Clinicopathological and genetic landscape of plasmablastic lymphoma in Taiwan.

Plasmablastic lymphoma (PBL) is an aggressive large B-cell lymphoma with a terminal B-cell differentiation phenotype and is frequently associated with immunodeficiency. We aimed to investigate the clinicopathological and immunophenotypic features, genetic alterations, and mutational landscape of PBL in Taiwan. We retrospectively recruited 26 cases. Five (5/18; 28%) patients were HIV-positive and 21 (81%) presented extranodally. There were two morphological groups: one with purely monomorphic large cells (85%) and the other comprising large cells admixed with plasmacytic cells (15%). Phenotypically, the tumors expressed MYC (8/10; 80%), CD138 (20/26; 77%), and MUM1 (20/20; 100%), but not CD20 (n = 26; 0%). Fourteen (54%) cases were positive for EBV by in situ hybridization; the EBV-positive cases were more frequently HIV infected (p = 0.036), with extranodal presentation (p = 0.012) and CD79a expression (p = 0.012), but less frequent light chain restriction (p = 0.029). Using fluorescence in situ hybridization, we identified 13q14 deletion, MYC rearrangement, and CCND1 rearrangement in 74%, 30%, and 5% cases, respectively, without any cases having rearranged BCL6 or IGH::FGFR3 fusion. In the 15 cases with adequate tissue for whole exome sequencing, the most frequent recurrent mutations were STAT3 (40%), NRAS (27%), and KRAS (20%). In conclusion, most PBL cases in Taiwan were HIV-unrelated. Around half of the cases were positive for EBV, with distinct clinicopathological features. Deletion of chromosome 13q14 was frequent. The PBL cases in Taiwan showed recurrent mutations involving JAK-STAT, RAS-MAPK, epigenetic regulation, and NOTCH signaling pathways, findings similar to that from the West.

Plasmablastic myeloma in Taiwan frequently presents with extramedullary and extranodal mass mimicking plasmablastic lymphoma.

Plasmablastic myeloma (PBM) is a blastic morphologic variant of plasma cell myeloma with less favorable prognosis than those with non-blastic morphology. PBM is rare, without clear-cut definition and detailed clinicopathologic features in the literature. PBM may mimic plasmablastic lymphoma (PBL) as they share nearly identical morphology and immunophenotype. Using the criteria of ≥ 30% plasmablasts in tissue sections, we retrospectively recruited PBM cases and analyzed their clinical, imaging, and pathologic findings, with emphasis on extramedullary involvement. We performed immunohistochemistry, in situ hybridization for Epstein-Barr virus (EBER), and fluorescence in situ hybridization (FISH) for lymphoma- and myeloma-associated genetic alterations. Of the 25 recruited cases, 15 (60%) had extramedullary involvement, which occurred as initial presentation in nine cases. The most common extramedullary sites were soft tissue and/or skin (10/15, 67%), followed by pleural effusion, the lungs, and lymph nodes. Immunohistochemically, tumor cells expressed MYC (74%; 17/23), CD56 (56%; 14/25), and cyclin D1 (16%; 4/25), while CD117 was all negative (n = 25). Of the 20 cases stained with p53, four (20%) cases were diffusely positive, and the remaining 16 cases showed a heterogeneous pattern. EBER was negative in all 24 cases examined. Of the 13 cases examined with FISH, the genetic aberrations identified included del(13q14)(92%; 12/13), gain of chromosome 1q (90%; 9/10), loss of chromosome 1p (60%; 6/10), IGH-FGFR3 reciprocal translocation (23%; 3/13), rearranged MYC (15%; 2/13), and rearranged CCND1 (8%; 1/13), while there were no cases with TP53 deletion (n = 10) or rearrangement of BCL2 (n = 13) or BCL6 (n = 13). The prognosis was dismal regardless of the presence or absence of extramedullary involvement. In conclusion, PBM in Taiwan frequently presented as extramedullary and extranodal lesions, particularly in soft tissue and/or skin, mimicking PBL. FISH for targeted genetic alterations such as del(13q14), gain of chromosome 1q, loss of chromosome 1p, and IGH-FGFR3 might be helpful for the differential diagnoses. Larger studies are warranted to investigate the genetic alterations between PBM and PBL.

Primary cutaneous gamma/delta T-cell lymphoma in Taiwan: A series of six cases with frequent solitary presentation and relatively indolent behavior.

