Expression of MYC, IgM, As Well As Non-Germinal Centre B-Cell Like Immunophenotype and Positive Immunofish Index Predict a Worse Progression Free Survival and Overall Survival in a Series of 670 De Novo Diffuse Large B-Cell Lymphomas Included in Clinical Trials: A GELA Study of the 2003 Program

Introduction: Diffuse large B-cell lymphomas (DLBCL) represent a heterogeneous disease with variable clinical outcome. Identifying phenotypic biomarkers of tumor cells on paraffin sections that predict different clinical outcome remain an important goal that may also help to better understand the biology of this lymphoma. Differentiating non-germinal centre B-cell-like (non-GCB) from Germinal Centre B-cell-like (GCB) DLBCL according to Hans algorithm has been considered as an important immunohistochemical biomarker with prognostic value among patients treated with R-CHOP although not reproducibly found by all groups. Gene expression studies have also shown that IgM expression might be used as a surrogate for the GCB and ABC subtypes with a strong preferential expression of IgM in ABC DLBCL subtype. ImmunoFISH index based on the differential expression of MUM-1, FOXP1 by immunohistochemistry and on the BCL6 rearrangement by FISH has been previously reported (C Copie-Bergman, J Clin Oncol. 2009;27:5573-9) as prognostic in an homogeneous series of DLBCL treated with R-CHOP. In addition, oncogenic MYC protein overexpression by immunohistochemistry may represent an easy tool to identify the consequences of MYC deregulation in DLBCL. Our aim was to analyse by immunohistochemistry the prognostic relevance of MYC, IgM, GCB/nonGCB subtype and ImmunoFISH index in a large series of de novo DLBCL treated with Rituximab (R)-chemotherapy (anthracyclin based) included in the 2003 program of the Groupe d'Etude des Lymphomes de l'Adulte (GELA) trials. Methods: The 2003 program included patients with de novo CD20+ DLBCL enrolled in 6 different LNH-03 GELA trials (LNH-03-1B, -B, -3B, 39B, -6B, 7B) stratifying patients according to age and age-adjusted IPI. Tumor samples were analyzed by immunohistochemistry using CD10, BCL6, MUM1, FOXP1 (according to Barrans threshold), MYC, IgM antibodies on tissue microarrays and by FISH using BCL6 split signal DNA probes. Considering evaluable Hans score, 670 patients were included in the study with 237 (35.4%) receiving intensive R-ACVBP regimen and 433 (64.6%) R-CHOP/R-mini-CHOP. Results: 304 (45.4%) DLBCL were classified as GCB and 366 (54.6%) as non-GCB according to Hans algorithm. 337/567 cases (59.4%) were positive for the ImmunoFISH index (i.e. two out of the three markers positive: MUM1 protein positive, FOXP1 protein Variable or Strong, BCL6 rearrangement). Immunofish index was preferentially positive in the non-GCB subtype (81.3%) compared to the GCB subtype (31.2%), (p<0.001). IgM was recorded as positive in tumor cells in 351/637 (52.4%) DLBCL cases with a preferential expression in non-GCB 195 (53.3%) vs GCB subtype 100(32.9%), p<0.001). MYC was positive in 170/577 (29.5%) cases with a 40% cut-off and in 44/577 (14.2%) cases with a cut-off of 70%. There was no preferential expression of MYC among GCB or non-GCB subtype (p>0.4) for both cut-offs. Progression-free Survival (PFS) was significantly worse among patients with high IPI score (p<0.0001), IgM positive tumor (p<0.0001), MYC positive tumor with a 40% threshold (p<0.001), ImmunoFISH positive index (p<0.002), non-GCB DLBCL subtype (p<0.0001). Overall Survival (OS) was also significantly worse among patients with high IPI score (p<0.0001), IgM positive tumor (p=0.02), MYC positive tumor with a 40% threshold (p<0.01), ImmunoFISH positive index (p=0.02), non-GCB DLBCL subtype (p<0.0001). All significant parameters were included in a multivariate analysis using Cox Model and in addition to IPI, only the GCB/non-GCB subtype according to Hans algorithm predicted significantly a worse PFS among non-GCB subgroup (HR 1.9 [1.3-2.8] p=0.002) as well as a worse OS (HR 2.0 [1.3-3.2], p=0.003). This strong prognostic value of non-GCB subtyping was confirmed considering only patients treated with R- CHOP for PFS (HR 2.1 [1.4-3.3], p=0.001) and for OS (HR 2.3 [1.3-3.8], p=0.002). Conclusion: Our study on a large series of patients included in trials confirmed the relevance of immunohistochemistry as a useful tool to identify significant prognostic biomarkers for clinical use. We show here that IgM and MYC might be useful prognostic biomarkers. In addition, we confirmed in this series the prognostic value of the ImmunoFISH index. Above all, we fully validated the strong and independent prognostic value of the Hans algorithm, daily used by the pathologists to subtype DLBCL.

