BACKGROUND: Fanconi anemia (FA) is a genetic disorder characterized by bone marrow failure typically developing in the first decade of life, congenital abnormalities, and an increased predisposition to malignancy. However, patients with FA can remain undiagnosed until adulthood and present with solid organ malignancies. Due to impaired DNA repair mechanisms, patients with FA are highly susceptible to severe bone marrow toxicity when treated with cisplatin.
METHODS: A 38-year-old woman, diagnosed with locally advanced squamous cell carcinoma (SCC) of the uterine cervix, underwent treatment with weekly cisplatin concurrent with radiotherapy. After the second week of cisplatin treatment, she presented with severe pancytopenia. The prolonged and severe pancytopenia following cisplatin and radiation, along with cervical SCC in the absence of risk factors and the presence of parental consanguinity, raised the possibility of FA as the underlying cause. Whole exome sequencing revealed a homozygous FANCI c.668A > C (p.Lys223Thr) missense variant confirming the diagnosis of FA.
METHODS: The pancytopenia exhibited a protracted course, necessitating admission and supportive treatment with antibiotics, red blood cell and platelet transfusions, as well as filgrastim and eltrombopag. Eventually, the pancytopenia improved after approximately 40 days of hospitalization.
CONCLUSIONS: SCC of the head and neck or gynecologic organs in a young adult without known risk factors should prompt consideration of FA. Cisplatin should be avoided in patients with FA.

BACKGROUND: Sn1-type alkylating agents methylate the oxygen atom on guanine bases thereby producing O6-methylguanine. This modified base could pair with thymine and cytosine, resulting in the formation of O6-methylguanine/thymine mismatch during DNA replication, recognized by the mismatch repair (MMR) complex, which then initiates the DNA damage response and subsequent apoptotic processes. In our investigation of the molecular mechanisms underlying MMR-dependent apoptosis, we observed FANCD2 modification upon the activity of alkylating agent N-methyl-N-nitrosourea (MNU). This observation led us to hypothesize a relevant role for FANCD2 in the apoptosis induction process.
RESULTS: We generated FANCD2 knockout cells using the CRISPR/Cas9 method in the human cervical cancer cell line HeLa MR. FANCD2-deficient cells exhibited MNU hypersensitivity. Upon MNU exposure, FANCD2 colocalized with the MMR complex. MNU-treated FANCD2 knockout cells displayed severe S phase delay followed by increased G2/M arrest and MMR-dependent apoptotic cell death. Moreover, FANCD2 knockout cells exhibited impaired CtIP and RAD51 recruitment to the damaged chromatin and DNA double-strand break accumulation, indicated by simultaneously observed increased γH2AX signal and 53BP1 foci.
CONCLUSIONS: Our data suggest that FANCD2 is crucial for recruiting homologous recombination factors to the sites of the MMR-dependent replication stress to resolve the arrested replication fork and counteract O6-methylguanine-triggered MMR-dependent apoptosis.

SERS (Surface Enhanced Raman Spectroscopy) is a new Raman spectroscopy which relies on Surface Plasmon Resonance (SPR) of metal nanoparticles. We have applied colloidal silver and gold nanoparticles as amplifier agents to enhance nucleotide Raman signals. It is observed that without these enhancing agents, it is impossible to investigate nucleotide spectrum due to weak Raman signals. Interaction mechanism of Melphalan, an anticancer drug with four nucleotides (Adenine, Cytosine, Guanine, Thymine) was investigated using SERS to detect and identify changes due to alkylating process in Raman spectra. After incubating Melphalan drug with nucleotides for 24 h at 37 °C, some changes occurred in SERS spectrum and interpretation of SERS spectra revealed the influence of the alkyl substitution on peaks and Raman shifts. After incubation of Melphalan with each nucleotide, intensity of relevant SERS signals assigned to Amid III group of Cytosine and Amid I of Thymine decreased significantly, confirming alkylating taking place. In this study, we also investigated the effect of nanoparticles type on nucleotide spectrum. We could not obtain useful information in the cases of guanine nucleotide. The SERS spectrum of Cytosine as an example of nucleotides in aqueous solution compared to solid state and results demonstrated that in solid state better signals were obtained than in liquid state.