BACKGROUND: Primary cutaneous gamma/delta T-cell lymphoma (PCDG-TCL) is aggressive, frequently presenting as multiple plaques, tumors, and/or subcutaneous nodules. METHODS: In this study, we conducted a retrospective study in a tertiary center in Taiwan to characterize this rare tumor. RESULTS: We identified six patients. Five presented with a solitary lesion, including two with clinical impression of epidermal inclusion cyst or lipoma. Two of four evaluable cases exhibited epidermotropism, with one mimicking Pautrier microabscess. The neoplastic cells were pleomorphic and mostly medium- to large-sized. In all cases, the neoplastic cells expressed T-cell receptor (TCR)-γ and/or TCR-δ, with four co-expressing ßF1. Two of these ßF1+ cases co-expressed TCR-γ but not TCR-δ (two different clones). All were negative for Epstein-Barr virus (EBV), low stage, and treated with radiotherapy alone or combined chemotherapy and radiotherapy. In two patients, lymphoma relapsed in 3 and 7 months, respectively, and one patient died of the disease in 7 months. Four other patients were free of disease for 6 to 126 months. CONCLUSION: PCGD-TCL cases in Taiwan are more commonly solitary, frequently with indolent courses. The two currently available TCR-δ clones alone might be insufficient to detect all tumors.

Lymphomatous effusion of monomorphic epitheliotropic intestinal T-cell lymphoma is characterized by azurophilic granules and is a dismal sign: Report of two new cases with literature review.

Lymphoma involving serous effusion is uncommon. The diagnosis of effusion lymphoma may be challenging, particularly when the lymphoid cells are small to medium-sized, which would be difficult for differentiating reactive effusions from low grade lymphomas. Monomorphic epitheliotropic intestinal T-cell lymphoma (MEITL) is an uncommon type of aggressive intestinal T cell lymphoma with a median survival of 7 months. MEITL rarely disseminates to the body cavities. To date, there are only three reported cases of MEITL with malignant effusion. Here we report two additional cases of MEITL with lymphoma cells involving the pleural effusion and the ascites, respectively. Review of the three literature cases and our two new cases of MEITL with malignant effusion, cytoplasmic azurophilic granules were identified in both the two cases with Liu stain. The median survival time was 1.5 months after the occurrence of malignant effusion, even shorter than the median survival in patients with MEITL. Although the case number is small, malignant effusion seems to be a poor prognostic factor of MEITL.

MYD88 L265P mutation analysis is a useful diagnostic adjunct for lymphoplasmacytic lymphoma with pleural effusion.

Lymphoplasmacytic lymphoma (LPL) is a marrow-based lymphoma, rarely involving extramedullary sites, particularly the pleural cavities. The distinction of lymphomatous pleural effusion (PE) in LPL patients from benign effusion is challenging. We conducted this study to examine whether MYD88 L265P mutation analysis is useful in distinguishing benign from lymphomatous PE in four patients with LPL, in which the initial marrow specimens were all positive for MYD88 mutation. In one case each with plasma cell- or lymphocyte-predominant PE, MYD88 mutation was positive, confirming lymphomatous effusion. The other lymphocyte-predominant PE was negative for MYD88 mutation, but was clonally related to a previous nodal biopsy and this PE was also considered to have LPL involvement. The fourth case developed large B-cell lymphoma in the PE 30 months later. The PE specimen was negative for MYD88 mutation but was clonally related to the diagnostic marrow tissue, indicating large cell transformation. Four cases of small lymphocyte-predominant benign PE from patients without history of lymphoma were examined and were all negative for MYD88 L265P mutation. In conclusion, in this small case series we showed that MYD88 L265P mutation analysis could serve as a useful adjunct in distinguishing benign from lymphomatous PE in patients with LPL.

Effusion-based lymphoma with morphological regression but with clonal genetic features after aspiration.

