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Simple Summary In this review, the authors propose a crosswise examination of cytarabine-related issues ranging from the spectrum of clinical activity and severe toxicities, through updated cellular pharmacology and drug formulations, to the genetic variants associated with drug-induced phenotypes. Cytarabine (cytosine arabinoside; Ara-C) in multiagent chemotherapy regimens is often used for leukemia or lymphoma treatments, as well as neoplastic meningitis. Chemotherapy regimens can induce a suboptimal clinical outcome in a fraction of patients. The individual variability in clinical response to Leukemia & Lymphoma treatments among patients appears to be associated with intracellular accumulation of Ara-CTP due to genetic variants related to metabolic enzymes. The review provides exhaustive information on the effects of Ara-C-based therapies, the adverse drug reaction will also be provided including bone pain, ocular toxicity (corneal pain, keratoconjunctivitis, and blurred vision), maculopapular rash, and occasional chest pain. Evidence for predicting the response to cytarabine-based treatments will be highlighted, pointing at their significant impact on the routine management of blood cancers. Abstract Cytarabine is a pyrimidine nucleoside analog, commonly used in multiagent chemotherapy regimens for the treatment of leukemia and lymphoma, as well as for neoplastic meningitis. Ara-C-based chemotherapy regimens can induce a suboptimal clinical outcome in a fraction of patients. Several studies suggest that the individual variability in clinical response to Leukemia & Lymphoma treatments among patients, underlying either Ara-C mechanism resistance or toxicity, appears to be associated with the intracellular accumulation and retention of Ara-CTP due to genetic variants related to metabolic enzymes. Herein, we reported (a) the latest Pharmacogenomics biomarkers associated with the response to cytarabine and (b) the new drug formulations with optimized pharmacokinetics. The purpose of this review is to provide readers with detailed and comprehensive information on the effects of Ara-C-based therapies, from biological to clinical practice, maintaining high the interest of both researcher and clinical hematologist. This review could help clinicians in predicting the response to cytarabine-based treatments.

Background: Drugs for targeted therapies are primarily Small Molecules Inhibitors (SMIs), monoclonal antibodies (mAbs), interfering RNA molecules and microRNA. The use of these new agents generates a multifaceted step in the pharmacokinetics (PK) of these drugs. Individual PK variability is often large, and unpredictability observed in the response to the pharmacogenetic profile of the patient (e.g. cytochome P450 enzyme), patient characteristics such as adherence to treatment and environmental factors. Objective: This review aims to overview the latest anticancer drugs eligible for targeted therapies and the most recent finding in pharmacogenomics related to toxicity/resistance of either individual gene polymorphisms or acquired mutation in a cancer cell. In addition, an early outline evaluation of the genotyping costs and methods has been taken into consideration. Future Outlook: To date, therapeutic drug monitoring (TDM) of mAbs and SMIs is not yet supported by heavy scientific evidence. Extensive effort should be made for targeted therapies to better define concentration-effect relationships and to perform comparative randomized trials of classic dosing versus PK-guided adaptive dosing. The detection of individual pharmacogenomics profile could be the key for the oncologists that will have new resources to make treatment decisions for their patients in order to maximize the benefit and minimize the toxicity. Based on this purpose, the clinician should evaluate advantages and limitations, in terms of costs and applicability, of the most appropriate pharmacological approach to performing a tailored therapy.

We describe a solitary extramedullary plasmacytoma of the breast in a 37-year-old woman. No other involvement was detected in the bone marrow or in any other site during a 15-month follow-up period. Extramedullary plasmacytomas of the breast are extremely rare, especially those that are not associated with multiple myeloma. We review the histologic features of the previously reported cases with an emphasis on differential diagnosis and the difficulties encountered in arriving at the correct diagnosis in frozen sections.

Tumor necrosis factor receptor-associated factor 1 (TRAF1) is unique among the members of the TRAF family, as it lacks the N-terminal RING/zinc-finger domain. Also the function of TRAF1 is not clearly established, with many papers reporting contradictory results. Here we show that TRAF1 interacts with BAFF receptor, a member of the TNF receptor family, and positively regulates activation of the alternative NF-kappaB pathway. Ectopic expression of TRAF1 causes degradation of TRAF3, stabilization of NIK, and processing of p100 to produce the mature form p52. In addition, we show that knocking-down expression of TRAF1 in the Hodgkin's disease derived cell line L1236, interfere with p100 processing and with p52 mediate gene transcription. Collectively these results support a role for TRAF1 as a positive regulator of the NF-kappaB alternative pathway.

