Whilst heterozygous germline mutations in the ABRAXAS1 gene have been associated with a hereditary predisposition to breast cancer, their effect on promoting tumourigenesis at the cellular level has not been explored. Here, we demonstrate in patient-derived cells that the Finnish ABRAXAS1 founder mutation (c.1082G>A, Arg361Gln), even in the heterozygous state leads to decreased BRCA1 protein levels as well as reduced nuclear localization and foci formation of BRCA1 and CtIP. This causes disturbances in basal BRCA1-A complex localization, which is reflected by a restraint in error-prone DNA double-strand break (DSB) repair pathway usage, attenuated DNA damage response and deregulated G2-M checkpoint control. The current study clearly demonstrates how the Finnish ABRAXAS1 founder mutation acts in a dominant-negative manner on BRCA1 to promote genome destabilisation in heterozygous carrier cells.

Abstract Whilst heterozygous germline mutations in the ABRAXAS gene have been associated with hereditary breast cancer predisposition, their initial effect on promoting tumorigenesis at the cellular level has not been explored. Here, we demonstrate in patient-derived cells that the Finnish ABRAXAS founder mutation (c.1082G>A, Arg361Gln), even in the heterozygous state leads to decreased BRCA1 protein levels as well as reduced nuclear localization and foci formation of BRCA1 and CtIP. This causes disturbances in basal BRCA1-A complex localization, which is reflected by a restraint in error-prone DNA double-strand break (DSB) repair pathway usage, attenuated DNA damage response, deregulated G2-M checkpoint control and apoptosis. Most importantly, mutation carrier cells display a change in their transcriptional profile, which we attribute to the reduced nuclear levels of BRCA1. The current study clearly demonstrates how the Finnish ABRAXAS founder mutation acts in a dominant-negative manner on BRCA1 to promote genome destabilisation in heterozygous carrier cells. Citation Format: Robert Winqvist, Muthiah Bose, Juliane Sachsenweger, Niina Laurila, Ann Christin Parplys, Jonas Willmann, Leila Eshraghi, Thomas W. Dunlop, Marco Groth, Katrin Rapakko, Pentti Nieminen, Thomas W. Friedl, Lisa Heiserich, Felix Meyer, Hanna Tuppurainen, Ganapathy Raman Devarajan, Hellevi Peltoketo, Heli Nevanlinna, Katri Pylkäs, Kerstin Borgmann, Lisa Wiesmuller, Helmut Pospiech. Heterozygous germline mutation in ABRAXAS causes BRCA1 mislocalization and DNA damage response defects [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 339.

Diffuse large B-cell lymphoma (DLBCL) is an aggressive lymphoma with diverse outcomes. Concurrent translocation of MYC and BCL-2 and/or BCL-6 , and concurrent immunohistochemical (IHC) high expression of MYC and BCL-2, have been linked to unfavorable treatment responses. TP53 -mutated DLBCL has also been linked to worse outcome. Our aim was to evaluate the aforementioned issues in a cohort of 155 patients uniformly treated with R-CHOP-like therapies. We performed direct sequencing of TP53 exons 5, 6, 7 and 8 as well as fluorescence in-situ hybridization (FISH) of MYC , BCL-2 and BCL-6 , and IHC of MYC, BCL-2 and BCL-6. In multivariate analysis, TP53 mutations in L3 and loop-sheet helix (LSH) associated with a risk ratio (RR) of disease-specific survival (DSS) of 8.779 (p = 0.022) and a RR of disease-free survival (DFS) of 10.498 (p = 0.011). In IHC analysis BCL-2 overexpression was associated with inferior DFS (p = 0.002) and DSS (p = 0.002). DLBCL with BCL-2 and MYC overexpression conferred inferior survival in all patients (DSS, p = 0.038 and DFS, p = 0.011) and in patients with non-GC phenotype (DSS (p = 0.013) and DFS (p = 0.010). Our results imply that in DLBCL, the location of TP53 mutations and IHC analysis of BCL-2 and MYC might have a role in the assessment of prognosis.

Several known breast cancer susceptibility genes encode proteins involved in DNA damage response (DDR) and are characterized by rare loss-of-function mutations. However, these explain less than half of the familial cases. To identify novel susceptibility factors, 39 rare truncating mutations, identified in 189 Northern Finnish hereditary breast cancer patients in parallel sequencing of 796 DDR genes, were studied for disease association. Mutation screening was performed for Northern Finnish breast cancer cases (n = 578–1565) and controls (n = 337–1228). Mutations showing potential cancer association were analyzed in additional Finnish cohorts. c.7253dupT in TEX15 , encoding a DDR factor important in meiosis, associated with hereditary breast cancer ( p = 0.018) and likely represents a Northern Finnish founder mutation. A deleterious c.2715 + 1G > A mutation in the Fanconi anemia gene, FANCD2 , was over two times more common in the combined Finnish hereditary cohort compared to controls. A deletion (c.640_644del5) in RNF168 , causative for recessive RIDDLE syndrome, had high prevalence in majority of the analyzed cohorts, but did not associate with breast cancer. In conclusion, truncating variants in TEX15 and FANCD2 are potential breast cancer risk factors, warranting further investigations in other populations. Furthermore, high frequency of RNF168 c.640_644del5 indicates the need for its testing in Finnish patients with RIDDLE syndrome symptoms.

