RAS mutation status predicts survival and patterns of recurrence in patients undergoing hepatectomy for colorectal liver metastases.

OBJECTIVE: To determine the impact of RAS mutation status on survival and patterns of recurrence in patients undergoing curative resection of colorectal liver metastases (CLM) after preoperative modern chemotherapy. BACKGROUND: RAS mutation has been reported to be associated with aggressive tumor biology. However, the effect of RAS mutation on survival and patterns of recurrence after resection of CLM remains unclear. METHODS: Somatic mutations were analyzed using mass spectroscopy in 193 patients who underwent single-regimen modern chemotherapy before resection of CLM. The relationship between RAS mutation status and survival outcomes was investigated. RESULTS: Detected somatic mutations included RAS (KRAS/NRAS) in 34 (18%), PIK3CA in 13 (7%), and BRAF in 2 (1%) patients. At a median follow-up of 33 months, 3-year overall survival (OS) rates were 81% in patients with wild-type versus 52.2% in patients with mutant RAS (P = 0.002); 3-year recurrence-free survival (RFS) rates were 33.5% with wild-type versus 13.5% with mutant RAS (P = 0.001). Liver and lung recurrences were observed in 89 and 83 patients, respectively. Patients with RAS mutation had a lower 3-year lung RFS rate (34.6% vs 59.3%, P < 0.001) but not a lower 3-year liver RFS rate (43.8% vs 50.2%, P = 0.181). In multivariate analyses, RAS mutation predicted worse OS [hazard ratio (HR) = 2.3, P = 0.002), overall RFS (HR = 1.9, P = 0.005), and lung RFS (HR = 2.0, P = 0.01), but not liver RFS (P = 0.181). CONCLUSIONS: RAS mutation predicts early lung recurrence and worse survival after curative resection of CLM. This information may be used to individualize systemic and local tumor-directed therapies and follow-up strategies.

Revisiting clinical trials using EGFR inhibitor-based regimens in patients with advanced non-small cell lung cancer: a retrospective analysis of an MD Anderson Cancer Center phase I population.

PURPOSE: Single-agent EGFR inhibitor therapy is effective mainly in patients with lung cancer and EGFR mutations. Treating patients who develop resistance, or who are insensitive from the outset, often because of resistant mutations, other aberrations or the lack of an EGFR mutation, probably requires rational combinations. We therefore investigated the outcome of EGFR inhibitor-based combination regimens in patients with heavily-pretreated non-small cell lung cancer (NSCLC) referred to a Phase I Clinic. METHODS: We reviewed the electronic records of patients with NSCLC treated with an EGFR inhibitor-based combination regimen: erlotinib and cetuximab; erlotinib, cetuximab and bevacizumab; erlotinib and dasatinib; erlotinib and bortezomib; or cetuximab and sirolimus. RESULTS: EGFR mutations were detected in 16% of patients (21/131). EGFR inhibitor-based combination regimens were administered to 15 patients with EGFR-mutant NSCLC and 24 with EGFR wild-type disease. Stable disease (SD) ≥6 months/partial remission (PR) was attained in 20% of EGFR-mutant patients (3/15; two with sensitive mutations and secondary resistance to prior erlotinib, and one with a resistant mutation), as well as 26% of evaluable patients (5/19) with wild-type disease. One of three evaluable patients with squamous cell histology achieved SD for 26.5 months (EGFR wild-type, TP53-mutant, regimen=erlotinib, cetuximab and bevacizumab). CONCLUSIONS: Eight of 34 evaluable patients (24%) with advanced, refractory NSCLC evaluable for response achieved SD ≥6 months/PR (PR=3; SD ≥6 months=5) on EGFR inhibitor-based combination regimens (erlotinib, cetuximab; erlotinib, cetuximab and bevacizumab; and, erlotinib, bortezomib), including patients with secondary resistance to single-agent EGFR inhibitors, resistant mutations, wild-type disease, and, squamous histology.

