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Background: Aberrant activity of the PI3K/mTOR pathway has been implicated in resistance to trastuzumab and anti-hormonal therapy for HER2-amplified and ER-positive breast cancer, respectively. Previous studies in our laboratory and others have shown that increased PI3K/mTOR signaling, either through PTEN loss or activating PIK3CA mutations, can confer resistance to trastuzumab therapy. In this study, we assessed the potential of targeting the PI3K/mTOR pathway in overcoming both de novo and acquired trastuzumab resistance. Materials and Methods: The in vitro activity of the pan-PI3K inhibitor BKM120, the mTORC1 inhibitor RAD001 and the dual PI3K/mTORC1/2 inhibitor BEZ235 were evaluated in a panel of 49 human breast cancer and immortalized cell lines. The in vivo activity of these molecules was assessed in six cell line xenografts models representing, ER+/HER2- (KPL-1, ZR75-1), ER+/HER2+ (UACC812, MDA361), ER-/HER2+ (SUM190), PIK3CA mutant (SUM190, MDA361), PTEN-null (ZR75-1) and trastuzumab resistant (BT-TR) breast cancer. Finally, the effect of PI3K/mTOR inhibition on feedback activation of PI3K signaling and compensatory pathways was measured by Western blot, immunohistochemistry (IHC) and reverse phase protein analysis (RPPA) of control and treated cell lysates/tumors. Results: Using a sensitivity cut-off of an IC50 of < 1 µmol/L, 16 of the 18 HER2-amplified breast cancer cell lines and 8 of 10 cell lines with activating mutations in PIK3CA were sensitive to the pan-PI3K inhibitor BKM120. BEZ235 showed the most potent efficacy across the panel with IC50s < 100 nmol/L for each of the 49 cell lines tested. The HER2- amplified/PIK3CA mutant cell lines were also unexpectedly sensitive to the mTORC1 inhibitor RAD001, this was despite the silencing of mTORC1 signaling being followed by a feedback increase in phospho-AKT signaling. Furthermore, each of these molecules showed remarkable in vivo activity across the panel of xenografts models. BKM120, RAD001 and BEZ235 induced both tumor stabilization and regression independent of PTEN, PI3K, ER and HER2 status. Pharmacodynamic analysis of tumor tissue revealed that BEZ235 and RAD001 both inhibited mTORC1 signaling as indicated by a reduction in the levels of phosphorylated ribosomal protein S6 (pS6). However, in contrast to BKM120 and BEZ235, RAD001 did not induce a reduction in the levels of pAKT (S473 or TH308) yet showed comparable in vivo efficacy to each of these molecules. Finally, combined targeting of HER2 and PI3K/mTOR in vitro increased the anti-proliferative activity of the molecules and led to an increased induction of apoptosis. The efficacy of these molecules (alone or in combination with trastuzumab) was assessed in a model of in vivo trastuzumab resistance generated through long-term treatment of the trastuzumab sensitive BT474 cells (BT-TR). All PI3Ki induced complete inhibition of tumor proliferation in monotherapy, while the combination of trastuzumab and each of these molecules induced tumor regression in the trastuzumab resistant tumors. Discussion: These pre-clinical data indicate that targeting the PI3K/mTOR pathway either alone or in combination with trastuzumab is effective strategy for overcoming trastuzumab resistance. [ABSTRACT FROM AUTHOR]