Breast cancer is strongly influenced by hereditary risk factors, a majority of which still remain unknown. Here, we performed a targeted next-generation sequencing of 796 genes implicated in DNA repair in 189 Finnish breast cancer cases with indication of hereditary disease susceptibility and focused the analysis on protein truncating mutations. A recurrent heterozygous mutation (c.904_916del, p.Arg304ValfsTer3) was identified in early DNA damage response gene, MCPH1, significantly associating with breast cancer susceptibility both in familial (5/145, 3.4%, P = 0.003, OR 8.3) and unselected cases (16/1150, 1.4%, P = 0.016, OR 3.3). A total of 21 mutation positive families were identified, of which one-third exhibited also brain tumors and/or sarcomas (P = 0.0007). Mutation carriers exhibited significant increase in genomic instability assessed by cytogenetic analysis for spontaneous chromosomal rearrangements in peripheral blood lymphocytes (P = 0.0007), suggesting an effect for MCPH1 haploinsufficiency on cancer susceptibility. Furthermore, 40% of the mutation carrier tumors exhibited loss of the wild-type allele. These findings collectively provide strong evidence for MCHP1 being a novel breast cancer susceptibility gene, which warrants further investigations in other populations.

Besides mutations in BRCA1/BRCA2, heterozygous defects in PALB2 are important in breast cancer predisposition. PALB2 heterozygosity increases the risk of malignancy about sixfold. PALB2 interacts with BRCA1 and BRCA2 to regulate homologous recombination and mediate DNA damage response. Here we show, by analysing lymphoblastoid cell lines from heterozygous female PALB2 mutation carriers, that PALB2 haploinsufficiency causes aberrant DNA replication/damage response. Mutation carrier cells show increased origin firing and shorter distance between consecutive replication forks. Carrier cell lines also show elevated ATR protein, but not phosphorylation levels, and a majority of them display aberrant Chk1-/Chk2-mediated DNA damage response. Elevated chromosome instability is observed in primary blood lymphocytes of PALB2 mutation carriers, indicating that the described mechanisms of genome destabilization operate also at the organism level. These findings provide a new mechanism for early stages of breast cancer development that may also apply to other heterozygous homologous recombination signalling pathway gene mutations in hereditary cancer predisposition.

A portion of familial breast cancer cases are caused by mutations in the same genes that are inactivated in the downstream part of Fanconi anemia (FA) signaling pathway. Here we have assessed the FANCA gene for breast cancer susceptibility by examining blood DNA for aberrations from 100 Northern Finnish breast cancer families using the MLPA method. We identified a novel heterozygous deletion, removing the promoter and 12 exons of the gene in one family. This allele was absent from 124 controls. We conclude that FANCA deletions might contribute to breast cancer susceptibility, potentially in combination with other germline mutations. To our knowledge, this is the first study reporting a large deletion in an upstream FA gene in familial breast cancer.

