed greater accuracy than IHC in defining HER2 status. QRT-PCR defined better than IHC the continuous spectrum of the expression of the assessed receptors. Moreover, PCR analysis demonstrated a strict correlation between HER2 status and higher levels of its transcript, correctly stratifying HER2+ and HER2- patients. Finally, there was a strongly significant agreement between HER2 GCN assessed on FNA specimens by qPCR and FISH data obtained on pathological tissue specimens. CONCLUSIONS The present results support a comprehensive approach to determine ER, PR, and HER2 status by PCR (QRT-PCR and qPCR) in FNA specimens, with high relevance for therapeutic strategies like neoadjuvant treatment.

Standard and variant Philadelphia translocation in a CML patient with different sensitivity to imatinib therapy

Most chronic myeloid leukemia (CML) patients show the Philadelphia chromosome (Ph) arising from the reciprocal t(9;22), but 5–10% present variants of this translocation involving different breakpoints besides 9q34 and 22q11. We report the non simultaneous occurrence of two different types of Ph translocation in a CML patient: a t(9;22)(q34;q11) standard and a three-way variant t(9;11;22)(q34;p15;q11). Bone marrow cells with standard translocation did not have BCR/ABL kinase domain (KD) mutations and were sensitive to imatinib therapy. In contrast, bone marrow cells with the variant translocation showed two BCR/ABL KD mutations and were resistant to imatinib, thus inducing transformation to the blast phase and karyotype evolution.

Tracking molecular relapse of chronic myeloid leukemia by measuring Hedgehog signaling status

Abstract Chronic myeloid leukemia (CML) is a clonal myeloproliferative disorder characterized by the expansion of a leukemic stem cell (LSC) clone, carring a Philadelphia translocation, able to overcome the non-malignant hematopoietic stem cells. The tyrosine kinase inhibitors (TKIs) imatinib, nilotinib and dasatinib are gold-standard for CML treatment. Each shows an impressive rate of complete cytogenetic response in chronic phase (CP)-CML. However, relapse and treatment failure are major problems with long-term use of TKIs. A polymerase chain reaction (PCR) assay to detect the mRNA expression of BCR–ABL1 represents the main molecular approach to monitoring response to treatment. However, using this analysis it is currently not possible to prospectively identify patients whose disease will relapse due to LSC reappearance. The aim of our study was to investigate whether the mRNA expression analysis of two Hedgehog (Hh) stemness signaling molecules, Smoothened (SMO) and Patched-1 (PTCH1), could predict upcoming molecular relapse. At the time of diagnosis, patients with high Sokal risk (n = 12) showed higher and lower levels of SMO and PTCH1, respectively (p = 0.0132), compared with patients with different Sokal scores (p = 0.0316 for intermediate risk and p = 0.0340 for low risk). These data suggest that Hh signaling was functionally more active in this risk group at the time of diagnosis. Furthermore, the kinetics of Hh signaling activity during the individual medical history correlated with BCR–ABL1 mRNA level and with upcoming molecular relapse. Also, mutation analysis of BCR–ABL1 revealed that activation of Hh signaling precedes molecular relapse by several months, mostly in patients carrying the gatekeeper mutation T315I. Importantly, in vitro data showed a synergistic effect of chemical inhibitors of Hh signaling and TKIs in both wild-type and resistant (T315I) CML cell lines. Collectively our data show that monitoring Hh pathway activity contemporaneously with BCR–ABL1 mRNA level may improve the chance of early detection of patients who will experience a relapse (mainly in the high Sokal risk group), paving the way for an innovative management of this hematologic malignancy.

Abstract 49: Agreement of immunohistochemistry, fluorescence in situ hybridization, real-time quantitative polymerase-chain reaction, and quantitative reverse transcriptase PCR for Her-2/Neu status assessment in breast cancer patients: a single center, retrospective m

Background: Immunohistochemistry (IHC) and fluorescence-in-situ hybridization (FISH) are the standard methods to assess human epidermal growth factor receptor 2 (HER-2) status in breast cancer (BC) patients. Real-time quantitative polymerase-chain-reaction (Q-PCR) and quantitative reverse transcriptase PCR (qRT-PCR) allow quantitative determination of gene copy number and gene expression on, respectively, DNA and RNA. These molecular methods are available to assess HER-2 amplification/overexpression but their use remains controversial. Here we performed a parallel comparison of these four methods to define their concordance rates and evaluate their relative role in HER-2 status determination. Patients and Methods: HER-2 status was determined by IHC, FISH, Q-PCR and qRT-PCR in a retrospectively assessed cohort of 130 BC patients. The studied set was enriched in cases scoring as 3+ by standard IHC analysis. Tests were performed blindly in parallel. Western blotting was performed on a subset of equivocal cases. Kappa statistics and ROC curves were used as appropriate, and concordance analyses were interpreted according to the ASCO/CAP guidelines. Results: Of the 130 enrolled patients, 47 (36%) were classified as positive and 27 (21%) as equivocal by IHC. With FISH, 50 patients (38%) were HER-2 amplified, while 80 (62%) were not. The overall agreement between FISH and Q-PCR was 98.5% (95% CI, 94.6% to 99.6%) with a k value of 0.97 (95% CI, 0.92 to 1). Assuming FISH as the standard reference, Q-PCR showed a sensitivity of 98% (95% CI, 89.5% to 99.6%) and a specificity of 98.8% (95% CI, 93.3% to 99.8%), with a global accuracy of 98%. The overall agreement between FISH and qRT-PCR was 96.9% (95% CI, 92.3% to 98.8%) with a k value of 0.94 (95% CI, 0.87 to 1). For both comparisons, the observed concordance values were greater than the 95% threshold required by the ASCO/CAP guidelines to validate novel approaches for HER-2 testing. 3% of samples showed HER-2 overexpression only by qRT-PCR analysis, in all cases belonging to the equivocal range as defined by either IHC or FISH. In these patients, WB confirmed higher HER-2 protein levels compared to healthy diploid controls. Conclusions: The high concordance between FISH and both Q-PCR and qRT-PCR supports the use of molecular tests as an alternative to current standard methods. Present results also suggest that qRT-PCR may be able to unequivocally classify patients belonging to the IHC/FISH equivocal range. Non-amplified HER-2 overexpressing BCs may account for the rare trastuzumab-responders observed in phase III trials, where trastuzumab response was assessed in HER-2-amplified vs. non-amplified cases. Citation Format: Gabriele Zoppoli, Anna Garuti, Ilaria Rocco, Claudia Palermo, Gabriella Cirmena, Enrico Carminati, Daniele Friedman, Ludovica Verdun di Cantogno, Anna Sapino, Franco Patrone, Alberto Ballestrero. Agreement of immunohistochemistry, fluorescence in situ hybridization, real-time quantitative polymerase-chain reaction, and quantitative reverse transcriptase PCR for Her-2/Neu status assessment in breast cancer patients: a single center, retrospective m [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 49. doi:10.1158/1538-7445.AM2013-49