The origin and underlying driving forces of the SARS-CoV-2 outbreak.

BACKGROUND: SARS-CoV-2 began spreading in December 2019 and has since become a pandemic that has impacted many aspects of human society. Several issues concerning the origin, time of introduction to humans, evolutionary patterns, and underlying force driving the SARS-CoV-2 outbreak remain unclear. METHOD: Genetic variation in 137 SARS-CoV-2 genomes and related coronaviruses as of 2/23/2020 was analyzed. RESULT: After correcting for mutational bias, the excess of low frequency mutations on both synonymous and nonsynonymous sites was revealed which is consistent with the recent outbreak of the virus. In contrast to adaptive evolution previously reported for SARS-CoV during its brief epidemic in 2003, our analysis of SARS-CoV-2 genomes shows signs of relaxation. The sequence similarity in the spike receptor binding domain between SARS-CoV-2 and a sequence from pangolin is probably due to an ancient intergenomic introgression that occurred approximately 40 years ago. The current outbreak of SARS-CoV-2 was estimated to have originated on 12/11/2019 (95% HPD 11/13/2019-12/23/2019). The effective population size of the virus showed an approximately 20-fold increase from the onset of the outbreak to the lockdown of Wuhan (1/23/2020) and ceased to increase afterwards, demonstrating the effectiveness of social distancing in preventing its spread. Two mutations, 84S in orf8 protein and 251 V in orf3 protein, occurred coincidentally with human intervention. The former first appeared on 1/5/2020 and plateaued around 1/23/2020. The latter rapidly increased in frequency after 1/23/2020. Thus, the roles of these mutations on infectivity need to be elucidated. Genetic diversity of SARS-CoV-2 collected from China is two times higher than those derived from the rest of the world. A network analysis found that haplotypes collected from Wuhan were interior and had more mutational connections, both of which are consistent with the observation that the SARS-CoV-2 outbreak originated in China. CONCLUSION: SARS-CoV-2 might have cryptically circulated within humans for years before being discovered. Data from the early outbreak and hospital archives are needed to trace its evolutionary path and determine the critical steps required for effective spreading.

Light chain multiple myeloma, clinic features, responses to therapy and survival in a long-term study.

BACKGROUND: We intended to investigate the long-term clinical characteristics, responses to therapy and survival in patients with lightchain multiple myeloma (MM). METHODS: Ninety-six patients were enrolled into the study. There were 42 κ-chain MM patients and 54 λ-chain MM patients. All the patients werestage III in the Durie-Salmonstaging system. Among them, 66 patients received Velcade (bortezomib) treatment and the other 30 did not. RESULTS: The main symptoms of these patients included bone pain (77.1%), weakness and fatigue (12.5%), foamy urine (8.3%) and extramedullaryplasmocytomas (33.3%). The overall response rate (ORR) was 95.5% in patients treated with Velcade and 60%in the patients without. The median survival times were 23 months in patients treated with Velcade and 12 months in patients without. The median time of progression-free survival (PFS) was nine months in patients treated with Velcade and five months in patients without. The one-year PFS and two-year PFS were 37% and 25%, 27% and 9% for patients treated with Velcade, or without, respectively. The three-year overall survival (OS) and five-year OS were 33% and 24%, 28% and 9% for patients treated with Velcade, or without, respectively. There was no significance in OS between the two groups (P = 0.335). But there was significant difference in PFS between the two groups (P = 0.036). CONCLUSIONS: Our long-term study demonstrated that patients with lightchain myeloma appeared to have more aggressive disease courses and poor outcomes, which could be improved by treatment with Velcade.

[Clinical analysis of multiple myeloma with extramedullary plasmacytomas].