Abstract Abstract 784 Background: In vitro studies have suggested that CML stem cells are resistant to tyrosine kinase inhibitors (TKIs), but in vivo effects in patients have not been prospectively assessed. Furthermore, the inter-individual variation of the leukemic stem cell pool at diagnosis and its possible prognostic value is unknown. Patients: 46 newly diagnosed CML-CP patients were randomized 1:1 to receive either dasatinib 100 mg or imatinib 400 mg QD. The primary endpoint was a comparison of the proportion of Ph+ cells in CD34+CD38− and CD34+CD38+ compartment at 6 months between the study arms. Key secondary endpoints were the fraction of Ph+ cells in the stem cell compartments at 1 and 3 months, and molecular and cytogenetic responses at 3, 6, 12 and 18 months. Experimental endpoints included the percentage of Ph+ cells in the stem cell compartment at diagnosis and its correlation with therapeutic response. Results: One patient in the imatinib arm and none in the dasatinib arm progressed to blast crisis within first 12 months. 4/22 of dasatinib patients have discontinued the treatment due to side-effects (mainly pleural effusion) and 1 patient due to insufficient response. 3/24 imatinib patients have discontinued the therapy (1 blast crisis, 1 side-effects, 1 other malignancy). Early cytogenetic responses were superior in the dasatinib arm: the median percentage of Ph+ cells in the bone marrow was 81% (imatinib) vs. 70% (dasatinib) at 1 month (p=0.15) and 5% vs. 0% at 3 months (p=0.0085). At 12 months all dasatinib (n=20) and 19/20 imatinib patients were in CCyR (results based on patients on treatment at 12 months). MMR rate was significantly higher in the dasatinib arm already at 6 months (70% vs. 20%, p=0.002) and similarly at 9 (75 vs. 26%, p=0.004) and 12 months (88% vs. 40%, p=0.009). Undetectable BCR-ABL1 transcripts (at least CMR4) were observed in 20% of the dasatinib patients at 6 months compared to none in the imatinib arm (p=0.11) and 44% in the dasatinib arm at 12 months compared to 7% in the imatinib arm (p=0.037). The median percentage of Ph+ cells, as measured by FISH (1000 cells analyzed), in the CD34+CD38− fraction at diagnosis was 79% (range 1–100%) compared to 96% (range 50–100%) in CD34+CD38+ fraction. The proportion of Ph+ cells in CD34+CD38− fraction correlated with WBC count (r=0.50, p<0.001), splenomegaly (r=0.43, p=0.0055), anemia (r=-0.44, p=0.004) and blood blast percentage (r=0.57, p=0.0001) at diagnosis. There was also a significant correlation between Ph+ cells in CD34+CD38− fraction at diagnosis and cytogenetic response at 1 (r=0.63, p<0.0001), 3 (r=0.48, p=0.0025) and 6 months (r=0.36, p=0.0271). Furthermore, leukemic stem cell burden at diagnosis correlated significantly with BCR-ABL1 transcript levels at 3 (r=0.54, p=0.0005), 6 (r=0.42, p=0.0088) and 9 months (r=0.40, p=0.0123). All patients who were not in MMR at 18 months, had >79% of Ph+ cells in CD34+CD38− fraction at diagnosis. During TKI therapy, the proportion of Ph+ cells decreased rapidly in the stem cell fractions. At 1 month, the median proportion of Ph+ cells was 14% and 56% in CD34+CD38− and CD34+CD38+ fractions compared to 69% in whole BM (p<0.0001, n=38). At 3 months, the respective numbers were 0.40%, 0.20% and 0.80% (p=0.087, n=33) and at 6 months 0%, 0% and 0.1% (p=0.23, n=41). Dasatinib-treated patients had significantly lower proportion of Ph+ cells in CD34+CD38+ fraction at 3 months than imatinib patients (0.05% vs. 0.68%, p=0.0318). A similar trend was also observed at 1 month (24% vs. 69%, p=0.05), but no difference existed at 6 months. In CD34+CD38− fraction the proportions of Ph+ cells did not differ significantly at 1 (11 vs. 17%, p=0.91), 3 (0.2 vs. 0.3%, p=0.44) or 6 months (0 vs. 0%, p=0.75). Conclusions: In comparison to imatinib, dasatinib induced superior therapeutic responses with remarkably high MMR and CMR rates. This was associated with a faster reduction of Ph+ cells at the progenitor CD34+CD38+ cell level. Surprisingly, both drugs rapidly depleted Ph+ cells from the more primitive CD34+CD38− compartment. The proportion of Ph+ stem cells at diagnosis varied significantly among individual patients and bore prognostic value. Patients with a low proportion of Ph+ leukemic stem cells at diagnosis achieved faster and better cytogenetic and molecular responses. The leukemic stem cell burden at diagnosis may be a biomarker predicting treatment outcome and reflecting key biological factors in CML. Disclosures: Mustjoki: Novartis: Honoraria; Bristol-Myers Squibb: Honoraria. Richter:Novartis: Honoraria; Bristol-Myers Squibb: Honoraria. Dybedal:Novartis: Travel support. Fioretos:Cantargia AB: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Qlucore AB: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Weiss Bjerrum:Novartis: Consultancy; Bristol-Myers Squibb: Consultancy. Simonsson:Novartis, BMS, Merck, Pfizer: Consultancy, Honoraria. Porkka:Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding. Hjorth-Hansen:Novartis: Honoraria; Bristol-Myers Squibb: Honoraria.

Abstract Abstract 667 Background: Targeted tyrosine kinase inhibitor (TKI) therapy efficiently induces rapid hematologic and cytogenetic responses in most CML patients. In vitro studies have suggested that CML stem cells are resistant to TKIs and therefore the treatment may need to be life-long. However, the in vivo effects of TKIs on the leukemic stem cell pool in a patient population have not been prospectively assessed. In addition, the biological impact and prognostic value of leukemic stem cell burden at diagnosis is unknown. Aim: To analyze the proportions of Ph+ cells in the stem cell compartment in newly diagnosed CP CML patients at diagnosis, and correlate the initial leukemic stem cell burden to biological variables and hematological toxicity during first 3 months of TKI therapy. Patients and Methods: 42 newly diagnosed CP CML patients within the Nordic countries were randomized to receive either dasatinib 100 mg (n=21) or imatinib 400 mg (n=21) once daily. Stem cell assays were performed at diagnosis and at 1, 3, and 6 months from start of TKI therapy. After pre-selection of CD34+ cells from large volume bone marrow (BM) aspirates with paramagnetic beads, the CD34+ cells were fractionated into CD38 positive and negative pools using a sorting flow cytometer. The proportion of Ph+ cells in the stem cell fractions was assayed by counting 1000 cells with interphase FISH for BCR-ABL1. Results: Measurement of Ph+ stem cells was feasible in most patients at diagnosis and results from 36 evaluable patients will be presented. The median volume of BM aspirate was 28, 37, 40 and 36 ml at diagnosis, 1, 3, and 6 months after therapy start, respectively. The median yield of BM mononuclear cells was 1000, 110, 93 and 79 ×106, respectively. The median proportion of Ph+ cells was significantly lower in the more primitive CD34+CD38neg fraction when compared to the CD34+CD38+ fraction or to unfractionated BM (79%, range 0.6–100%; 96%, 50–100%; and 96%, 57–100%, respectively, p=0.0001). The proportion of Ph+ cells in the CD34+CD38neg fraction at diagnosis correlated with high leukocyte count (r=0.59, p<0.001), enlarged spleen size (r=0.46, p=0.005), low hemoglobin concentration (r=-0.46, p=0.006) and high blast percentage in peripheral blood (r=0.62, p<0.001). No correlation was found to age, gender, platelets, eosinophils or basophils. The proportion of Ph+ cells in the CD34+CD38+ fraction correlated only to the leukocyte count (r=0.33, p=0.049). Patients with high Sokal risk had a significantly higher proportion of Ph+ CD34+CD38neg stem cells at diagnosis as compared to low and intermediate risk patients (94% vs. 75%, respectively, p=0.036). Patients who had a higher leukemic stem cell burden (more than the median value of 79% of Ph+ CD34+CD38neg cells, Table 1) at diagnosis experienced more grade ≥2 hematological toxicity (neutropenia in particular) during first 3 months of TKI therapy as compared to patients with a lower (<79%) stem cell burden (62% vs. 19%, respectively, p=0.027). Conclusions: The proportion of Ph+ stem cells at the time of diagnosis varied from 1 to 100% between individual CML patients. It was correlated with hemoglobin concentration, leukocyte count, blast percentage and spleen size at diagnosis and with hematological toxicity during early course of treatment, mirroring paucity of healthy hematopoietic stem cell reservoir. The size of the leukemic stem cell pool at diagnosis may be a powerful prognostic marker and a major biological determinant for the high Sokal risk group. The effect of TKI therapy on the malignant stem cell pool size and correlation to therapy responses will be evaluated when all patients have reached the primary study endpoint of 6 months. Disclosures: Mustjoki: BMS, Novartis: Honoraria. Richter:BMS, Novartis: Consultancy, Honoraria. Simonsson:BMS, Novartis: Consultancy, Honoraria, Research Funding. Porkka:BMS, Novartis: Consultancy, Honoraria, Research Funding. Hjorth-Hansen:BMS, Novartis: Consultancy, Honoraria, Research Funding.