Patients with Hodgkin lymphoma (HL) failing salvage stem cell transplantation are candidates to investigational strategies. Lenalidomide represents an attractive option as it targets several signaling pathways, which regulate survival of HL cells and their microenvironmental interactions. We report the occurrence of Grades 2 and 3 tumor flare reactions in the first three patients entered a lenalidomide-based compassionate program for treatment-refractory HL. Flares occurred in concomitance of the scheduled week-off lenalidomide and upon withdrawal of symptomatic steroid treatment, and were associated with changes in B-cell regulatory cytokines and the concurrent expansion of polyclonal B-cells. Flares mimicked tumor progression but were effectively managed with anti-inflammatory treatment and followed by a clinical response, suggesting that they may mirror the pleiotropic actions of lenalidomide on HL microenvironment.

Background: Given the consistent antitumor activity and favorable toxicity profile in heavily pretreated patients, BDM represents a suitable platform for combination with target-based agents in the setting of recurrent HL. The anti-CD30 antibody-monomethyl auristatin E (MMAE) conjugate brentuximab vedotin (BV) appears a most valuable candidate by coupling an impressive clinical activity with the lack of serious overlapping toxicities with BDM. While the combination of BDM and BV is being evaluated in Phase II studies, no information is available as to possible mechanisms through which BDM may regulate the antitumor efficacy of BV towards HL cells. Methods: We evaluated the effects of acute and extended exposure to BDM on CD30 expression and sensitivity to the cytotoxic activity BV in the HL cell line L1236. Cells were cultured in the presence of BDM at its IC50 for different time points of acute exposure. Through continuous exposure to increasing concentrations (25.0, 50.0, 75.0, 100 micromol/L) of BDM, we then performed a serial in vitro selection for BDM-resistant (R) L1236 cell clones and determined their CD30 expression by flow cytometry, qRT-PCR, and Western analysis. Results: Acute exposure to BDM (48 to 72 hrs) of L1236 cells led to a sizeable upregulation of CD30 as shown by flow cytometry. This effect was unsustained since CD30 intensity returned to baseline levels upon culturing in BDM-free medium for one week. Expression of other surface antigens, i.e. HLA-DR, was unaffected by BDM while acute exposure to doxorubicin and other cytotoxic drugs did not modify the CD30 expression. We established four L1236-derived cell clones (R25, R50, R75 and R100) able to proliferate across the different concentrations of BDM with growth/viability curves superimposable to native cells. Clonal identity among clones and with parental cells was confirmed by sequencing of V3-21 (FR2/FR3) and JH3-JH4 Ig DNA regions. All R-clones displayed an up to 900% increase in CD30 median fluorescence intensity, relative to native L1236 cells (Figure 1A). The sustained CD30 upregulation was confirmed by qRT-PCR and Western blotting since R-clones expressed up to 10-fold higher levels of CD30-specific mRNA and CD30-specific 120 kDa components as compared to parental cells. To rule out a general upregulation of surface molecules as a result of the chronic exposure to BDM we evaluated the expression of other surface antigens. As opposed to CD30, HLA-DR and PDL-1 relative transcript levels and cell surface expression were significantly reduced in R-cells. Intriguingly, BDM-induced CD30 upregulation was specific to L1236 cells since extended exposure to BDM did not modify expression levels of CD30 in other lymphoma cell types (SUDHL1, JEKO). BDM-induced CD30 overexpression significantly enhanced the sensitivity of HL cells to the cytotoxic effects of BV. MTS assay showed the IC50 of R100 cells to BV shifted to 0.21 ± 0.06 mcg/mL (48 hrs) and 0.19 ± 0.05 mcg/mL (72 hrs) vs. 3.16 ± 0.75 mcg/mL (48 hrs) and 3.87 ± 0.68 mcg/mL (72 hrs) of parental cells, a 15- and 20-fold increase, respectively (p Conclusions: Whileacute exposure to BDM induced a transient upregulation of CD30, extended exposure to the agent resulted in the selection of L1236 subclones with a stable overexpression of surface CD30 and reduced levels of HLA-DR and PDL-1. Upregulation of CD30 was associated with a significantly enhanced sensitivity to cytotoxic effects of BV. Translated into a clinical context these findings suggest that HL patients progressing upon BDM treatment may turn exquisitely sensitive to BV and prompt the exploration of a sequential treatment with BDM and BV in late and earlier disease phases. Results also provide a mechanistic insight as to the efficacy of the BDM-BV combination in HL, highlighting non-overlapping resistance pathways between BDM and MMAE. BDM-induced selection of CD30hi, HLA-DRlow/PDL-1low tumor cell clones might be relevant to the design of novel treatment strategies for HL based on the combination of BDM with BV and anti-PD1/PDL-1 antibodies. Figure 1. Expression of CD30 and HLA-DR in parental (L1236) and BDM-resistant (R) HL cells Figure 1. Expression of CD30 and HLA-DR in parental (L1236) and BDM-resistant (R) HL cells Figure 2. Sensitivity to BV of parental (L1236) and BDM-resistant (R100) HL cells Figure 2. Sensitivity to BV of parental (L1236) and BDM-resistant (R100) HL cells Disclosures Pinto: Takeda: Research Funding; Takeda, Celgene, Roche, TEVA: Honoraria.

