Lessons learnt from implementation of a Lynch syndrome screening program for patients with gynaecological malignancy.

Despite a trend towards universal testing, best practice to screen patients presenting with gynaecological malignancy for Lynch syndrome (LS) is uncertain. We report our institutional experience of a co-ordinated gynaecological LS screening program. All patients with endometrial carcinoma or carcinosarcoma, or gynaecological endometrioid or clear cell carcinomas undergo reflex four panel immunohistochemistry (IHC) for MLH1, PMS2, MSH2 and MSH6 followed by cascade somatic hypermethylation analysis of the MLH1 promoter locus for dual MLH1/PMS2 negative tumours. On the basis of these results, genetic counselling and targeted germline mutation testing is then offered to patients considered at high risk of LS. From 1 August 2013 to 31 December 2015, 124 patients were screened (mean age 64.6 years). Thirty-six (29.0%) demonstrated abnormal MMR IHC: 26 (72.2%) showed dual loss of MLH1/PMS2, five (13.9%) dual loss of MSH2/MSH6, three (8.3%) isolated loss of MSH6, and two (5.6%) isolated loss of PMS2. Twenty-five of 26 (96.1%) patients with dual MLH1/PMS2 loss demonstrated MLH1 promoter methylation. Therefore, 11 (8.9%) patients screened were classified as high risk for LS, of whom nine (81.8%) accepted germline mutation testing. Three (2.4% of total screened) were confirmed to have LS, two with germline PMS2 and one with germline MSH2 mutation. Massive parallel sequencing of tumour tissue demonstrated somatic mutations which were concordant with the IHC results in the remainder. Interestingly, the one MLH1/PMS2 IHC negative but not hypermethylated tumour harboured only somatic MLH1 mutations, indicating that universal cascade methylation testing in MLH1/PMS2 IHC negative tumours is very low yield and could be reconsidered in a resource-poor setting. In conclusion, universal screening for LS in patients presenting with gynaecological malignancy using the algorithm described above identified LS in three of 124 (2.4%) of our population. Only three of nine (33.3%) patients considered at high risk for LS by combined IHC and hypermethylation analysis were proven to have LS. Only one of the LS patients was less than 50 years of age and none of these patients would have been identified had more restrictive Amsterdam or Bethesda criteria been applied.

Poor response to second-line kinase inhibitors in chronic myeloid leukemia patients with multiple low-level mutations, irrespective of their resistance profile.

Specific imatinib-resistant BCR-ABL1 mutations (Y253H, E255K/V, T315I, F317L, and F359V/C) predict failure of second-line nilotinib or dasatinib therapy in patients with chronic myeloid leukemia; however, such therapy also fails in approximately 40% of patients in the chronic phase of this disease who do not have these resistant mutations. We investigated whether sensitive mutation analysis could identify other poor-risk subgroups. Analysis was performed by direct sequencing and sensitive mass spectrometry on 220 imatinib-resistant patients before they began nilotinib or dasatinib therapy. Patients with resistant mutations by either method (n = 45) were excluded because inferior response was known. Of the remaining 175 patients, 19% had multiple mutations by mass spectrometry versus 9% by sequencing. Compared with 0 or 1 mutation, the presence of multiple mutations was associated with lower rates of complete cytogenetic response (50% vs 21%, P = .003) and major molecular response (31% vs 6%, P = .005) and a higher rate of new resistant mutations (25% vs 56%, P = .0009). Sensitive mutation analysis identified a poor-risk subgroup (15.5% of all patients) with multiple mutations not identified by standard screening.

Sensitive detection of BCR-ABL1 mutations in patients with chronic myeloid leukemia after imatinib resistance is predictive of outcome during subsequent therapy.

PURPOSE: BCR-ABL1 mutation analysis is recommended to facilitate selection of appropriate therapy for patients with chronic myeloid leukemia after treatment with imatinib has failed, since some frequently occurring mutations confer clinical resistance to nilotinib and/or dasatinib. However, mutations could be present below the detection limit of conventional direct sequencing. We developed a sensitive, multiplexed mass spectrometry assay (detection limit, 0.05% to 0.5%) to determine the impact of low-level mutations after imatinib treatment has failed. PATIENTS AND METHODS: Mutation status was assessed in 220 patients treated with nilotinib or dasatinib after they experienced resistance to imatinib. RESULTS: Mutations were detected by sequencing in 128 patients before commencing nilotinib or dasatinib therapy (switchover). In 64 patients, 132 additional low-level mutations were detected by mass spectrometry alone (50 of 132 mutations were resistant to nilotinib and/or dasatinib). When patients received the inhibitor for which the mutation confers resistance, 84% of the low-level resistant mutations rapidly became dominant clones detectable by sequencing, including 11 of 12 T315I mutations. Subsequent complete cytogenetic response rates were lower for patients with resistant mutations at switchover detected by sequencing (0%) or mass spectrometry alone (16%) compared with patients with other mutations or no mutations (41% and 49%, respectively; P < .001). Failure-free survival among the 100 patients with chronic phase chronic myeloid leukemia when resistant mutations were detected at switchover by sequencing or mass spectrometry alone was 0% and 0% compared with 51% and 45% for patients with other mutations or no mutations (P = .003). CONCLUSION: Detection of low-level mutations after imatinib resistance offers critical information to guide subsequent therapy selection. If an inappropriate kinase inhibitor is selected, there is a high risk of treatment failure with clonal expansion of the resistant mutant.