To the Editor: Tyrosine kinase inhibitors (TKIs) efficiently induce rapid hematologic and cytogenetic responses in most chronic myeloid leukemia (CML) patients. TKI therapy has been considered to be life long, as rapid leukemia relapses are often observed in patients who discontinue treatment. The cause for relapse is unknown, but has been related to TKI resistance of the leukemic stem cells (LSCs) and/or immune tolerance of CML cells. Accordingly, in vitro experiments have suggested that TKIs have an antiproliferative, but not a proapoptotic or cytotoxic effect on the most primitive CML stem cells (Ph+ CD34+CD38neg cells).1, 2 Second generation TKI dasatinib has been shown to have a more profound effect on the stem cell compartment when compared to imatinib or nilotinib, but the drug was still unable to kill the most primitive CD34+CD38neg LSCs in vitro.2 The causes for stem cell resistance are unclear. In a recent study, primitive leukemic cells had higher BCR-ABL1 and CRKL kinase activity levels when compared with more mature cells. In addition, mRNA expression level of the imatinib uptake pump OCT-1 was lower and the drug efflux transporter protein ABCB1 (MDR) and ABCG2 levels were higher.3 The stem cell persistence has also been implied in previous publications showing small numbers of Ph+ CD34+ progenitor cells in bone marrow (BM) of CML patients even after years on TKI therapy.4, 5 However, recent observations suggest that in a substantial proportion of CML patients who have reached complete molecular remission during TKI treatment, therapy can be discontinued without imminent relapse of the disease.6 The molecular or cellular mechanisms for ‘cure’ are unknown, but either the therapy has eradicated the LSCs or they are under a transcriptional control by the immune system preventing stem cell proliferation and expansion. Thus far there are no reports describing the prevalence of the most primitive CML stem cells (CD34+CD38neg) in patients during TKI therapy. The aim of this project was to analyze the Philadelphia chromosome-positive (Ph+) LSCs in CML patients who have achieved a good response to TKI therapy. A total of 25 chronic phase CML patients were included in the study; 17 patients were treated with imatinib (3 of them had discontinued treatment as per a study protocol), 5 with dasatinib, and 3 with bosutinib. The median time of TKI treatment was 21 months (range 3–72 months, Table 1). The study was conducted in accordance with the principles of the Helsinki declaration and was approved by the Helsinki University Central Hospital Ethics committee. Written informed consents were obtained from all patients as either part of clinical drug studies or separately. Large volume (range 5–55 ml) BM aspirates were collected in EDTA anticoagulated tubes. The mononuclear cells were isolated with Ficoll (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) gradient centrifugation. CD34+ cells were separated from mononuclear cell fraction with paramagnetic beads using AutoMACS separator (Miltenyi Biotech, Bergisch Gladbach, Germany). CD34+ cells were stained with FITC-conjugated CD34 antibody and PE-conjugated CD38 antibody. Cells were sorted in three different fractions based on the expression of CD38 antigen: CD34+CD38high, CD34+CD38med, and CD34+CD38neg (see Figure 1a). The lowest 5% of cells were regarded CD38 negative. Cytospin slides were prepared from sorted cells (15 000–45 000 cells/slide). From a proportion of samples additional staining was carried out with CD19, CD117, CD133, and CD13/33 antibodies to characterize the phenotype of different fractions. All antibodies were purchased from BD Biosciences (San Jose, CA, USA). The FACS sorting and analysis were performed with FACSAria flowcytometer (BD). Interphase FISH analysis was carried out on cytospin slides using locus-specific dual color, dual fusion BCR-ABL1 probe mixture (Vysis, Downers Crove, IL, USA) (Figures 1a and b). Hybridizations were performed according to the manufacturer's instructions. Up to 1000 cells were aimed to be analyzed from every slide. The false positivity rate of the method is considered to be close to 0%. A highly sensitive metaphase fluorescence in situ hybridization (FISH) from a median of 1000 BM cells (instead of conventional G-banding of 20 metaphases) was used to define complete cytogenetic remission (CCyR). Molecular genetic analyses of BCR-ABL1 transcripts were carried out with standardized real-time quantitative PCR and the results were expressed on the international scale. The median volume of collected BM aspirates was 30 ml (range 5–55 ml) with a median yield of 280 × 106 mononuclear cells per sample. The median amount of CD34+CD38neg cells obtained was 32 000 (range 1000–91 000) denoting that 1–3 cytospin slides of the most CD38-negative cell fraction were available for FISH analysis. The median number of interphase nuclei analyzed was 1005 with no difference in the different stem cells fractions (median number of 1004, 1002, and 1010 cells analyzed in CD34+CD38high, CD34+CD38med, and CD34+CD38neg cell fractions, respectively). Although the analysis required large volume BM aspirates, it was feasible with minimal additional patient discomfort and representative samples were obtained from each patient with good quality FISH results (Figures 1a and b). Of the 25 patients studied, 18 were in CCyR when assessed by whole BM metaphase FISH (1000 cells evaluated). Surprisingly, from only 2/18 (11%) patients in CCyR could residual leukemic cells be detected in any of the sorted CD34+ cell fractions and in very small amounts (less than 1% Ph+ cells) (Table 1). In the remaining 16/18 (89%) patients, all the stem cell fractions, including the most primitive CD34+CD38neg cells, were negative for Ph+ cells. In previous publications assessing the entire unfractionated CD34+ stem cell population, Ph+ residual cells could be detected in approximately 50% of CML patients in CCyR, albeit in minute amounts 4, 5. As we used more stringent criteria for CCyR (1000 Ph-negative cells by FISH vs 20 Ph-negative cells by G-banding), differences in patient population may partly account for rarity of residual Ph+ stem cells in our study cohort. The relatively insensitive G-banding technique used in previous studies may miss up to 5–10% of residual disease. However, paucity of LSCs in the most primitive (CD34+CD38neg) fraction in our study was still an unexpected finding. Seven patients in the study cohort were not in CCyR and had detectable Ph+ cells in the non-fractionated BM sample when studied with metaphase FISH (1000 cells counted). Three of them had minimal residual disease only (less than 1% of Ph+ cells, patients 19, 20, and 22, respectively; Table 1). On fractionation, one patient (number 19, Table 1) had Ph+ cells also in CD34+CD38med (0.50%) and CD34+CD38neg (0.19%) fractions, whereas in the other two patients no Ph+ cells could be found, even when up to 1000 cells were analyzed in each case. Four out of seven non-CCyR patients had significant residual disease on sample analysis (5–20% of Ph+ cells in non-fractionated BM sample). All of them had previously been in CCyR, but three had lost CCyR during discontinuation of imatinib treatment (per a study protocol) and one patient developed secondary resistance because of emergence of an imatinib resistant BCR-ABL1 kinase domain mutation L248V (patient 21, Table 1). The proportion of Ph+ cells was decreased in the most primitive CD34+CD38neg cell fraction, in contrast to what was expected based on the in vitro data showing resistance of CD34+CD38neg cells to TKI therapy. Interestingly, patients who had discontinued imatinib treatment and relapsed had markedly lower levels of Ph+ cells in different CD34+ fractions when compared with non-fractionated BM (patients 23–25, see Table 1). This suggests that in the patients relapsing after the cessation of TKI therapy, the transcriptional control of the rare existing LSCs is nonexistent and on drug discontinuation the LSCs proliferate and differentiate rapidly without any control from the immune system producing abundant amount of Ph+ cells in the more mature fractions. In healthy normal individuals, the majority of BM CD34+CD38+ cells are CD19-expressing progenitor B-cells, whereas the CD34+CD38neg cells do not express CD19. In contrast, in untreated CML patients, only a minority of CD34+CD38+ cells express the CD19 antigen (results not shown), reflecting dominance of granulopoiesis typical of the disease. However, earlier publications have shown that in CML, the progenitor B-cells also belong to the malignant Ph+ clone and only mature T cells are Ph negative.7 In the patient cohort from which stem cell sorting was carried out (CML patients in CCyR), CD34+ cells expressing the highest CD38 antigen level (Figures 1a and c) were predominantly CD19 positive, thus resembling the situation seen in normal healthy BM. The CD34+ cells with low CD38 antigen level expressed CD117 (C-KIT) and CD133 (Figures 1a and c) confirming their more primitive nature. Our data of chronic phase CML patients indicated that TKI therapy eradicates most Ph+ CD34+ progenitor cells when analyzed from standard clinical BM aspirates. Surprisingly, residual LSCs could not be detected even in the most primitive CD34+CD38neg cell fraction in vivo. On the basis of the estimates of stem cell frequency, our high-resolution FISH method is adequately sensitive and comparable to real-time quantitative PCR. Increasing the sensitivity of the assay (FISH or PCR) would require aspiration of impractical amounts of BM. Furthermore, FISH analysis gives more precise estimate of the actual number of residual Ph+ LSCs when compared with PCR as higher BCR-ABL1 transcript level in CD34+CD38neg cells3 and possible amplification of RNA because of small cell number can affect the results of PCR assay. These results differ from the in vitro studies, in which CD34+CD38neg cells have been shown to be resistant to TKIs.1, 2 However, recent observations imply that TKI therapy can be discontinued in a proportion of patients in complete molecular remission.6 Our data suggest mechanistic explanation to this observation and support the theory that in vivo, TKIs eradicate majority of LSCs and therefore for successful imatinib discontinuation the restoration of anti-CML immune control is likely a more determining factor than the level of residual LSCs. The difference between in vivo and in vitro settings could partly be becuse of non-physiological conditions (growth factor sensitivity, other cytokines) in cell culture assays. Some residual Ph+cells can exist, but our observations indicate that they are very rare and not enriched in the most primitive CD34+CD38neg fraction. Further, recent mouse studies have shown that both normal and LSCs reside in the hypoxic endosteal niche separate from the medullary central BM.8 The fact that matrix-bound subendosteal cells are rarely, if ever, aspirated with conventional BM puncture methods, may partly explain the paucity of LSCs in our patient samples. By definition, cure of leukemia requires eradication or transcriptional control of LSCs. This can be achieved by high-dose chemotherapy in a proportion of patients with acute leukemia, but with significant therapy-related morbidity and mortality. The successful therapy outcome can also be affected by the number of LSCs at diagnosis and based on our unpublished data, the proportion of Ph+ CD34 cells vary significantly (median 92%, range 52–99%, n=9) between different CML patients at diagnosis. Recently, several agents, such as a farnesyltransferase inhibitor (BMS 214662)9 or parthenolide,10 have been shown to selectively target and kill CML and acute myeloid leukemia LSCs in vitro, respectively. Future clinical studies with these novel stem cell targeting drugs will necessitate accurate assessment of LSC burden in patients. LSC analysis may also have prognostic significance in evaluating the response to cytotoxic therapy in acute leukemia. However, novel technical innovations and techniques for assessing the BM microenvironment as a whole, such as tracking LSCs in vivo with high-resolution positron emission tomography, would be most useful to make response evaluation more comprehensive. In conclusion, in chronic phase CML, successful TKI therapy eradicates most Ph+CD34+CD38neg cells from the central BM. Prospective studies analyzing the kinetics of LSC disappearance after the start of TKI therapy will shed more light on the depth of the therapy response at the stem cell level. On the basis of our data, significant reduction in the number of LSCs by TKI therapy is feasible and when accompanied with restoration of anti-CML immune control, can explain the prolonged remissions observed in a proportion of patients discontinuing TKI therapy. S Mustjoki has received honoraria from Bristol-Myers Squibb. K Porkka has received research funding and honoraria from Novartis and Bristol-Myers Squibb. The other authors have no financial relationships to disclose. This work was supported by the Finnish special governmental subsidy for health sciences, research, and training, by the Finnish Cancer Societies, Emil Aaltonen Foundation, Academy of Finland, Finnish Medical Foundation, Blood Disease Foundation, Biomedicum Helsinki Foundation, Gyllenberg Foundation, IGA NS 9949-3 and KA Johansson foundation. The authors would like to thank the personnel at the Hematology Research Unit at Helsinki and flow cytometry laboratory, HUSLAB for their expert technical assistance.

