Noninvasive Monitoring of Mantle Cell Lymphoma by Immunoglobulin Gene Next-Generation Sequencing in a Phase 2 Study of Sequential Chemoradioimmunotherapy Followed by Autologous Stem-Cell Rescue.

BACKGROUND: Minimal residual disease (MRD) monitoring has been used to identify early molecular relapse and predict clinical relapse in mantle cell lymphoma (MCL). Few published data exist in MCL on the performance of next-generation sequencing-based assay of immunoglobulin gene rearrangements for MRD assessment. PATIENTS AND METHODS: In a prospective clinical trial (NCT01484093) with intensive induction chemotherapy and autologous stem-cell transplantation, posttreatment peripheral blood samples were collected from 16 MCL patients and analyzed with an earlier version of the Adaptive Biotechnologies MRD assay. RESULTS: Of the 7 patients whose disease remained in remission, the MRD test remained negative in 5 (71%). Of the 9 patients who experienced relapse, the MRD test was positive at least 3 months before relapse in 6 patients (67%) and positive at the time of relapse in 1 patient (11%). All patients with at least 2 positive MRD tests experienced relapse. CONCLUSION: The next-generation sequencing-based MRD assay identified early molecular relapse, and we observed more sensitivity in the cellular (circulating leukocytes) versus acellular (plasma cell-free DNA) compartment. This observation may be due to availability of tumor target or a limitation of the assay.

90Y Resin Microspheres Radioembolization for Colon Cancer Liver Metastases Using Full-Strength Contrast Material.

OBJECTIVES: To assess safety and efficacy of 90Y resin microspheres administration using undiluted non-ionic contrast material (UDCM) {100% Omnipaque-300 (Iohexol)} in both the "B" and "D" lines. MATERIALS AND METHODS: We reviewed all colorectal cancer liver metastases patients treated with 90Y resin microspheres radioembolization (RAE) from 2009 to 2017. As of April 2013, two experienced operators started using UDCM (study group) instead of standard sandwich infusion (control group). Occurrence of myelosuppression (leukopenia, neutropenia, erythrocytopenia or/and thrombocytopenia), stasis, nontarget delivery (NTD), median fluoroscopy radiation dose (FRD), median infusion time (IT), liver progression-free (LPFS) and overall survivals (OS) was evaluated. Complications within 6 months post-RAE were reported according to CTCAE v3.0 criteria. RESULTS: Study and control groups comprised 23(28%) and 58(72%) patients, respectively. Median follow-up was 9.1 months. There was no statistically significant difference in myelosuppression incidence within 6 months post-RAE between groups. Median FRD and IT for study and control groups were 44.6 vs. 97.35 Gy/cm2 (p = 0.048) and 31 vs. 39 min (p = 0.006), respectively. A 38% lower stasis incidence in study group was not significant (p = 0.34). NTD occurred in 1/27(4%) study vs. 5/73(7%) control group procedures (p = 1). Grade 1-2 and grade 3-4 toxicities between study and control group patients were 36%(8/22) vs. 45%(26/58), p = 0.61 and 9%(2/22) vs. 16%(9/58), p = 0.72, respectively. There was no difference in LPFS and OS between groups. CONCLUSION: Administration of 90Y resin microspheres using UDCM in both lines is safe and effective, resulting in lower fluoroscopy radiation dose and shorter infusion time, without evidence of myelosuppression or increased stasis incidence.

Tissue-specific alternate splicing of human telomerase reverse transcriptase (hTERT) influences telomere lengths during human development.

Direct genetic manipulations have shown that telomerase-mediated telomere elongation plays a key role in determining cellular replicative capacity and senescence. The mechanisms regulating the production of an active telomerase enzyme are still predominantly unknown, although roles for transcriptional control of hTERT, alternative-splicing of hTERT transcripts, and post-translational phosphorylation of hTERT protein have been advocated. Here we show that hTERT is alternatively spliced in specific patterns by different tissue types during human development. Alternative splicing often prohibits the expression of hTERT protein containing functional reverse transcriptase domains. In these instances, telomerase activity is absent, leading to shortening of telomeres. The specific pattern of hTERT mRNA variants in human development provides evidence that alternative splicing is non-random and participates in the regulation of telomerase activity.