UNASSIGNED: Medulloblastoma is the most common malignant brain tumor in children, often requiring intensive multimodal therapy, including chemotherapy with alkylating agents. However, therapy-related complications, such as therapy-related myeloid neoplasms (t-MNs), can arise, particularly in patients with genetic predisposition syndromes. This case report presents three pediatric cases of medulloblastoma with subsequent development of t-MNs, highlighting the potential role of genetic predisposition and the importance of surveillance for hematological abnormalities in long-term survivors.
UNASSIGNED: We describe three cases of pediatric medulloblastoma who developed t-MNs after receiving chemotherapy, including alkylating agents. Two of the patients had underlying genetic predisposition syndromes (TP53 pathologic variants). The latency period between initial diagnosis of medulloblastoma and the development of secondary cancer varied among the cases, ranging from 17 to 65 months. The three cases eventually succumbed from secondary malignancy, therapy-related complications and progression of primary disease, respectively.
UNASSIGNED: This report highlights the potential association between genetic predisposition syndromes and the development of therapy-related myeloid neoplasms in pediatric medulloblastoma survivors. It underscores the importance of surveillance for hematological abnormalities among such patients.

An efficient and convenient method for the synthesis of 1-hydroxyalkylphosphonium salts is described. Reactions were carried out at room temperature, in a short time, and without chromatography for product isolation. The properties of the obtained phosphonium salts were examined and discussed. In this paper, primary attention was paid to the stability of phosphonium salts, depending on the structure of the aldehydes used as substrates in their preparation. Other conditions such as the type of solvent, temperature, and molar ratio of the substrates were also investigated. Finally, the high reactivity of 1-hydroxyalkylphosphonium salts was demonstrated in reactions with amide-type substrates and (hetero)aromatic compounds. The developed step-by-step procedure (with the isolation of 1-hydroxyphosphonium salts) was compared to the one-pot protocol (in situ formation of such phosphonium salts).

BACKGROUND: The cell cycle is tightly regulated by checkpoints, which play a vital role in controlling its progression and timing. Cancer cells exploit the G2/M checkpoint, which serves as a resistance mechanism against genotoxic anticancer treatments, allowing for DNA repair prior to cell division. Manipulating cell cycle timing has emerged as a potential strategy to augment the effectiveness of DNA damage-based therapies.
METHODS: In this study, we conducted a forward genome-wide CRISPR/Cas9 screening with repeated exposure to the alkylating agent temozolomide (TMZ) to investigate the mechanisms underlying tumor cell survival under genotoxic stress.
RESULTS: Our findings revealed that canonical DNA repair pathways, including the Ataxia-telangiectasia mutated (ATM)/Fanconi and mismatch repair, determine cell fate under genotoxic stress. Notably, we identified the critical role of PKMYT1, in ensuring cell survival. Depletion of PKMYT1 led to overwhelming TMZ-induced cytotoxicity in cancer cells. Isobologram analysis demonstrated potent drug synergy between alkylating agents and a Myt1 kinase inhibitor, RP-6306. Mechanistically, inhibiting Myt1 forced G2/M-arrested cells into an unscheduled transition to the mitotic phase without complete resolution of DNA damage. This forced entry into mitosis, along with persistent DNA damage, resulted in severe mitotic abnormalities. Ultimately, these aberrations led to mitotic exit with substantial apoptosis. Preclinical animal studies demonstrated that the combination regimen involving TMZ and RP-6306 prolonged the overall survival of glioma-bearing mice.
CONCLUSIONS: Collectively, our findings highlight the potential of targeting cell cycle timing through Myt1 inhibition as an effective strategy to enhance the efficacy of current standard cancer therapies, potentially leading to improved disease outcomes.

Combinations of nucleoside analog(s) and DNA alkylating agent(s) are used for cancer treatment as components of pre-transplant regimens used in hematopoietic stem cell transplantation. Their efficacies are enhanced by combining drugs with different mechanisms of action, which also allows a reduction in the individual drug dosages and thus potentially in toxicity to the patient. We hypothesized that addition of SAHA and olaparib, an HDAC- and a PARP-inhibitor, respectively, to the established combination of fludarabine, clofarabine and busulfan would enhance AML cell cytotoxicity. Exposure of the AML cell lines KBM3/Bu2506, MV4-11, MOLM14 and OCI-AML3 to the 5-drug combination resulted in synergistic cytotoxicity with combination indexes < 1. Increased protein acetylation and decreased poly(ADP-ribosyl)ation were observed, as expected. Activation of apoptosis was suggested by cleavage of Caspase 3 and PARP1, DNA fragmentation, increased reactive oxygen species, and decreased mitochondrial membrane potential. The reduction in poly(ADP-ribosyl)ation was independent of caspase activation. Several proteins involved in DNA damage response and repair were downregulated, which may be contributing factors for the observed synergism. The increased phosphorylation of DNAPKcs suggests inhibition of its kinase activity and diminution of its role in DNA repair. A similar synergism was observed in patient-derived cell samples. These findings will be important in designing clinical trials using these drug combinations as pre-transplant conditioning regimens for AML patients.