The cyclinD:CDK4/6:Rb axis is dysregulated in a variety of human cancers. Targeting this pathway has proven to be a successful therapeutic approach in ER + breast cancer. In this study, in vitro and in vivo preclinical breast cancer models were used to investigate the expanded use of the CDK4/6 inhibitor, abemaciclib. Using a panel of 44 breast cancer cell lines, differential sensitivity to abemaciclib was observed and was seen predominately in the luminal ER + /HER2 - and ER + /HER2 + subtypes. However, a subset of triple-negative breast cancer (TNBC) cell lines with intact Rb signaling were also found to be responsive. Equivalent levels of tumor growth inhibition were observed in ER + /HER2 - , ER + /HER2 + as well as biomarker selected TNBC xenografts in response to abemaciclib. In addition, abemaciclib combined with hormonal blockade and/or HER2-targeted therapy induced significantly improved antitumor activity. CDK4/6 inhibition with abemaciclib combined with antimitotic agents, both in vitro and in vivo , did not antagonize the effect of either agent. Finally, we identified a set of Rb/E2F-regulated genes that consistently track with growth inhibitory response and constitute potential pharmacodynamic biomarkers of response to abemaciclib. Taken together, these data represent a comprehensive analysis of the preclinical activity of abemaciclib, used alone or in combination, in human breast cancer models. The subtypes most likely to respond to abemaciclib-based therapies can be identified by measurement of a specific set of biomarkers associated with increased dependency on cyclinD:CDK4/6:Rb signaling. These data support the clinical development of abemaciclib as monotherapy or as a combination partner in selected ER + /HER2 - , HER2 + /ER + , and TNBCs. Mol Cancer Ther; 17(5); 897-907. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Poly (ADP-ribose) polymerase 1 (PARP1) is an enzyme involved in DNA repair under investigation as a chemotherapeutic target. Current randomized phase three trials of PARPi in metastatic breast cancer are limited to patients with documented BRCA1/2 mutations and no biomarker of PARPi beyond BRCA status is available. In an effort to identify novel biomarkers for PARP inhibition, we created a cell line (HCC1187/TALRES) resistant to the PARP1 inhibitor talazoparib. Herein we show by array-CGH that HCC1187/TALRES has a selective loss of the proteasome ubiquitin receptor PSMD4 amplicon resulting in significant down-regulation of PSMD4. Conversely, we find that breast cancer cell lines that have copy number gain or amplification for PSMD4 are significantly more sensitive to talazoparib. Functional studies reveal that knock-down of PSMD4 in amplified breast cancer cells and loss of the PSMD4 amplicon result in knock-down of PARP1 protein. We show that PSMD4 is amplified and overexpressed in breast cancer and its overexpression correlates with poor survival. Knock-down of PSMD4 results in a significant decrease in cell growth. We provide evidence that PSMD4 is a proteasomal amplification target in breast cancer that PSMD4 amplification confers sensitivity to PARP inhibition, and that PSMD4 amplification is lost in the process of acquiring resistance to PARPi. Finally, this study shows not only that PSMD4 copy number correlates with PARPi sensitivity, but also, that it may be a better predictor of sensitivity to PARPi than BRCA1/2 mutation., (© 2017 Wiley Periodicals, Inc.)

Everolimus (RAD001, Afinitor(®)) is an oral, selective mTOR inhibitor recently approved by the US-FDA in combination with exemestane for treatment of hormone receptor positive advanced breast cancer. To date, no molecular predictors of response to everolimus in breast cancer have been identified. We hypothesized predictive markers could be identified using preclinical models. Using a molecularly characterized panel of human breast cancer and immortalized breast epithelial cell lines, we determined sensitivity to everolimus alone or in combination with ER- or HER2- targeted therapy. Gene expression microarrays and comparative genomic hybridization were performed on the cell lines to identify predictors of response to everolimus. Among 13 everolimus-sensitive cell lines, 10/13(77 %) were luminal, while in 26 resistant cell lines, 16/26(62 %) were non-luminal, and 10/26(38 %) were luminal. Only 3/24 non-luminal lines were sensitive, two of which were HER2+. Everolimus enhanced the anti-proliferative effect of both tamoxifen (TAM) and fulvestrant (FUL) in ER+ breast cancer cell lines, as well as trastuzumab in HER2+ cell lines. Everolimus + FUL but not everolimus + TAM reversed acquired resistance to TAM. Everolimus inhibited mTOR in tested cell lines by decreasing S6 phosphorylation, mediating its anti-proliferative effect by G0/G1 cell cycle arrest and induction of apoptosis. Chromosomal amplifications of AURKA (p value = 0.04) and HER2 (p value = 0.03) were each associated with increased sensitivity to everolimus. Transcript expression microarrays identified GSK3A, PIK3R3, KLF8, and MAPK10 among the genes overexpressed in sensitive luminal lines, while PGP, RPL38, GPT, and GFAP were among the genes overexpressed in resistant luminal cell lines. These preclinical in vitro data provide further support for continued clinical development of everolimus in luminal (ER+ or HER2+) breast cancer in combination with targeted therapies. We identified several potential molecular markers associated with response to everolimus that will require validation in clinical material.

