BackgroundPigmentary mosaicism constitutes a heterogeneous group of skin pigmentation alterations associated with multisystem involvement. The aim of this study was to establish a complete cytogenetic and molecular characterization of PM patients, emphasizing on searching for possible low chromosomal mosaicism and on establishing an accurate genotype-phenotype correlation.ResultsA total of 73 patients were included (3 months to 18 years of age), 52% male and 48% female. Observed in 69 (95%) patients, the most frequent pattern of pigmentation was fine and whorled BL, which was associated with disseminated skin extent in 41 (59%) patients. Central nervous system (84%) alterations were the most frequent observed in the group of patients, followed by the musculoskeletal (53%) and ophthalmologic (27%) alterations. Considering the pattern of pigmentation, no significant differences in association with skin extent or extracutaneous manifestations were detected. Following a strict cytogenetic analysis strategy, screening metaphases from three different tissues (peripheral blood, hyperpigmented and hypopigmented skin) we found that 23/73 patients had chromosomal abnormalities classified as follows: 1) Mosaic with 2 or more different cell lines with structural alterations n = 19; 2) Polyploidy (mosaic) n = 1 and 3) Alterations in all cells in three different tissues n = 3. SNP array, array CGH and FISH were useful for the complete characterization of the chromosomal aberrations, for the detection of microdeletions in patients with normal karyotype but with strong clinical suspicious of chromosomal alteration, and for a better establishment of genotype-phenotype correlation. In 2 patients we found genes associated with some of the extracutaneous manifestations (SHH, MNX1, PPP2R2C).ConclusionsThis group of 73 patients finely described is the largest series of patients with pigmentary mosaicism reported worldwide. As we showed in this study, the followed analysis strategy allowed the detection of cytogenetic and molecular abnormalities, and made possible the establishment of genotype-phenotype associations in some patients. An important limitation of our study was the analysis of fibroblasts cultures instead of melanocytes and keratinocytes. In some cases the direct molecular DNA analysis of skin biopsy could be another choice.

Ph-like subtypes with CRLF2 abnormalities are frequent among Hispano–Latino children with pre-B ALL. Therefore, there is solid ground to suggest that this subtype is frequent in Mexican patients. The genomic complexity of Ph-like subtype constitutes a challenge for diagnosis, as it requires diverse genomic methodologies that are not widely available in diagnostic centers in Mexico. Here, we propose a diagnostic strategy for Ph-like ALL in accordance with our local capacity. Pre-B ALL patients without recurrent gene fusions (104) were classified using a gene-expression profile based on Ph-like signature genes analyzed by qRT-PCR. The expressions of the CRLF2 transcript and protein were determined by qRT-PCR and flow cytometry. The P2RY8::CRLF2, IGH::CRLF2, ABL1/2 rearrangements, and Ik6 isoform were screened using RT-PCR and FISH. Surrogate markers of Jak2-Stat5/Abl/Ras pathways were analyzed by phosphoflow. Mutations in relevant kinases/transcription factors genes in Ph-like were assessed by target-specific NGS. A total of 40 patients (38.5%) were classified as Ph-like; of these, 36 had abnormalities associated with Jak2-Stat5 and 4 had Abl. The rearrangements IGH::CRLF2,P2RY8::CRLF2, and iAMP21 were particularly frequent. We propose a strategy for the detection of Ph-like patients, by analyzing the overexpression/genetic lesions of CRLF2, the Abl phosphorylation of surrogate markers confirmed by gene rearrangements, and Sanger sequencing.