8083 Background: Since early identification of patients (pts) at risk of failure is the mainstay of a risk-adapted therapy, we explored the prognostic impact of the sFLC assay in cHL, whose biology involves ongoing activation of polyclonal B-cells. Methods: Serum samples from 248 untreated cHL pts were tested by the Freelite assay. Median age was 32 yrs (r 15-85), males 47%, stages: I (5%), II (51%), III (17%), IV (27%); B-sympt. 60%, E-disease, 38%; bulky >10 cm, 44%; ESR > 65, 42%; IPS ≥3, 39%. Early unfavorable disease (GHLSG/ EORTC) was respectively found in 33% and 42% of cases. ABVD was given to 89% of pts. Results: Absolute FLC levels were summed into a sFLC(κ+λ) variable and ROC analysis indicated 57.1 mg/mL as the threshold to discriminate outcomes. CR rates were 96% and 67% for pts below and above the cutoff, respectively (p1 (HR 4.2), IPS ≥3 (HR 2.8) and all other predictors (HR 0.54-2.4). In a multivariaye model only sFLC(κ+λ) and PET2 remained independent predictors. A dismal 8-yrs EFS characterized pts with sFLC(κ+λ) above threshold (20% vs 89%; Χ2 119, p2 65.4; p2 51 p

Abstract 5168 Background: Myeloproliferative neoplasms (MPNs) are a group of diseases characterized by clonal expansion of single or multiple lineages of myeloid subset (i.e. granulocytic, erythroid, megakaryocytic and mast cell). Since 2008, the WHO included the detection of JAK2 mutations (the common V617F and the less common exon 12 mutations), into the diagnostic criteria of MPNs. The specific pathogenic implication of JAK2 mutations in MPNs is still under investigation. Preliminary data emerging from the treatment of Idiopathic Myelofibrosis patients with JAK2 inhibitors have shown only clinically significant benefits (improvement of splenomegaly and constitutional symptoms) but not a clear evidence of disease-modifying activity. Several techniques (e.g. ASO-PCR, ARMS-PCR, Direct sequencing, HRM, DHPLC, etc) have been used to detect the JAK2 V617F mutation, but all of them were low sensitive and time-consuming. We developed a PNA-clamping competitive PCR assay able to detect JAK2 V617F mutation with a very high level of sensitivity and specificity. Methods: In order to promote the selective amplification of the JAK2 V617F mutant allele, a specific PNA oligonucleotide, full matching with the wild-type (wt) sequence of the portion of JAK2 gene containing the V617F mutation, was designed to compete with the reverse primer used in the PCR assay. It was experimentally demonstrated that a PNA concentration of 6 μmol/L occurred for the complete clamping of 100 ng of wt genomic DNA used as template for the PCR reaction. The sensitivity of the assay was determined by a serial dilution (100% through 0.01%) of genomic DNA containing JAK2 V617F mutation, obtained from HEL cell line, with wt DNA obtained from healthy donors. The specificity of PCR products was assessed by sequencing in both forward and reverse directions. The thermal dissociation profile of PNA/DNA and DNA/DNA duplexes was studied by monitoring the Enthalpy as function of the temperature (range 20–100°C), with an heating/cooling rate of 1°C/min. Results: PNA-clamping competitive PCR was able to detect JAK2 V617F mutation with a sensitivity of 0.01%. Thermodynamic studies clearly showed that the melting temperature (Tm) of fully matched PNA/DNA duplex is always higher than Tm of the corresponding DNA/DNA duplex. The enthalpy values for the hybrid PNA/DNA are always greater than the corresponding DNA/DNA duplex and a single mismatch has an energetic cost higher in a PNA/DNA than in DNA/DNA duplex. This energetic cost is more evident in the melting temperature with a ΔTm of 16°C and 7°C for the PNA/DNA and