Systemic mastocytosis with associated clonal hematological non-mast cell lineage disease: clinical significance and comparison of chomosomal abnormalities in SM and AHNMD components.

Some patients with systemic mastocytosis have concurrent hematological neoplasms, designated in the World Health Organization (WHO) classification as systemic mastocytosis with associated clonal hematological non-mast cell lineage disease (SM-AHNMD). In this study, we analyzed 29 patients with SM-AHNMD and compared them to 40 patients with pure SM. The AHNMDs were classified as chronic myelomonocytic leukemia (CMML) (n = 10), myelodysplastic syndrome (MDS) (n = 7), myeloproliferative neoplasms (n = 4), B-cell lymphoma/leukemia/plasma cell neoplasms (n = 7), and acute myeloid leukemia (n = 1). Patients with SM-AHNMD were older, more frequently had constitutional symptoms and hematological abnormalities, less often had skin lesions, and had an inferior overall survival compared with pure SM patients (48 months vs. not-reached, P < 0.001). Karyotypic abnormalities were detected in 9/28 (32%) patients with SM-AHNMD but not in pure SM patients (P < 0.001). Combined imaging/ fluorescence-in-situ hybridization performed in four SM-AHNMD cases revealed shared abnormal signals in mast cells and myeloid cells in two patients with SM-CMML and one patient with SM-MDS, but not in the mast cells of a case SM-associated with chronic lymphocytic leukemia with ATM-deletion. Quantitative mutation analysis showed higher levels of mutant KIT D816V in SM-CMML and SM-MDS than in pure SM (P < 0.001). Our data indicate that the SM-AHNMD category in the WHO classification is heterogeneous, including clonally related and unrelated forms of AHNMD. The presentation, treatment, and outcome of patients with SM-AHNMD is often dictated by the type of AHNMD.

Phase II trial of erlotinib plus concurrent whole-brain radiation therapy for patients with brain metastases from non-small-cell lung cancer.

PURPOSE: Brain metastasis (BM) is a leading cause of death from non-small-cell lung cancer (NSCLC). Reasoning that activation of the epidermal growth factor receptor (EGFR) contributes to radiation resistance, we undertook a phase II trial of the EGFR inhibitor erlotinib with whole-brain radiation therapy (WBRT) in an attempt to extend survival time for patients with BM from NSCLC. Additional end points were radiologic response and safety. PATIENTS AND METHODS: Eligible patients had BM from NSCLC, regardless of EGFR status. Erlotinib was given at 150 mg orally once per day for 1 week, then concurrently with WBRT (2.5 Gy per day 5 days per week, to 35 Gy), followed by maintenance. EGFR mutation status was tested by DNA sequencing at an accredited core facility. RESULTS: Forty patients were enrolled and completed erlotinib plus WBRT (median age, 59 years; median diagnosis-specific graded prognostic assessment score, 1.5). The overall response rate was 86% (n = 36). No increase in neurotoxicity was detected, and no patient experienced grade ≥ 4 toxicity, but three patients required dose reduction for grade 3 rash. At a median follow-up of 28.5 months (for living patients), median survival time was 11.8 months (95% CI, 7.4 to 19.1 months). Of 17 patients with known EGFR status, median survival time was 9.3 months for those with wild-type EGFR and 19.1 months for those with EGFR mutations. CONCLUSION: Erlotinib was well tolerated in combination with WBRT, with a favorable objective response rate. The higher-than-expected rate of EGFR mutations in these unselected patients raises the possibility that EGFR-mutated tumors are prone to brain dissemination.

Adjuvant chemotherapy with FOLFOX for primary colorectal cancer is associated with increased somatic gene mutations and inferior survival in patients undergoing hepatectomy for metachronous liver metastases.