Effusion-based lymphoma (EBL) is a rare but distinct entity of large B-cell lymphoma in effusion without association with human herpes virus-8 (HHV-8). Spontaneous regression after pleurocentesis has been observed; but to our knowledge, there are no reports on the morphological and molecular features of subsequent aspirations in regressing cases. Here, we report the case of a 92-year-old male with chronic obstructive pulmonary disease, who presented with right pleural effusion. He had no human immunodeficiency virus, hepatitis B virus, or hepatitis C virus infection, and CT scans revealed no mass lesion. The first pleural effusion aspiration cytology revealed large lymphoma cells with vesicular nuclei, irregular nuclear contours, and prominent nucleoli, consistent with EBL. The second aspiration cytology showed a few slightly enlarged lymphocytes in a background of small lymphocytes. Immunohistochemical study on cell block of the second aspiration revealed equal amounts of CD3-positive and CD20-positive cells. All these cells on the section tested negative for HHV-8 through immunohistochemistry and Epstein-Barr virus by in situ hybridization. Our initial impression was EBL in regression. However, flow cytometric immunophenotyping showed monotypic light chain expression of the gated B-cells. B-cell receptor gene rearrangement study showed a clonal result. Furthermore, fluorescence in situ hybridization revealed rearrangement of IGH gene. The diagnosis of the second aspiration was EBL with morphological regression but retained clonal genetic features. The patient passed away one month after diagnosis without chemotherapy. This case illustrated the importance of ancillary studies in confirming the clonal nature of a morphologically regressing EBL.

Primary effusion lymphoma in Taiwan shows two distinctive clinicopathological subtypes with rare human immunodeficiency virus association.

AIMS: To investigate the clinicopathological and molecular features of primary effusion lymphoma (PEL) in Taiwan and the association with human immunodeficiency virus (HIV), human herpesvirus 8 (HHV8) and Epstein-Barr virus (EBV). METHODS AND RESULTS: We investigated retrospectively 26 cases with a median age of 76.5. Only one (4%) patient was infected with HIV. Cytologically, all lymphoma cells revealed typical immunoblastic to plasmablastic morphology. Immunohistochemically, HHV8 was positive in eight (32%) tumours and negative in 17 (68%) cases. All 23 tested cases examined were of the non-germinal-centre B cell phenotype. MYC proto-oncogene (MYC) and Epstein-Barr encoding mRNA (EBER) were positive in 43% (nine of 21) and 17% (four of 23) cases, respectively. Immunoglobulin heavy chain (IGH), B cell lymphoma (BCL)2, BCL6 and MYC were rearranged in 71%, 11%, 12% and 18% cases, respectively. By univariate analysis, the overall survival (OS) was associated statistically with MYC expression (P = 0.012) and BCL2 rearrangement (P = 0.035), but not with the others. By multivariate analysis, no factor was statistically significant. Compared to the HHV8-negative cases, the HHV8-positive cases were mainly of the plasmablastic immunophenotype expressing CD30 and CD138, and with a less frequent expression of pan-B cell markers. CONCLUSIONS: Apart from the phenotypical difference, our HHV8-positive neoplasms were not distinct from the HHV8-negative group. Literature review of 256 cases, including our cases, revealed that HHV8-positive cases were associated more frequently with HIV and EBV infection, with rare MYC rearrangement, and a poorer prognosis than HHV8-negative cases. We propose to name the HHV8-positive cases as 'classical' or 'type I PEL' and the HHV8-negative cases as 'type II PEL', stressing the similarities and the distinctive features between these two groups.

Evaluation of PIMA point-of-care CD4 analyzer in Yunnan, China.

BACKGROUND: CD4 count is used to determine antiretroviral therapy (ART) eligibility. In China, flow cytometers are mostly located in urban areas with limited access by patients residing in remote areas. In an attempt to address this issue, we conducted a study to validate the performance of Alere PIMA point-of-care CD4 analyzer. METHODS: Venous and finger-prick blood specimens were collected from HIV-positive participants from two voluntary counseling and testing sites in Yunnan Province. Both venous and finger-prick blood specimens were tested with the PIMA analyzer. Venous blood specimens tested with the Becton Dickinson FACSCalibur were used as a reference. RESULTS: Venous specimens from 396 and finger-prick specimens from 387 persons were available for analysis. CD4 counts by PIMA correlated well with those from FACSCalibur with an R2 of 0.91 for venous blood and 0.81 for finger-prick blood. Compared to FACSCalibur, the PIMA analyzer yielded lower counts with a mean bias of - 47.0 cells/µl (limit of agreement, [LOA]: -204-110 cells/µl) for venous blood and -71.0 cells/µl (LOA: -295-153 cells/µl) for finger-prick blood. For a CD4 threshold of 350 cells/µl, the positive predictive value (PPV) of PIMA was 84.2% and 75.7% and the negative predictive value (NPV) was 97.6% and 95.8% for venous and finger-prick blood, respectively. For an ART threshold of 500 cells/µl, the corresponding PPV was 90.3% and 84.0% and NPV was 94.3% and 93.4%, respectively. CONCLUSIONS: CD4 counting using venous blood with PIMA analyzers is a feasible alternative to a large flow cytometer to determine ART eligibility.

