Effect of low-level BCR-ABL1 kinase domain mutations identified by next-generation sequencing in patients with chronic myeloid leukaemia: a population-based study.

BACKGROUND: Kinase domain mutations in BCR-ABL1 are associated with resistance to tyrosine kinase inhibitors in patients with chronic myeloid leukaemia. Next-generation sequencing (NGS) allows detection of low-level kinase domain mutations, but its relevance in clinical practice remains debated. We aimed to examine the clinical effects of low-level kinase domain mutations identified using NGS in patients with chronic myeloid leukaemia. METHODS: In this population-based study, we included consecutive patients newly diagnosed with chronic myeloid leukaemia treated with first-line tyrosine kinase inhibitors, and patients identified at the time of resistance to first-line treatment with imatinib at six institutions (teaching hospitals and district hospitals) in southeast England. We screened patients for BCR-ABL1 kinase domain mutations using NGS, irrespective of patient response to tyrosine kinase inhibitor therapy. When we detected a mutation with NGS, we retrospectively analysed all previous samples to establish the date of first occurrence and subsequent kinetics of the mutant subclone (or subclones). The primary endpoints of this study were progression-free and event-free survival at 5 years. FINDINGS: Between Feb 1, 2007, and Dec 31, 2014, we screened 121 patients with chronic myeloid leukaemia for BCR-ABL1 kinase domain mutation. 99 consecutive patients were newly diagnosed, with available sequential RNA stored. The remaining 22 patients were diagnosed between June 1, 1999, and June 30, 2006, and were screened at the time of resistance to first-line treatment with imatinib. Imatinib was the first-line treatment for 111 patients, nilotinib for seven patients, and dasatinib for three patients. We detected a kinase domain mutation in 25 (21%) of 121 patients. Low-level kinase domain mutations were first identified in 17 (68%) of 25 patients with mutation. For patients with a complete cytogenetic response, 13 (14%) of 93 patients screened had a mutation. Five (71%) of the seven patients with a clinically relevant mutation lost complete cytogenetic response compared with 15 (17%) of 86 patients without a clinically relevant mutation (80 patients without mutation and six patients with a tyrosine kinase inhibitor-sensitive mutation, p=0·0031). Patients harbouring a mutant clone had poorer 5-year progression-free survival (65·3% [95% CI 40·5-81·8] vs 86·9% [75·8-93·2]; p=0·0161) and poorer 5-year event-free survival (22·2% [CI 5·6-45·9] vs 62·0% [50·4-71·6]; p<0·0001) than did patients without a mutation. We identified a kinase domain mutation in four (10%) of 41 patients with samples available at 3 months after starting first-line tyrosine kinase inhibitor treatment; all four subsequently progressed to accelerated phase disease compared with only three (8%) of 37 without a mutation (p<0·0001). INTERPRETATION: NGS reliably and consistently detected early appearance of kinase domain mutations that would not otherwise be detected by Sanger sequencing. For the first time, to our knowledge, we report the presence of kinase domain mutations after only 3 months of therapy, which could have substantial clinical implications. NGS will allow early clinical intervention and our findings will contribute to the establishment of new recommendations on the frequency of kinase domain mutation analysis to improve patient clinical care. FUNDING: None.

Primary systemic amyloidosis presenting as idiopathic inflammatory colitis.

A 75-year old-female was referred with chest pain. She was fully investigated and it was felt that her symptoms were non-cardiac. Four months later, she was seen in gastroenterology outpatients with bloody diarrhoea and abdominal pain. Colonoscopy demonstrated inflammation up to the splenic flexure and histology confirmed inflammatory colitis. Later, she developed dyspepsia and weight loss. An oesophagogastroduodenoscopy (OGD) showed Helicobacter pylori negative erosive gastritis with a benign duodenal ulcer. Whole body CT scan was normal. Ten months later, she was admitted with dyspnoea due to severe heart failure. The admission ECG had significantly changed, now showing low voltage complexes and repeat echocardiography showed restrictive cardiomyopathy. Specific congo red staining on the biopsy specimens from the previous OGD and colonoscopy confirmed amyloid deposits. Further investigations detected an underlying light chain myeloma causing systemic (AL) amyloidosis. Unfortunately, her condition deteriorated rapidly and she died shortly afterwards.

Severe folate-deficiency pancytopenia.

Folate-deficiency anaemia occurs in about 4 per 100 000 people, although severe cases causing moderate pancytopenia are rarer. We present the case of a significant folate deficiency in a 50-year-old alcoholic with a background of mild liver impairment and recurrent nasal and rectal bleeding. Her blood tests showed profound macrocytic anaemia with haemoglobin 2.6 g/dl, leucopoenia with white cell count 3.2 × 10(9)/litre and thrombocytopenia with platelets 17 × 10(9)/litre. Serum folate was 0.8 ng/ml (normal 2.5-13.5 ng/ml) confirming severe deficiency. Despite these life-threatening results, the patient was stable, alert and was keen to avoid admission. Medical management of the anaemia included slow transfusion of red cells and one unit of platelets in view of haemorrhagic symptoms, two injections of vitamin B12 while awaiting assays and oral folic acid. A rapid improvement in the leucopoenia and thrombocytopenia resulted and no additional complications were encountered.

Translocation t(2;7)(p12;q21-22) with dysregulation of the CDK6 gene mapping to 7q21-22 in a non-Hodgkin's lymphoma with leukemia.

BACKGROUND AND OBJECTIVES: A female patient presented with splenomegaly and lymphocytosis with atypical lymphoid cell morphology. We identified t(2;7)(p12;q21) prompting studies of the translocation breakpoint and its consequences on protein expression to confirm or otherwise the recently reported involvement of CDK6 and IG k genes in the t(2;7) leading to over-expression of CDK6 protein. DESIGN AND METHODS: A variety of clinical and laboratory techniques including cell marker, cytogenetic and histologic studies were applied in order to establish the diagnosis. Fluorescence in situ hybridization (FISH) and Southern blotting were used for mapping the translocation breakpoint and Western blotting for assessing protein expression. RESULTS: Immunophenotyping showed the presence of a B-cell population with strong expression of FMC7, CD22, CD79b, CD5 and k restricted surface immunoglobulins. Based on morphology and immunophenotypic markers the diagnosis of B-cell non-Hodgkin's lymphoma was made. Karyotyping revealed a clone with t(2;7)(p12;q21-22). Evidence for clonal evolution with additional abnormalities including a deletion of the TP53 was present. We established by FISH and Southern blotting that the breakpoint on 7q21-22 fell in a region 66kb telomeric to the previously reported breakpoint for the t(2;7) and was the same as that observed in a t(7;21). CDK6 protein was over-expressed. The patient received alkylating agents and splenectomy and is alive but the lymphocytosis persists with evidence of disease progression. INTERPRETATIONS AND CONCLUSIONS: We have demonstrated that CDK6 expression is dysregulated even when the breakpoint on 7q21-22 is located 66kb upstream from the coding region. Interestingly, the precise assignment of the lymphoma type in our case was not possible even when the splenic histology was analyzed.