Purpose: Altered PI3K/mTOR signaling is implicated in the pathogenesis of a number of breast cancers, including those resistant to hormonal and HER2-targeted therapies., Experimental Design: The activity of four classes of PI3K/mTOR inhibitory molecules, including a pan-PI3K inhibitor (NVP-BKM120), a p110α isoform-specific PI3K inhibitor (NVP-BYL719), an mTORC1-specific inhibitor (NVP-RAD001), and a dual PI3K/mTORC1/2 inhibitor (NVP-BEZ235), was evaluated both in vitro and in vivo against a panel of 48 human breast cell lines., Results: Each agent showed significant antiproliferative activity in vitro, particularly in luminal estrogen receptor-positive and/or HER2(+) cell lines harboring PI3K mutations. In addition, monotherapy with each of the four inhibitors led to significant inhibition of in vivo growth in HER2(+) breast cancer models. The PI3K/mTOR pathway inhibitors were also effective in overcoming both de novo and acquired trastuzumab resistance in vitro and in vivo. Furthermore, combined targeting of HER2 and PI3K/mTOR leads to increased apoptosis in vitro and induction of tumor regression in trastuzumab-resistant xenograft models. Finally, as previously shown, targeting mTORC1 alone with RAD001 leads to consistent feedback activation of AKT both in vitro and in vivo, whereas the dual mTOR1-2/PI3K inhibitor BEZ235 eliminates this feedback loop. However, despite these important signaling differences, both molecules are equally effective in inhibiting tumor cell proliferation both in vitro and in vivo., Conclusion: These preclinical data support the findings of the BOLERO 3 trial that shows that targeting of the PI3K/mTOR pathway in combination with trastuzumab is beneficial in trastuzumab-resistant breast cancer., (©2014 American Association for Cancer Research.)

Aurora kinases play important roles in cell division and are frequently overexpressed in human cancer. AMG 900 is a novel pan-Aurora kinase inhibitor currently being tested in Phase I clinical trials. We aimed to evaluate the in vitro activity of AMG 900 in a panel of 44 human breast cancer and immortalized cell lines and identify predictors of response. AMG 900 inhibited proliferation at low nanomolar concentrations in all cell lines tested. Response was further classified based on the induction of lethality. 25 cell lines were classified as highly sensitive (lethality at 10 nM of AMG 900 >10 %), 19 cell lines as less sensitive to AMG 900 (lethality at 10 nM of AMG 900 <10 %). Traditional molecular subtypes of breast cancer did not predict for this differential response. There was a weak association between AURKA amplification and response to AMG 900 (response ratio = 2.53, p = 0.09). mRNA expression levels of AURKA, AURKB, and AURKC and baseline protein levels of Aurora kinases A and B did not significantly associate with response. Cell lines with TP53 loss of function mutations (RR = 1.86, p = 0.004) and low baseline p21 protein levels (RR = 2.28, p = 0.0004) were far more likely to be classified as highly sensitive to AMG 900. AMG 900 induced p53 and p21 protein expression in cell lines with wt TP53. AMG 900 caused the accumulation of cells with >4 N DNA content in a majority of cell lines independently of sensitivity and p53 status. AMG 900 induced more pronounced apoptosis in highly sensitive p53-dysfunctional cell lines. We have found that AMG 900 is highly active in breast cancer cell lines and that TP53 loss of function mutations as well as low baseline expression of p21 protein predict strongly for increased sensitivity to this compound in vitro.

HSP90 enables the activation of many client proteins of which the most clinically validated is HER2. NVP-AUY922, a potent HSP90 inhibitor, is currently in phase II clinical trials. To explore its potential clinical use in HER2-amplified breast and gastric cancers, we evaluated the effect of AUY922 alone and in combination with trastuzumab in both trastuzumab-sensitive and -resistant models. A panel of 16 human gastric and 45 breast cancer cell lines, including 16 HER2-amplified (3 and 13, respectively) cells, was treated with AUY922 over various concentrations. In both breast and gastric cancer, we used cell lines and xenograft models with conditioned trastuzumab-resistance to investigate the efficacy of AUY922 alongside trastuzumab. Effects of this combination on downstream markers were analyzed via Western blot analysis. AUY922 exhibited potent antiproliferative activity in the low nanomolar range (<40 nmol/L) for 59 of 61 cell lines. In both histologies, HER2-amplified cells expressed greater sensitivity to AUY than HER2-negative cells. In conditioned trastuzumab-resistant models, AUY922 showed a synergistic effect with trastuzumab. In vitro, the combination induced greater decreases in HER2, a G2 cell-cycle arrest, and increased apoptosis. In a trastuzumab-resistant gastric cancer xenograft model, the combination of AUY922 and trastuzumab showed greater antitumor efficacy than either drug alone. These data suggest that AUY922 in combination with trastuzumab has unique efficacy in trastuzumab-resistant models. The combination of HSP90 inhibition and direct HER2 blockade represents a novel approach to the treatment of HER2-amplified cancers and clinical trials based on the above data are ongoing.