DNA/DNA duplex, respectively. In the PCR assay the PNA complementary to the JAK2 wild type sequence strongly compete with the primer resulting in a complete knock out of the wild type allele. In a blind screening of samples obtained from 308 MPN patients, PNA clamping competitive PCR was able to detect JAK2 V617F mutation in 61.4% cases of Polycythemia Vera, 54.4% of Essential Thrombocythemia, 57.9% of Idiopathic Myelofibrosis and in 21.4% of Ph negative-MPNs, respectively. In 40 unselected patients, PNA clamping PCR results were confirmed by other techniques such as ASO-PCR, ARMS-PCR and direct sequencing. Conclusions: The PNA-clamping competitive PCR assay could be used as convenient, high-sensitive and reliable diagnostic test for detection of JAK2 V617F mutation both on genomic DNA and cDNA samples. As suggested from the thermodynamic studies, a similar technique could be developed for the detection of any known single point mutation, widely contributing to the improvement of molecular diagnostic tests usable in clinical practice. Disclosures: No relevant conflicts of interest to declare.

Abstract 2437 Clinical course in individual CLL patients (pts) is highly heterogeneous. Several pts show an indolent disease and other experience an aggressive course rapidly succumbing to disease-related events. Different biomarkers have been developed to identify pts with evolving disease but their application is mostly limited to clinical trials. It has been shown that measurement of clonal serum free light chains (sFLC) levels, through a straightforward assay validated for plasma cell disorders, could independently identify CLL pts with progressive disease. By analyzing the largest cohort of CLL pts ever reported, we wished to: i) determine the independent predictive value of sFLC abnormalities in the context of established biomarkers; ii) define the predictive hierarchy of these markers; iii) incorporate sFLC into a novel prognostic system. Cryopreserved sera at diagnosis from 449 untreated pts (282 males and 167 females, median age 65 yrs; r 33–89) were collected for analysis at two reference laboratories. After quantization (Freelite, The Binding Site, Ltd., UK) of sFLC k and l levels (r, k: 3.3–19.4 mg/mL; l: 5.71–26.3 mg/mL), their ratio was calculated as sFLC k/l (r: 0.26–1.65). An abnormal sFLCk/l was found in 150/449 cases (132 k/18 l). A significant correlation emerged between sFLCk/l and CD38, ZAP70, unmutated IgHV and unfavourable FISH but not with Binet stage (Table). At a median follow up of 3 yrs (r 1–20), 149 of 449 pts were treated due to progressive disease (NCI Guidelines). Treatment-free survival (TFS) was significantly shorter (p60.6 mg/mL (HR=2.5, C.I. 2.6–3.9), remained the strongest independent predictor of TFS together with ZAP70 (HR=2.8, C.I. 1.8–4.6) and stage (HR=2.5, C.I. 1.6–3.8), while CD38 and IgVH status lost their predictive power. Most intriguingly, it emerged that sFLCk/l is irrelevant in predicting TFS if sFLCk+l is above cut-off and that cytogenetic risk remains significantly associated with sFLCk+l (P=0.047) but not FLCk/l. sFLCk+l retained an independent association with TFS together with cytogenetics, ZAP70 and stage. A novel prognostic model was then constructed with 1 point for each of these unfavorable marker and a score given by their sum. On 260 pts scores were: 104=0, 79=1, 56=2, 19=3, 4=4. The 3 year probability of avoiding treatment was 94.8%, 84.5%, 61.6% and 21.1% for pts scoring 0, 1, 2 and 3+4, respectively (P sFLC k/l in 449 untreated CLL pts Table 1. Death in complete remission by year sFLC k/l P Normal (%) Abnormal (%) CD38 − 234 (70.9) 96 (29.1) Disclosures: Amoroso: The Binding Site, LtD: Consultancy.