OBJECTIVE: To explore the clinical features, treatment and prognosis of multiple myeloma (MM) with extramedullary plasmacytomas (EM). METHODS: A total of 43 patients were enrolled and divided into 2 groups. Group-1 had 12 patients of EM occurring after the diagnosis of MM while Group-2 included 31 EM patients at the initial diagnosis of MM. RESULTS: The male-to-female proportion was 23:20 and there was a median age of 53 years. The distribution of different isotypes was IgG (n = 15), IgA (n = 9), IgD (n = 2), κ light chain (n = 6), λ light chain (n = 6), biclonal myeloma (n = 3) and nonsecretory myeloma (n = 2). The sites of complicating plasmacytoma included skin, muscle and spinal canal. Nine patients received bortezomib plus DECP (cisplatin, etoposide, cyclophosphamide and prednisone) and 2 patients underwent traditional chemotherapy in Group-1. The outcomes were as follows: complete remission (CR, n = 2), partial remission (PR, n = 4) and death (n = 5). And 11 patients received traditional chemotherapy in Group-2, 7 attained PR and 4 died. Twenty patients received bortezomib plus other chemotherapeutic drugs in Group-2. The outcomes were as follows: CR (n = 12), PR (n = 7) and death (n = 1). The median overall survival (OS) was 36 months (range: 10 - 120) in Group-1 and 23 months (range: 5 - 52) in Group-2 respectively. In Group-1, the estimated 3- and 5-year OS were 54.21% and 27.10% respectively. And in Group-2, the estimated 3- and 4-year OS were 39.83% and 13.28% respectively. CONCLUSIONS: The EM patients show aggressive, complicated and diverse clinical courses and the unusual manifestation of multiple organ involvement by plasma cells. Traditional chemotherapy has a poor efficacy and the prognosis is unfavorable, especially for EM with concurrent MM. The combined treatment of bortezomib with second-line chemotherapy may achieve curative effects.

[The efficacy and safety of bortezomib plus thalidomide in treatment of newly diagnosed multiple myeloma].

OBJECTIVE: The aim of this phase II study was to determine the efficacy and safety of combined bortezomib and thalidomide (VT) regime as initial treatment for newly diagnosed multiple myeloma (MM) in China. METHODS: Thirty-four patients were enrolled in this study and received VT regime up to 21-day cycles. Bortezomib (1.3 mg/m(2)) was administered intravenously on days 1, 4, 8, and 11, while oral thalidomide (100 mg/day) was given from days 1 to 21. The primary end point was clinical response. The secondary end point was safety. RESULTS: Among the 34 patients, 20 were male, 14 were female, with a median age of 59 years, and 15 in international stage system (ISS) III, 18 in ISS II, 1 in ISS I. Among them, 28 completed 2 cycles' treatment and achieved an overall response rate (ORR) of 92.9%; 26 were able to complete the planned 8 cycles of therapy. After 8 cycles, the ORR was 100% (complete response 30.8%, near-complete response 23.1%, partial response 42.3%, minimal response 3.8%). After followed up with a median time of 12 months, the estimated rate without progress of disease was 62%, and the estimated continuous remission rate of 12 months was 62%. The median survival time was not achieved. The most common adverse events were mild to moderate (grades 1, 2). The main toxicities were hematologic (53.3%), gastrointestinal (40.0%), peripheral neuropathy (38.0%), fatigue (36.6%) and fever (32.0%). CONCLUSIONS: VT regime provides a very high ORR and complete response rate in the treatment of newly diagnosed MM patients. No patients experienced deep venous thrombosis. In conclusion, bortezomib in combination with thalidomide is a very effective regimen for newly diagnosed MM patients and the toxicities are manageable.

[A clinical analysis of 25 cases of multiple myeloma complicated by extramedullary plasmacytomas].