Targeted tyrosine kinase inhibitors (TKIs) efficiently induce rapid hematologic and cytogenetic remission in most chronic myeloid leukemia (CML) patients. However, in vitro experiments have suggested that the most primitive CML stem cells residing in the CD34posCD38neg fraction are relatively resistant to TKIs. The prevalence of these stem cells in vivo in patients under TKI therapy is unclear. The aim of this project was to analyze the effect of TKI therapy on Ph+ leukemia stem cell pool in patients and to analyze the proportion of Ph+ cells in different stem cell fractions. A total of 26 chronic phase CML patients were included in the study. 18 patients were treated with imatinib, 5 with dasatinib, and 3 with bosutinib. The median time of TKI treatment was 20 months (range 3–72 months). Large volume (median 30 ml, range 5–55 ml) of bone marrow (BM) aspirate was collected and mononuclear cells (MNC) were isolated. CD34pos cells were separated with paramagnetic beads and further sorted into CD34posCD38pos and CD34posCD38neg cell populations with multicolor flow cytometry in order to analyze progenitor cell fractions of different maturation stage. Proportion of Ph+ cells was determined with interphase FISH by counting 1000 cells in each fraction. The median yield of MNCs from 30 ml of BM aspirate was 280x106 cells resulting in a median of 32 000 CD34posCD38neg cells (range 1000–91000). High-sensitivity counting of the proportion of Ph+ cells was feasible with a median number of counted interphase nuclei of 1005. During TKI therapy the CD34pos cells expressing highest CD38 antigen level were already mostly differentiated into B-cell lineage (CD19 positive). The CD34pos cells expressing low CD38 antigen levels expressed markers of more primitive cells such as C-kit (CD117) and CD133. Of 26 patients with CML, 19 were in complete cytogenetic remission (CCyR) when assessed by metaphase FISH of non-fractionated BM cells (1000 cells analyzed). Only 3 patients had single Ph+ cells in CD34pos cell fractions (less than 1%). In remainder of patients, all progenitor cell fractions, including the most primitive CD34posCD38neg cells, were negative for Ph+ cells. 3 patients had 0–1% of Ph+ cells in non-fractionated BM sample. One of them had 0.2% of Ph+ cells in CD34posCD38neg fraction, but the other 2 patients had 0/1000 Ph+ stem cells. 4 patients had a partial cytogenetic response (5–20% of Ph+ cells in non-fractionated BM sample). Again, the proportion of Ph+ cells was not increased in the most primitive CD34posCD38neg cell fraction. Interestingly, patients who had discontinued imatinib treatment had lower level of Ph+ cells in different CD34pos fractions (median 0.1%) when compared to non-fractionated BM (median 9.3%). Based on our data, in chronic phase CML patients, TKI therapy eradicates most Ph+ CD34pos progenitor cells. Unexpectedly, leukemic stem cells were not enriched in the most primitive CD34posCD38neg cell fraction in vivo. These results differ from the in vitro studies, where CD34posCD38neg cells have been shown to be resistant to TKIs. This could be due to non-physiological conditions (growth factor sensitivity, other cytokines) in cell culture assays. In addition, leukemic stem cells in vivo may be located in the subcortical hypoxic stem cell niche in the BM and are less likely to be aspirated. Our data underline the tremendous proliferative potential of very rare stem cells in CML patients in CCyR, as is evident after discontinuation of TKI therapy. Future studies evaluating the kinetics of disappearance of Ph+ cells from stem cell fractions during TKI therapy and the location of residual Ph+ stem cells in the BM are warranted and may give important information on the depth of the therapy response. Furthermore, this knowledge may aid in targeting therapy to these cells and finding curative treatment strategies in CML.

