ALBERT

Description: Background: The prognosis of APL has improved markedly with the use of All-Trans-Retinoic Acid (ATRA), but many patients in developing countries are in the high-risk group at presentation and because of inadequate infrastructure die during aggressive induction therapy due to either sepsis or bleeding and/or ATRA syndrome. There is growing appreciation of pivotal role of angiogenesis in APL (Blood2001; 97: 3919). The objective of our study was to evaluate the efficacy of low-dose oral metronomic CT during induction and maintenance therapy along with ATRA in APL. Materials and Methods: From 2003, 23 patients (ages 1 yr to 43 yrs; median 24 yrs; 13 females, 10 males; median WBC count 5.09 X 109/L, range 0.5 to 113 X 109 /L; 7 low risk, 7 intermediate risk & 9 high risk) with APL who could not go for standard CT were included. ATRA 45 mg/m2/d was given for 90 days. Oral CT was given with Prednisolone 40 mg/m2/d, Etoposide 50 mg/m2/d and 6-TG 40 mg/m2/d (PET) for 21 days as induction therapy with ATRA. Consolidation was with 3 cycles of single agent Daunorubicin at 45 mg/m2/d on d 1, 2, 3 every 21 days. This was followed by 6 cycles of oral ET/PET. ATRA was continued for 15 days every 3 months for total of 18 months. Results: The protocol was well tolerated on out-patient basis and after the initial admission at presentation, only 6 patients required readmission. Complete morphological, cytogenetic and molecular remission was achieved in 21 patients (91.3%) at a median of 40 days. There were 2 septic deaths during induction; only 1 patient developed ATRA-syndrome once started on PET. There was 1 remission death because of disseminated chickenpox and 1 patient had CNS relapse. Projected Event Free Survival (EFS) and Overall Survival (OAS) at 24 months is 73% and 84% respectively which is significantly better than our previous results (Am J Hematol1999; 60:87). Interestingly the CR rate and OAS for the 9 high risk patients is 100% so far. Conclusions: There is a valuable role of single hospital trials in exploring innovative therapies in APL. This pilot study contributes to discussion of 4 more debated issues (1) Is there a role for Ara-C in the treatment of APL. (2) How can one improve the therapy in high risk patients without increasing toxicity, (3) What is the ideal duration of maintenance therapy, and (4) What is the ideal therapy for elderly patients and for those who are known to have appreciable mortality from aggressive chemotherapy.

Description: Introduction: Patients with recurrent or refractory Hodgkin (HL) and non-Hodgkin lymphoma (NHL) treated with high dose chemotherapy and autologous stem-cell rescue (ASCR) commonly relapse (post-ASCR) (70–90%) in sites of previous disease. Although adjuvant involved field radiation therapy (IFRT) to sites of previous recurrence might be expected to enhance local control, translation of this into improved disease-free (DFS) or overall survival (OS) is controversial. We sought to evaluate IFRT following ASCR in terms of patterns of recurrence, OS and DFS. Methods and Materials: All 281 patients with recurrent or refractory HL and NHL who underwent ASCR between 5/92 and 7/03 were analyzed. Disease stratification at the time of transplant included HL, 24%, aggressive NHL (ANHL) 62%, and indolent NHL (INHL) 14%. Most, 46% underwent ASCR after 1st relapse, 18% after 2nd relapse, 4% after 3rd relapse and 26% had refractory disease at ASCR. IFRT was administered to 129 patients (46%). Physician and patient choice determined which patients received IFRT. Dose ranged from 20–36 Gy depending on response to salvage therapy before ASCR and the presence of visible imaging abnormalities following ASCR. For end point analysis 39 patients (14%) including 11 HL, 23 ANHL, and 5 INHL had insufficient data and were excluded. Results: Mean follow-up was 3 years (.3 – 12). The median age at ASCR was 45 years (8 – 73). Male to female ratio was 1.3:1. Thirty five percent of patients had prior RT. On univariate analysis, OS and DFS following IFRT for ANHL was superior (or approached this statistically) at 5 years. For HL, improved OS approached significance, whereas DFS did not despite an apparent benefit by disease-free percentages. IFRT appears to be disadvantageous in INHL, but patient numbers were very small. OS and DFS at 5 years are in the table. Survival Table 5-year OS (%) 5-year DFS (%) HL With IFRT 62 79 Without IFRT 37 68 p-value .07 .41 Aggressive NHL With IFRT 57 65 Without IFRT 37 49 p-value .02 .07 Indolent HNL With IFRT 50 67 Without IFRT 85 88 p-value .06 .30 On multivariate analysis, for ANHL, IFRT was protective (p = .002, Hazard Ratio = 0.39) and bulky disease was adverse (p = .04, HR = 2.04). For HL, an advantage of IFRT did not reach statistical significance (p = .63, HR = .8). For INHL, IFRT was associated with an inferior outcome (p = .23, HR = 4.9). Conclusion: Recognizing that bias exists in patient selection for IFRT post-ASCR (in both directions), a survival benefit appears to exist for patients with ANHL, and potentially for those with HL. The absence of a difference in DFS may relate to relapses in other sites or competing events with censored data. Longer follow-up or larger patient numbers are necessary to confirm a long- term improvement. INHL did not benefit from IFRT as might be expected since this group is more likely to have occult disseminated, chemotherapy insensitive disease. Determination of specific patterns of disease recurrence is in progress.

