Small heat shock proteins (sHSPs) participate in numerous cellular functions including cell signaling, differentiation, and apoptosis. Deregulation of the physiological expression level of sHSPs has been associated with several malignancies. Heat shock protein beta 3 (HSPB3) is the third member of the sHSP family in human and is mainly expressed in skeletal and smooth muscles. In this study, we investigated the potential prognostic significance of HSPB3 expression in colorectal adenocarcinoma, the most frequent type of colorectal cancer. For this purpose, we isolated total RNA from 188 colorectal adenocarcinoma specimens and 68 paired noncancerous ones. After reverse transcription of 2 μg total RNA, we quantified HSPB3 levels by using an in-house-developed real-time quantitative polymerase chain reaction method, based on the SYBR Green chemistry. Comparison of HSPB3 levels among 68 pairs of colorectal tumors and their adjacent noncancerous mucosae uncovered the downregulation of HSPB3 expression in the majority of malignant colorectal tumors. More importantly, high HSPB3 expression is associated with poor relapse-free survival (RFS) and overall survival (OS) of patients with colorectal adenocarcinoma. Multivariable Cox regression analysis revealed that HSPB3 overexpression could serve as an adverse prognostic biomarker in colorectal adenocarcinoma, independent of tumor location, histological grade, and TNM stage. Patients' stratification according to tumor location, histological grade, and TNM stage revealed that high HSPB3 messenger RNA expression retains its unfavorable prognostic potential regarding OS, in particular groups of patients with substantially different prognosis. In conclusion, high HSPB3 expression is associated with poor RFS and OS of patients with colorectal adenocarcioma, independently of clinicopathological prognosticators. K E Y W O R D S colorectal cancer, HSPB3, molecular tumor markers, prognosis, prognostic biomarkers. Abbreviations: B2M, Beta-2-microglobulin; cDNA, Complementary DNA; CRC, Colorectal cancer; HSPB3, Heat shock protein beta 3; mRNA, Messenger RNA; OS, Overall survival; qPCR, Quantitative polymerase chain reaction; RFS, Relapse-free survival; RQU, Relative quantification unit; sHSP, Small heat shock protein. Maria-Anna Kalioraki and Pinelopi I. Artemaki equally contributed to the study. How to cite this article: Kalioraki M-A, Artemaki PI, Sklirou AD, et al. Heat shock protein beta 3 (HSPB3) is an unfavorable molecular biomarker in colorectal adenocarcinoma. Molecular Carcinogenesis. 2020;59:116-125.

Multiple myeloma (MM) is a hematologic malignancy arising from the clonal proliferation of malignant plasma cells. tRNA-derived RNA fragments (tRFs) constitute a class of small non-coding RNAs, deriving from specific enzymatic cleavage of tRNAs. To the best of our knowledge, this is one of few studies to uncover the potential clinical significance of tRFs in MM. Total RNA was extracted from CD138+ plasma cells of MM and smoldering MM patients, and in vitro polyadenylated. First-strand cDNA synthesis was performed, priming from an oligo-dT-adaptor sequence. Next, real-time quantitative PCR (qPCR) assays were developed for the quantification of six tRFs. Biostatistical analysis was performed to assess the results and in silico analysis was conducted to predict the function of one of the tRFs. Our results showed that elevated levels of five out of six tRFs are indicators of favorable prognosis in MM, predicting prolonged overall survival (OS), while two of them constitute potential molecular biomarkers of favorable prognosis in terms of disease progression. Moreover, three tRFs could be used as surrogate prognostic biomarkers along with the R-ISS staging system to predict OS. In conclusion, tRFs show molecular biomarker utility in MM, while their mechanisms of function merit further investigation.