OBJECTIVE: To investigate the clinical features of multiple myeloma (MM) complicated by extramedullary plasmacytomas (EM). METHODS: Twenty five patients were enrolled into the study. The proportion male to female was 15:10 and the median age 55.2 years. The distribution of different isotypes was IgA ten, IgG nine and light chain lambda five. The sites of complicating plasmacytoma included muscle, bone, skin, rectum, and testicles. The most common site was muscle. RESULTS: Patients with complicated extramedullary plasmacytomas at the time of diagnosis received traditional treatment, including vincristine adriamycin, dexamethasone, medphalan, prednisone, thalidomide and bortezomib. Rates of overall response (ORR) were 80%. Plasmacytomas occurring after the diagnosis of MM received cisplatin, etoposide, cyclophosphamide, prednisone, or bortezomib ORR were 66.7%, 50.0%. CONCLUSION: These results lend support to the efficacy of bortezomib in the treatment of plasmacytoma. MM cases with unconventional disease recurrence are likely to be seen due to sub-clinical seeding of tumour cells suggestive of the presence of an EM clone of plasma cells with a high degree of chemoresistance. Available data in the literature concerning the optimal therapy for patients with EM relapse were reviewed.

[Clinical analysis of 15 patients with Waldenstrom macroglobulinemia].

OBJECTIVE: To explore the clinical characteristics of Waldenstrom macroglobulinemia (WM) and enhance the level of diagnosis and treatment. METHOD: The data of 15 patients with WM in our hospital from November 1995 to October 2007 were retrospectively analyzed. RESULTS: The median age was 68.5 (60 - 79) years, male/female = 2.75/1. Main clinical manifestations were fatigue, loss of weight, splenomegaly and lymphadenopathy. All the patients accepted the treatment of alkylating agents, purine nucleoside analogs, bortezomib or thalidomide respectively. The follow-up period for the patients was 4 months to 10 years and the median follow-up time was 82 months. CONCLUSION: WM may often be seen in old male patients with varied clinical manifestations. The primary treatment is chemotherapy, but the disease is incurable. Bortezomib and thalidomide may improve the therapeutic effect.

[Osteonecrosis of the jaw associated with the use of bisphosphonates in multiple myeloma: report of four cases and literature review].

OBJECTIVE: To summarize the clinical features of osteonecrosis of the jaw (ONJ) in multiple myeloma (MM) patients treated with bisphosphonate. METHODS: The clinical data of 4 patients with bisphosphonate-related (ONJ) in MM were reported and literatures were reviewed. RESULTS: In these patients, 3 males and 1 female, aged 59-73, pamidronate combined with chemotherapy, high dose glucocorticosteroid, and anti-angiogenic drug had been used for 12-44 months before the occurrence of ONJ. In treatment of ONJ conservative debridement of necrotic bone, systematic use of antibiotics, use of chlorhexidine acetate gargle, and withdrawal of bisphosphonates were preferable to aggressive surgical measures. CONCLUSION: Long-term use of bisphosphonates combined with chemotherapy in MM may cause ONJ that involves both mandible and maxilla. No satisfactory therapy is currently available.

[The effect of cyclophosphamide, thalidomide and dexamethasone combination therapy in relapsed/refractory multiple myeloma].

OBJECTIVE: To investigate the effect of the combination of thalidomide, cyclophosphamide and dexamethasone for the treatment of relapsed/refractory multiple myeloma. METHODS: 20 relapsed/refractory multiple myeloma patients received thalidomide (200 mg/d), cyclophosphamide (300 mg.m(-2).d(-1), d1-4, every 4 weeks) and pulsed dexamethasone (20-40 mg/d, d1-4, every 4 weeks). RESULTS: After 3 cycles of therapy, 9 cases obtained partial remission (PR), 4 had minimal responses (MR), 5 were of no change and 2 had progressive disease. After 6 cycles of therapy, 13 patients achieved PR and 5 had MR. CONCLUSION: The combination of thalidomide, cyclophosphamide and dexamethasone is a promising treatment regime for relapsed/refractory multiple myeloma.

[A study of cellular telomerase activity in different stages of multiple myeloma cells].