Approximately 25,000 ovarian cancers are diagnosed in the U.S. annually, and 75% are in the advanced stage and largely incurable. There is critical need for early detection tools and novel treatments. Proteasomal ubiquitin receptor ADRM1 is a protein that is encoded by the ADRM1 gene. Recently, we showed that among 20q13-amplified genes in ovarian cancer, ADRM1 overexpression was the most highly correlated with amplification and was significantly upregulated with respect to stage, recurrence, and metastasis. Its overexpression correlated significantly with shorter time to recurrence and overall survival. Array-CGH and microarray expression of ovarian cancer cell lines provided evidence consistent with primary tumor data that ADRM1 is a 20q13 amplification target. Herein, we confirm the ADRM1 amplicon in a second ovarian cancer cohort and define a minimally amplified region of 262 KB encompassing seven genes. Additionally, using RNAi knock-down of ADRM1 in naturally amplified cell line OAW42 and overexpression of ADRM1 via transfection in ES2, we show that (1) ADRM1 overexpression increases proliferation, migration, and growth in soft agar, and (2) knock-down of ADRM1 results in apoptosis. Proteomic analysis of cells with ADRM1 knock-down reveals dysregulation of proteins including CDK-activating kinase assembly factor MAT1. Taken together, the results indicate that amplified ADRM1 is involved in cell proliferation, migration and survival in ovarian cancer cells, supporting a role as an oncogene and novel therapeutic target for ovarian cancer.

The human EGF (HER) family of receptors has been pursued as therapeutic targets in breast cancer and other malignancies. Trastuzumab and lapatinib are standard treatments for HER2-amplified breast cancer, but a significant number of patients do not respond or develop resistance to these drugs. Here we evaluate the in vitro activity of dacomitinib (PF-00299804), an irreversible small molecule pan-HER inhibitor, in a large panel of human breast cancer cell lines with variable expression of the HER family receptors and ligands, and with variable sensitivity to trastuzumab and lapatinib. Forty-seven human breast cancer and immortalized breast epithelial lines representing the known molecular subgroups of breast cancer were treated with dacomitinib to determine IC(50) values. HER2-amplified lines were far more likely to respond to dacomitinib than nonamplified lines (RR, 3.39; P < 0.0001). Furthermore, HER2 mRNA and protein expression were quantitatively associated with response. Dacomitinib reduced the phosphorylation of HER2, EGFR, HER4, AKT, and ERK in the majority of sensitive lines. Dacomitinib exerted its antiproliferative effect through a combined G(0)-G(1) arrest and an induction of apoptosis. Dacomitinib inhibited growth in several HER2-amplified lines with de novo and acquired resistance to trastuzumab. Dacomitinib maintained a high activity in lines with acquired resistance to lapatinib. This study identifies HER2-amplified breast cancer lines as most sensitive to the antiproliferative effect of dacomitinib and provides a strong rationale for its clinical testing in HER2-amplified breast cancers resistant to trastuzumab and lapatinib., (©2012 AACR.)

Purpose: Fenretinide (4-HPR) is a cytotoxic retinoid with minimal systemic toxicity that has shown clinical activity against recurrent high-risk neuroblastoma. To identify possible synergistic drug combinations for future clinical trials, we determined whether ABT-737, a small-molecule BH3-mimetic that inhibits most proteins of the antiapoptotic Bcl-2 family, could enhance 4-HPR activity in neuroblastoma., Experimental Design: Eleven neuroblastoma cell lines were tested for the cytotoxic activity of 4-HPR and ABT-737 as single agents and in combination using the DIMSCAN fluorescence digital imaging cytotoxicity assay. The effect of these agents alone and in combination on mitochondrial membrane depolarization and apoptosis (by flow cytometry), cytochrome c release, caspases, Bax-α, t-Bid, and Bak activation, and subcutaneous xenografts in nu/nu mice was also determined., Results: Multilog synergistic cytotoxicity was observed for the drug combination in all of the 11 neuroblastoma cell lines tested, including MDR lines and those insensitive to either drug as single agents. 4-HPR + ABT-737 induced greater mitochondrial membrane depolarization and mitochondrial cytochrome c release, greater activation of caspases, Bax-α, t-Bid, and Bak, and a higher level of apoptosis than either drug alone. In vivo, 4-HPR + ABT-737 increased the event-free survival of the MDR human neuroblastoma line CHLA-119 implanted subcutaneously in nu/nu mice (194.5 days for the combination vs. 68 days for ABT-737 and 99 days for 4-HPR)., Conclusion: Thus, the combination of 4-HPR with a BH3-mimetic drug warrants clinical trials in recurrent neuroblastoma.