Angiopep-pluronic F127-conjugated superparamagnetic iron oxide nanoparticles as nanotheranostic agents for BBB targeting.

Pluronic® F127-modified water-dispersible poly(acrylic acid)-bound iron oxide (PF127-PAAIO) nanoparticles have been prepared as diagnostic agents. A blood-brain-barrier penetrating peptide, angiopep-2 (ANG), was further conjugated onto the surface of the PF127-PAAIO (ANG-PF127-PAAIO) for brain targeting. The ANG-PF127-PAAIO shows negligible cell cytotoxicity, better cellular uptake, and higher T2-weighted image enhancement than the PF127-PAAIO in U87 cells. Using an ex vivo blood-brain barrier (BBB) model, we showed that the ANG-PF127-PAAIO shows better permeability to bypass the BBB. This is because the ANG-PF127-PAAIO has a dual-targeting ability, recognition of the low-density lipoprotein receptor-related protein and clathrin-mediated receptor on the U87 surface. Thus, the ANG-PF127-PAAIO is a potential nanotheranostic agent for brain dysfunction.

Rapidly fatal leukemia comprising pleomorphic large granular lymphocytes: a report of 2 cases.

BACKGROUND: Large granular lymphocytes (LGLs) are either cytotoxic T or natural killer (NK) cells exhibiting round nuclei and azurophilic cytoplasmic granules. Morphologically, neoplastic LGLs of T cell lineage (T-LGLLs) are usually indistinguishable from normal LGLs, while there is a wide morphological range of aggressive NK cell leukemia (ANKL). CASES: We present 2 consecutive cases of leukemia comprising pleomorphic LGLs. One patient presented with drowsy consciousness and unstable hemodynamics. Her peripheral blood smear disclosed a significant number of LGLs with pleomorphic nuclei expressing CD2, CD56 and HLA-DR but not surface or cytoplasmic CD3 (cCD3). The second patient, previously healthy, presented with a sudden death. Her peripheral blood revealed LGLs ranging from round to pleomorphic nuclei with a CD2+ cCD3+ surface CD3- CD56+ phenotype and clonally rearranged T cell receptor gene. The findings of the first patient were consistent with ANKL and the second, T-LGLL. Both patients passed away shortly before treatment. CONCLUSION: The 2 cases highlight the importance of a multidisciplinary approach in addition to cytological examination to reach accurate diagnoses of such rare leukemia cases.

[Estimation on the HIV-1 incidence among high risk groups using the pooling RNA technique].

OBJECTIVE: To timely identify the HIV-1 infection in window-period and to estimate the HIV-1 incidence among people who came for voluntary counseling and testing (VCT) service as well as men who have sex with men (MSM), respectively. METHODS: HIV antibody negative samples that were determined by screening tests between January and October 2012, were collected and tested with pooling HIV-1 RNA testing technique (2-staged pooling by 50:1, 10:1). Positive cases were followed-up for HIV antibody testing while HIV incidence was calculated under Ron Brookmeyer' s method, among VCT and MSM populations. RESULTS: Among 1400 HIV antibody negative samples of VCT, two showed HIV-1 RNA positive during the antibody window period with the HIV-1 incidence as 1.87% per year (95% CI: 1.23%-2.65% ). Among 500 HIV antibody negative samples from MSM population, two showed HIV-1 RNA positive in the antibody window period, with HIV-1 incidence as 5.31% per year (95% CI: 3.52%-7.45% ). CONCLUSION: Pooling HIV-1 RNA testing seemed a powerful tool for HIV antibody testing in the window-period. Measures should be taken to strengthen the HIV diagnostic programs among MSM and other high risk groups,during the HIV antibody window-period. More frequent detection approach as pooling HIV-1 RNA testing might be a good choice.

[Evaluation of PIMA analyzer detecting CD4 cell count of venous and capillary blood in HIV-infected individuals].