OBJECTIVE: To evaluate the clinical significance of telomerase in multiple myeloma (MM) and explore its relationship with cell cycle. METHODS: Marrow plasma cells were collected form 21 patients with newly diagnosed or relapsed MM, 2 patients with monoclonal gammopathy of undetermined significance (MGUS), 15 MM patients in complete remission and 10 donors as control. PC isolation from the marrow mononuclear cell fraction was performed using immunomagnetic bead selection with CD(138) antibodies. Telomerase activity was detected with TRAP (Telomerase repeats amplification protocol)-fluorescence. Cell cycle was assayed by flow cytometry. RESULTS: (1) The results showed that the positive rate of telomerase in all the newly diagnosed and relapsed patients was 90.5%, which was higher than that in the control group (10.0%) and the patients in remission (13.3%) (P < 0.01). There was no significant difference between the patients in remission and control group (P > 0.05), as well as between newly diagnosed and relapsed patients (P > 0.05). (2) The average percentage of S period in the telomerase positive patients was (18.78 +/- 8.02)%, while that in the negative patients was (5.64 +/- 4.03)%; there was statistics difference between them (P < 0.05). CONCLUSIONS: (1) Telomerase plays an important role in the development of MM, it may be used to evaluate disease progression, therapeutic curative effect and prognosis; (2) Telomerase activity is expressed in the proliferation period of MM cells. Telomerase activity is correlated with cell cycle.

[The interaction of vascular endothelial growth factor and interleukin-6 in multiple myeloma].

OBJECTIVE: To study the interaction between human multiple myeloma (MM) cell line and MM-bone marrow stromal cells (BMSCs) modulated by mutual stimulation of vascular endothelial growth factor (VEGF) and interleukin-6 (IL-6), and to analyze the role of VEGF and IL-6 in the pathogenesis of MM. METHODS: MM patient (MM-BMSCs) and normal donor-BMSCs (N-BMSCs) were established from the MNCs of MM and normal bone marrow. ELISA was performed to detect the expression of VEGF and IL-6 in culture supernatants of human MM cell lines U266, MM-BMSCs, N-BMSCs, and co-cultures of U266 and BMSCs in vitro. VEGF and IL-6 were detected in culture supernatants with or without IL-6, anti-IL-6, VEGF, anti-VEGF antibody. RESULTS: Human MM cell line U266 secreted VEGF, but did not secrete IL-6. BMSCs from MM patients and normal donors secreted both VEGF and IL-6. BMSCs stimulated with recombinant human VEGF induced a time and dose-dependent increase in IL-6 secretion. The effects of VEGF were canceled by monoclonal anti-VEGF antibody. Exogenous IL-6 stimulated VEGF secretion of BMSCs. Importantly, when U266 cell were adhered to BMSCs, there was significant increase of VEGF (2.5 - 5.0 fold, P < 0.05) and IL-6 (5.5 - 9.0 fold, P < 0.05) secretion. The secretion of VEGF or IL-6 in BMSCs alone or co-cultures of BMSCs with U266 were inhibited by anti-human IL-6 or anti-human VEGF antibody. U266 cell stimulated with recombinant human IL-6 induced a dose-dependent increase in VEGF secretion, which was inhibited in the presence of a monoclonal antihuman IL-6 antibody. CONCLUSIONS: The interaction between myeloma cells and marrow stromal cells modulates the secretion of VEGF and IL-6, facilitates myeloma cells growth and angiogenesis in MM. It plays an important role in the pathogenesis of MM. This study provides a theoretic basis for target therapy in the treatment of bone marrow microenvironment.

[The changes of gene expression in multiple myeloma treated with thalidomide].

