Javier Laurini

MYC and BCL2 protein expression predicts survival in patients with diffuse large B-cell lymphoma treated with rituximab

Diffuse large B‐cell lymphoma (DLBCL) is a heterogeneous disease and “double‐hit” DLBCL, with both MYC and BCL2 translocations has a poor prognosis. In this study, we investigated whether MYC and BCL2 protein expression in tissue would predict survival in DLBCL. The study included 106 cases of de novo DLBCL treated with rituximab and cyclophosphamide, doxorubicin, vincristine and prednisone (R‐CHOP) or CHOP‐like regimens. The results were validated on an independent cohort of 205 DLBCL patients. Patients with low expression of BCL2 (≤30%) and MYC (≤50%) had the best prognosis, whereas those with high BCL2 (>30%) and MYC (>50%) had the worst outcome. In multivariate analysis, the combination of the BCL2 and MYC was an independent predictor of overall survival (OS) and event‐free survival (EFS) (P = 0·015 and P = 0·005, respectively). The risk of death was nine times greater for patients with high BCL2 and MYC compared to those with low expression. High BCL2 and MYC was a strong predictor of poor OS (P < 0·001) and EFS (P = 0·0017) in patients with the germinal centre B‐cell (GCB) type, but not in the non‐GCB type. In DLBCL, high co‐expression of MYC and BCL2 was an independent predictor of poor survival, and could be used to stratify patients for risk‐adapted therapies.

Gastrointestinal Stromal Tumors A Review of the Literature

Gastrointestinal stromal tumors are mesenchymal neoplasms with a spectrum of histologic appearances and biologic activity. The morphologic classification of these lesions has evolved over time, and molecular analysis has led to a better understanding of their nature. The histologic differential diagnosis for these lesions is broad and includes many spindle cell lesions of the gastrointestinal tract, including neoplasms of true smooth muscle and neural origin, proliferating fibrous lesions, metastatic neoplasms, and primary sarcomas of vascular and adipose origin. Immunohistochemical studies that include CD117 have become invaluable in the classification of mesenchymal lesions arising in the gastrointestinal tract. Treatment of gastrointestinal stromal tumors has historically been involved surgery, but the use of the chemotherapeutic agent imatinib mesylate for advanced disease has made accurate classification even more important. The molecular features have not only allowed us to understand the pathogenesis of these tumors but also have proven to be associated with response to kinase inhibitors.

Non-integrating episomal plasmid-based reprogramming of human amniotic fluid stem cells into induced pluripotent stem cells in chemically defined conditions

ABSTRACT Amniotic fluid stem cells (AFSC) represent an attractive potential cell source for fetal and pediatric cell-based therapies. However, upgrading them to pluripotency confers refractoriness toward senescence, higher proliferation rate and unlimited differentiation potential. AFSC were observed to rapidly and efficiently reacquire pluripotency which together with their easy recovery makes them an attractive cell source for reprogramming. The reprogramming process as well as the resulting iPSC epigenome could potentially benefit from the unspecialized nature of AFSC. iPSC derived from AFSC also have potential in disease modeling, such as Down syndrome or β-thalassemia. Previous experiments involving AFSC reprogramming have largely relied on integrative vector transgene delivery and undefined serum-containing, feeder-dependent culture. Here, we describe non-integrative oriP/EBNA-1 episomal plasmid-based reprogramming of AFSC into iPSC and culture in fully chemically defined xeno-free conditions represented by vitronectin coating and E8 medium, a system that we found uniquely suited for this purpose. The derived AF-iPSC lines uniformly expressed a set of pluripotency markers Oct3/4, Nanog, Sox2, SSEA-1, SSEA-4, TRA-1-60, TRA-1-81 in a pattern typical for human primed PSC. Additionally, the cells formed teratomas, and were deemed pluripotent by PluriTest, a global expression microarray-based in-silico pluripotency assay. However, we found that the PluriTest scores were borderline, indicating a unique pluripotent signature in the defined condition. In the light of potential future clinical translation of iPSC technology, non-integrating reprogramming and chemically defined culture are more acceptable.

Lymphoma diagnosis at an academic centre: rate of revision and impact on patient care.