Background: Philadelphia-like (Ph-like) subtype occurs in 15-20% of children patients diagnosed with precursor B cell (PCB) acute lymphoblastic leukemia (ALL). It is characterized by alterations in kinases and their receptors, affecting signaling pathways such as Jak2-Stat5, class Abl and Ras. It is particularly frequent (35%) in Hispano-Latino high risk PCB-ALL patients, residing in the USA. In Mexico, based on our ethnicity and the poor response to treatment of PCB-ALL pediatric patients, the Ph-like subtype could be frequent. However, this entity is widely variable and the diagnosis is based on genomic methods. In our country the incidence of leukemia is high, 78.1/1,000,000, and 83% of these cases are ALL. Therefore, it is important to detect Ph-like patients using diagnostic methods accessible to Institutions that treat patients with leukemia in our country. Aims: To develop an algorithm adapted to our local capacity for the diagnosis of Ph-like ALL children in Mexico. To determine the frequency of Ph-like patients in two Pediatric Institutions in our country Hypothesis: Ph-like ALL frequency in Mexican children will be higher than that reported in Hispano-Latino PCB-ALL patients. Methods, study design and laboratory studies: Bone marrow samples from 119 PCB-ALL patients (18≤ years old) at diagnosis were studied. Samples were collected at the Instituto Nacional de Pediatría and the Hospital Infantil de México Federico Gómez in Mexico City. Patients with recurrent gene fusions, ETV6-RUNX1, TCF3-PBX1,KMT2A-var and BCR-ABL1, were excluded. The Ph-like status of each patient was determined using molecular and biochemical methods. We analyzed a gene expression signature conformed by the genes: CRLF2, TSPN7, IGJ, PON2, SEMA6A and BMPR1B (q-RT-PCR), and detected the P2RY8-CRLF2 deletion (RT-PCR) in all patients. In those patients with overexpression of CRLF2, but negative to P2RY8-CRLF2, the IGH-CRLF2 rearrangement (FISH) was examined. The positive cases to P2RY8-CRLF2, without overexpression of CRLF2, were analyzed looking for minor subclones (FISH) with the deletion. IKZF1 deletions (nested RT-PCR) were analyzed in all patients. The biochemical studies included CRLF2 protein expression, phosphoflow analysis of Jak2-Stat5, class Abl and Ras targets (flow cytometry). All these assays were performed whenever the cell sample allowed it. Patients with two or more of the following characteristics were considered as Ph-like cases: with overexpression of 50% or more of the expression signature, positive to P2RY8-CRLF2 or IGH-CRLF2, alteration of one or more signaling pathways, with IKZF1 deletions. Results: In 50% (n=74) of patients CRLF2 overexpression was detected, within them 15% were positive to P2RY8-CRLF2 deletion, 17% to IGH-CRLF2 rearrangement, and 18% had an unidentified CRLF2 alteration that cause the overexpression. Within the 50% of cases without CRLF2 overexpression, 18% were positive to the P2RY8-CRLF2 rearrangement. IKZF1 deletion was evaluated in 55 patients and 72% were positive. Results obtained revealed a high frequency of CRLF2 and IKZF1. In 68 patients the gene expression profile was evaluated, 60% presented overexpression of 50% or more genes. The biochemical analysis revealed that 67% (n=42) of patients were positive to CRLF2 protein, 29% (n=21) had abnormal Jak2/Stat5 activation, 34% (n=32) presented abnormal Abl activation, and in 43% (n=7) abnormal Ras activation was detected. Not all the CRLF2 protein positive patients presented Jak2-Stat5 pathway activation (2%). Patients with more than one abnormal pathway were detected (18% with Jak2-Stat5/Abl or Abl/Ras, n=21). Based on our algorithm of analysis, 53% of the analyzed patients present genetic and biochemical characteristics of the Ph-like subtype. Conclusions: The combined methods contributed to identify Ph-like patients, the results demonstrate that the frequency of PCB-ALL children with Ph-like characteristics in Mexican patients is higher compared to other populations. This study demonstrates high frequency of CRLF2 alterations and IKZF1 deletions in PCB-ALL in Mexican population. In our experience, analysis of the pathway activation assays and the identification of CRLF2 and IKZF1 alterations are required to detect Ph-like patients. These results could be useful to stratify the childhood PCB-ALL patients in our country. Acknowledgments: CONACYT: PDCPN-2004/248591; Cátedra 2038. Disclosures No relevant conflicts of interest to declare.

CRLF2 is a member of type I cytokine receptor family. CRLF2 forms a functional complex with IL-7 receptor α chain and thymic stromal lymphopoietin, this complex induces the activation of signal transducers and activators of transcription proteins. The overexpression of CRLF2 induced by genetic rearrangements has been described in acute lymphoblastic leukemia.

Background To date, only twenty-one cases diagnosed postnatally with mosaic trisomy 12 have been reported. The most frequent phenotypic manifestations are developmental delay, dysmorphic facial features, congenital heart defects, digital alterations, and pigmentary disorders. In the present report, detailed clinical and genetic profiles of three unrelated new patients with mosaic trisomy 12 are described and compared with previously reported cases. Case presentation In the present report, we include the clinical, cytogenetic, and molecular description of three Mexican patients diagnosed postnatally with mosaic trisomy 12. At phenotypic level, the three patients present with developmental delay, dysmorphic facial features, congenital heart defects and skin pigmentary anomalies. Particularly, patient 1 showed unique eye alterations as bilateral distichiasis, triple rows of upper lashes, and digital abnormalities. In patient 2 redundant skin, severe hearing loss, and hypotonia were observed, and patient 3 presented with hypertelorism and telecanthus. Hyperpigmentation with disseminated pigmentary anomalies is a common trait in all of them. The cytogenetic study was carried out under the strict criteria of analysis, screening 50–100 metaphases from three different tissues, showing trisomy 12 mosaicism in at least one of the three different tissues analyzed. With SNParray, the presence of low-level mosaic copy number variants not previously detected by cytogenetics, and uniparental disomy of chromosome 12, was excluded. STR markers allowed to confirm the absence of uniparental disomy as well as to know the parental origin of supernumerary chromosome 12. Conclusions The detailed clinical, cytogenetic, and molecular description of these three new patients, contributes with relevant information to delineate more accurately a group of patients that show a heterogeneous phenotype, although sharing the same chromosomal alteration. The possibility of detecting mosaic trisomy 12 is directly associated with the sensitivity of the methodology applied to reveal the low-level chromosomal mosaicism, as well as with the possibility to perform the analysis in a suitable tissue.