OBJECTIVE: To investigate the effect of thalidomide on bone marrow cells gene expression in multiple myeloma (MM) patients with suppression subtractive hybridization (SSH) and explore the molecular mechanism of thalidomide therapy for MM. METHODS: In a MM patient receiving thalidomide therapy and bone marrow cell from himself, total RNA extraction, mRNA isolation and cDNA synthesis were carried out respectively with routine procedures. SSH were performed in A and B group respectively. The subtracted cDNA was selectively amplified by suppression PCR. The product was inserted into T vector, and then transfected into the competent host JM109. So two subtractive libraries were constructed. After blue-white screening, colonies were selected and plasmids extracted. Homologous comparation was conducted in GenBank. RESULTS: In group A, seven clones were isolated, including ribosomal protein L19 (HUMAN), IgG lambda chain v-v region (HUMAN), contains Alu repetitive element, NADH-ubiquinone oxidoreductase chain 2, elongation factor 1-gamma, human beta globin region, and 40S ribosomal protein S4 (HUMAN). In group B, six clones were isolated, including cytochrome B, up-regulated by 1,25-dihydroxyvitamin D(3) (VDUP1), NADH- ubiquinone oxidoreductase 20 Kd subunit, mu-calpain large subunit, tumor protein, translationally-controlled 1 (TPT1) and COATOMER alpha subunit. CONCLUSION: Thalidomide induces apoptosis and antiangiogenesis by down-regulating some genes and up-regulating some others genes.

[Application of flow cytometry in the differential diagnosis of lymphoma/leukemia with aberrant antigen expression].

OBJECTIVE: To investigate the application of flow cytometry in the differential diagnosis of lymphoma/leukemia with aberrant antigen expression. METHODS: The results of flow cytometry of 30 lymphoma/leukemia cases with aberrant antigen expression, of which 3 cases being lymphomas, 8 B-cell leukemia, 1 T-cell leukemia, 17 acute non-lymphoid leukemia and 1 acute non-lymphoid leukemia involving lymph nodes were analyzed. Immunohistochemistry (EnVision) for CD79a, CD3 and MPO was performed on all cases. RESULTS: Eleven cases of B-cell lymphoma/leukemia were cytoplasmic CD79a (cCD79a)-positive, cytoplasmic CD3 (cCD3epsilon) and cytoplasmic MPO (cMPO)-negative. Five of these cases were positive for CD5 and 2 for CD5, 1 or 2 for myeloid marker(s). The T-cell leukemia cases were cCD3epsilon-positive, cCD79a and cMPO-negative, they also co-expressed CD13 and CD33. The mantle cell lymphoma cases were positive for CD3, CD13 and CD33. Of the 8 B-cell leukemia cases, 4 were positive for CD5, 3 for CD13 and 1 for CD13 and CD33. The 18 acute non-lymphoid leukemia cases (including 1 acute non-lymphoid leukemia case involving lymph nodes) were cMPO-positive and cCD79a and cCD3epsilon-negative. Eight of the 18 expressed T-cell markers (including 1 case of acute non-lymphoid leukemia involving lymph nodes), 8 expressed B-cell markers, 2 expressed both T and B-cell markers. CONCLUSIONS: Flow cytometry can demonstrate aberrant antigen expression in lymphoma/leukemia cells and is helpful in delineating their cell origin. The technique is thus useful in the differential diagnosis of lymphoma/leukemia.

[Effects of pamidronate disodium (Bonin) combined with chemotherapy on bone pain in multiple myeloma].

Objective. To evaluate the therapeutic effects of Disodium Pamidronate (Bonin) on bone pain in multiple myeloma. Method. 18 patients received only chemotherapy and 16 patients with addition of Bonin were compared. Result. The bone pain was significantly relieved both in chemotherapy alone group and in the combination group of Bonin with chemotherapy after treatment (P<0.01, as compared with before therapy). However, the effects of combination group were more dramatical than that of the other group (P<0.05). No obvious side-effects were observed except mild fever in one patient in the combination group. Conclusion. Bonin, as a safe and effective Bisphosphonates preparation, could relieve bone pain in multiple myeloma more effectively when combined with chemotherapy.

[Effects of bortezomib combined with methylprednisolone in treatment of 33 cases of relapsed or refractory multiple myeloma].