Special Surface Preparation Prior to Bituminous Overlays, HR-231, 1987

Research project HR-231, "Special Surface Preparation Prior to Bituminous Overlay", was initiated in 1982 to study the effectiveness of three different crack fillers in extending pavement life. In particular, this project was designed to determine if any of the fillers could substantially reduce the rate of subsurface deterioration and general deterioration of an asphalt pavement at crack locations. This project also sought to determine the effects of the various crack filling procedures on different thicknesses of bituminous overlays. The three fillers, a fly ash slurry, an emulsion, and a rubberized asphalt mixture, were used along with a control section with no crack filler material on a 2.5 mile section of Cerro Gordo Trunk Route S-25 south of the town of Thornton. This report discusses the construction and performance of each filler material and makes recommendations concerning future use of any of the materials used.

Effects of Deicing Salt Trace Compounds on Deterioration of Portland Cement Concrete, HR-271, 1987

A study was made of the detrimental effects of trace amounts of calcium sulfate (occurring naturally in halite deposits used for deicing) on portland cement concrete pavements. It was found that sulfate introduced as gypsum with sodium chloride in deicing brines can have detrimental effects on portland cement mortar. Concentrations of sulfate as low as 0.5% of the solute rendered the brine destructive. Conditions of brine application were critical to specimen durability. The mechanisms of deterioration were found to be due to pore filling resulting from compound formation and deposition. A field evaluation of deteriorating joints suggests that the sulfate phenomena demonstrated in the laboratory also operates in the field. A preliminary evaluation was made of remedies: limits on sulfates, fly ash admixtures, treatment of existing pavement, and salt treatments. This report gives details of the research objectives, experimental design, field testing, and possible solutions. Recommendations for further study are presented.

Control of Concrete Deterioration Due to Trace Compounds in Deicers, HR-299 Phase I, 1988

This report presents the results of research on the influence of trace compounds from rock salt deicers on portland cement mortar and concrete. An evaluation of the deicers in stock throughout the state showed that about ninety-five percent contained enough sulfate to cause accelerated deterioration of concrete. Of the impurities found in rock salts, sulfate compounds of calcium and magnesium were found to be equally deleterious. Magnesium chloride was found to be innocuous. Introduction of fly ash eliminated the damage to portland cement mortar caused by sulfates. When used with frost resistant Alden aggregate in fly ash concrete and exposed to a variety of deicer brine compositions, the concrete did not deteriorate after exposure. With the exception of a high calcium brine, the behavior of the frost-prone Garrison aggregate was independent of deicer treatment; the high calcium brine reduced frost damage with this aggregate. Two approaches to reducing sulfate deterioration from deicers are suggested as (1) limiting the amount of sulfate to about 0.28 percent, and (2) making concrete sulfate-resistant by using fly ash. Techniques for making existing concrete deicer-sulfate-resistant are essential to a practical solution.