OBJECTIVE: We hypothesized that metachronous colorectal liver metastases (CLM) have different biology after failure of oxaliplatin (FOLFOX) compared to 5-fluorouracil (5-FU) or no chemotherapy for adjuvant treatment of colorectal cancer (CRC). BACKGROUND: It is unclear whether patients treated with liver resection for metachronous CLM after adjuvant FOLFOX for CRC have worse outcomes than those who received 5-FU or no chemotherapy. METHODS: We identified 341 patients who underwent hepatectomy for metachronous CLM (disease-free interval ≥12 months, 1993-2010). Mass-spectroscopy genotyping for somatic gene mutations in CLM was performed in a subset of 129 patients. RESULTS: Adjuvant treatment for primary CRC was FOLFOX in 77 patients, 5-FU in 169 patients, and no chemotherapy in 95 patients. Node-positive primary was comparable between FOLFOX and 5-FU but lower in the no-chemotherapy group (P < 0.0001). Median metastasis size was smaller in the FOLFOX group (2.5 cm) than in the 5-FU (3.0 cm) or no-chemotherapy (3.5 cm) groups, (P = 0.008) although prehepatectomy chemotherapy utilization, metastases number, and carcinoembryonic antigen levels were similar. Disease-free survival (DFS) and overall survival (OS) rates after hepatectomy were worse in patients treated with adjuvant FOLFOX [DFS at 3 years: 14% vs 38% (5-FU) vs 45% (no-chemo), OS at 3 years: 58% vs 70% (5-FU) vs 84% (no-chemo)]. On multivariate analysis, adjuvant FOLFOX was associated with worse DFS (P < 0.0001) and OS (P < 0.0001). Mutation analysis revealed ≥1 mutations in 57% of patients (27/47) after FOLFOX, 29% (12/41) after 5-FU, and 32% (13/41) after no chemotherapy (P = 0.011). CONCLUSIONS: Adjuvant FOLFOX for primary CRC is associated with a high rate of somatic mutations in liver metastases and inferior outcomes after hepatectomy for metachronous CLM.

CD117 expression is a sensitive but nonspecific predictor of FLT3 mutation in T acute lymphoblastic leukemia and T/myeloid acute leukemia.

Others have suggested that CD117, or an immunophenotypic profile including CD117, can serve as surrogate for FLT3 mutation in T acute lymphoblastic leukemia (ALL), thereby guiding targeted therapy. We report the results of flow cytometry immunophenotypic analysis in 42 cases of T-ALL and T/myeloid acute leukemia also assessed for FLT3 mutation. CD117 was expressed in 21 (50%), and FLT3 was mutated in 8 cases (19%; 1 T-ALL and 7 T/myeloid). FLT3-mutated cases were terminal deoxynucleotidyl transferase (TdT)+/CD2+ (7/8), cytoplasmic CD3+/CD5+ (5/8), CD7+/CD13+/CD15+ (4/6), CD33+ (4/8), CD34+, and CD117+ (bright). Cytochemistry showed myeloperoxidase-positive cells in all T/myeloid acute leukemias (3%-50%). We conclude that FLT3 mutation is rare in T-ALL, and its presence supports T/myeloid lineage. CD117 expression alone is sensitive but not specific for FLT3 mutation. The immunophenotypic profile of TdT, CD7, CD13, CD34, and CD117 (bright) is helpful for predicting FLT3 mutation, with a sensitivity of 100% and specificity of 94%.

Monitoring of minimal residual disease in acute myeloid leukemia: methods and best applications.

Posttherapy minimal residual disease assessment in acute myeloid leukemia is an important prognostic indicator as well as a potential early surrogate measure of therapeutic effectiveness. The goal of this brief review is to discuss the broad classes of targets for molecular diagnostic assays, with specific examples of each, and to describe the analytic approach to flow cytometry testing for minimal residual disease in acute myeloid leukemia. Several of the most significant recent contributions to this field are highlighted.

