Flow cytometric expression of Bcl-2, Mcl-1, and their ratios correlates with primary and secondary cytogenetic changes and their combinations in multiple myeloma.

Response to BH3 mimetics in multiple myeloma (MM) correlates with CCND1-rearrangement or expression of anti-apoptotic molecules, particularly Bcl-2 and Mcl-1. Our study investigates the relationship between cytogenetic abnormalities (CGAs) and intracellular Bcl-2 and Mcl-1 expression in myeloma plasma cells (MPCs) using flow cytometry (FCM). We measured median fluorescence intensity (MFI) of Bcl-2 and Mcl-1 in 163 bone marrow samples (143 MM, 20 controls) across various cell types. Both Bcl-2MFI and Mcl-1MFI were significantly higher in MPCs compared to other cells, with Bcl-2 MFI exceeding Mcl-1 MFI in MPCs. Bcl-2 expression peaked in CCND1-rearranged cases, while Mcl-1 expression was highest in cases with 1q21 gain/amplification. Notably, 65-74% of cases with other CGAs exhibited moderate to strong Bcl-2 or Mcl-1 expression, indicating potential utility of BH3 mimetics in this group, while 25% showed dim to absent expression of one or both markers, suggesting potential futility in these patients. Our study highlights FCM's potential for rapid Bcl-2 and Mcl-1 quantification, surpassing traditional methods. We propose that direct measurement of Bcl-2 and Mcl-1 expression in PCs by FCM, combined with cytogenetic characterization, could improve therapeutic decision-making regarding the use of BH3 mimetics in MM, potentially enhancing outcomes and overcoming resistance.

The frequency and clinical outcome of mono-hit and multi-hit TP53 aberrations in newly diagnosed multiple myeloma.

We investigated the frequency and outcome of mono-hit and multi-hit TP53 aberrations [biallelic or ≥1 TP53 mutations (TP53mut) or TP53mut with variant allele frequency (VAF) ≥55%] in an Indian cohort of newly diagnosed multiple myeloma (NDMM) patients. We employed fluorescence insitu hybridisation (FISH; n=457) and targeted next-generation sequencing (NGS; n=244) on plasma cell-enriched samples. We also studied the impact of TP53mut in cases with and without TP53 deletions (TP53del). In our cohort with a median age of 60 years, TP53del and TP53mut were seen in 12.9% (n=59/457; 14-95% cells) and 10.2% (n=25/244; 30 variants; VAF 3.4-98.2%; median 38.2%) respectively. Mono-hit and multi-hit-TP53 aberrations were observed in 10.2% and 7.8%, respectively. Compared to TP53-wild-type (TP53wt), mono-hit and multi-hit TP53 aberrations were associated with significantly poorer progression-free survival (PFS) (22.6 vs 12.1 vs 9.5 months; p=0.004) and overall survival (OS) [not reached (NR) vs 13.1 vs 15.6 months respectively; p=0.024]. However, multi-hit TP53 did not significantly differ in OS/PFS compared to mono-hit cases. Compared to TP53wt, PFS and OS were significantly poorer in patients with TP53mut only (9.5 vs 22.6 months and 12.1 months vs NR, respectively; p=0.020/0.004). TP53mut retained its significance even in the presence of any Revised International Staging System (HR 2.1; 95% CI 1.1-3.8; p=0.015) for OS. The detection of additional cases with TP53 aberrations, as well as poor survival associated with the presence of mutation alone, supports TP53mut testing in NDMM at least in patients without TP53del and other high-risk cytogenetic abnormalities.

Impact of Organ Impairment on the Pharmacokinetics of Therapeutic Peptides and Proteins.