OBJECTIVE: This study is aimed at evaluating the utility of the portable CD4 analyzers (PIMA). METHODS: The paired finger prick blood (25 µl) and 5 ml venous blood samples were collected from 196 HIV infected patients, who came to Yunnan CDC voluntary counseling and testing (VCT) clinic for CD4 test services, from May to August, 2012. The absolute CD4 cell counts were measured by PIMA (using venous and finger-prick blood) and by Calibur (using venous blood) as the reference. The PIMA and Calibur CD4 results were compared using the Wilcoxon matched-pairs test, and the Spearman's rank correlation coefficients were estimated. The Bland-Altman plots were used to assess the consistency of the two methods. RESULTS: The median absolute CD4 counts of 196 venous blood samples obtained by PIMA and by Calibur were 268 (range:169-403) cells/µl and 302 (range:181-474) cells/µl respectively, which showed significant difference (Z = -7.31, P < 0.01). The median absolute CD4 counts measured by PIMA and by Calibur (using 188 finger-prick and venous blood samples respectively) were 271 (range: 165-450) cells/µl and 304 (range:188-476) cells/µl, which also showed significant difference (Z = -7.60, P < 0.01). The CD4 counts obtained by PIMA CD4 analyzer (using venous and finger-prick blood) showed strong positive correlation with the CD4 counts obtained by the reference method (using venous blood), and the r values were 0.94 and 0.92 respectively (P < 0.01) . The mean biases (limit of agreement) were -38.7 (-210.9-133.5)cells/µl and -45.4 (-221.8-131.0) cells/µl, respectively.Using 350 CD4 counts as the threshold for ART treatment initiation, the sensitivity and specificity of PIMA were 99.1% and 79.3% for venous blood samples, and 97.2%and 78.5% for finger-prick blood samples, respectively. CONCLUSION: The CD4 counts obtained by PIMA are lower than that obtained by Calibur, while the sensitivity is high.

[Early detection of HIV infection with Dried Blood Spot testing among infants in Yunnan province].

OBJECTIVE: To explore the application of Dried Blood Spot (DBS) testing for early detection of HIV infection among infants. METHODS: All of the infants aged between 6 weeks and 18 months and born by HIV positive mothers from 14 Maternity and Child Health Care Hospitals in Kunming, Dali, Dehong, Lincang of Yunnan province were investigated from 2010 to 2011. By using DBS and Roche HIV-1 DNA test techniques, 286 infants were tested for HIV early diagnosis and compared with HIV antibody results of 18 months infants. DBS from uninfected infants were taken periodically and screened of HIV antibody to find their time of antibody-disappearing. The information of treatment for pregnant women and feeding methods for infants was also investigated. RESULTS: A total of 286 infants were tested with HIV-1 DNA among which 148 infants were male and 138 infants female, and 8 infants were HIV-1 DNA positive and the infection rate was 2.8% (8/286) that was in accord with their antibodies results in 18 months old; the other 278 infants whose HIV-1 DNA was negative was also negative with their antibodies. By following up the antibody test of 143 HIV negative infants the cumulate rates of antibody-disappearing at the age of 6, 9, 12 and 18 months were 14.0% (20/143), 61.5% (88/143), 88.1% (126/143) and 100.0% (143/143), respectively. Among 286 HIV positive pregnant women, the group with anti-viral treatment had a lower rate of HIV infection with their infants that was 2.14% (6/280) while the group without anti-viral treatment had a high rate of HIV infection with their infants that was 33.33% (2/6). There was significantly different in the rates of two groups (P < 0.01). The HIV infection rate of infants fed with milk powder was 2.55% (7/274) and the rate was 8.33% (1/12) with breast milk. CONCLUSION: The HIV-1 DNA detection techniques with DBS sample was effective for the early diagnosis of HIV in infants from 6 weeks to 18 months.

[Survey on the transmission of HIV drug resistance in Kunming, Yunnan province in 2010].

OBJECTIVE: To study the HIV drug resistance (HIVDR) transmission in Kunming city of Yunnan province in 2010. METHODS: Referring to the guidelines for HIV drug resistance threshold survey (HIVDR-TS) set by WHO, 62 plasma samples of recently reported HIV-infected individuals who were older than 25 years of age, were collected from January to August 2010. Genotyping of pol genetic mutations associated with HIVDR with reverse transcriptional PCR was performed and the prevalence of HIV-1 drug resistance transmission was evaluated. RESULTS: Of the 62 plasma samples, 54 were successfully sequenced and genotyped on pol sequence. Based on the pol sequences, HIV subtypes including CRF08_BC (53.2%), CRF07_BC (25.5%), CRF01_AE (19.1%) and C (2.1%) were identified. According to the time of sampling, the first 47 sequenced samples were used for drug resistance prevalence analysis. A protease inhibitor (PI) relative mutation was found in one sample. Based on the WHO standard, the prevalence of transmitted HIV-1 drug resistance was < 5%. CONCLUSION: HIV-1 drug resistant strains transmission was still catalogued as low prevalence level in Kunming. To prevent the increase of HIVDR prevalence, normative treatment and scientific management to AIDS patients seemed to be quite important.