Chromosome 20q deletion: a recurrent cytogenetic abnormality in patients with chronic myelogenous leukemia in remission.

del(20q) can be observed in hematologic neoplasms, including chronic myelogenous leukemia (CML), and has been reported in patients undergoing blast transformation. We describe 10 patients with CML in hematologic and cytogenetic remission with del(20q) detected by conventional cytogenetics. There were 6 men and 4 women with a median age of 56 years. All patients initially had BCR-ABL1 and t(9;22) (q34;q11.2) and achieved morphologic and cytogenetic remission after therapy. del(20q) was identified before (2/10 [20%]), at the time of (3/10 [30%]), or after (5/10 [50%]) cytogenetic remission and was not associated with morphologic evidence of dysplasia. At last follow-up, no patients had a myelodysplastic syndrome (MDS). Leukocyte and platelet counts were normal; 4 of 10 patients had mild anemia. Nine patients have remained in morphologic and cytogenetic remission with stable del(20q). BCR-ABL1 fusion transcript levels were absent or low (median, 0.01%). Recently, in 1 patient, recurrent CML developed and del(20q) was lost. We conclude that del(20q) in the setting of CML in remission is not predictive of MDS or blast transformation.

De novo acute myeloid leukemia with inv(3)(q21q26.2) or t(3;3)(q21;q26.2): a clinicopathologic and cytogenetic study of an entity recently added to the WHO classification.

Acute myeloid leukemia with inv(3)(q21q26.2) or t(3;3)(q21;q26.2) is a rare type of leukemia recently added to the World Health Organization (WHO) classification scheme. In this study, we analyzed the clinicopathologic and cytogenetic features of 30 cases of de novo acute myeloid leukemia with inv(3)/t(3;3). The median patient age was 53 years (range, 27-77 years). The platelet count was variable (range, 21-597 × 10(9)/l, median: 128 × 10(9)/l), and two (6.7%) patients presented with thrombocytosis (>450 × 10(9)/l). Morphologically, these neoplasms showed a spectrum of findings. Myelomonocytic differentiation was most common in 11 (37%) cases. Morphological evidence of dysplasia was observed in at least one lineage in 23 of 25 (92%) cases in which maturing elements could be assessed. In all, 5 (17%) patients had isolated inv(3) or t(3;3) and 25 (83%) patients had additional cytogenetic abnormalities, most often monosomy 7 (40%). Eleven (37%) patients had a complex karyotype (≥ 3 additional abnormalities). FLT3 gene mutation by internal tandem duplication was identified in 2 of 23 (9%) cases assessed. No clinical, pathological, or cytogenetic features independent of inv(3) or t(3;3) correlated with a worse outcome. However, patients treated with allogeneic stem cell transplantation (n=11) had a significantly better survival than did those treated with chemotherapy alone (n=17) (13.8 vs 8.0 months, P=0.041). We conclude that de novo acute myeloid leukemia associated with inv(3)/t(3;3) is an aggressive type of leukemia regardless of morphological or karyotypic findings, supporting the inclusion of this disease as a specific entity defined by inv(3)/t(3;3) in the WHO classification. Allogeneic stem cell transplantation seems to improve outcome in patients with this disease.

A pathway-based gene signature correlates with therapeutic response in adult patients with Philadelphia chromosome-positive acute lymphoblastic leukemia.

Biomarkers to predict response to therapy in adults with Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL) are not yet established. In this study, we performed a meta-analysis of earlier genome-wide gene expression studies to identify pathway-based genes that are associated with therapeutic response. The predictive power of these genes was validated by transcript profiling in diagnostic bone marrow samples from Ph+ ALL patients using a quantitative real-time PCR array. Gene expression was correlated with cytogenetic and molecular characteristics, including presence of ABL1 mutations and IKZF1 deletion. A total of 43 de novo Ph+ ALL patients treated uniformly with tyrosine kinase inhibitors combined with chemotherapy were selected to validate 46 identified genes. A 9-gene signature was established to distinguish optimal responders from patients with persistent residual disease and early molecular recurrence. The signature was subsequently validated with 87% predictive accuracy in an independent validation set of patients. When initially optimal responders relapsed, their gene expression patterns also shifted. Optimal responders showed upregulation of genes involved in proliferation and apoptosis pathways, whereas poor responders had higher expression of genes that facilitate tumor cell survival in hypoxic conditions as well as development of drug resistance. This unique 9-gene signature may better enable stratification of patients to proper therapeutic regimens and provides new insights into mechanisms of Ph+ ALL response to therapy.

