application of molecular tests in hematolymphoid pathology

1、The 4th Seminar on Good Laboratory Practice and Operating Training Course on Molecular PathologyApplication of Molecular Tests inHematolymphoid PathologyXiao-Qiu Li, M.D., Ph.D.Fudan University Shanghai Cancer CenterAugust 30, 2015, ShanghaiClinical featuresMorphologyLymphomaEntityImmunophenotypeGen

2、eticsALCLM123CD30ALK1IHCMolecularFCMCase 1ClinicalinformationA 62-year-old man complained of dysphagia Physical examination revealed a left tonsillar massPET/CT scan revealed bilateral cervical lymphadenopathy in addition to the tonsillar massExcisional biopsy of left tonsil assure the diagnosis of

3、lymphoma, with an IPI score of 1Six cycles of RCHOP chemotherapy were instituted, achieving CR, and then HDT+ASCT wasadministeredYour diagnosis?A. Mantle cell lymphomaB. Marginal zone lymphomaC. Burkitt lymphomaD. Lymphoblatic lymphomaE. Diffuse large B-cell lymphomaCD20kappalambdaCD23CD10IgDIgMCD5c

4、yclin D1Ki-67BCL2t(11;14)(q13;q32)/CCND1-IGHPathological diagnosisLeft tonsil, excisional biopsy: Mantle cell lymphomaUnusualclinicopathologic features ofthe present MCL case Morphologically aggressive (blastoid cytology, very high proliferation activity), but clinically not so bad (limited stage, l

5、ow IPI, and sensitive to RCHOP therapy) A peculiar, non-neoplastic mantle zone separating lymphomatous infiltrates andresidual germinal centersProposed model of molecular pathogenesis in the development and progression of MCLDistinction between in situ MCL and MCL with a mantle zone growth patternIn

6、 situMCLMCL with a MZ patternCarbone A, et al. Blood 2011;117(15):3954-60.Carvajal-Cuenca A, et al. Haematologica 2012; 97(2):270-8Normal cell counterpart of MCL: Updated knowledgeMCL has been traditionally regarded as aneoplasm of nave B -cell (pre- t(11;14) occurs at the pre-B stage- Expressing na

7、ve B-cell markerserivation):- Unmutated IG genes (homology with germline sequence)Nevertheless, recent data indicate 15-30% of MCL cases carry a large number of IG gene somatic hypermutations, suggesting a possibleorigin of post-GC cells as wellWhat is somatic hypermutation (SHM) of IG genes?Involvi

8、ng a process of random mutation affecting the variable regions of IG gene (e.g., IGVH)A major component of the process of affinity maturation, leading to the diversity of Ig protein Taking place in GC, and T-cell dependent Germline IG sequence available onlineUsually employing a 98% homology to germ

9、lineIG as the cut-off of SHMSomatichypermutationSHMPre-GCGCPost-GCUnmutatedMutatedLymphomas derived from Pre-GCPre-GCGCPost-GCUnmutatedMutatedLymphomas derived from Post-GCSHMPre-GCGCPost-GCUnmutatedMutatedLymphomas derived from GCSHMyPre-GCGCPost-GCUnmutatedMutatedIntraclonal Heterogeneit Ongoing h

10、ypermutationIntraclonal heterogeneityIntraclonal heterogeneity indicating ongoing hypermutation process of IG gene Demonstration of intraclonal heterogeneity inlymphomas suggestingerivationPost-GC B-cells lacking intraclonal heterogeneityMethodology for detecting intraclonal heterogeneityWhole tissu

11、e DNA extractionPCR amplification of rearranged IG gene (containing multiple subclones) Transfection plasmid habouring PCR products into E. coli Selecting 8 individual colonies (containing single subclone), sequencing and alignmentSubclone 1from 8859349499911042to 93394899010411137length 4915425196m

12、atches 4914425196mismatches 01000Gaps 00000identity(%) 10093.3100100100FWR1 CDR1 FWR2 CDR2 FWR3CDR3 (Vregion only)Total1138113922001002552541099.6Subclone 2fromtolengthmatchesmismatchesgapsidentity(%)FWR1 CDR1 FWR2 CDR2 FWR3CDR3 (Vregion only)Total5410211715921010111615820930548154251964814415195011

13、010000010093.397.61009930630722001002542513098.8Contradictory findings on the molecular changes of the present case IGVH seemed unmutated (98% homology)- Pre-erivation? But intraclonal heterogeneity was demonstrated- Originating from GC B-cells?cyclin D1MZL of MALTMZL of MALTSplenic MZLNodal MZLIs i

14、t likely a MCL deriving from outer mantle/marginal zone?Inspiration drawn from splenic marginal zone lymphoma (SMZL)Ongoing mutation of IG gene has already been demonstrated in SMZLSMZL cases showing ongoing mutation are not restricted to those with hypermutated IG ones, tooZhu D, et al. Blood 2002,

15、 100(7):2659-61Tierens A, et al. Am J Pathol 2003,162(2):681-9Traverse-GlehenA, et al. Haematologica 2005, 90(4):470-8Updated notion on SHM of IGTaking place in GCLow level mutation can happen in ectopic site outside GCT-cell dependent Sometimes unnecessary 98% homology to germline indicxating unmut

16、atedAny mutation leading to animo acid replacement makes sense biologicallyPrecursor B-cell neoplasmsB-LBL/LPre-GC neoplasmMCL, CLL/SLLGC neoplasmsFL, BL DLBCL (some), HLPost-GC neoplasmsMZLs, LPL, CLL/SLLDLBCL (some), myelomaCase 2Male, 44 yrs, bilateral tonsillar massBCL2BCL2BCL2Your diagnosis?A. Follicular lymphomaB. Mantle cell lymphomaC. Marginal zone lymphomaD. CLL/SLLE. Other than above listedCD20BCL2IgDBCL6CD20CD10BCL2BCL6CD5IgDcyclin D1Ki-67BCL2t(14;18)(q32;q21)/IGH-BCL2DiagnosisA. Follicular lymphomaB. Mantle cell lymphomaC. Marginal zone lymphomaD. CLL/