Description: Background: It is well established that recombinant human G-CSF accelerates neutrophil recovery following hematopoietic stem cell transplantation. However, the optimal timing of G-CSF following transplantation remains unknown. We have done a Phase II non randomized open label study on effects of adding an extra dose of G-CSF (filgrastim 5μgm/kg) on Day -1 of HSCT. Patients and methods: Thirty nine consecutive patients who underwent HSCT in one year period were given G-CSF on Day -1(Day before stem cell infusion) in addition to the regular doses from Day +1 till engraftment. Results: Among the 39, males were 31 and females 8 with 15 allograft and 24 autografts. The type of harvest was PBSC in 35, BM alone in 1and both in 3 cases. Disease wise distribution was AML-11, MM-8, NHL-6, HL-5, CML-4, MDS-2 and Aplastic Anemia, ALL and Ca nasopharynx 1 each. The median MNC and CD 34 doses infused were 5.4X108/Kg and 2.26X106/Kg respectively. Median days of Grade IV neutropenia was 11 (range 5–19) and that of ANC〈 100 was 6 (range 2–19). Neutropenic fever was present for a median of 10 days (range 4–19). Neutrophil engraftment and platelet engraftment occurred at Day +11(range 8–13) and Day +21(range 11–73) respectively. Median number of packed cells and platelets (SDP) transfused were 4 and 5 respectively. Mucositis was present for a median of 9 days with Grade III in 51% (n=20) and Grade IV in 23% (n=9) patients. TPN was used in 76% (n=30) patients for a median days of 6 days. Median number of days of antibiotics use was 10 and 57% (n=22) needed 3 lines of antibiotics. Antifungals were used in 57% (n=22) and 95% of use was empirical. Infections were documented in 42% (n=16). Median days of hospitalization was 22 (range 13–38). Transplant related mortality was 10% (2 Auto and 2 Allo). After a median follow up of 113 days 79.71% (n=34) patients are alive with 71% (n=28) in complete remission. One patient with Ca nasopharynx had progressive disease on evaluation and one patient with peripheral T cell lymphoma developed a secondary leukemia (myeloid) post transplant. Conclusions: Addition of G-CSF on Day -1 of HSCT is safe and provides stable engraftment and acceptable results in terms of neutropenic fever, requirement of blood products, antibiotics, TPN and duration of hospital stay. It needs to be compared in randomized trials with use of G-CSF from Day +1 onwards to prove its superiority over that schedule.