Distinct patterns of cytogenetic and clinical progression in chronic myeloproliferative neoplasms with or without JAK2 or MPL mutations.

Chronic myeloproliferative neoplasms (MPN), including essential thrombocythemia (ET) and primary myelofibrosis (PMF), result from interactions between initiating growth factor mutations and secondary genomic changes. Codon 617 mutation of the JAK2 kinase is found in 40-50% of ET/PMF, whereas the mutation of codon 515 in the JAK2-linked thrombopoietin receptor MPL is found in approximately 20% of JAK2-unmutated cases of ET and PMF. Using quantitative mutation assays, we compared patterns of clinical and cytogenetic progression in MPL-mutated MPN (n=21) to those with JAK2 V617F mutation (n=383) or neither mutation (n=109). Among patients with MPL mutations, ET was seen in 9 and PMF in 12. Median mutation levels in pretreatment ET samples were significantly higher for MPL-mutated cases (60%) than for JAK2-mutated cases (24%; P=0.01), as was presentation with anemia. Differential genomic changes included +9 in JAK2-mutated cases and chromosome 1 alterations in MPL-mutated ones, implicating dosage effects related to gene copy number. Decreases in the levels of MPL mutation were seen in sequential marrow samples from some patients under treatment with biologic therapies, but not in those treated with kinase inhibitors, consistent with selective response of the MPL-mutated clone similar to the responses seen in JAK2-mutated MPN.

Flow cytometric detection of peripheral blood involvement by mycosis fungoides and Sézary syndrome using T-cell receptor Vbeta chain antibodies and its application in blood staging.

Peripheral blood involvement has been recognized as an adverse prognostic factor in patients with mycosis fungoides and Sézary syndrome. However, accurate identification and enumeration of the neoplastic cells in these diseases can be challenging. We assessed the clinical utility of flow cytometric immunophenotypic analysis of T-cell receptor Vbeta expression in 82 mycosis fungoides and 6 Sézary syndrome patients, with an atypical T-cell immunophenotype, or abnormal CD4:CD8 ratio, identified from peripheral blood specimens of 723 patients submitted for routine mycosis fungoides/Sézary syndrome blood staging. To improve detection sensitivity, Vbeta expression was analyzed on gated CD3+CD4+ T cells or T cells with an aberrant immunophenotype, if present. The flow cytometric results were compared with traditional morphologic assessment (n=88) and molecular methods to assess the T-cell receptor gamma or beta genes (n=41 tested in parallel). Flow cytometric immunophenotyping yielded a clonal Vbeta pattern in 60/82 mycosis fungoides and 6/6 Sézary syndrome patients. By contrast, flow cytometric Vbeta was negative in all 10 healthy donors and 18 control patients, showing a specificity of 100% and concordance with molecular testing of 86%. Using flow cytometric Vbeta results instead of morphologic assessment, 12 patients were upstaged from B1 to B2, and 20 patients from B0 to B1 (P<0.0001). The 12 upstaged B2 patients had no morphologic evidence of involvement, but had an aggressive clinical course similar to those staged by traditional morphologic assessment (median survival 27 vs 41 months, log-rank P=0.701). In 30/44 patients with a tumor-associated Vbeta expression, a single Vbeta tube was used to monitor treatment response. In conclusion, flow cytometric Vbeta analysis is rapid and convenient, can assess T-cell clonality and tumor quantity simultaneously, and is useful both in initial blood staging and monitoring tumor burden during therapy in patients with mycosis fungoides or Sézary syndrome.

t(10; 12) (q24; p13) as the sole abnormality in a case with refractory acute myeloid leukemia: The first case report and literature review.