Allelic expression of the putative tumor suppressor gene p73 in human fetal tissues and tumor specimens.

p73, a proposed tumor suppressor, shares significant amino acid sequence homology with p53. However, p73 is rarely mutated in tumors but it has been suggested that p73 is monoallelically expressed in some tissues. This latter feature would predispose p73 to gene inactivation because a single genetic 'hit' or the loss of the expressed parental allele would leave the cell without p73 activity. We examined the allelic expression of p73 in normal fetal tissues and in ovarian cancer and Wilms' tumor. We found that p73 was biallelically expressed in all fetal tissues, except in brain, where differential expression of the two parental alleles was observed. Biallelic expression of p73 was also observed in paired samples of ovary cancer and Wilms' tumor. Loss of heterozygosity of p73 occurred at relatively low rates in tumors: one of 11 informative samples (9.1%) of ovarian cancer and two of 19 (10.1%) Wilms' tumors. These data demonstrate that p73 is biallelically expressed in most tissues, thus excluding genomic imprinting as a molecular mechanism to predispose to allelic inactivation of p73 in human tumors.

Regulation of telomerase by alternate splicing of human telomerase reverse transcriptase (hTERT) in normal and neoplastic ovary, endometrium and myometrium.

Telomerase extends telomeric repeats at the ends of linear chromosomes, thereby prolonging the replicative capacity of cells. To investigate possible regulatory mechanisms of telomerase, we measured telomerase enzyme activity, human telomerase RNA (hTR) and human telomerase reverse transcriptase (hTERT) mRNA in normal and neoplastic ovary, endometrium and myometrium. Telomerase activity was detected in most malignancies and in normal endometrium but not in myometrial leiomyoma, normal myometrium or normal ovary. hTR was expressed in all tissue samples. hTERT mRNA was expressed in many tissue samples, and no tissue sample exhibited telomerase activity without expressing hTERT mRNA. However, the presence of hTR and hTERT mRNA was not sufficient for telomerase activity. Alternate splicing of hTERT produced mRNAs lacking critical reverse transcriptase (RT) motifs in both normal and neoplastic tissues. Only tissues expressing hTERT containing complete A and B RT motifs demonstrated telomerase activity. Finally, several normal ovarian tissues and myometrial leiomyomas lacked telomerase activity despite expressing hTR and hTERT containing complete A and B RT motifs. This was not seen in ovarian and myometrial malignancies, where the expression of hTR and hTERT containing complete A and B RT motifs was sufficient for telomerase activity. We conclude that in ovarian and uterine tissues, the presence of a functional telomerase complex is regulated at multiple levels, including hTERT transcription and alternative splicing of hTERT transcripts. The lack of telomerase activity in several normal but not malignant tissues expressing hTR and hTERT containing complete A and B RT motifs suggests that there are further mechanisms for suppressing telomerase activity downstream of hTERT transcription and mRNA splicing, and these mechanisms have been lost during neoplastic transformation.

Desensitization and resensitization of lutropin receptors expressed in transfected Y-1 adrenal cells.