Description: Purpose/Objective: To evaluate patterns of recurrence in patients with Hodgkin’s (HL) and non-Hodgkin’s lymphoma (NHL) who subsequently undergo autologous stem cell transplant (ASCT). In this population that has declared itself as high risk, we evaluated time to and sites of relapse relative to initial sites of disease and radiation therapy (RT). This information might enhance understanding of the natural history of these diseases in the setting of modern therapy, influence treatment strategies, and assist in screening decisions. Materials/Method: We analyzed the records of all 281 consecutive patients with refractory or recurrent HL and NHL (indolent and aggressive, as defined at initial diagnosis) who underwent ASCT in our center between 5/92–7/03. Patients were initially diagnosed between 1979–2003 at a median age of 44 years (8–70). 25 patients were unevaluable due to insufficient data, and 68 patients were excluded from analysis because their disease was refractory to initial and salvage therapy. HL patients were segregated according to initial staging (I/II vs. III/IV). Results: Early stage HL patients relapsed at a median of 2.0 years (0.5–10.3) with 87% relapsing in initial disease site(s); 13% (95% CI 3.8–30.1%) relapsed only in new sites. Advanced stage HL patients relapsed at a median of 1.4 years (0.6–10.5) with 96% relapsing in initial site(s); 4% (95% CI 0.1–21.9%) relapsed only in new sites. Indolent histology NHL patients relapsed at a median of 2.1 years (0.5–14.9) with 83% relapsing in initial site(s); 17% (95% CI 7.3–32.8%) relapsed only in new sites. Aggressive histology NHL patients relapsed at a median of 1.0 year (0.3–8.0) with 64% relapsing in initial site(s); 36% (95% CI 26.2–46.2%) relapsed only in new sites. For early stage HD patients, recurrences were predominantly local, and uniformly so in those unirradiated. For all other groups, fewer patients were irradiated than unirradiated and local recurrences predominated regardless of therapy. Conclusions: Almost all patients with HL who relapse and subsequently undergo ASCT initially recur in previous disease sites. Although patients with aggressive histology NHL are more likely to relapse in new sites than patients with indolent NHL, local recurrences predominate in both groups. The median time to recurrence is brief (1–2.1 years). In a population defined by recurrent disease, it is expected that relapses will occur in irradiated sites. Relative protection by RT of local recurrence cannot be determined until all patients, regardless of relapse status, are analyzed. However, these data support an emphasis on local control and suggest that the frequency of screening be most rapid in the early post-therapy years. Comparison of Site(s) of Relapse to Site(s) of Initial Presentation HL NHL Early (n=30) Adv. (n=23) Ind. (n=40) Agg. (n=95) Characteristic % % % % New Site(s) 13 4 18 36 Previous site(s) only 63 61 60 44 Previous site(s) + new site(s) 23 35 23 20 Characteristic n n n n Radiated patients relapsing in previous site(s) 15/19 6/6 5/7 20/34 Unradiated patients relapsing in previous site(s) 11/11 16/17 26/33 42/61

Description: The pivotal role of tumor-associated macrophages (TAMs) in tumor progression is now well established. TAMs have been shown to influence multiple steps in tumor development including the growth, survival, invasion, and metastasis of tumor cells as well as angiogenesis and lymphangiogenesis in tumors. The molecular circuits that polarize TAMs toward such a protumoral phenotype are now the focus of intense investigation. The transcription factor, nuclear factor–κB (NF-κB), is a master regulator of many cellular processes and been shown to regulate various pathways that impact on the function of TAMs. Much evidence for this has come from the use of elegant transgenic murine tumor models in which modification of single components of the NF-κB signaling pathway has been shown to regulate the pro-tumor repertoire of TAMs. Here, we outline this evidence and attempt to reconcile the various views that have emerged recently over the exact role of NF-κB in this phenomenon.

