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Posterior polymorphous corneal dystrophy (PPCD) is a rare, bilateral autosomal dominant disorder affecting primarily the corneal endothelium and descemet membrane (DM). The aim of this study was to establish the origin of abnormal endothelium in a patient with PPCD exhibiting cornea graft failure after keratoplasty surgery. A sex-mismatched graft obtained from a patient with PPCD who underwent repeat penetrating keratoplasty and the patient's original cornea were investigated. Combined fluorescent immunohistochemistry for cytokeratin (CK) 19 (a marker of aberrant PPCD endothelium) with fluorescence in situ hybridization (FISH) of the sex chromosomes were used in order to characterize the cells on the posterior graft surface. The pathological endothelium of the failed PPCD cornea revealed strong positivity for CK19 using fluorescent immunohistochemistry. In all the CK19-positive cells, both X and Y chromosomes were simultaneously detected using FISH. The results clearly showed the original cells of the patient (XY), within 3.5 years, almost totally overgrown the posterior corneal surface of the graft (XX). Moreover, an abnormal posterior collagenous layer populated by fibroblast-like cells was observed between DM and the endothelium in the failed graft, but its exact origin could not be established due to the low number of cells. Simultaneous detection of CK19 using fluorescent immunohistochemistry together with the detection of gonosomes using FISH was performed for the first time in the cornea and allowed us to prove that the recurrence of PPCD was caused by pathological abnormal proliferation and migration of recipient cells into donor graft.

Monosomy 7 and/or deletion of the long arm of chromosome 7 is a common cytogenetic aberration in children with myelodysplastic syndrome (MDS) and is associated with poor outcome. In this report, we present an unusual cytogenetic abnormality leading to loss of both the whole short and whole long arms of chromosome 7, which was found in the bone marrow cells of three pediatric patients with MDS. Using a combination of conventional and molecular cytogenetic methods, a tiny "dot-like" marker chromosome was found and described as der(7)del(7)(p11)del(7)(q11). Together with one previously published case, this chromosomal aberration represents a new rare recurrent karyotypic abnormality involving chromosome 7 in children with MDS.

Gene amplification is a frequent genetic abnormality in solid tumors, and many oncogenes are activated in this way. In acute myeloid leukemia (AML), a frequent target of gene amplification is chromosome 11, particularly chromosome region 11q23, including the MLL (myeloid/lymphoid leukemia) gene. However, the number of other amplicons from the long arm of chromosome 11 has also been described. Duplication/amplification of chromosome 11 was found by cytogenetic methods in 10 of 119 newly diagnosed patients with AML. The amplification was presented as: amplification including only the 5' segment of the MLL gene (1 patient), trisomy 11 (3 patients), partial trisomy 11q (2 patients), isochromosome 11q (1 patient), and multiple amplification of specific regions (3 patients). In two cases, amplification involved parts of not only long arm but also of short arm of the chromosome 11: 11p15 and 11p11.1 to 11p13.

Unusual MLL gene rearrangements were found in bone marrow cells of four patients with acute myeloid leukemia. A combination of conventional and molecular cytogenetic methods were used to describe translocations t(9;12;11)(p22;p13;q23), t(11;19)(q23;p13.3), and t(10;11)(p12;23) and inverted insertion ins(10;11)(p12;q23.3q23.1). Partial nontandem duplication of the MLL gene was identified by reverse transcriptase-polymerase chain reaction in all cases. The duplication, which included MLL exons 2 through 8-9, was interrupted by a cryptic insertion of one or two exons from the respective MLL partner gene: MLLT10, MLLT3, or MLLT1.

Background Both high hyperdiploidy (HeH) and the translocation t(9;22)(q34;q11) are recurrent abnormalities in childhood B-cell acute lymphoblastic leukemia (ALL) and both are used in current classification to define different genetic and prognostic subtypes of the disease. The coexistence of these two primary genetic aberrations within the same clone is very rare in children with ALL. Here we report a new case of a 17-year-old girl with newly diagnosed ALL and uncommon cytogenetic and clinical finding combining high hyperdiploidy and a cryptic BCR/ABL1 fusion and an inherited Charcot-Marie-Tooth neuropathy detected during the induction treatment. Results High hyperdiploid karyotype 51,XX,+X,+4,+14,+17,+21 without apparent structural aberrations was detected by conventional cytogenetic analysis and multicolor FISH. A cryptic BCR/ABL1 fusion, which was caused by the insertion of part of the ABL1 gene into the 22q11 region, was proved in HeH clone by FISH, RT-PCR and CGH-SNP array. In addition, an abnormal FISH pattern previously described as the deletion of the 3′BCR region in some BCR/ABL1 positive cases was not proved in our patient. Conclusion A novel case of extremely rare childhood ALL, characterized by HeH and a cryptic BCR/ABL1 fusion, is presented and to the best of our knowledge described for the first time. The insertion of ABL1 into the BCR region in malignant cells is supposed. Clearly, further studies are needed to determine the genetic consequences and prognostic implications of these unusual cases.