Stimulation of gonadal cells by lutropins such as human chorionic gonadotropin (hCG) is often transient and followed by down-regulation and/or desensitization of lutropin receptors (LHR). Here we describe desensitization/resensitization of LHR in Y-1 adrenal cell lines (termed Y-1L) expressing a rat cDNA lacking most 5' and 3' LHR untranslated regions under the control of a metallothionein promoter. Using a simple morphological assay in which stimulated cells are round and unstimulated cells are flat, we identified clones that rounded and remained round and others that became insensitive to lutropin stimulation and reverted to their flat appearance within 2-4 h. Flattened cells were insensitive to further hormonal stimulation but rounded after treatments with cholera toxin, forskolin, or cyclic AMP, showing that loss of responsiveness was associated with an early step in signal transduction, not loss of rounding potential. Removing the lutropin stimulus for at least 90-120 min reversed hormone insensitivity, even in the presence of the protein synthesis inhibitor puromycin. The number of surface bound receptors did not change during a cycle of rounding/flattening and hCG bound to rounded or flattened cells was replaced equally by radioiodinated hCG during incubations at 4 degrees C. Thus, desensitization/resensitization of LHR in Y-1L cells occurred in the absence of new receptor synthesis, receptor degradation, or receptor recycling. These observations suggest that LHR desensitization/resensitization in Y-1L cells was closely coupled to receptor occupancy and that this cell line may be useful for identifying factors that modulate the activities of occupied receptors.

Telomerase activity in human development is regulated by human telomerase reverse transcriptase (hTERT) transcription and by alternate splicing of hTERT transcripts.

The correlation between telomerase activity, telomere lengths, and cellular replicative capacity has led to the theory that maintenance of telomere lengths by telomerase acts as a molecular clock to control replicative capacity and senescence. Regulation of this molecular clock may have applications in the treatment of cell aging and tumorigenesis, although little is presently known about the regulation of telomerase activity. To investigate possible mechanisms of regulation, we examined telomerase activity and the expression of the human telomerase RNA subunit and the human telomerase reverse transcriptase protein (hTERT) during human fetal heart, liver, and kidney development. The human telomerase RNA subunit is expressed in all three tissues at all gestational ages examined. hTERT expression correlates with telomerase activity in the liver, where both are expressed at all ages surveyed, and in the heart, where both are present until the 11th gestational week but not thereafter. However, although telomerase activity in the kidney is suppressed after the 15th gestational week, the hTERT transcript can be detected until at least the 21st week. Reverse transcription-PCR using primers within the reverse transcriptase domain of hTERT show the presence of multiple alternately spliced transcripts in these tissues, corresponding to full-length message as well as spliced messages with critical reverse transcriptase motifs deleted. Of note, telomerase activity in the kidney is only present at those gestational ages when full-length hTERT message is expressed (until approximately week 15), with spliced transcripts continuing to be expressed at later stages of development. The tissue-specific and gestational-age dependent expression of hTERT mRNA seen in human development suggests the presence of at least two regulatory mechanisms controlling the activity of telomerase: transcriptional control of the hTERT gene and alternate splicing of hTERT transcripts.

Developmental regulation of telomerase activity in human fetal tissues during gestation.

Telomerase is a ribonucleoprotein that adds hexanucleotide repeats (telomeres) to the ends of linear chromosomes, compensating for the loss of telomeric DNA which occurs with DNA replication. In humans, telomerase has been previously detected in germ-line tissues, blastocysts, 16-20 week old fetal tissue, and most cancers, but not in mature sperm or ova, or in most normal somatic tissues. It has been hypothesized that telomerase is suppressed during somatic development and reactivated in malignancy. To test the hypothesis that telomerase is suppressed during somatic development, human fetal tissues of 8-21 weeks gestational age were assayed for telomerase activity. All tissues expressed telomerase at the earliest ages examined. Lung, liver, spleen, and testis maintained telomerase activity through the latest age assayed, namely 21 weeks. Brain and kidney telomerase activity was present up to the 16th week and was undetectable thereafter. Heart tissue did not display activity beyond the 12th week. Lysates of heart, brain, and kidney without telomerase activity did not inhibit the activity of known telomerase-positive cells, suggesting that suppression of telomerase activity during gestational development is due to a lack of active telomerase rather than to the presence of an inhibitor. These findings demonstrate tissue-specific and developmental regulation of telomerase in the human fetus, suggesting an important role for this ribonucleoprotein in human fetal tissue differentiation and development.