The aim of this study was to explore the clinical effect and toxicity of bortezomib combined with methylprednisolone in treatment of relapsed or refractory multiple myeloma (MM). Clinical data of 33 patients (23 male, 10 female; aged from 38 to 85 years old) were analyzed retrospectively. The median diagnosis time was 25 (2 - 120) months. 33 patients received bortezomib (0.9 - 1.1) mg/m(2) on days 1, 4, 8, 11, in combination with methylprednisolone 40 mg/d (4 cases), 80mg/d (13 cases), 120 mg/d (2 cases), 200 mg/d (9 cases), 300 mg/d (5 cases) respectively. The median follow-up time was 10(3-60) months. The used therapy courses were 1 - 8 (mean 4 courses). The results indicated that 24 cases showed the response of different degree, the overall response rate (ORR) was 72.7% (24/33). 32 patients received ≥ 2 therapy courses, and ORR was 71.9% (23/32). 16 patients received 4 therapy courses, and ORR was 93.8% (15/16 cases). 7 patients received 6 therapy courses and the ORR was 100% (7/7 cases). Main side-effects were thrombocytopenia, infection and peripheral neuropathy. The median survival time was 41.5 (2 - 120) months and the 2-year, 3-year and 5-year overall survival rate were 80%, 59.1% and 21.1%, respectively. It is concluded that bortezomib combined with methylprednisolone is an effective therapy with higher response rate, and safe in treatment of relapsed or refractory multiple myeloma.

[Multiple myeloma complicated with spinal infiltration].

This study was aimed to investigate the clinical features of multiple myeloma (MM) complicated by spinal infiltration (extramedullary plasmacytoma) so as to enhance the understanding of this kind of MM and reduce the missed diagnosis for these MM patients. 10 patients with MM complicated by spinal infiltration were enrolled in this study. Bone marrow, blood, hepatic and renal function, electrolyte, beta2 microglobulin, C-reactive protein and immunoglobulin (M-protein) were detected. The involved spinal sites were examined by using magnetic resonance imaging (MRI) or computerized tomography (CT). 10 patients were staged according to International Stage System (ISS) and Duric-Salmon stage system. The clinical data of patients were analyzed. The results showed that among 10 cases of MM complicated by spinal infiltration, involved pectoral cord was observed in 7 cases, involved lumbar cord in 2 cases and sacral cord in 1 case. The treatment with operation and protocol containing cisplatin, ifosfamide etoposide, prednisone, and bortezomib in combination with other drugs indicated that the total remission rate was 80% (8/10), no serious adverse responses was observed. In conclusion, the patients with MM complicated by spinal infiltration must be diagnosed and treated as early as possible. Once paraplegia happened in patients, the therapeutic efficacy and prognosis would be very poor.

Bortezomib plus thalidomide for newly diagnosed multiple myeloma in China.

The aim of this Phase II study was to determine the efficacy and safety of combined bortezomib and thalidomide (VT) regime as initial treatment for newly diagnosed patients with multiple myeloma (MM) in China. Thirty-four patients have been enrolled in this study and were planned to receive VT regime up to eight 21-day cycles. Bortezomib (1.3 mg/m(2) ) was given intravenously on days 1, 4, 8, and 11, while oral thalidomide (100 mg/day) was given on days 1 to 21. The primary end point was clinical response; the secondary end point was safety. Among 34 patients enrolled, 26 patients were able to complete the planned eight cycles of therapy. After eight cycles, the overall response rate was 100% (complete response 31%; near-complete response 23%; partial response 42%; minimal response 4%). The best response occurred within the first four cycles in 96% of patients. Adverse events included hematologic (53%), peripheral neuropathy (38%), fatigue (35%), gastrointestinal (45%), and fever (32%). Grade 3 nonhematologic adverse events included four patients (12%) with renal failure associated with tumor lysis syndrome, one patient (3%) with peripheral sensory and motor neuropathy that improved with VT dose reduction, and one patient (3%) with hypotension. One patient (3%) experienced Grade 4 thrombocytopenia. No patient experienced deep venous thrombosis, while 1 patient (3%) died due to acute renal failure. In conclusion, Bortezomib in combination with thalidomide is a very effective regimen for newly diagnosed MM patients and the toxicities are manageable.