Purpose To establish the origin of cells causing the recurrence of posterior polymorphous corneal dystrophy (PPCD). Methods A corneal explant obtained from a male patient suffering from PPCD, who had undergone repeat penetrating keratoplasty (female donor), was examined. Cytokeratin (CK) 19 was detected using indirect immunofluorescence to confirm the presence of abnormal epithelial-like cells on the endothelial layer. The origin of these CK19-positive aberrant cells was then determined using fluorescence in situ hybridization of the X and Y chromosomes. Results Two out of 559 cells evaluated (0.4%) were XX positive and CK19-negative throughout the pathological explant. 99.6% of the endothelial cells were positive for CK19 and exhibited XY staining. These results indicate that the abnormal endothelial cells, which overgrew the posterior surface of the explant within 3.5 years, originated from the patient (XY) but not from the donor tissue (XX). Extreme aggressivity of the cells could be clinically correlated with the finding of retrocorneal membrane on the iris, anterior synechiae, corectopia and secondary glaucoma. Conclusion PPCD recurrence is caused by the abnormal proliferation and migration of the patient’s aberrant endothelium, which remained localized at the periphery of the original cornea, into the donor graft. The presence of retrocorneal membrane prior to the surgery should be considered as an indication of potential aggressive behavior of these cells and it should be taken into consideration prior to the indication of corneal grafting.

UNLABELLED Telomere length was evaluated by terminal repeat fragment method in 66 previously untreated patients with B-chronic lymphocytic leukemia (B-CLL) to ascertain whether telomere shortening was associated with genomic aberrations, immunoglobulin variable heavy chain (IgVH) mutational status, CD38 and ZAP-70 expression, and telomerase activity. Chromosomal aberrations were present in peripheral blood cells of 73% patients (48/66), no difference in telomere length between patients with good and intermediate prognosis according to cytogenetics was found. Association between telomere length and IgVH mutational status, ZAP-70 and CD38 expression was proved as significantly shorter telomeres in patients with unmutated IgVH status (p=0.01) and ZAP-70 positivity (p=0.01) and CD38 positivity (p=0.05) were detected. Telomerase activity was positive in 11 patients out of 21 examined, correlation between telomere length and telomerase activity was found (p=0.05). Telomere length and telomerase activity in combination with other prognostic parameters complete the risk profile of B-CLL patients and might serve for an easy decision on optimal treatment strategy. KEYWORDS B-chronic lymphocytic leukemia, telomere length, telomerase activity, chromosomal aberrations, prognosis.

Abstract 1306 Introduction Acute lymphoblastic leukemia (ALL) can rarely be manifested before its diagnosis as pancytopenia and bone-marrow aplasia followed by spontaneous transient hematological remission. A leukemic clone can be detected during this particular period, making it possible to follow its dynamics in size, as well as independent changes leading to the overt disease. Patients and methods We initially selected 5 cases of ALL, in which the pre-diagnostic period of anemia had occurred. In all pre-diagnostic samples, the immunoglobulin (Ig) and/or T-cell receptor (TCR) gene rearrangements were investigated using quantitative real-time PCR. In addition, SNP array and FISH were performed in some cases in order to reveal other aberrations specific to leukemia. To backtrack the preleukemic cells three different approaches were employed - qPCR, FISH and next generation sequencing (NGS). Results Immunoreceptor gene rearrangements were detected in all 5 patients in the diagnostic sample. At least one of the diagnostic rearrangements was found in all preleukemic bone-marrow samples. The levels of positivity differed from 10−1 to 10−6. In 3 patients more than one pre-diagnostic sample was made available for our examination. A surprising dynamics in the size of the leukemic clone was revealed by the investigation. Although a steady increase of blasts was expected, particularly high levels occurred in the first available preleukemic samples (10% and more clonal Ig/TCR positive cells compared to the diagnosis, in 4/5 of patients), followed by remarkable decrease 1 to 3 months before the diagnosis in 3 patients with more than one sample available. This dynamics points to possible immune system interventions during preleukemic phase. In 2 patients NGS (targeted to Ig/TCR gene rearrangements, Adaptive Biotechnologies, Seattle) was applied. The acquired data correlated with the results of qPCR backtracking. In addition, more detailed information was gained about gene rearrangement spectra as well as about possible immunological background of the leukemia progression. In both these patients, the examination revealed a significant fraction of non-malignant γΔ T cell-clone carrying an identical Vg9-Jg1.2 gene rearrangement, which has been reported previously to play an important role in anti-tumour and anti-microbe immune responses. Interestingly, the dynamics of this particular clone seemed to correlate negatively with the leukemic cell levels, pointing out its putative role in tumour surveillance. Consequently, SNP array was performed to define the aberrations acquired, followed by FISH investigation to confirm the above conclusions. While some aberrations were confirmed already in the pre-diagnostic samples (namely ETV6 deletion in the patient with ETV6/RUNX1 positive ALL), others occurred in the diagnostic sample, yet not in the preleukemic cells several months before the diagnosis. Conclusion Preleukemic cells present during the aplastic phase preceding leukemia onset are not just a gradually growing clone. Their specific dynamics has been shown, which supports our previous data acquired during the investigation of secondary leukemias. Furthermore, at least in some cases, this clone indeed should be considered ‘preleukemic’ as it is to undergo at least one more hit or subclonal selection to progress into an active disease. Disclosures: No relevant conflicts of interest to declare.