Few studies have examined the value of a mandatory second review of outside pathology material for haematological malignancies. Therefore, we compared diagnoses on biopsies referred to an academic medical centre to determine the rate and therapeutic impact of revised diagnoses resulting from a second review. We reviewed 1010 cases referred for lymphoma during 2009–2010. For each case, referral diagnosis and second review diagnosis were compared. Revised diagnoses were grouped into major and minor discrepancies and all major discrepancies were reviewed by a haematologist to determine the effect the diagnostic change would have on therapy. There was no change in diagnosis in 861 (85·2%) cases. In 149 (14·8%) cases, second review resulted in major diagnostic change, of which 131 (12·9%) would have resulted in a therapeutic change. The highest rates of revision were for follicular, high‐grade B‐cell, and T‐cell lymphomas. We found higher rates of major discrepancy in diagnoses from non‐academic centres (15·8%) compared to academic centres (8·5%; P = 0·022), and in excisional biopsies (17·9%) compared to smaller biopsies (9·6%; P = 0·0003). Mandatory review of outside pathology material prior to treatment of patients for lymphoma will identify a significant number of misclassified cases with a major change in therapy.

Neonatal Candida parapsilosis meningitis and empyema related to epidural migration of a central venous catheter

Candida parapsilosis is an extremely rare cause of meningitis. We report the case of a neonate born at 26+4 weeks of gestation who was admitted to the neonatal intensive care unit at our institution due to respiratory immaturity. During the course of a 3-month hospitalization, the neonate developed fever and lethargy. A lumbar puncture revealed milky-white, turbid cerebrospinal fluid which contained many nucleated cells, mostly neutrophils. Microscopic examination of the cerebrospinal fluid revealed marked acute inflammation and fungal yeast forms, and cultures of the cerebrospinal fluid and peripheral blood yielded C. parapsilosis. Imaging studies subsequently revealed a subdural empyema related to epidural migration of a central venous catheter (CVL). The neonate received extended therapy with amphotericin B and fluconazole. He responded favorably to therapy and was discharged 3 months after birth. This case underscores the clinical importance of the recognition and treatment of a potentially lethal fungal pathogen of the central nervous system and the need for awareness of complications resulting from CVL malposition.

Kluyvera infections in the pediatric population.

In pediatric patients, Kluyvera spp. has emerged as a cause of disease ranging from soft tissue infections to sepsis with multiorgan failure. Successful treatment options include third-generation cephalosporins, tetracycline, aminoglycosides, and fluoroquinolones, but resistance to first- and second-generation cephalosporins persists. Clinicians should be aware of the spectrum of disease and increasing clinical importance associated with this emerging pathogen.

Exosomal Markers (CD63 and CD9) Expression Pattern Using Immunohistochemistry in Resected Malignant and Nonmalignant Pancreatic Specimens.

OBJECTIVES Exosomes are important mediators in intercellular communications and play a role in cancer progression and metastasis. Exosomal membranes are enriched in endosome-specific tetraspanins (CD9 and CD63). Here, we explored the expression of CD63 and CD9 utilizing immunohistochemistry in malignant and nonmalignant cells in 29 resected pancreatic specimens (RPSs) of mixed racial background. METHODS The pathologic tissues (PTs) and adjacent normal tissues (ANTs) in each RPS were stained for CD63 and CD9. Two pathologists independently scored the expression of CD63 and CD9. Staining intensity was graded from 1 to 3. Staining percentage was estimated in 10% increments. An average Quick score (Q score) (intensity × percentage of staining) was calculated. Unpaired t test was used for statistical analysis. RESULTS The mean multiplicative Q score for CD63 and CD9 expression is higher in PTs (209 and 72) compared with ANTs (154 and 24) (P = 0.0041, P = 0.0018), respectively. The Mean Q score for CD63 and CD9 expression is higher in the malignant PTs (231 and 85) compared with ANTs (129 and 25) (P < 0.0001 and P < 0.0124). CONCLUSIONS Exosomal markers (CD63 and CD9) expression assessment using immunohistochemistry is feasible in RPS. The expression of CD63 and CD9 is higher in PTs and malignant PTs compared with their ANTs.Objectives Exosomes are important mediators in intercellular communications and play a role in cancer progression and metastasis. Exosomal membranes are enriched in endosome-specific tetraspanins (CD9 and CD63). Here, we explored the expression of CD63 and CD9 utilizing immunohistochemistry in malignant and nonmalignant cells in 29 resected pancreatic specimens (RPSs) of mixed racial background. Methods The pathologic tissues (PTs) and adjacent normal tissues (ANTs) in each RPS were stained for CD63 and CD9. Two pathologists independently scored the expression of CD63 and CD9. Staining intensity was graded from 1 to 3. Staining percentage was estimated in 10% increments. An average Quick score (Q score) (intensity × percentage of staining) was calculated. Unpaired t test was used for statistical analysis. Results The mean multiplicative Q score for CD63 and CD9 expression is higher in PTs (209 and 72) compared with ANTs (154 and 24) (P = 0.0041, P = 0.0018), respectively. The Mean Q score for CD63 and CD9 expression is higher in the malignant PTs (231 and 85) compared with ANTs (129 and 25) (P < 0.0001 and P < 0.0124). Conclusions Exosomal markers (CD63 and CD9) expression assessment using immunohistochemistry is feasible in RPS. The expression of CD63 and CD9 is higher in PTs and malignant PTs compared with their ANTs.