Description: HIb-1 is a single chain antibody (ScFv) originally obtained from the Griffin.1 library. ScFv from this library are derived from the in vitro recombination of germline human immunoglobulin VH and VL chain segments whose diversity has been greatly increased by mutagenesis directed at the CDR3 regions. HIb-1 was selected from the library by sequential biopanning first against CHO cells surface-expressing human GPIbα, and then against human platelets. Previous studies using Western blot analysis have shown that HIb-1 binds to an epitope within the 45 kDa amino-terminal portion of GPIbα (Lapan, Lyle, and Miller, 1999, Thromb. Haemost. 82S:393). HIb-1 displays inhibitory activity against both ristocetin-induced and shear-induced platelet aggregation. Biacore surface plasmon resonance analysis was used to establish dissociation constants between HIb-1and either the extracellular domain of GPIbα (i.e., “glycocalicin” or GC) derived from normal human platelets or the CHO-cell secreted GPIbα1–483 recombinant extracellular protein. With native GC immobilized on dextran sulfate, the KD for binding by analyte HIb-1 was ~600nM. With the wild-type recombinant GPIbα1–483 bound to dextran sulfate, the KD was only slightly higher at ~900 nM. We additionally studied a double mutant increase-of-function GPIbα1-483 containing the Gly233→Val233 and Met239→Val239 substitituions. The KD of HIb-1 binding to the double mutant was virtually identical to that of the wild-type, at ~900 nM. Binding of HIb-1 to native GC immobilized in an ELISA assay format was also performed. HIb-1 showed saturable binding, with a half-maximal binding concentration of approximately 170 nM. Flow cytometric analysis was also performed on platelets from murine GPIbα-null (i.e., “Bernard-Soulier”) mice that had been rescued with the human GPIbα transgene (Ware, Russell, and Ruggeri, 2000, PNAS, 97:2803), as well as upon normal mice. Murine platelets were gated using a rat anti-murine GPIIb/IIIa antibody. HIb-1 did not bind to normal murine platelets, but showed strong binding to platelets from the transgenic animals, comparable in intensity to that seen with the GPIbα mab SZ-2 (for which quantitative bead analysis indicated approximately 8,000 GPIbα receptors per platelet). It should be noted that the binding capabilities of HIb-1 to human GPIbα are those of a monovalent ScFv molecule of approximately 30 kDa, and may be sufficient for studies of in vivo GPIbα inhibition either in the case of acute injury model or of longer-term models involving repeated dosing. Model systems of more chronic inhibition, however, may require conversion of the ScFv into IgG molecules, in order to achieve greater avidity.

Description: The extracellular domain of human platelet GPIbα contains the binding site for von Willebrand factor (vWF) within the amino-terminal 45 kDa region comprising approximately 300 amino acids. In the present studies, we have used CHO cells to synthesize a longer fragment of His1-Phe483, representing nearly the complete extracellular domain of GPIbα, with an additional 8 histidine residues added at the carboxyl end to provide recognition by his-tag reagents. Both wild-type and the increase-of-function mutants Gly233→Val233 and/or Met239→Val239 were employed. For studies of antibody binding, modulator-induced vWF binding, or antibody inhibition of this vWF binding, capture of the recombinant GPIbα could be accomplished by nickel chelate binding of the his-tag. Both botrocetin and ristocetin induced saturable binding of vWF by ELISA, as demonstrated by HRP-conjugated anti-vWF polyclonal antibody. In the case of the different increase-of-function mutants, significant vWF binding was seen even in the absence of added modulator. Inhibition of botrocetin-induced vWF binding was greater for wild-type than for the Met239→Val239 GPIbα mutant with some mabs (e.g., AS-2), whereas the degree of inhibition was essentially equivalent with other mabs (e.g., C-34). When a rabbit polyclonal anti-his antibody was used to immobilize the recombinant proteins instead of the nickel chelate system, binding of vWF and of conformation-dependent mabs was impaired. However, when this same antibody was biotinylated and then captured on a streptavidin plate, excellent capture and subsequent binding characteristics of the recombinant GPIbα were restored. With both this latter system and with the nickel chelate system, a highly sensitive functional vWF activity assay could be established, using botrocetin-induced binding of vWF from human plasma. Linearity of binding was observed between 0.1–0.6% normal plasma, with saturation occurring around 2%. Whereas higher concentrations of plasma actually appeared to displace bound GPIbα to the nickel chelate, the streptavidin-biotin-anti-his attachment method appeared more resistant to such effects. The ELISA-based assay systems developed in this study accordingly provide an and effective approach to study functional interactions of both wild-type and mutant forms of GPIbα with its ligand vWF and also with antibodies directed against different epitopes within the entire extracellular domain of this receptor molecule. The high sensitivity, reproducibility, and rapidity of the assay system also offer the possibility of a useful test for functional assay of plasma vWF.

