Molecular monitoring of response to imatinib (Glivec) in chronic myeloid leukemia patients: experience at a tertiary care hospital in Saudi Arabia.

AIM: The aim of this study was to evaluate the response and resistance of cases to chronic myeloid leukemia (CML) therapy with tyrosine kinase (TK) inhibitors (imatinib mesylate) and to search for mutations in the breakpoint cluster region (BCR)-Abelson murine leukemia (ABL) kinase domain prior to and during therapy. METHODS: Molecular response was assessed with real-time quantitative reverse transcription-polymerase chain reaction and was expressed as the ratio between BCR-ABL and ABL (k562 cell line) x 100. In addition, we searched for mutations in BCR-ABL kinase domain by amplification and direct sequencing of cDNA products of archived RNA samples. RESULTS: There were 85 cases of CML Philadelphia-chromosome-positive patients. Major molecular response [corrected] (MMR) of 0.05% was achieved in 40 (47%) of 85 patients and 3-log reduction was achieved in 37 (44%) after 6 months of imatinib therapy. When molecular monitoring was extended to 12 months in a subset of delayed responsive cases (17 cases) who did not achieve an MMR at 6 months, significant changes in BCR-ABL/ABL ratio were noticed. Fifteen de novo CML patients were started directly on treatment and were monitored for BCR-ABL/ABL ratio for a further period of up to 24 months. Their median of BCR-ABL/ABL ratio was 18% at diagnosis, 0.3% after 6 months, 0.2% after 12 months, and 0.01% after 18 and 24 months. Four (27%) of 15 patients achieved MMR as 3-log reduction after 6 months, 6 (40%) after 12 months, 9 (60%) after 18 months, and 7 (46%) after 24 months. No mutation(s) or polymorphism(s) were detected in all tested patients at diagnosis, at 6 months following imatinib and following 12 months for patients showing delayed response. CONCLUSION: BCR-ABL mutations are rare in early chronic phase and increases with CML disease progression. Therefore, search for other causes in resistant cases at this phase should be sought.

The applicability of T-cell receptor gamma gene rearrangement as an adjuvant diagnostic tool in skin biopsies for cutaneous T-cell lymphoma.

OBJECTIVE: The diagnosis of cutaneous T-cell lymphoid infiltrates may be difficult based on clinical and routine immunohistologic findings. In this situation, an ancillary technique demonstrating the presence of a monoclonal cell proliferation could help to rule in or out cutaneous T-cell lymphoma (CTCL) in cases that clinically and histopathologically do not allow a definitive diagnosis. Southern blot analysis is a time-consuming method with low sensitivity that should not be considered for the routine diagnosis of cutaneous lymphoid infiltrates. Moreover, it can be used only when fresh tissue is available. New assays based on the amplification of the T-cell receptor gamma (TCR gamma) chain gene rearrangement by polymerase chain reaction (PCR) have been proposed to overcome these limitations. METHODS: We retrospectively studied 124 biopsies from 104 patients (66 biopsies with the clinical and histological diagnosis or suspicious of CTCL and 58 biopsies with histological diagnosis of benign reactive dermatological conditions who presented to the Dermatology Unit at King Faisal Specialist Hospital and Research Center, Riyadh, Kingdom of Saudi Arabia between 1996 and 2004. The specimens were morphologically examined and then analyzed by PCR for the gamma chain of the TCR gamma followed by gel electrophoresis. RESULTS: The results showed 87.1% sensitivity and 92% specificity in detecting clonal T-cell gene rearrangements among CTCL cases with a positive predictive value of 93.1% and negative predictive value of 85.2%. Therefore, negative TCR gamma results in CTCL should be taken with caution. CONCLUSION: The detection of clonal TCR gamma gene rearrangement by PCR based method is an adjuvant diagnostic marker for CTCL, although it can be seen in some benign dermatoses.

Molecular hematology. Qualitative to quantitative techniques.

Over the last decade molecular diagnostics technology has developed dramatically from the most laborious, time- consuming southern blot methodology through the revolution of polymerase chain reaction PCR technology to the most reliable, fast, and contamination free molecular analyzer, the real-time quantitative-PCR. The Section of Hematology, Department of Pathology and Laboratory Medicine at King Faisal Specialist Hospital and Research Center has shared this experience during the last 10 years with more than 6,546 samples submitted for the analysis of different gene rearrangements, fusion gene transcripts and gene mutations including Ig heavy chain gene rearrangement for B-cell malignancies, T-cell receptor gamma chain gene rearrangement for T-cell malignancies, BCR/ABL-P210 and P190 fusion gene transcripts, for chronic myeloid leukemia and Philadelphia positive acute lymphoblastic leukemia, PML/RARalpha fusion gene for promyelocytic leukemia, AML1/ETO for acute myeloid leukemia AML-M2 with t8;21, CBFB/MYH11 for AML M4E0 with inv 16, BCL-2 for follicular lymphoma, and BCL-1 for mantle cell lymphoma. Hence, most molecular assays are qualitative in nature, quantitative assays are deemed necessary in the monitoring and follow-up of minimal residual disease in leukemia and lymphoma, and proved in our experience to serve as an essential tool to confirm complete remission CR post-chemotherapy and bone marrow transplantation, and to detect signs of early relapse for proper clinical intervention. In this manuscript, we retrospectively review our experience in molecular hematology and propose our recommended guidelines at King Faisal Specialist Hospital and Research Center.

Bone marrow examination in staging of Lymphoma: Revisited.

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