Melphalan flufenamide (melflufen), a first-in-class alkylating peptide-drug conjugate, plus dexamethasone demonstrated superior progression-free survival (PFS), but not overall survival (OS), versus pomalidomide plus dexamethasone in relapsed/refractory multiple myeloma in the OCEAN study. Time to progression (TTP) <36 months after a prior autologous stem cell transplantation (ASCT) was a negative prognostic factor for OS with melflufen. This post hoc exploratory analysis evaluated patients refractory to prior alkylators (e.g., cyclophosphamide and melphalan) in OCEAN. In 153 patients refractory to prior alkylators (melflufen, n = 78; pomalidomide, n = 75), the melflufen and pomalidomide arms had similar median PFS (5.6 months [95% CI, 4.2-8.3] vs. 4.7 months [95% CI, 3.1-7.3]; hazard ratio [HR], 0.92 [95% CI, 0.63-1.33]) and OS (23.4 months [95% CI, 14.4-31.7] vs. 20.0 months [95% CI, 12.0-28.7]; HR, 0.92 [95% CI, 0.62-1.38]). Among alkylator-refractory patients with a TTP ≥ 36 months after a prior ASCT or no prior ASCT (melflufen, n = 54; pomalidomide, n = 53), the observed median PFS and OS were longer in the melflufen arm than the pomalidomide arm. The safety profile of melflufen was consistent with previous reports. These results suggest that melflufen is safe and effective in patients with alkylator-refractory disease, suggesting differentiated activity from other alkylators.

Ifosfamide, which is widely used as a chemotherapeutic agent in various kinds of malignancies, sometimes causes neurotoxicity known as ifosfamide-induced encephalopathy (IIE). Herein, we report the case of a three-year-old girl who developed IIE during chemotherapy for Ewing\'s sarcoma and was treated with methylene blue as a prophylactic agent for IIE, after which she continued with ifosfamide and completed treatment without IIE recurrence. This case suggests that methylene blue may be effective in preventing the recurrence of IIE in pediatric patients. Further studies, including clinical trials, are needed to confirm the efficacy and safety of methylene blue in pediatric patients.

BACKGROUND: A growing number of centers worldwide are preserving testicular tissue (TT) of young boys at risk of fertility loss to preserve their fertility. Data in this regard are scarce and experience sharing is essential to the optimization of the process.
OBJECTIVE: This report of our 10-year activity of pediatric fertility preservation (FP) has the objective to (1) improve knowledge regarding the feasibility, acceptability, safety, and potential usefulness of the procedure; (2) analyze the impact of chemotherapy on spermatogonia in the cryopreserved TT.
METHODS: For this retrospective study of data prospectively recorded, we included all boys under 18 years of age referred to the FP consultation of our academic network between October 2009 and December 2019. Characteristics of patients and cryopreservation of testicular tissue (CTT) were extracted from the clinical database. Univariate and multivariate analyses were used to assess factors associated with the risk of absence of spermatogonia in the TT.
RESULTS: Three hundred and sixty-nine patients (7.2 years; 0.5-17.0) were referred to the FP consultation for malignant (70%) or non-malignant (30%) disease, of whom 88% were candidates for CTT, after a previous chemotherapy exposure (78%). The rate of recorded immediate adverse events was 3.5%, with painful episodes dominating. Spermatogonia were detected in the majority of TTs: 91.1% of those exposed to chemotherapy and 92.3% of those not exposed (p = 0.962). In multivariate analysis, the risk of absence of spermatogonia was almost three-fold higher in boys > 10 years of age ([OR] 2.74, 95% CI 1.09-7.26, p = 0.035) and four-fold higher in boys exposed to alkylating agents prior to CTT ([OR] 4.09, 95% CI 1.32-17.94, p = 0.028).
CONCLUSIONS: This large series of pediatric FP shows that this procedure is well accepted, feasible, and safe in the short term, strengthening its place in the clinical care pathway of young patients requiring a highly gonadotoxic treatment. Our results demonstrate that CTT post-chemotherapy does not impair the chance to preserve spermatogonia in the TT except when the treatment includes alkylating agents. More data on post-CTT follow-up are still required to ensure the long-term safety and usefulness of the procedure.