Rearrangements involving chromosome region at 12p13 are common abnormalities in hematological malignancies, including myeloid and lymphoid types. ETV6 gene is usually involved in the 12p13 region. ETV6 rearrangements are more often observed in acute lymphoblastic leukemia than in acute myeloid leukemia (AML), where ETV6 gene deletions are more common than rearrangements. Here, we report an AML case with the recurrent t(10; 12) (q24; p13) as the sole abnormality. Fluorescence in situ hybridization with mapping back to metaphases confirmed that the ETV6 gene splits, and rearranges with a locus at 10q24. In review of the literature, this is the first report of AML case with the novel abnormality as the sole change. Complete laboratory findings from bone marrow examination, flow cytometry analysis, cytogenetic studies, molecular analysis, and clinical features are also described in the report.

Application of COLD-PCR for improved detection of KRAS mutations in clinical samples.

KRAS mutations have been detected in approximately 30% of all human tumors, and have been shown to predict response to some targeted therapies. The most common KRAS mutation-detection strategy consists of conventional PCR and direct sequencing. This approach has a 10-20% detection sensitivity depending on whether pyrosequencing or Sanger sequencing is used. To improve detection sensitivity, we compared our conventional method with the recently described co-amplification-at-lower denaturation-temperature PCR (COLD-PCR) method, which selectively amplifies minority alleles. In COLD-PCR, the critical denaturation temperature is lowered to 80 degrees C (vs 94 degrees C in conventional PCR). The sensitivity of COLD-PCR was determined by assessing serial dilutions. Fifty clinical samples were used, including 20 fresh bone-marrow aspirate specimens and the formalin-fixed paraffin-embedded (FFPE) tissue of 30 solid tumors. Implementation of COLD-PCR was straightforward and required no additional cost for reagents or instruments. The method was specific and reproducible. COLD-PCR successfully detected mutations in all samples that were positive by conventional PCR, and enhanced the mutant-to-wild-type ratio by >4.74-fold, increasing the mutation detection sensitivity to 1.5%. The enhancement of mutation detection by COLD-PCR inversely correlated with the tumor-cell percentage in a sample. In conclusion, we validated the utility and superior sensitivity of COLD-PCR for detecting KRAS mutations in a variety of hematopoietic and solid tumors using either fresh or fixed, paraffin-embedded tissue.

Therapy-related acute myeloid leukemia with t(8;21) (q22;q22) shares many features with de novo acute myeloid leukemia with t(8;21)(q22;q22) but does not have a favorable outcome.

To determine if therapy-related acute myeloid leukemia (t-AML) with t(8;21)(q22;q22) [t-AML-t(8;21)] harbors similar characteristic clinicopathologic features as de novo AML-t(8;21) (q22;q22), we studied 13 cases of t-AML-t(8;21) and 38 adult cases of de novo AML-t(8;21) diagnosed and treated at our hospital (1995-2008). Of 13 t-AML-t(8;21) cases, 11 had previously received chemotherapy with or without radiation for malignant neoplasms and 2 received radiation alone. The median latency to t-AML onset was 37 months (range, 11-126 months). Compared with patients with de novo AML-t(8;21), patients with t-AML-t(8;21) were older (P = .001) and had a lower WBC count (P = .039), substantial morphologic dysplasia, and comparable CD19/CD56 expression. The AML1-ETO (RUNX1-RUNX1T1) fusion was demonstrated in all 10 cases assessed. Class I mutations analyzed included FLT3 (0/10 [0%]), RAS (0/10 [0%]), JAK2 V617 (0/11 [0%]), and KIT (4/11 [36%]). With a median follow-up of 13 months, 10 patients with t-AML-t(8;21) died; the overall survival was significantly inferior to that of patients with de novo AML-t(8;21) (19 months vs not reached; P = .002). These findings suggest that t-AML-t(8;21) shares many features with de novo AML-t(8;21)(q22;q22), but affected patients have a worse outcome.