Biallelic mutations in the ataxia-telangiectasia mutated (ATM) gene result in ataxia-telangiectasia (A-T). Studies on A-T families have shown that obligate female carriers have increased risk of developing breast cancer. Here we have evaluated the role of known Finnish ATM germ line mutations as possible breast cancer predisposing alleles outside A-T families by analyzing their prevalence in large cohorts of familial and unselected breast cancer cases. Of seven different alterations, two were observed in the studied breast cancer material. ATM 6903insA (causing protein truncation) was seen in 3/541 familial and 5/1124 unselected cases, but not among healthy population controls (0/1107). 7570G>C (Ala2524Pro) occurred in 1/541 familial and 2/1124 unselected cases compared with 1/1107 in controls. Additionally, 8734A>G (Arg2912Gly) associated previously with breast cancer susceptibility and suggested to be causative also for A-T was detected in 2/541 of familial cases, but not in unselected cases (0/1124) or controls (0/1107). In total, heterozygous ATM mutation carriers were observed in 6/541 familial [P = 0.006, odds ratio (OR) 12.4, 95% confidence interval (CI) 1.5-103.3) and 7/1124 unselected cases (P = 0.07, OR 6.9, 95% CI 0.9-56.4), compared with 1/1107 in controls, suggesting an apparent yet overall limited contribution to predisposition to cancer. The current results also provided evidence for founder effects in the geographical distribution of these mutations. Interestingly, results from functional analysis of the breast cancer-associated ATM mutations indicated that cancer susceptibility is not restricted to mutations with dominant-negative effect on kinase activity, displayed only by 7570G>C, whereas 8734A>G showed only a partial defect in the phosphorylation of ATM substrates, and 6903insA seemed to be a null allele.