Development of an Economic Dust Palliative for Limestone Surfaced Secondary Roads, HR-297, 1990

This research project was directed at laboratory and field evaluation of sodium montmorillonite clay (bentonite) as a dust palliative for limestone surfaced secondary roads. It had been postulated that the electrically charged surfaces of the clay particles could interact with the charged surfaces of the limestone and act as a bonding agent to agglomerate fine (-#200) particulates and also to band the fine particulates to larger (+#200) limestone particles. Laboratory testing using soda ash dispersed bentonite treatment of limestone fines indicated significant improvement of compressive strength and slaking characteristics. It was recommended that the project proceed to field trials and test roads were constructed in Dallas and Adair counties in Iowa. Soda ash dispersed bentonite solutions can be field mixed and applied with conventional spray distribution equipment. A maximum of 1.5% bentonite(by weight of aggregate)can be applied at one time. Higher applications would have to be staged allowing the excess moisture to evaporate between applications. Construction of higher application treatments can be accomplished by adding dry bentonite to the surfacing material and then by dry road mixing. The soda ash water solution can then be spray applied and the treated surfacing material wet mixed by motor graders to a consistency of 3 to 4 inch slump concrete. Two motor graders working in tandem can provide rapid mixing for both methods of construction. Calcium and magnesium chloride treatments are 2 to 3 times more effective in dust reduction in the short term (3-4 months) but are prone to washboarding and potholing due to maintenance restrictions. Bentonite treatment at the 2-3% level is estimated to provide a 30-40% dust reduction over the long term(18-24 months). Normal maintenance blading operations can be used on bentonite treated areas. Vehicle braking characteristics are not adversely affected up to the 3.0% treatment level. The bentonite appears to be functioning as a banding agent to bind small particulates to larger particles and is acting to agglomerate fine particles of limestone. This bonding capability appears recoverable from environmental effects of winter, and from alternating wet and dry periods. The bentonite appears to be able to interact with new applications of limestone maintenance material and maintains a dust reduction capability. Soda ash dispersed bentonite treatment is approximately 10 times more cost effective per percent dust reduction than conventional chloride treatments with respect to time. However,the disadvantage is that there is not the initial dramatic reduction in dust generation as with the chloride treatment. Although dust is reduced 30-40% after treatment there is still dust being generated and the traveling public or residents may not perceive the reduction.

Control of Concrete Deterioration Due to Trace Compounds in Deicers, Phase II, HR-299, 1989

This report presents results of research on ways to reduce the detrimental effects of sulfate-tainted rock salt deicers on portland cement concrete used for highway pavements. Repetitious experiments on the influence of fly ash on the mortar phase of concrete showed significant improvement in resistance to deicing brines is possible. Fifteen to twenty percent by weight of fly ash replacement for portland cement was found to provide optimum improvement. Fly ashes from five sources were evaluated and all were found to be equally beneficial. Preliminary results indicate the type of coarse aggregate also plays an important role in terms of concrete resistance to freeze-thaw in deicing brines. This was particularly true for a porous ferroan dolomite thought to be capable of reaction with the brine. In this case fly ash improved the concrete, but not enough for satisfactory performance. An intermediate response was with a porous limestone where undesirable results were observed without fly ash and adequate performance was realized when 15% fly ash was added. The best combination for making deicer-resistant concrete was found to be with a non-porous limestone. Performance in brines was found to be adequate without fly ash, but better when fly ash was included. Consideration was given to treating existing hardened concrete made with poor aggregate and no fly ash to extend pavement life in the presence of deicers, particularly at joints. Sodium silicate was found to improve freeze-thaw resistance of mortar and is a good candidate for field usage because of its low cost and ease of handling.