Introduction: Multiple myeloma (MM) is the second most common hematological malignancy arising from terminally differentiated plasma cells. The diagnosis of symptomatic ΜΜ is usually based on the presence of end-organ damage, as defined by the CRAB criteria. ΜΜ is part of a spectrum of disorders characterized as monoclonal gammopathies. Smoldering multiple myeloma (SMM) is a plasma cell dyscrasia preceding MM. The risk of sMM progression to active MM is determined by risk stratification models, such as the Mayo Clinic and the Spanish models. The clinical course of MM is quite heterogenous; patients survival is ranging from months to more than a decade. MM risk is determined using the international staging system (ISS) and the revised-ISS (R-ISS), which incorporates LDH and cytogenetics to ISS. tRNA-derived RNA fragments suggest a class of small non-coding RNAs, which derive from either pre- or mature tRNAs. tRNA fragments have only recently emerged and therefore have not been broadly studied. These fragments are not random degradation products, yet occur through specific enzymatic activity. Two large groups of these molecules are currently known and are distinguished based on their length; tiRNAs or tRNA halves consist of 30-50 nucleotides, while tRNA-derived fragments (tRFs) consist of 16-28 nucleotides. Both categories are further subgrouped according to their site of origin, in 5′-, 3′-, and internal fragments. Although their role is still under evaluation, studies have linked them to pathological situations, such as neurodegenerative diseases, various cancer types and hematological malignancies. In this study, the clinical significance of 6 of these fragments was evaluated in MM, and three of them showed interesting results. These molecules are 3′-tRFs or internal tRFs (i-tRFs), which occur from the tRNAs bearing leucine, glutamic acid and proline anticondons, namely 3′-tRF-LeuAAG/TAG, i-tRF-GluCTC, and i-tRF-ProTGG, respectively. Methods: CD138+ plasma cells were collected from 80 patients at the time of diagnosis: 65 with MM and 15 with SMM. Total RNA was extracted from CD138+ cells using TRIzol and thereafter was polyadenylated by Escherichia coli poly(A) polymerase. First-strand cDNA synthesis was performed by MMLV transcriptase, using an oligo-dT-adaptor primer. Subsequently, we used and in-house real-time quantitative PCR assay, based on SYBR Green chemistry, in order to quantify tRFs in all samples. Specific forward primers were designed for all tested molecules, along with a common reverse primer, complementary to the adaptor used during reverse transcription. The small nucleolar RNAs RNU43 and RNU48 were used as reference genes, in order to normalize qPCR data. Biostatistical analysis was carried out to assess these results. Results: Out of 65 MM patients 13 were classified as having ISS I stage, 22 as ISS II stage, and 30 as ISS III stage. Similarly, 13 of them were grouped in R-ISS I stage, 35 as R-ISS II, and 17 as R-ISS III. The Mann Whitney U test revealed that the levels of 3′-tRF-LeuAAG/TAG, itRF-GluCTC, and i-tRF-ProTGG differed significantly between MM and SMM cases (P=0.001, P=0.047 and P=0.033, respectively). Specifically, these molecules had higher levels in the CD138+ cells of the SMM cases. These results were validated by logistic regression analysis, which showed that patients with lower expression levels of these molecules were at a higher risk of suffering from MM (P=0.003 for 3′-tRF-LeuAAG/TAG , P=0.036 for itRF-GluCTC, and P=0.032 for i-tRF-ProTGG). Furthermore, 3′-tRF-LeuAAG/TAG was related with translocation t(4;14). Conclusions: 3′-tRF-LeuAAG/TAG, i-tRF-GluCTC, and i-tRF-ProTGG may represent novel molecular biomarkers for the differential diagnosis of MM from SMM in ambiguous cases, in which the diagnostic work-up does not provide a clear diagnosis. These novel biomarkers could also play a role in the prediction of sMM cases at high risk of evolution to active multiple myeloma given their overexpression in asymptomatic cases. A possible correlation of these biomarkers to prognosis in MM patients may be indicated by their relationship with the t(4;14) translocation. Disclosures Gavriatopoulou: Amgen: Honoraria; Janssen: Honoraria, Other: Travel expenses; Takeda: Honoraria, Other: Travel expenses; Genesis: Honoraria, Other: Travel expenses. Kastritis:Genesis: Honoraria; Prothena: Honoraria; Amgen: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Takeda: Honoraria; Pfizer: Honoraria. Terpos:Genesis: Honoraria, Other: Travel expenses, Research Funding; Takeda: Honoraria, Other: Travel expenses, Research Funding; Celgene: Honoraria; Medison: Honoraria; Janssen: Honoraria, Other: Travel expenses, Research Funding; Amgen: Honoraria, Research Funding. Dimopoulos:Sanofi Oncology: Research Funding.

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