[Distribution of HIV-1 subtypes among different populations in Dehong prefecture, Yunnan province, in 2011].

OBJECTIVE: To investigate the distribution of HIV-1 subtypes in Dehong prefecture, Yunnan province, in 2011. METHODS: 300 HIV-1 positive plasma samples were collected from Jan. 2011 to May 2011 in Dehong prefecture. HIV-1 gag genes and env genes were amplified by nested-polymerase chain reaction (PCR) from viral RNA. After sequencing, the HIV-1 subtypes were determined by phylogenetic analysis. RESULTS: Based on the phylogenetic trees of gag gene and env gene fragments, a total of 222 samples were genotyped. Subtype C was the predominant strain in Dehong (43.2%, 96/222), followed by unique recombinant forms (URFs, 27.0%, 60/222), CRF01_AE (21.2%, 47/222), CRF08_BC (5.0%, 11/222), B' (2.3%, 5/222) and CRF07_BC (1.4%, 3/222). Subtype C strains were predominant in both heterosexually transmitted population and intravenous drug users (IDUs), but different subtype distribution patterns were found in these two populations. All 6 genotypes including subtype C (40.7%, 70/172), CRF01_AE (25.0%, 43/172), and URFs (25.0%, 43/172) found in this area among heterosexually transmitted population, which showed the diversity of genotypes in this population. Except subtype B' and CRF07_BC, the other 3 subtypes and URFs were detected among IDUs, mainly including subtype C (54.8%, 23/42) and URFs (38.1%, 16/42), which showed the concentration trend of genotypes distribution among IDUs. The proportion of URFs increased significantly in this area, including the new BC recombinants (41.7%, 25/60) and CRF01_AE relative URFs (58.3%, 35/60). However, the distributions of these two URFs among heterosexually transmitted population and IDUs showed no statistical significance. CONCLUSION: The distribution of HIV-1 strains prevailing in Dehong prefecture was diversity, including 5 subtypes and a variety of URFs, of which subtype C was the predominant strain. The distribution patterns of subtype were different among different populations.

Flavokawain B, a novel chalcone from Alpinia pricei Hayata with potent apoptotic activity: Involvement of ROS and GADD153 upstream of mitochondria-dependent apoptosis in HCT116 cells.

Flavonoids synthesized from chalcone precursors in plants have been shown to possess cytotoxic activities with therapeutic potential. We have isolated the novel chalcone flavokawain B from Alpinia pricei Hayata, a plant native to Taiwan that is used in food and traditional Chinese medicine. Here, we report for the first time that flavokawain B significantly inhibits the growth of colon cancer cells and provide novel insight into the molecular mechanisms that underlie its apoptotic activity. Flavokawain B exerts its apoptotic action through ROS generation and GADD153 up-regulation, which lead to mitochondria-dependent apoptosis characterized by release of cytochrome c and translocation of Bak. The up-regulation of GADD153 in flavokawain B-treated HCT116 cells is associated with mitochondrial dysfunction and altered expression of Bcl-2 family members. Moreover, pretreatment with the ROS scavenger N-acetylcysteine abolishes flavokawain B-induced ROS generation, GADD153 up-regulation, and apoptosis. Similarly, RNAi-mediated gene silencing reduced flavokawain B-enhanced expression of GADD153 and apoptotic Bim, leading to diminished apoptosis. Interestingly, flavokawain B provokes G2/M accumulation as well as autophagy, in addition to apoptosis, suggesting that multiple pathways are activated in flavokawain B-mediated anticancer activity. Taken together, our data provide evidence for a molecular mechanism to explain the apoptotic activity of Alpinia plants, showing that flavokawain B acts through ROS generation and GADD153 up-regulation to regulate the expression of Bcl-2 family members, thereby inducing mitochondrial dysfunction and apoptosis in HCT116 cells.