Bentonite Treatment for Economical Dust Reduction on Limestone Surfaced Secondary Roads, HR-351, 1995

This research project was directed at laboratory and field evaluation of sodium montmorillonite clay (Bentonite) as a dust palliative for limestone surfaced secondary roads. It was postulated that the electrically charged surfaces (negative) of the clay particles could interact with the charged surfaces (positive) of the limestone and act as a bonding agent to agglomerate fine (-#200) particulates, and also to bond the fine particulates to larger (+#200) limestone particles. One mile test roads were constructed in Tama, Appanoose, and Hancock counties in Iowa using Bentonite treatment levels (by weight of aggregate) ranging from 3.0 to 12.0%. Construction was accomplished by adding dry Bentonite to the surfacing material and then dry road mixing. The soda ash/water solution (dispersing agent) was spray applied and the treated surfacing material wet mixed by motor graders to a consistency of 2 to 3 inch slump concrete. Two motor graders working in tandem provided rapid mixing. Following wet mixing the material was surface spread and compacted by local traffic. Quantitative and qualitative periodic evaluations and testing of the test roads was conducted with respect to dust generation, crust development, roughness, and braking characteristics. As the Bentonite treatment level increased dust generation decreased. From a cost/benefit standpoint, an optimum level of treatment is about 8% (by weight of aggregate). For roads with light traffic, one application at this treatment level resulted in a 60-70% average dust reduction in the first season, 40-50% in the second season, and 20-30% in the third season. Crust development was rated at two times better than untreated control sections. No discernible trend was evident with respect to roughness. There was no evident difference in any of the test sections with respect to braking distance and braking handling characteristics, under wet surface conditions compared to the control sections. Chloride treatments are more effective in dust reduction in the short term (3-4 months). Bentonite treatment is capable of dust reduction over the long term (2-3 seasons). Normal maintenance blading operations can be used on Bentonite treated areas. Soda ash dispersed Bentonite treatment is estimated to be more than twice as cost effective per percent dust reduction than conventional chloride treatments, with respect to time. However, the disadvantage is that there is not the initial dramatic reduction in dust generation as with the chloride treatment. Although dust is reduced significantly after treatment there is still dust being generated. Video evidence indicates that the dust cloud in the Bentonite treated sections does not rise as high, or spread as wide as the cloud in the untreated section. It also settles faster than the cloud in the untreated section. This is considered important for driving safety of following traffic, and for nuisance dust invasion of residences and residential areas. The Bentonite appears to be functioning as a bonding agent.

Fast Track Portland Cement Concrete Pavement, HR-538, 1997

In 1987, 1.5 km (0.935 mi.) of Spruce Hill Drive in Bettendorf, Iowa was reconstructed. It is an arteriel street with commercial usage on both termini with single family residential dwellings along most of the project. A portland cement concrete (PCC) pavement design was selected, but a 14 day curing period would have been an undue hardship on the residents and commercial businesses. An Iowa DOT Class F fast track concrete was used so the roadway could be used in 7 to 10 days. The Class F concrete with fly ash was relatively sticky and exhibited early stiffening problems and substantial difficulty in obtaining the target entrained air content of 6.5%. These problems were never completely resolved on the project. Annual visual field reviews were conducted through 1996. In November 1991, severe premature distress was identified on the westbound two lanes of the full width replacement. The most deteriorated section in a sag vertical, 152 m (500 ft.) of the westbound roadway, was replaced in 1996. Premature distress has been identified on a dozen other conventional PCC Iowa pavements constructed between 1983 and 1989, so the deterioration may not be related to the fact that it was fast track pavement.

Pooled Fund Study of Premature Concrete Pavement Deterioration, HR-1063, Supplementary Study, 1997

Recently, a number of roads have begun to exhibit the onset of deterioration at relatively early ages. Since this deterioration appears to be the result of materials issues, data concerning raw materials, design, and paving conditions have been collected and analyzed for correlation between independent variables and deterioration. This analysis shows that there is a positive and statistically significant correlation between deterioration and the following variables: alkali and sulfate content of the cementitious materials, impermeable base course, paving temperature, and the presence of fly ash. This study also concludes that there is a significant need for improvement in data collection and maintenance by many organizations responsible for the production of concrete.