Abstract 267 Introduction: The tumor cells of HL, the Hodgkin and Reed-Sternberg (H-RS) cells, derive from germinal center B-cells with a deranged B-cell transcription program due to epigenetic silencing and acquired genetic lesions. Tissue H-RS cells are surrounded by a preponderant infiltrate of mixed non-malignant reactive cells, including B-lymphocytes, which provide essential signals for their survival and proliferation. Small percentages of B-cells, clonally related to H-RS cells, were found in the blood of HL patients (pts), suggesting they may represent putative HL-initiating cells (Jones, 2009). Since the serum free light chain (sFLC) assay detects and quantifies monoclonal and polyclonal B-cell populations expanding in lymphohemopoietic tissues, we exploited the sFLC testing to further explore the biologic significance of B-lymphocytes in HL. Patients and Methods: Frozen (-80°C) serum samples from 119 untreated cHL pts (48% males), with normal renal function and serum immunochemistry, were assayed by immunonephelometry (Freelite, The Binding Site, Ltd., UK). After quantization of free κ and λ concentrations (normal ranges, κ: 3.3-19.4 mg/L; λ: 5.71-26.3 mg/L), sFLC κ/λratio was calculated (reference range 0.26-1.65). The median age was 31 years (r, 15–70), with 22% aged ≥ 45 years. Histology was nodular sclerosis in 85 pts (71.4%) and 72 (60.5%) were in stage I–II. According to GHSG criteria, 16% had early favorable, 29% early unfavorable and 55% advanced disease. The International Prognostic Score (IPS) was of 0-2 and ≥ 3 in 66.4% and 33.6% of pts, respectively. Following ABVD (4-6 courses ± RT), the median Event-free survival (EFS) was 78 mo.s [95% CI, 68-88] for the entire population and 75 mo.s [95% CI, 60-90] and 64 mo.s [95% CI, 50-77] for pts in early and advanced stage, respectively. Results: Elevated κor λsFLC concentrations were found in 47% (median 29.55 mg/L; r, 19.44 - 64.70) and 29.4% (median 32.70 mg/L; r, 26.60 - 79.77) of pts, respectively. In 30 pts (25.2%) levels of polyclonal κ and λ sFLC were concurrently elevated. The sFLC κ/λratio was abnormal in only 7.5% of pts (clonal λ: 8/119; clonal λ: 1/119). The presence of high sFLC levels correlated with lymphopenia (< 0.6 × 109/L; p= 0.04), leukocytosis (WBC > 15 × 109/L; p=0.03), ESR (> 50; p=0.04) and unfavorable IPS (≥ 3; p=0.03), but not with EBV status and risk factors such as stage, B symptoms, bulky and extra nodal disease, LDH and albumin. The association with leukocytosis may in part result from inhibition of spontaneous neutrophils apoptosis exerted by FLC (Cohen, 2003). Most interestingly, while we found no significant association with response rate, baseline elevation in sFLC predicted for EFS in 51 evaluable pts with early stage disease. Patients were divided into tertiles and best break point value for predicting EFS coincided with the upper limit of the highest tertile for both κ (>25.53 mg/L) or λsFLC (>26.66 mg/L) (Figure 1). The pts in the top tertile had the worst outcome compared with the 2 lower tertiles (κ, p=0.015; λ, p=0.002). Outcomes for the 2 lower tertiles were comparable. Data remained significant after stratification for the IPS. In contrast, baseline sFLC levels, κ or λ, were not predictive for EFS in pts with advanced disease. Interestingly, sFLC levels in a control group of 30 pts in continuous CR from 2 years, were within the normal ranges. Conclusions: We have shown that about 50% of cHL pts displays elevated levels of sFLC mirroring the presence of a consistent polyclonal B-cell expansion at diagnosis. The role of reactive B-cells in HL is poorly understood. While some data indicate that high intratumoral B-cell counts may predict for a better outcome, the activity of rituximab in cHL, regardless of CD20 expression on H-RS cells, was suggested to result by depletion from the HL microenvironment of normal B lymphocytes required for tumor cell growth (Younes, 2003). Our study support that elevated sFLC levels may reflect an increased polyclonal B cell activity in cHL microenvironment which appears to negatively influence the outcome of pts in early stage disease. This effect is lost in advanced disease, suggesting that rituximab might result more active for pts in early than advanced stages. That putative HL-initiating small B-cells may emerge from the expanded polyclonal B-cell population present from the early phases of disease development, is an intriguing possibility. Disclosures: Marchei: Radim, Italy: Employment. Amoroso:The Binding Site, Ltd: Consultancy.