Germline pharmacogenomics of DPYD*9A (c.85T>C) variant in patients with gastrointestinal malignancies treated with fluoropyrimidines

Background The correlation between DPYD*9A (c.85T>C) genotype and dihydropyrimidine dehydrogenase (DPD) deficiency clinical phenotype is controversial. Reference laboratories either did not perform DPYD*9A genotyping or have stopped DPYD*9A genotyping and limited genotyping to high-risk variants (DPYD*2A, DPYD*13 and DPYD*9B) only. This study explored DPYD*9A genotype and clinical phenotype correlation in patients with gastrointestinal (GI) malignancies treated with fluoropyrimidines. Methods Between 2011 and 2017, 67 patients with GI malignancies were genotyped for DPYD variants. Fluoropyrimidines-associated toxicity was graded according to the National Cancer Institute Common Terminology Criteria for Adverse Events (version 3.0). Fishers exact test was used for statistical analysis. Results DPYD variants were identified in 17 out of 67 (25%) patients. One patient was homozygous for DPYD*9A variant and one patient was double heterozygous for DPYD*9A and DPYD*9B variants. In patients with identified DPYD variants, 13/17 (76%) patients had DPYD*9A variant, 3/17 (18%) patients had DPYD*2A variant and 2/17 (12%) patient had DPYD*9B variant. Only patients genotyped prior to 2015 were genotyped for DPYD*9A variant (N=28). Of those, 13/28 patients (46%) had DPYD*9A variant. Grade 3-4 diarrhea was associated with DPYD*9A variant in patients treated with full dose fluoropyrimidines (P=0.0055). Conclusions In our cohort, DPYD*9A variant was the most common diagnosed variant. The correlation between DPYD*9A genotype and DPD deficiency in clinical phenotype was noticeable in patients who received full dose fluoropyrimidines as they all experienced grade 3-4 toxicities (diarrhea).

Follicular large cleaved cell (centrocytic) lymphoma: An unrecognized variant of follicular lymphoma

The World Health Organization classification of lymphoma recommends the subdivision of follicular lymphoma (FL) into 3 grades (FL1-3) based on the average number of centroblasts per high-power field in the neoplastic follicles, but does not recognize a form of FL characterized by a predominance of large cleaved cells (centrocytes) without enough centroblasts to meet the World Health Organization criteria for FL3. We have classified such cases as follicular large cleaved cell lymphoma (FLC) and, herein, describe the pathologic and clinical features of 72 cases of this entity. The features of FLC include a follicular growth pattern with pale follicles at low magnification and frequent follicular and/or interfollicular fibrosis. Cytologically, the cells are predominantly large cleaved cells with moderately coarse to fine chromatin, absent or inconspicuous nucleoli, and small to moderate amounts of pale cytoplasm. The mean nuclear diameter of the large cleaved cells was 10.1μ, approximately twice that of small lymphocytes and similar to centroblasts. The t(14;18) was present in 83% of the cases, and a high proportion expressed BCL2 (84%), BCL6 (100%), and CD10 (88%) and had high Ki67 proliferation (81%). The clinical features of patients with FLC were similar to those with other types of FL, and survival was excellent with anthracycline-based chemotherapy plus rituximab. FLC is a variant of follicular lymphoma which should be recognized in future lymphoma classifications because the diagnosis of FLC may be important for the selection of therapy.

Induced pluripotent stem cells derived from human amnion in chemically defined conditions

ABSTRACT Fetal stem cells are a unique type of adult stem cells that have been suggested to be broadly multipotent with some features of pluripotency. Their clinical potential has been documented but their upgrade to full pluripotency could open up a wide range of cell-based therapies particularly suited for pediatric tissue engineering, longitudinal studies or disease modeling. Here we describe episomal reprogramming of mesenchymal stem cells from the human amnion to pluripotency (AM-iPSC) in chemically defined conditions. The AM-iPSC expressed markers of embryonic stem cells, readily formed teratomas with tissues of all three germ layers present and had a normal karyotype after around 40 passages in culture. We employed novel computational methods to determine the degree of pluripotency from microarray and RNA sequencing data in these novel lines alongside an iPSC and ESC control and found that all lines were deemed pluripotent, however, with variable scores. Differential expression analysis then identified several groups of genes that potentially regulate this variability in lines within the boundaries of pluripotency, including metallothionein proteins. By further studying this variability, characteristics relevant to cell-based therapies, like differentiation propensity, could be uncovered and predicted in the pluripotent stage.