Laboratory Evaluation of Roller-Compacted Concrete, MLR-86-6, 1988

Fine limestone aggregate is abundant in several areas of the state. The aggregate is a by-product from the production of concrete stone. Roller compacted concrete (RCC) is a portland cement concrete mixture that can be produced with small size aggregate. The objective of the research was to evaluate limestone screenings in RCC mixes. Acceptable strength and freeze/thaw durability were obtained with 300 pounds of portland cement and 260 pounds of Class C fly ash. The amount of aggregate passing the number 200 sieve ranged from 4.6 to 11 percent. Field experience in Iowa indicates that the aggregate gradation is more critical to placeability and compactibility than laboratory strength and durability.

Drying Shrinkage in P.C. Concrete, MLR-89-5, 1990

Crack formation has been a problem on some recently constructed bridges in Iowa. Drying shrinkage has been considered a contributing factor in that cracking. The study was undertaken to evaluate some of those material properties that contribute to the magnitude of drying shrinkage. Cement content, cement composition, fly ash and retarding admixture were the factors studied. Concrete prisms were cast for seven mixes and, after curing, were exposed to 100 deg F heat at ambient humidity for 280 days. The following were observed from the testing: (1) Higher C3A content cement concrete produced larger shrinkage; (2) Use of fly ash increased shrinkage; (3) Use of retarder increased shrinkage; and (4) Lowering the cement content reduced the shrinkage.

Evaluation of Rapid Determination of the Chloride Permeability of Portland Cement Concrete by AASHTO T277-83, MLR-86-11, 1987

An Iowa D.O.T. Laboratory built machine was constructed for the chloride permeability testing of concrete by measuring electric current through a specimen between a salt solution and a base solution. This study had two purposes. The first was to evaluate the machine's performance. To do this, three concrete mixes were made consisting of different cement factors and water/cement ratios. Each mix was tested for chloride ion content by the 90- day salt ponding method and for chloride permeability at a 28-day cure by the permeability machine. The results from each test were evaluated to see if there was correlation between chloride ion content and the chloride permeability. It was determined that there was a correlation and that the permeability machine was satisfactory for determining chloride permeability in concrete. The second purpose of this study was to examine the effects that pozzolans have on the chloride permeability of concrete. Four mixes were made: one without any pozzolans as a control, one with class C fly ash, one with class F fly ash, and one with silica fume. Specimens from each mix were evaluated for chloride ion content by the 90-day salt ponding test and by the laboratory built machine for chloride permeability after curing 28 days. Specimens from these mixes were also taken from the salt ponding slabs after completion of the ponding test to examine the effect chloride ion content has on the operation of the chloride permeability machine. Specimens containing pozzolans were also examined for chloride permeability after a cure of 180 days. It was determined that the addition of pozzolans to concrete lowers the chloride permeability as measured by the permeability machine. Class F fly ash and silica fume in the concrete had a major effect in lowering the chloride permeability in concrete as measured by the permeability machine.

Fine Sand for Use in PC Concrete, MLR-88-6, 1989

In several locations of Iowa, it is becoming more difficult to produce concrete sand consistently at a reasonable cost. Both ASTM and AASHTO have specifications for concrete sands that allow a finer, poorer graded sand than Iowa specifications. The objective of the study was to develop standard mix designs to permit the use of finer graded sand for PC concrete. Three hundred cylinders were made from five sands available in the state. Based on the results of the study, the following are recommended: (1) Create another class of concrete sand by: (a) lowering the current mortar strength ratio from 1.5 to 1.3, (b) raising the allowance for the percent passing one sieve and retained on the next from 40 to 45, and (c) including a provision that 25 to 60 percent passing the number 30 sieve is required for the sand; and (2) Modify the standard paving mixes with and without fly ash for use with the finer sand as follows: (a) 8% more cement and fly ash for B-2 to B-5 mixes, (b) 7% more cement and fly ash for A-2 to A-5 mixes, and (c) 5% more cement and fly ash for C-2 to C-5 mixes and water reduced mixes.