Introduction: Alterations in cell cycle regulators have been implicated in human malignancies including breast cancer. PD 0332991 is an orally active, highly selective inhibitor of the cyclin D kinases (CDK)4 and CDK6 with ability to block retinoblastoma (Rb) phosphorylation in the low nanomolar range. To identify predictors of response, we determined the in vitro sensitivity to PD 0332991 across a panel of molecularly characterized human breast cancer cell lines., Methods: Forty-seven human breast cancer and immortalized cell lines representing the known molecular subgroups of breast cancer were treated with PD 0332991 to determine IC50 values. These data were analyzed against baseline gene expression data to identify genes associated with PD 0332991 response., Results: Cell lines representing luminal estrogen receptor-positive (ER+) subtype (including those that are HER2 amplified) were most sensitive to growth inhibition by PD 0332991 while nonluminal/basal subtypes were most resistant. Analysis of variance identified 450 differentially expressed genes between sensitive and resistant cells. pRb and cyclin D1 were elevated and CDKN2A (p16) was decreased in the most sensitive lines. Cell cycle analysis showed G0/G1 arrest in sensitive cell lines and Western blot analysis demonstrated that Rb phosphorylation is blocked in sensitive lines but not resistant lines. PD 0332991 was synergistic with tamoxifen and trastuzumab in ER+ and HER2-amplified cell lines, respectively. PD 0332991 enhanced sensitivity to tamoxifen in cell lines with conditioned resistance to ER blockade., Conclusions: These studies suggest a role for CDK4/6 inhibition in some breast cancers and identify criteria for patient selection in clinical studies of PD 0332991

Purpose: We determined if the potentially otoprotective agent sodium thiosulfate (STS) could be given 6 h after cisplatin without diminishing the antineuroblastoma activity of cisplatin in human neuroblastoma cell lines in vitro (including cisplatin-resistant cell lines) and in neuroblastoma xenografts in vivo., Experimental Design: We determined the antineuroblastoma activity of cisplatin with or without the addition of STS at 0 or 6 h after cisplatin in six neuroblastoma cell lines, both in standard cell culture conditions (20% O(2)) and in physiologic hypoxia (2% O(2)). Drug cytotoxicity was measured using the DIMSCAN fluorescence/digital imaging microscopy assay. In vivo studies of cisplatin combined with STS used a human neuroblastoma subcutaneous xenograft model (SMS-SAN) in athymic nu/nu mice., Results: A significant protection against cisplatin cytotoxicity was seen when the neuroblastoma cells were exposed to cisplatin directly combined with STS. However, when cisplatin was given first and STS exposure occurred 6 h later, no effect on cisplatin cytotoxicity was observed. In a subcutaneous neuroblastoma xenograft model in nu/nu mice, mice receiving cisplatin alone or cisplatin + STS at 6 h had significantly better progression-free survival rates (P < 0.03) compared with controls or mice treated with cisplatin + STS concurrently. There was no statistically significant difference in outcomes between mice treated with cisplatin alone and the group treated with cisplatin followed by STS 6 h later (P = 0.9)., Conclusion: These preclinical data suggest that the use of STS 6 h after cisplatin for otoprotection is unlikely to compromise the antineuroblastoma activity of cisplatin.

Purpose: Fenretinide [N-(4-hydroxyphenyl)retinamide (4-HPR)] is a cytotoxic retinoid that suffers from a wide interpatient variation in bioavailability when delivered orally in a corn oil capsule. The poor bioavailability of the capsule formulation may have limited responses in clinical trials, and the large capsules are not suitable for young children. To support the hypothesis that a novel organized lipid matrix, LYM-X-SORB, can increase the oral bioavailability of fenretinide, fenretinide in LYM-X-SORB matrix and in a powderized LYM-X-SORB formulation was delivered to mice., Experimental Design: Fenretinide was delivered orally to mice as the contents of the corn oil capsule, in LYM-X-SORB matrix (4-HPR/LYM-X-SORB matrix) or in a LYM-X-SORB matrix powderized with sugar and flour (4-HPR/LYM-X-SORB oral powder). Levels of 4-HPR, and its principal metabolite, N-(4-methoxyphenyl)retinamide, were assayed in plasma and tissues., Results: In a dose-responsive manner, from 120 to 360 mg/kg/d, delivery to mice of 4-HPR in LYM-X-SORB matrix, or as 4-HPR/LYM-X-SORB oral powder, increased 4-HPR plasma levels up to 4-fold (P<0.01) and increased tissue levels up to 7-fold (P<0.01) compared with similar doses of 4-HPR delivered using capsule contents. Metabolite [N-(4-methoxyphenyl)retinamide] levels mirrored 4-HPR levels. Two human neuroblastoma murine xenograft models showed increased survival (P<0.03), when treated with 4-HPR/LYM-X-SORB oral powder, confirming the bioactivity of the formulation., Conclusions: 4-HPR/LYM-X-SORB oral powder is a novel, oral drug delivery formulation, suitable for pediatric use, which warrants further development for the delivery of fenretinide in the treatment of cancer. A phase I clinical trial in pediatric neuroblastoma is in progress.