The serum free light chain (sFLC) assay represents a predictor of prognosis and therapeutic response in monoclonal gammopathies and multiple myeloma (MM). Since sFLC testing detects and quantifies monoclonal and polyclonal B-cell populations expanding in lymphohemopoietic tissues, we evaluated the potential value of sFLC abnormalities as a novel disease marker for patients (pts) with B-cell non-Hodgkin’s lymphomas (NHL). Frozen (−80°C) serum samples from 354 untreated pts with NHL categorized according to WHO criteria (Table 1) and with normal renal function and serum immunochemistry, were assayed by immunonephelometry (Freelite, The Binding Site, Ltd., Birmingham, UK). After quantitation of serum free k and l concentrations (normal ranges, k: 3.3–19.4 mg/mL; l: 5.71–26.3 mg/mL), FLC ratio was calculated as k/l (free k concentration divided by free l, reference range 0.26–1.65). In cases with a ratio >1.65, k was considered the ‘involved’ (inv.) FLC and l the ‘uninvolved’ FLC, and vice versa if the ratio was less than 0.26. Sera from 45 MM pts, 43 pts with solid tumors and 22 pts with reactive lymphadenopathy (LAD) were also analyzed. Elevated sFLC concentrations, i.e. both k and/or l, were found in 53% to 80% of cases while abnormal FLC (k/l) ratios were detected in a restricted fraction of pts with a histotype-related fashion (Table). Tumors mostly arising from pre-germinal centre (GC) B-cells, i.e. Small Lymphocytic Lymphoma (SLL) and Mantle Cell Lymphoma (MCL), and those deriving from late post-GC cells, i.e. Burkitt lymphoma (BL), displayed the higher rates of abnormal FLC ratio (57%, 42% and 33% for MCL, SLL and BL, respectively), due to free k chain involvement in 86% to >90% of cases. Accordingly, these tumors showed a high median inv. k chain concentration (33.5 to 53 mg/L). In Follicular Cell (FCL) and Diffuse Large B Cell (DLBC) lymphomas the FCL ratio was abnormal in 23% and 25% of pts respectively, due to k FLC involvement in > 90% of cases, and a median serum inv. k concentration of 26 mg/mL in FCL (G1 20.5; G2, 16.3; G3, 16.9; G3b, 23,6 mg/L) and 25 mg/L in DLBCL. Interestingly, 8 cases of localized DLBCL of bone and CNS showed a normal sFLC test. Pts with disseminated Marginal Zone (MZ) lymphoma displayed abnormal FLC ratio in 16% of cases and the highest median inv. k concentration (66.5 mg/L). sFLC testing was positive > 80% of MM pts while no solid tumors and reactive LADs cases displayed abnormal sFLC ratio. In15 NHL pts (FCL, MCL, MZL) given upfront treatment with Rituximab (R) or Zevalin, sFLC test was explored as a tool for therapeutic monitoring. A stepwise decrease in inv. k chains levels was observed in all responders, followed by a rise at relapse. Similarly, disease control in SLL (n=14) and MCL (n=10) pts by first line R-immunochemotherapy was associated to normalization of sFCL test. Our results, on the largest serie reported, indicate that NHL pts display a high frequency of monoclonal sFLC. Differences among WHO histotypes at presentation may mirror specific biologic features, including pre/post-GC derivation and propensity to dissemination. sFCL testing represents a furher tool to dissect biologic heterogeneity of NHL and a new marker for therapeutic monitoring. SLL (n= 33) MCL (n=28) MZL (n=37) FCL (n=105) DLBCL (n=123) BL (n=20) * median concentration (mg/L) sFLC+ (%) 20 (60.6) 23 (82) 28 (75.6) 56 (53.3) 71 (57.7) 13 (65) Abnormal k/l (%) 14 (42.4) 16 (57) 6 (16) 24 (23) 31 (25.2) 6 (33) Involved k (%) 12 (85.7) 14 (88) 5 (83.3) 23 (95.8) 29 (93.5) 4 (66.6) Involved l (%) 2 (14.2) 2 (12.5) 1 (16.6) 1 (4.2) 2 (6.45) 2 (33) Involved k* (range) 53.0 (7.2–83.5) 33.5 (10.6–168.4) 65.5 (25.6–269.6) 25.96 (9.3–108.3) 25.2 (6.8–207.6) 47.6 (12.3–117.2) Involved l* (range) 133.7 (74.6–192.8) 75.3 (39.7–111) 318.28 215.4 97.5 (95–100) 135.4 (127.2–143.5)