OBJECTIVE:The purpose of this study was to compare the efficacy of both separate and combined maternal serum testing and fetal nuchal translucency measurement in the first trimester screening for Down syndrome in northern Finland. STUDY DESIGN:The following screening tests were evaluated: measurement of nuchal translucency (NT) alone; serum screening (pregnancy-associated plasma protein A [PAPP-A] and free beta subunit of human chorionic gonadotropin [beta-hCG]) alone; and combined screening (NT plus PAPP-A and free beta-hCG). RESULTS:The participants comprised 7534 pregnant women during the 10+0-12+6 weeks of pregnancy. All 7534 women participated in serum screening, and 4765 women participated in combined screening. In the serum screening-alone group, there were 30 cases of trisomy 21, of which 23 (76%) were detected. In the combined-screening group, there were 24 cases of trisomy 21 and 21 (87.5%) were detected. In the combined-screening group NT alone detected 15 cases of Down syndrome (62%). CONCLUSION:Combined screening is the method of choice for Down syndrome screening.

Breast cancer BRCA1 , BRCA2 and other established susceptibility genes account for less than half of the known hereditary predisposition to breast cancer. So there are other relevant genes to be discovered. A new study in a Finnish population has identified a recurrent mutation in the PALB2 gene that is associated with increased breast cancer risk. PALB2 acts with BRCA2 in DNA repair, and the newly identified mutations impair this function.

The conserved TP53-binding protein 1 (53BP1) is a central mediator of the DNA damage checkpoint and appears to be one of the sensors of DNA double-strand breaks (DSBs). Improper processing of DSBs can result in loss or rearrangement of genetic information, leading to cell death or tumorigenesis. 53BP1 interacts with both TP53 and ATM, key proteins involved in the monitoring of genomic integrity and regulation of apoptosis. 53BP1 is also required for the formation of BRCA1 foci and the C-terminal part of these two proteins display significant homology. Based on its biological function, the 53BP1 gene is a good candidate for being involved in cancer susceptibility. Consequently, in the current study patients belonging to 126 breast and/or ovarian cancer families were screened for germline mutations in the entire coding region of the 53BP1 gene. A number of sequence variants were found, but none of them appeared to associate with cancer predisposition. To our knowledge this is the first comprehensive screening of 53BP1 mutations in familial breast and ovarian cancer cases.