Purpose: Cytotoxicity assays in 96-well tissue culture plates allow rapid sample handling for multicondition experiments but have a limited dynamic range. Using DIMSCAN, a fluorescence digital image system for quantifying relative cell numbers in tissue culture plates, we have developed a 96-well cytotoxicity assay with a >4-log dynamic range., Methods: To overcome background fluorescence that limits detection of viable cells with fluorescein diacetate, we used 2'4'5'6'-tetrabromofluorescein (eosin Y) to quench background fluorescence in the medium and in nonviable cells to enhance the reduction of background fluorescence achieved with digital image thresholding. The sensitivity and linearity of the new assay were tested with serial dilutions of neuroblastoma and leukemia cell lines. DIMSCAN was compared with other in vitro cytotoxicity assays: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, colony formation, and trypan blue dye exclusion., Results: Without background fluorescence reduction, scans produced a nearly flat curve across various cell concentrations from 100 to 10(6) cells per well. Either digital image thresholding or eosin Y dramatically reduced background fluorescence, and combining them achieved a linear correlation (r > 0.9) of relative fluorescence to viable cell number over >4 logs of dynamic range, even in the presence of 4 x 10(4) nonviable cells per well. Cytotoxicity of deferoxamine for neuroblastoma cell lines measured by the DIMSCAN assay achieved dose-response curves similar to data obtained by manual trypan blue counts or colony formation in soft agar but with a wider dynamic range. Long-term cultures documented the clonogenic ability of viable cells detected by DIMSCAN over the entire dynamic range. The cytotoxicity of two drug combinations (buthionine sulfoximine + melphalan or fenretinide + safingol) was tested using both DIMSCAN and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays, and the wider dynamic range of DIMSCAN facilitated detection of synergistic interactions., Conclusion: DIMSCAN offers the ability to rapidly and efficiently conduct cytotoxicity assays in 96-well plates with a dynamic range of >4 logs. This assay enables rapid testing of anticancer drug combinations in microplates.

Bone marrow and peripheral blood stem cell transplantations, despite their curative potential, still carry significant risks for patients. Toxicity of high-dose chemotherapy is one of the leading causes of peritransplant mortality. Busulfan is a frequently used chemotherapeutic agent in conditioning regimens prior to transplantations. The choice of the optimal busulfan dose and prediction of its clinical effect may be very difficult. Absorption of busulfan from gastrointestinal tract and its metabolism can vary considerably. Several studies published recently showed that the busulfan plasma concentrations correlate better with the clinical effect and extramedullary toxicity caused by this drug than with the actual dose administered to the patient. Approximate target plasma concentrations of busulfan, which meet the optimal balance between the clinical effect and the risk of severe side effects of chemotherapy, were proposed. Almost twenty chromatographic methods were published, which make the quantitative measurement of busulfan possible. The maintenance of certain busulfan plasma concentration during its whole administration with the help of the repeated adjustments of its dosage can reduce the toxicity caused by this agent and improve the results of bone marrow or peripheral blood stem cell transplantations. This method is easily applicable, has low financial and personal demands, and technical appliances, which it requires are usually accessible in most transplant center laboratories.

Authors present a historical overview of multiple myeloma. The first well-known case of multiple myeloma was that of Mr. McBean described in 1846, 1847, and 1850 by John Darlympe, Henry Bence Jones, and William MacIntyre. The term multiple myeloma dates from 1873, and was introduced by von Rusitzky. In 1889, Otto Kahler published the case report about Dr. Loos, his patient with multiple myeloma. In 1895, Marschalkó described the essential characteristics of plasma cells. Authors present other interesting early cases of multiple myeloma and diagnostic advances in this disease.