The kidneys and liver are major organs involved in eliminating small-molecule drugs from the body. Characterization of the effects of renal impairment (RI) and hepatic impairment (HI) on pharmacokinetics (PK) have informed dosing in patients with these organ impairments. However, the knowledge about the impact of organ impairment on therapeutic peptides and proteins is still evolving. In this study, we reviewed how often therapeutic peptides and proteins were assessed for the effect of RI and HI on PK, the findings, and the resulting labeling recommendations. RI effects were reported in labeling for 30 (57%) peptides and 98 (39%) proteins and HI effects for 20 (38%) peptides and 55 (22%) proteins. Dose adjustments were recommended for RI in 11 of the 30 (37%) peptides and 10 of the 98 (10%) proteins and for HI in 7 of the 20 (35%) peptides and 3 of the 55 (5%) proteins. Additional actionable labeling includes risk mitigation strategies; for example, some product labels have recommended avoid use or monitor toxicities in patients with HI. Over time, there is an increasing structural diversity of therapeutic peptides and proteins, including the use of non-natural amino acids and conjugation technologies, which suggests a potential need for reassessing the need to evaluate the effect of RI and HI. Herein, we discuss scientific considerations for weighing the risk of PK alteration due to RI or HI for peptide and protein products. We briefly discuss other organs that may affect the PK of peptides and proteins administered via other delivery routes.

The success rate of interphase fluorescence in situ hybridization in plasma cell disorders can be improved using unconventional sources of plasma cells.

BACKGROUND: Immunomagnetic cell sorting (IMCS) is a preferred technique for the enrichment of plasma cells (PC) before fluorescence in situ hybridization (FISH). Here, we share our real-world experience regarding the success rate of IMCS, its limitations, and the utility of alternate sources to obtain a successful FISH in various PC disorders. MATERIALS AND METHODS: A retrospective analysis was performed in patients with a PC neoplasm, who underwent bone marrow (BM) examination, and FISH testing over 30 months. In all cases with an unsuccessful IMCS, an attempt was made to identify the cause of failure. RESULTS: Immunomagnetic cell sorting of PCs was successful in 395/450 cases (87.8%; 77/98 cases (78.6%) with <10% PCs and 318/352 (90.3%) with ≥10% PCs in BM aspirate; P = .003). Among cases with unsuccessful IMCS (<10% PCs; n = 21 and ≥10% PCs; n = 34), an alternate source could be used successfully in 34 (62%) patients and includes air-dried trephine biopsy imprint smears (n = 28) with aggregates or sheets of PCs, fine-needle aspiration smears/biopsy from plasmacytoma (n = 5), and ascitic fluid (n = 1). 284/395 (71.9%) patients with successful IMCS and all 34 cases with an alternate source of PCs showed at least one cytogenetic abnormality on four-probe FISH. CONCLUSION: Variations in the sample quality together with significant variation in the number of PCs between BM aspirate and the trephine biopsy imprint smears/biopsy reduce the success rate of IMCS in a real-world scenario and necessitate utilization of patient-specific alternate sources of PCs like a trephine biopsy imprint or cytology smears from extramedullary sources for successful FISH testing in PC neoplasms.

Withania somnifera targets interleukin-8 and cyclooxygenase-2 in human prostate cancer progression.

BACKGROUND: Prostate cancer (PC) is a common noncutaneous malignancy in men. The incidence of PC is increasing at an alarming rate across the globe. Progression of PC is associated with elevated levels of interleukin-8 (IL-8) and cyclooxygenase-2 (COX-2) in malignant cells. Overexpression of these players is accompanied by chronic inflammation, increased angiogenesis, proliferation, migration, and inhibition of apoptosis. Moreover, their elevated circulating levels promote the disease progression from androgen-dependent to androgen-independent state. Thus, inhibiting the expression of IL-8 and COX-2 would be a promising target in the development of PC therapeutics. In this study, we investigated the inhibitory effects of Withania somnifera extract on highly metastatic, androgen-independent prostate cancer cell line (PC3). Additionally, we compared the real-time expression of IL-8 and COX-2 in prostate tissue samples. MATERIALS AND METHODS: The cell viability and cytotoxicity of W. somnifera extract in PC3 cells was quantified colorimetrically by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide and lactate dehydrogenase leakage assay, respectively. Hematoxylin and eosin staining for histological examination, trypan blue, and acridine orange dyes to enumerate apoptotic and live cells, quantitative real-time polymerase chain reaction to determine the expression and flow cytometry to study the cell cycle analysis were used. RESULTS: We observed a significant decrease in the cell viability with a half-maximal inhibitory concentration (IC50) of 10 µg/mL. The expression levels of IL-8 and COX-2 in prostate tissue samples and in PC3 cells were predominantly high; however, the lowest dose of W. somnifera significantly inhibited the enhanced expression of IL-8 and COX-2 in PC3 cells in 24 hours. Furthermore, W. somnifera extract (10 µg/mL) irreversibly arrested the cell cycle in G2/M phase, which was evident from the rapid accumulation of PC3 cells significantly. CONCLUSION: Our results indicate that inherent metastatic and selective inhibitory potential of W. somnifera against PC. W. somnifera may be a good therapeutic agent in addition to the existing drugs for PC. Further studies with more prostate tissue samples are warranted.