Together, germline mutations in the two major susceptibility genes BRCA1 and BRCA2 account for approximately 20-30% and 70-80% of the familial breast and ovarian cancer cases, respectively. This indicates involvement of additional susceptibility genes, perhaps in combination with a polygenic effect. However, it is also possible that part of the mutations disrupting BRCA1 and BRCA2 function still remains to be discovered. In response to double-strand DNA damage the co-operation between RAD51 and BRCA2 is of great importance, and the conserved BRC repeat motifs in BRCA2 are crucial for this interaction. In the current study, patients belonging to 126 breast and/or ovarian cancer families were screened for RAD51 and BRCA2 BRC repeat mutations in order to uncover aberrations that may contribute to hereditary cancer susceptibility. The performed study revealed several novel alterations, however, none of them appeared to be disease-related. Thus, it seems likely that germline mutations in the highly conserved RAD51 gene are extremely rare and generally poorly tolerated.

Besides BRCA1 and BRCA2 other genes are also likely to be involved in hereditary predisposition to breast and/or ovarian cancer. TopBP1 (topoisomerase IIbeta binding protein 1) displays sequence homology as well as functional similarities with BRCA1, and the two proteins have been suggested to function partly in the same cellular processes. TopBP1 is crucial for DNA damage and replication checkpoint controls. Based on its biological significance, we reasoned that TopBP1 is a plausible susceptibility gene for hereditary breast and/or ovarian cancer and therefore screened affected index cases from 125 Finnish cancer families for germline changes by conformation sensitive gel electrophoresis (CSGE). Altogether 19 different sequence alterations were detected. A novel heterozygous Arg309Cys variant was observed at elevated frequency in the familial cancer cases compared to healthy controls (15.2% versus 7.0%; P=0.002). Current results suggest that Arg309Cys is a commonly occurring germline alteration possibly associated with a slightly increased breast and/or ovarian cancer risk. This is the first study reporting mutation screening of the TopBP1 gene in a familial cancer material.

The Mre11 complex, composed of RAD50, NBS1 and MRE11, has an essential role in the maintenance of genomic integrity and preventing cells from malignancy. Here we report the association of three Mre11 complex mutations with hereditary breast cancer susceptibility, studied by using a case-control design with 317 consecutive, newly diagnosed Northern Finnish breast cancer patients and 1000 geographically matched healthy controls (P = 0.0004). RAD50 687delT displayed significantly elevated frequency in the studied patients (8 out of 317, OR 4.3, 95% CI 1.5-12.5, P = 0.008), which indicates that it is a relatively common low-penetrance risk allele in this cohort. Haplotype analysis and the screening of altogether 512 additional breast cancer cases from Sweden, Norway and Iceland suggest that RAD50 687delT is a Finnish founder mutation, not present in the other Nordic cohorts. The RAD50 IVS3-1G > A splicing mutation leading to translational frameshift was observed in one patient, and the NBS1 Leu150Phe missense mutation affecting a conserved residue in the functionally important BRCA1 carboxyterminal (BRCT) domain in two patients, both being absent from 1000 controls. Microsatellite marker analysis showed that loss of the wild-type allele was not involved in the tumorigenesis in any of the studied mutation carriers, but they all showed increased genomic instability assessed by cytogenetic analysis of peripheral blood T-lymphocytes (P = 0.006). In particular, the total number of chromosomal rearrangements was significantly increased (P = 0.002). These findings suggest an effect for RAD50 and NBS1 haploinsufficiency on genomic integrity and susceptibility to cancer.

INTRODUCTION:Mutations in BRCA1, BRCA2, ATM, TP53, CHK2 and PTEN account for only 20-30% of the familial aggregation of breast cancer, which suggests the involvement of additional susceptibility genes. The ATR (ataxia-telangiectasia- and Rad3-related) kinase is essential for the maintenance of genomic integrity. It functions both in parallel and cooperatively with ATM, but whereas ATM is primarily activated by DNA double-strand breaks induced by ionizing radiation, ATR has been shown to respond to a much broader range of DNA damage. Upon activation, ATR phosphorylates several important tumor suppressors, including p53, BRCA1 and CHK1. Based on its central function in the DNA damage response, ATR is a plausible candidate gene for susceptibility to cancer.METHODS:We screened the entire coding region of the ATR gene for mutations in affected index cases from 126 Finnish families with breast and/or ovarian cancer, 75 of which were classified as high-risk and 51 as moderate-risk families, by using conformation sensitive gel electrophoresis and direct sequencing.RESULTS:A large number of novel sequence variants were identified, four of which -- Glu254Gly, Ser1142Gly, IVS24-48G>A and IVS26+15C>T -- were absent from the tested control individuals (n = 300). However, the segregation of these mutations with the cancer phenotype could not be confirmed, partly because of the lack of suitable DNA samples.CONCLUSION:The present study does not support a major role for ATR mutations in hereditary susceptibility to breast and ovarian cancer.