Description: Ischemia exists in many diseased tissues, including arthritic joints, atherosclerotic plaques, and malignant tumors. Macrophages accumulate in these sites and up-regulate hypoxia-inducible transcription factors (HIFs) 1 and 2 in response to the hypoxia present. Here we show that the gene expression profile in primary human and murine macrophages changes markedly when they are exposed to hypoxia for 18 hours. For example, they were seen to up-regulate the cell surface receptors, CXCR4 and GLUT1, and the potent, tumor-promoting cytokines, vascular endothelial growth factor A, interleukin (IL)-1β and IL-8, adrenomedullin, CXCR4, and angiopoietin-2. Hypoxia also stimulated their expression and/or phosphorylation of various proteins in the nuclear factor-κB (NF-κB) signaling pathway. We then used both genetic and pharmacologic methods to manipulate the levels of HIFs-1α and 2α or NF-κB in primary macrophages to elucidate their role in the hypoxic induction of many of these key genes. These studies showed that both HIF-1 and -2, but not NF-κB, are important transcriptional effectors regulating the responses of macrophages to such a period of hypoxia. Further studies using experimental mouse models are now warranted to investigate the role of such macrophage responses in the progression of various diseased tissues, such as malignant tumors.

Description: Background: PICC is a well known central venous access device that plays an important role in successful administration of chemotherapeutic agents and other supportive medications. This system is very popular not only because of its low incidence of complications and cost compared with other central venous catheters, but also it can be easily inserted by personnel trained in the procedure. Except for very small children, anti-cubital fossa of upper arm is the most common point of insertion and its distal end terminates into the SVC. Long term I.V. therapy, hyper-osmolar solution infusion, repeated chemotherapy administration, difficulty in accessing peripheral venous system in obese individuals are common indications for the procedure. Due to low incidence of procedure related infection, it is now becoming the method of choice in dealing patients with hemato-oncological conditions. Considering all the merits mentioned above and the demerits like risk of infection, thrombosis, venous damage, stenosis, veno-spasm, non-thrombosis occlusion, catheter mal position, removal difficulty with “stuck-PICC”, we did a prospective study to compare beneficial effects versus complications in our pool of patients with hematological malignancies in this centre. Materials & Methods: For this study we recruited a total of 76 patients with hematological malignancies admitted in our hospital from July 2005 to June 2008. The age range was between 2 and 65 years, median was 20 years, irrespective of gender status. To eliminate bias in the study we selected the PICC from Bard Access System, USA, with traditional peel-away cannula insertion technique and placed only in the vein in the ante-cubital fossa under strict aseptic condition and performed by doctors & registered nurses in our study team. Result : We evaluated the results of first 50 cases of insertion outcome. Out of them, 37 patients came out without complication with regard to PICC insertion, while the PICC was in-situ, and also during removal phase after successful chemotherapy administration. 5 patients developed severe inflammation along with elevated ANC. Coagulase negative staphylococcus was the most common pathogen responsible for the catheter induced sepsis. We initiated vancomycin therapy for those patients who developed these type of infection and the success rate was 100%. While 4 of them developed blood oozing from insertion site, there were neither documented incidence of thrombus formation nor catheter malposition. One of the patients developed unexplained pain in the upper extremity where the PICC was inserted without any sign of inflammation or elevated ANC. This could be attributable to short lasting veno-spasm. Unfortunately, there were three incidence of catheter damage possibly due to defective products or improper care. Conclusion: Our study revealed that 74% of the patients did not face any complications, 10% developed catheter induced sepsis. Six (6%) percent PICC required removal. We can conclude that PICC is the method of choice for long term accessing of central venous system for patients with hematological malignancies which has the merits like minimum discomfort, easy to implant, well tolerated, fewer complications rate and without any need for repeated venipuncture.