In vitro p-glycoprotein inhibition assays for assessment of clinical drug interaction potential of new drug candidates: a recommendation for probe substrates.

Because modulation of P-glycoprotein (Pgp) through inhibition or induction can lead to drug-drug interactions by altering intestinal, central nervous system, renal, or biliary efflux, it is anticipated that information regarding the potential interaction of drug candidates with Pgp will be a future regulatory expectation. Therefore, to be able to utilize in vitro Pgp inhibition findings to guide clinical drug interaction studies, the utility of five probe substrates (calcein-AM, colchicine, digoxin, prazosin, and vinblastine) was evaluated by inhibiting their Pgp-mediated transport across multidrug resistance-1-transfected Madin-Darby canine kidney cell type II monolayers with 20 diverse drugs having various degrees of Pgp interaction (e.g., efflux ratio, ATPase, and calcein-AM inhibition). Overall, the rank order of inhibition was generally similar with IC(50) values typically within 3- to 5-fold of each other. However, several notable differences in the IC(50) values were observed. Digoxin and prazosin were the most sensitive probes (e.g., lowest IC(50) values), followed by colchicine, vinblastine, and calcein-AM. Inclusion of other considerations such as a large dynamic range, commercially available radiolabel, and a clinically meaningful probe makes digoxin an attractive probe substrate. Therefore, it is recommended that digoxin be considered as the standard in vitro probe to investigate the inhibition profiles of new drug candidates. Furthermore, this study shows that it may not be necessary to generate IC(50) values with multiple probe substrates for Pgp as is currently done for cytochrome P450 3A4. Finally, a strategy integrating results from in vitro assays (efflux, inhibition, and ATPase) is provided to further guide clinical interaction studies.

Development of stably transfected monolayer overexpressing the human apical sodium-dependent bile acid transporter (hASBT).

PURPOSE: The human apical sodium-dependent bile acid transporter (hASBT) represents a potential target for prodrug design to increase oral drug absorption. Unfortunately, available monolayer cell culture models do not reliably express hASBT, and nonpolarized cells only allow for uptake assessment, which limits prodrug development efforts. The objective of this study was to develop and characterize a stably transfected hASBT-MDCK cell line. METHODS: cDNA encoding hASBT was cloned into pcDNA3.1-V5-polyHis-B to generate an expression plasmid that was then transfected into MDCK-II cells. Clonal populations were chosen based on high hASBT activity and monolayer integrity. Western blot confirmed the expression of the recombinant hASBT; functionality was characterized using taurocholic acid. RESULTS: In the selected clone, hASBT-mediated taurocholate permeability across hASBT-MDCK monolayers was almost 25-fold higher with sodium, than without sodium where hASBT is not functional. In the presence of sodium, taurocholate and mannitol permeabilities were 23.0x10(-6) cm/sec and 2.60x10(-6) cm/s, respectively, indicating high hASBT functionality and monolayer integrity. hASBT-MDCK monolayer properties were stable over 6 months and demonstrated low within-day variability. Taurocholate uptake and inhibition kinetic parameters from hASBT-MDCK were similar to those obtained from hASBT-COS7 model, confirming hASBT functionality in hASBT-MDCK. CONCLUSIONS: Results indicate that the developed hASBT-MDCK system is a competent, high-expression, stable assay for hASBT transport and inhibition studies.